CIRCULAR RNA COMPOSITIONS AND METHODS

Abstract

Circular RNA, along with related compositions and methods are described herein. In some embodiments, the inventive circular RNA comprises post splicing group I in iron fragments, spacers, an IRES, optional duplex forming regions, and more than one expression sequence. In some embodiments, the expression sequences are separated by one or more polynucleotide sequences encoding a cleavage site. In some embodiments, circular RNA of the invention has improved expression, functional stability, immunogenicity, ease of manufacturing, and/or half-life when compared to linear RNA. In some embodiments, inventive methods and constructs result in improved circularization efficiency, splicing efficiency, and/or purity when compared to existing RNA circularization approaches.

Claims

1-126. (canceled)

127. A circular RNA polynucleotide comprising, in the following order, a post splicing 3 group I intron fragment, a first Internal Ribosome Entry Site (IRES), a first expression sequence, a second expression sequence, and a post splicing 5 group I intron fragment.

128. The circular RNA polynucleotide of claim 127, further comprising a second IRES between the first expression sequence and the second expression sequence.

129. The circular RNA polynucleotide of claim 127, further comprising a cleavage site between the first expression sequence and the second expression sequence.

130. The circular RNA polynucleotide of claim 129, wherein the cleavage site is a self-cleaving spacer or a 2A self-cleaving peptide.

131. The circular RNA polynucleotide of claim 127, wherein the first IRES and/or the second IRES consists of or comprises a sequence according to any of SEQ ID NO: 1-72.

132. The circular RNA polynucleotide of claim 127, wherein the first expression sequence encodes a first therapeutic protein, and the second expression sequence encodes a second therapeutic protein.

133. The circular RNA polynucleotide of claim 132, wherein the first expression sequence or the second expression sequence encodes a chimeric antigen receptor (CAR), an antibody, a transcription factor, a cytokine, an immune inhibitory molecule, an agonist of a costimulatory molecule, or an inhibitor of an immune checkpoint molecule.

134. The circular RNA polynucleotide of claim 127, wherein: a. the first expression sequence encodes an alpha chain of a T cell receptor (TCR) and the second expression sequence encodes a beta chain of a TCR; b. the first expression sequence encodes a beta chain of a TCR and the second expression sequence encodes an alpha chain of a TCR; c. the first expression sequence encodes a gamma chain of a TCR and the second expression sequence encodes a delta chain of a TCR; d. the first expression sequence encodes a delta chain of a TCR and the second expression sequence encodes a gamma chain of a TCR; e. the first expression sequence encodes a TCR, and the second expression sequence encodes a cytokine; or f. the first expression sequence encodes a cytokine, and the second expression sequence encodes a TCR; g. the first expression sequence encodes a TCR, and the second expression sequence encodes a chemokine; h. the first expression sequence encodes for a chemokine, and the second expression sequence encodes for a TCR; i. the first expression sequence encodes for a TCR, and the second expression sequence encodes for a transcription factor; j. the first expression sequence encodes for a transcription factor, and the second expression sequence encodes for a TCR; k. the first expression sequence encodes a CAR, and the second expression sequence encodes a PD1 or PDL1 antagonist; l. the first expression sequence encodes a PD1 or PDL1 antagonist and the second expression sequence encodes a CAR; m. the first expression sequence encodes a CAR, and the second expression sequence encodes a cytokine; n. the first expression sequence encodes a cytokine, and the second expression sequence encodes a CAR; o. the first expression sequence encodes for a CAR, and the second expression sequence encodes a chemokine; p. the first expression sequence encodes for a chemokine, and the second expression sequence encodes for a CAR; q. the first expression sequence encodes a transcription factor, and the second expression sequence encodes a cytokine; r. the first expression sequence encodes a cytokine, and the second expression sequence encodes a transcription factor; s. the first expression sequence encodes a transcription factor, and the second expression sequence encodes a chemokine; or t. the first expression sequence encodes a chemokine, and the second expression sequence encodes a transcription factor.

135. The circular RNA polynucleotide of claim 134, wherein: a. the cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-10, IL-12, and TGF?; b. the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and CD25; and/or c. the chemokine is selected from a CC chemokine, CXC chemokine, C chemokine, CX3C chemokine, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXLC6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1.

136. The circular RNA polynucleotide of claim 127, wherein: a. the first expression sequence encodes a tumor antigen, and the second expression sequence encodes a cytokine; b. the first expression sequence encodes a cytokine, and the second expression sequence encodes a tumor antigen; c. the first expression sequence encodes a CAR, and the second expression sequence encodes a CAR; d. the first expression sequence encodes a cytokine, and the second expression sequence encodes a cytokine; e. the first expression sequence encodes a TCR, and the second expression sequence encodes a TCR; f. first expression sequence encodes for a chemokine, and the second expression sequence encodes for a chemokine; g. the first expression sequence or second expression sequence encodes for an immunosuppressive enzyme; h. the first expression sequence encodes a rate-limiting enzyme, and the second expression sequence encodes a flux-limiting enzyme; i. the first expression sequence encodes a flux-limiting enzyme, and the second expression sequence encodes a rate-limiting enzyme; j. the first expression sequence encodes a transcription factor, and the second expression sequence encodes a survival factor; k. the first expression sequence encodes a survival factor, and the second expression sequence encodes a transcription factor; l. the first expression sequence or second expression sequence encodes a chaperone protein or complex; m. the first expression sequence encodes a transcription factor, and the second expression sequence encodes a chaperone protein or complex; n. the first expression sequence encodes a chaperone protein or complex, and the second expression sequence encodes for a transcription factor; o. the first expression sequence and/or second expression sequences each independently encode a signaling protein; p. the first expression sequence encodes for an enzyme, and the second expression encodes for a negative regulatory inhibitor of the first expression sequence; q. the first expression sequence encodes for a negative regulatory inhibitor protein of an enzyme encoded in the second expression sequence; r. the first expression sequence encodes a dominant negative protein, and the second expression sequence encodes an immune protein; s. the first expression sequence encodes an immune protein, and the second expression sequence encodes a dominant negative protein; t. the first expression sequence or second expression sequence encodes an anti-inflammatory protein; u. the first expression sequence encodes a transcription factor, and the second expression sequence is capable of converting 5-fluorocytosinde (5-FC) into 5-fluorouracil (5-FU); or v. the first expression sequence is capable of converting 5-FC into 5-FU, and the second expression sequence is a transcription factor.

137. The circular RNA polynucleotide of claim 136, wherein: a. the tumor antigen is a neoantigen; b. the cytokine is IFN?, IL-2, IL-7, IL-15, IL-18, IL-10, TGF?, or IL-35; c. the transcription factor is selected from FOXP3, STAT5B, HELIOS, Tbet, GATA3, RORgt, and CD25; d. the survival factor is BCL-XL; e. the chaperone protein or complex is selected from Skp, Spy, FkpA, SurA, Hsp60, Hsp70, GroEL, GroES, Hsp90, HtpG, Hsp100, ClpA, ClpX, ClpP, and Hsp104; f. the negative regulatory inhibitor is selected from a p57kip2, BAX inhibitor, and TIPE2; and/or g. the expression sequence capable of converting 5-FC into 5-fluorouracil 5-FU is cytosine deaminase.

138. The circular RNA polynucleotide of claim 127, comprising, in the following order, a post splicing 3 group I intron fragment, a 5 duplex forming region, a first spacer, a first IRES, a first expression sequence, a second expression sequence, a second spacer, a 3 duplex forming region, and a post splicing 5 group I intron fragment.

139. The circular RNA polynucleotide of claim 138, wherein the first and second spacers each has a length of about 10 to about 60 nucleotides, or about 9 to about 19 nucleotides.

140. The circular RNA polynucleotide of claim 128, wherein the first IRES or second IRES has a sequence of an IRES from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G.

141. The circular RNA polynucleotide of claim 127, wherein the circular RNA consists of natural nucleotides.

142. The circular RNA polynucleotide of claim 127, wherein the first expression sequence and/or the second expression sequence is codon optimized.

143. The circular RNA polynucleotide of claim 127, wherein the circular RNA is optimized to lack: a. at least one microRNA binding site present in an equivalent pre-optimized polynucleotide; b. at least one endonuclease-susceptible site present in an equivalent pre-optimized polynucleotide; and/or c. at least one RNA-editing-susceptible site present in an equivalent pre-optimized polynucleotide.

144. The circular RNA polynucleotide of claim 127, wherein the circular RNA polynucleotide is from about 100 nucleotides to about 15 kilobases in length.

145. The circular RNA polynucleotide of claim 127, wherein the circular RNA polynucleotide has an in vivo duration of therapeutic effect in humans of at least about 20 hours and/or a functional half-life of at least about 20 hours.

146. The circular RNA polynucleotide of claim 127, wherein the circular RNA polynucleotide has: a. a duration of therapeutic effect in a human cell greater than or equal to that of an equivalent linear RNA polynucleotide comprising the same expression sequence; b. a functional half-life in a human cell greater than or equal to that of an equivalent linear RNA polynucleotide comprising the same expression sequence; c. an in vivo duration of therapeutic effect in humans greater than that of an equivalent linear RNA polynucleotide having the same expression sequence; and/or d. an in vivo functional half-life in humans greater than that of an equivalent linear RNA polynucleotide having the same expression sequence.

147. A pharmaceutical composition comprising a circular RNA polynucleotide of claim 127, and a nanoparticle.

148. The pharmaceutical composition of claim 147, comprising a targeting moiety operably connected to the nanoparticle.

149. The pharmaceutical composition of claim 147, wherein the nanoparticle is a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymer nanoparticle, or a biodegradable polymer nanoparticle.

150. The pharmaceutical composition of claim 148, wherein the targeting moiety mediates receptor-mediated endocytosis or direct fusion into selected cells of a selected cell population or tissue in the absence of cell isolation or purification.

151. The pharmaceutical composition of claim 148, wherein the targeting moiety is a scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof.

152. The pharmaceutical composition of claim 147, wherein: a. less than 1%, by weight, of the polynucleotides in the pharmaceutical composition are double stranded RNA, DNA splints, or triphosphorylated RNA; and/or b. less than 1%, by weight, of the polynucleotides and proteins in the pharmaceutical composition are double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes.

153. The pharmaceutical composition of claim 147, wherein the nanoparticle comprises: a. one or more cationic lipids, ionizable lipids, or poly ?-amino esters; b. one or more non-cationic lipids; c. one or more PEG-modified, polyglutamic acid lipids, or hyaluronic acid lipids; d. cholesterol; and/or e. arachidonic acid or oleic acid.

154. The pharmaceutical composition of claim 147, wherein the nanoparticle comprises more than one circular RNA polynucleotide.

155. A method of treating a cancer and/or an immune disorder using the RNA polynucleotide of claim 127.

156. The method of claim 155, wherein: a. the cancer is selected from the group consisting of acute lymphocytic leukemia; acute myeloid leukemia (AML); alveolar rhabdomyosarcoma; B cell malignancies; bladder cancer; bone cancer; brain cancer; breast cancer; cancer of the anus, anal canal, or anorectum; cancer of the eye; cancer of the intrahepatic bile duct; cancer of the joints; cancer of the neck; gallbladder cancer; cancer of the pleura; cancer of the nose, nasal cavity, or middle ear; cancer of the oral cavity; cancer of the vulva; chronic lymphocytic leukemia; chronic myeloid cancer; colon cancer; esophageal cancer, cervical cancer; fibrosarcoma; gastrointestinal carcinoid tumor; head and neck cancer; Hodgkin lymphoma; hypopharynx cancer; kidney cancer; larynx cancer; leukemia; liquid tumors; liver cancer; lung cancer; lymphoma; mesothelioma; mastocytoma; melanoma; multiple myeloma; nasopharynx cancer; non-Hodgkin lymphoma; B-chronic lymphocytic leukemia; hairy cell leukemia; acute lymphocytic leukemia (ALL); Burkitt's lymphoma; ovarian cancer; pancreatic cancer; cancer of the peritoneum; cancer of the omentum; mesentery cancer; pharynx cancer; prostate cancer; rectal cancer; renal cancer; skin cancer; small intestine cancer; soft tissue cancer; solid tumors; synovial sarcoma; gastric cancer; testicular cancer; thyroid cancer; and ureter cancer; and/or b. the autoimmune disorder is selected from scleroderma, Grave's disease, Crohn's disease, Sjogren's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyendocrinopathy syndromes, Type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, thyroiditis, and the generalized autoimmune diseases typified by human Lupus.

157. A vector for making a circular RNA polynucleotide, comprising, in the following order, a 3 Group I intron fragment, 5 duplex forming region, an Internal Ribosome Entry Site (IRES), a first expression sequence, a second expression sequence, a 3 duplex forming region, and a 5 Group I intron fragment.

158. The vector of claim 157, comprising: a. a polynucleotide sequence encoding a cleavage site between the first expression sequence and the second expression sequence; and/or b. a first spacer between the 5 duplex forming region and the IRES, and a second spacer between the second expression sequence and the 3 duplex forming region.

159. A eukaryotic cell comprising a circular RNA polynucleotide according to claim 127.

160. The eukaryotic cell of claim 159, wherein the eukaryotic cell is an immune cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0091] FIG. 1 depicts luminescence in supernatants of HEK293 (FIGS. 1A, 1D, and 1E), HepG2 (FIG. 1B), or 1C1C7 (FIG. 1C) cells 24 hours after transfection with circular RNA comprising a Gaussia luciferase expression sequence and various IRES sequences.

[0092] FIG. 2 depicts luminescence in supernatants of HEK293 (FIG. 2A), HepG2 (FIG. 2B), or 1C1C7 (FIG. 2C) cells 24 hours after transfection with circular RNA comprising a Gaussia luciferase expression sequence and various IRES sequences having different lengths.

[0093] FIG. 3 depicts stability of select IRES constructs in HepG2 (FIG. 3A) or 1C1C7 (FIG. 3B) cells over 3 days as measured by luminescence.

[0094] FIG. 4A and FIG. 4B depict protein expression from select IRES constructs in Jurkat cells, as measured by luminescence from secreted Gaussia luciferase in cell supernatants.

[0095] FIG. 5A and FIG. 5B depict stability of select IRES constructs in Jurkat cells over 3 days as measured by luminescence.

[0096] FIG. 6 depicts comparisons of 24 hour luminescence (FIG. 6A) or relative luminescence over 3 days (FIG. 6B) of modified linear, unpurified circular, or purified circular RNA encoding Gaussia luciferase.

[0097] FIG. 7 depicts transcript induction of IFN? (FIG. 7A), IL-6 (FIG. 7B), IL-2 (FIG. 7C), RIG-I (FIG. 7D), FN-?1 (FIG. 7E), and TNF? (FIG. 7F) after electroporation of Jurkat cells with modified linear, unpurified circular, or purified circular RNA.

[0098] FIG. 8 depicts a comparison of luminescence of circular RNA and modified linear RNA encoding Gaussia luciferase in human primary monocytes (FIG. 8A) and macrophages (FIG. 8B and FIG. 8C).

[0099] FIG. 9 depicts relative luminescence over 3 days (FIG. 9A) in supernatant of primary T cells after transduction with circular RNA comprising a Gaussia luciferase expression sequence and varying IRES sequences or 24 hour luminescence (FIG. 9B).

[0100] FIG. 10 depicts 24 hour luminescence in supernatant of primary T cells (FIG. 10A) after transduction with circular RNA or modified linear RNA comprising a Gaussia luciferase expression sequence, or relative luminescence over 3 days (FIG. 10B), and 24 hour luminescence in PBMCs (FIG. 10C).

[0101] FIG. 11 depicts HPLC chromatograms (FIG. 11A) and circularization efficiencies (FIG. 11B) of RNA constructs having different permutation sites.

[0102] FIG. 12 depicts HPLC chromatograms (FIG. 12A) and circularization efficiencies (FIG. 12B) of RNA constructs having different introns and/or permutation sites.

[0103] FIG. 13 depicts HPLC chromatograms (FIG. 13A) and circularization efficiencies (FIG. 13B) of 3 RNA constructs with or without homology arms.

[0104] FIG. 14 depicts circularization efficiencies of 3 RNA constructs without homology arms or with homology arms having various lengths and GC content.

[0105] FIGS. 15A and 15B depict HPLC chromatograms showing the contribution of strong homology arms to improved splicing efficiency, the relationship between circularization efficiency and nicking in select constructs, and combinations of permutations sites and homology arms hypothesized to demonstrate improved circularization efficiency.

[0106] FIG. 16 shows fluorescent images of T cells mock electroporated (left) or electroporated with circular RNA encoding a CAR (right) and co-cultured with Raji cells expressing GFP and firefly luciferase.

[0107] FIG. 17 shows bright field (left), fluorescent (center), and overlay (right) images of T cells mock electroporated (top) or electroporated with circular RNA encoding a CAR (bottom) and co-cultured with Raji cells expressing GFP and firefly luciferase.

[0108] FIG. 18 depicts specific lysis of Raji target cells by T cells mock electroporated or electroporated with circular RNA encoding different CAR sequences.

[0109] FIG. 19 depicts luminescence in supernatants of Jurkat cells (left) or resting primary human CD3+ T cells (right) 24 hours after transduction with linear or circular RNA comprising a Gaussia luciferase expression sequence and varying IRES sequences (FIG. 19A), and relative luminescence over 3 days (FIG. 19B).

[0110] FIG. 20 depicts transcript induction of IFN-?1 (FIG. 20A), RIG-I (FIG. 20B), IL-2 (FIG. 20C), IL-6 (FIG. 20D), IFN? (FIG. 20E), and TNF? (FIG. 20F) after electroporation of human CD3+ T cells with modified linear, unpurified circular, or purified circular RNA.

[0111] FIG. 21 depicts specific lysis of Raji target cells by human primary CD3+ T cells electroporated with circRNA encoding a CAR as determined by detection of firefly luminescence (FIG. 21A), and IFN? transcript induction 24 hours after electroporation with different quantities of circular or linear RNA encoding a CAR sequence (FIG. 21B).

[0112] FIG. 22 depicts specific lysis of target or non-target cells by human primary CD3+ T cells electroporated with circular or linear RNA encoding a CAR at different E:T ratios (FIG. 22A and FIG. 22B) as determined by detection of firefly luminescence.

[0113] FIG. 23 depicts specific lysis of target cells by human CD3+ T cells electroporated with RNA encoding a CAR at 1, 3, 5, and 7 days post electroporation.

[0114] FIG. 24 depicts specific lysis of target cells by human CD3+ T cells electroporated with circular RNA encoding a CD19 or BCMA targeted CAR.

[0115] FIG. 25 depicts total Flux of organs harvested from CD-1 mice dosed with circular RNA encoding FLuc and formulated with 50% Lipid 15 (Table 10b), 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0116] FIG. 26 shows images highlighting the luminescence of organs harvested from CD-1 mice dosed with circular RNA encoding FLuc and formulated with 50% Lipid 15 (Table 10b), 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

[0117] FIG. 27 depicts molecular characterization of Lipids 26 and 27 from Table 10a. FIG. 27A shows the proton nuclear magnetic resonance (NMR) spectrum of Lipid 26. FIG. 27B shows the retention time of Lipid 26 measured by liquid chromatography-mass spectrometry (LC-MS). FIG. 27C shows the mass spectrum of Lipid 26. FIG. 27D shows the proton NMR spectrum of Lipid 27. FIG. 27E shows the retention time of Lipid 27 measured by LC-MS. FIG. 27F shows the mass spectrum of Lipid 27.

[0118] FIG. 28 depicts molecular characterization of Lipid 22-S14 and its synthetic intermediates. FIG. 28A depicts the NMR spectrum of 2-(tetradecylthio)ethan-1-ol. FIG. 28B depicts the NMR spectrum of 2-(tetradecylthio)ethyl acrylate. FIG. 28C depicts the NMR spectrum of bis(2-(tetradecylthio)ethyl) 3,3-((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)dipropionate (Lipid 22-S14).

[0119] FIG. 29 depicts the NMR spectrum of bis(2-(tetradecylthio)ethyl) 3,3-((3-(1H-imidazol-1-yl)propyl)azanediyl)dipropionate (Lipid 93-S14).

[0120] FIG. 30 depicts molecular characterization of heptadecan-9-yl 8-((3-(2-methyl-1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 54 from Table 10a). FIG. 30A shows the proton NMR spectrum of Lipid 54. FIG. 30B shows the retention time of Lipid 54 measured by LC-MS. FIG. 30C shows the mass spectrum of Lipid 54.

[0121] FIG. 31 depicts molecular characterization of heptadecan-9-yl 8-((3-(1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 53 from Table 10a).

[0122] FIG. 31A shows the proton NMR spectrum of Lipid 53. FIG. 31B shows the retention time of Lipid 53 measured by LC-MS. FIG. 31C shows the mass spectrum of Lipid 53.

[0123] FIG. 32A depicts total flux of spleen and liver harvested from CD-1 mice dosed with circular RNA encoding firefly luciferase (FLuc) and formulated with ionizable lipid of interest, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio. FIG. 32B depicts average radiance for biodistribution of protein expression.

[0124] FIG. 33A depicts images highlighting the luminescence of organs harvested from CD-1 mice dosed with circular RNA encoding FLuc and formulated with ionizable Lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio. FIG. 33B depicts whole body IVIS images of CD-1 mice dosed with circular RNA encoding FLuc and formulated with ionizable Lipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio.

[0125] FIG. 34A depicts images highlighting the luminescence of organs harvested from CD-1 mice dosed with circular RNA encoding FLuc and formulated with ionizable Lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio. FIG. 34B depicts whole body IVIS images of CD-1 mice dosed with circular RNA encoding FLuc and formulated with ionizable Lipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio.

[0126] FIG. 35A depicts images highlighting the luminescence of organs harvested from CD-1 mice dosed with circular RNA encoding FLuc and formulated with ionizable Lipid 26 from Table 10a, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio. FIG. 35B depicts whole body IVIS images of CD-1 mice dosed with circular RNA encoding FLuc and formulated with ionizable Lipid 26, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio.

[0127] FIG. 36 depicts images highlighting the luminescence of organs harvested from c57BL/6J mice dosed with circular RNA encoding FLuc and encapsulated in lipid nanoparticles formed with Lipid 15 from Table 10b (FIG. 36A), Lipid 53 from Table 10a (FIG. 36B), or Lipid 54 from Table 10a (FIG. 36C). PBS was used as control (FIG. 36D).

[0128] FIG. 37A and FIG. 37B depict relative luminescence in the lysates of human PBMCs after 24-hour incubation with testing lipid nanoparticles containing circular RNA encoding firefly luciferase.

[0129] FIG. 38 shows the expression of GFP (FIG. 38A) and CD19 CAR (FIG. 38B) in human PBMCs after incubating with testing lipid nanoparticle containing circular RNA encoding either GFP or CD19 CAR.

[0130] FIG. 39 depicts the expression of an anti-murine CD19 CAR in 1C1C7 cells lipotransfected with circular RNA comprising an anti-murine CD19 CAR expression sequence and varying IRES sequences.

[0131] FIG. 40 shows the cytotoxicity of an anti-murine CD19 CAR to murine T cells. The CD19 CAR is encoded by and expressed from a circular RNA, which is electroporated into the murine T cells.

[0132] FIG. 41 depicts the B cell counts in peripheral blood (FIG. 40A and FIG. 40B) or spleen (FIG. 40C) in C57BL/6J mice injected every other day with testing lipid nanoparticles encapsulating a circular RNA encoding an anti-murine CD19 CAR.

[0133] FIGS. 42A and 42B compares the expression level of an anti-human CD19 CAR expressed from a circular RNA with that expressed from a linear mRNA.

[0134] FIGS. 43A and 43B compares the cytotoxic effect of an anti-human CD19 CAR expressed from a circular RNA with that expressed from a linear mRNA

[0135] FIG. 44 depicts the cytotoxicity of two CARs (anti-human CD19 CAR and anti-human BCMA CAR) expressed from a single circular RNA in T cells.

[0136] FIG. 45A shows representative FACS plots with frequencies of tdTomato expression in various spleen immune cell subsets following treatment with LNPs formed with Lipid 27 or 26 from Table 10a or Lipid 15 from Table 10b. FIG. 45B shows the quantification of the proportion of myeloid cells, B cells, and T cells expressing tdTomato (mean+std. dev., n=3), equivalent to the proportion of each cell population successfully transfected with Cre circular RNA. FIG. 45C illustrates the proportion of additional splenic immune cell populations, including NK cells, classical monocytes, nonclassical monocytes, neutrophils, and dendritic cells, expressing tdTomato after treatment with Lipids 27 and 26 (mean+std. dev., n=3).

[0137] FIG. 46A depicts an exemplary RNA construct design with built-in polyA sequences in the introns. FIG. 46B shows the chromatography trace of unpurified circular RNA. FIG. 46C shows the chromatography trace of affinity-purified circular RNA. FIG. 46D shows the immunogenicity of the circular RNAs prepared with varying IVT conditions and purification methods. (Commercial=commercial IVT mix; Custom=customized IVT mix; Aff=affinity purification; Enz=enzyme purification; GMP:GTP ratio=8, 12.5, or 13.75).

[0138] FIG. 47A depicts an exemplary RNA construct design with a dedicated binding sequence as an alternative to polyA for hybridization purification. FIG. 47B shows the chromatography trace of unpurified circular RNA. FIG. 46C shows the chromatography trace of affinity-purified circular RNA.

[0139] FIG. 48A shows the chromatography trace of unpurified circular RNA encoding dystrophin. FIG. 48B shows the chromatography trace of enzyme-purified circular RNA encoding dystrophin.

[0140] FIG. 49 compares the expression (FIG. 49A) and stability (FIG. 49B) of purified circRNAs with different 5 spacers between the 3 intron fragment/5 internal duplex region and the IRES in Jurkat cells. (AC=only A and C were used in the spacer sequence; UC=only U and C were used in the spacer sequence.)

[0141] FIG. 50 shows luminescence expression levels and stability of expression in primary T cells from circular RNAs containing the original or modified IRES elements indicated.

[0142] FIG. 51 shows luminescence expression levels and stability of expression in HepG2 cells from circular RNAs containing the original or modified IRES elements indicated.

[0143] FIG. 52 shows luminescence expression levels and stability of expression in 1C1C7 cells from circular RNAs containing the original or modified IRES elements indicated.

[0144] FIG. 53 shows luminescence expression levels and stability of expression in HepG2 cells from circular RNAs containing IRES elements with untranslated regions (UTRs) inserted or hybrid IRES elements. Scr means Scrambled, which was used as a control.

[0145] FIG. 54 shows luminescence expression levels and stability of expression in 1C1C7 cells from circular RNAs containing an IRES and variable stop codon cassettes operably linked to a Gaussia luciferase coding sequence.

[0146] FIG. 55 shows luminescence expression levels and stability of expression in 1C1C7 cells from circular RNAs containing an IRES and variable untranslated regions (UTRs) inserted before the start codon of a gaussian luciferase coding sequence.

[0147] FIG. 56 shows expression levels of human erythropoietin (hEPO) in Huh7 cells from circular RNAs containing two miR-122 target sites downstream from the hEPO coding sequence.

[0148] FIG. 57 shows CAR expression levels in the peripheral blood (FIG. 57A) and spleen (FIG. 57B) when treated with LNP encapsulating circular RNA that expresses anti-CD19 CAR. Anti-CD20 (aCD20) and circular RNA encoding luciferase (oLuc) were used for comparison.

[0149] FIG. 58 shows the overall frequency of anti-CD19 CAR expression, the frequency of anti-CD19 CAR expression on the surface of cells and effect on anti-tumor response of IRES specific circular RNA encoding anti-CD19 CARs on T-cells. FIG. 58A shows anti-CD19 CAR geometric mean florescence intensity, FIG. 58B shows percentage of anti-CD19 CAR expression, and FIG. 58C shows the percentage target cell lysis performed by the anti-CD19 CAR. (CK=Caprine Kobuvirus; AP=Apodemus Picornavirus; CK*=Caprine Kobuvirus with codon optimization; PV=Parabovirus; SV=Salivirus.)

[0150] FIG. 59 shows CAR expression levels of A20 FLuc target cells when treated with IRES specific circular RNA constructs.

[0151] FIG. 60 shows luminescence expression levels for cytosolic (FIG. 60A) and surface (FIG. 60B) proteins from circular RNA in primary human T-cells.

[0152] FIG. 61 shows luminescence expression in human T-cells when treated with IRES specific circular constructs. Expression in circular RNA constructs were compared to linear mRNA. FIG. 61A, FIG. 61B, and FIG. 61G provide Gaussia luciferase expression in multiple donor cells. FIG. 61C, FIG. 61D, FIG. 61E, and FIG. 61F provides firefly luciferase expression in multiple donor cells.

[0153] FIG. 62 shows anti-CD19 CAR (FIG. 62A and FIG. 62B) and anti-BCMA CAR (FIG. 62B) expression in human T-cells following treatment of a lipid nanoparticle encompassing a circular RNA that encodes either an anti-CD19 or anti-BCMA CAR to a firefly luciferase expressing K562 cell.

[0154] FIG. 63 shows anti-CD19 CAR expression levels resulting from delivery via electroporation in vitro of a circular RNA encoding an anti-CD19 CAR in a specific antigen-dependent manner. FIG. 63A shows Nalm6 cell lysing with an anti-CD19 CAR. FIG. 63B shows K562 cell lysing with an anti-CD19 CAR.

[0155] FIG. 64 shows transfection of LNP mediated by use of ApoE3 in solutions containing LNP and circular RNA expressing green fluorescence protein (GFP). FIG. 64A showed the live-dead results. FIG. 64B, FIG. 61C, FIG. 61D, and FIG. 64E provide the frequency of expression for multiple donors.

DETAILED DESCRIPTION

[0156] The present invention provides, among other things, methods and compositions for treating an autoimmune disorder or cancer based on circular RNA therapy. In particular, the present invention provides methods for treating an autoimmune disorder or cancer by administering to a subject in need of treatment a composition comprising an RNA encoding 2 therapeutic proteins at an effective dose and an administration interval such that at least one symptom or feature of an autoimmune disorder or cancer is reduced in intensity, severity, or frequency or is delayed in onset.

[0157] In certain embodiments, provided herein is a vector for making circular RNA, the vector comprising an optional 5 duplex forming region, a 3 group I intron fragment, optionally a first spacer, an Internal Ribosome Entry Site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, optionally a second spacer, a 5 group I intron fragment, and an optional 3 duplex forming region. In certain embodiments, provided herein is a vector for making circular RNA, the vector comprising an optional 5 duplex forming region, a 3 group I intron fragment, optionally a first spacer, a first Internal Ribosome Entry Site (IRES), a first expression sequence, a second IRES, a second expression sequence, optionally a second spacer, a 5 group I intron fragment, and an optional 3 duplex forming region. In some embodiments, these elements are positioned in the vector in the above order. In some embodiments, a polynucleotide contains a 3 duplex forming region and a 5 duplex forming region. In some embodiments, the vector further comprises an internal 5 duplex forming region between the 3 group I intron fragment and the IRES and an internal 3 duplex forming region between the expression sequences and the 5 group I intron fragment. In some embodiments, the internal duplex forming regions are capable of forming a duplex between each other but not with the external duplex forming regions. In some embodiments, the internal duplex forming regions are part of the first and second spacers. Additional embodiments include circular RNA polynucleotides, including circular RNA polynucleotides made using the vectors provided herein, compositions comprising such circular RNA, cells comprising such circular RNA, methods of using and making such vectors, circular RNA, compositions and cells.

[0158] In some embodiments, provided herein are methods comprising administration of circular RNA polynucleotides provided herein into cells for therapy or production of useful proteins. In some embodiments, the method is advantageous in providing the production of a desired polypeptide inside eukaryotic cells with a longer half-life than linear RNA, due to the resistance of the circular RNA to ribonucleases.

[0159] Circular RNA polynucleotides lack the free ends necessary for exonuclease-mediated degradation, causing them to be resistant to several mechanisms of RNA degradation and granting extended half-lives when compared to an equivalent linear RNA. Circularization may allow for the stabilization of RNA polynucleotides that generally suffer from short half-lives and may improve the overall efficacy of exogenous mRNA in a variety of applications. In an embodiment, the functional half-life of the circular RNA polynucleotides provided herein in eukaryotic cells (e.g., mammalian cells, such as human cells) as assessed by protein synthesis is at least 20 hours (e.g., at least 80 hours).

1. Definitions

[0160] As used herein, the terms circRNA or circular polyribonucleotide or circular RNA are used interchangeably and refers to a polyribonucleotide that forms a circular structure through covalent bonds.

[0161] As used herein, the term 3 group I intron fragment refers to a sequence with 75% or higher similarity to the 3-proximal end of a natural group I intron including the splice site dinucleotide and optionally a stretch of natural exon sequence.

[0162] As used herein, the term 5 group I intron fragment refers to a sequence with 75% or higher similarity to the 5-proximal end of a natural group I intron including the splice site dinucleotide and optionally a stretch of natural exon sequence.

[0163] As used herein, the term permutation site refers to the site in a group I intron where a cut is made prior to permutation of the intron. This cut generates 3 and 5 group I intron fragments that are permuted to be on either side of a stretch of precursor RNA to be circularized.

[0164] As used herein, the term splice site refers to a dinucleotide that is partially or fully included in a group I intron and between which a phosphodiester bond is cleaved during RNA circularization.

[0165] The expression sequences in the polynucleotide construct may be separated by a cleavage site sequence which enables polypeptides encoded by the expression sequences, once translated, to be expressed separately by the cell.

[0166] A self-cleaving peptide refers to a peptide which is translated without a peptide bond between two adjacent amino acids, or functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately cleaved or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.

[0167] As used herein, the term therapeutic protein refers to any protein that, when administered to a subject directly or indirectly in the form of a translated nucleic acid, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.

[0168] The ? and ? chains of ?? TCR's are generally regarded as each having two domains or regions, namely variable and constant domains/regions. The variable domain consists of a concatenation of variable regions and joining regions. In the present specification and claims, the term TCR alpha variable domain therefore refers to the concatenation of TRAV and TRAJ regions, and the term TCR alpha constant domain refers to the extracellular TRAC region, or to a C-terminal truncated TRAC sequence. Likewise the term TCR beta variable domain refers to the concatenation of TRBV and TRBD/TRBJ regions, and the term TCR beta constant domain refers to the extracellular TRBC region, or to a C-terminal truncated TRBC sequence.

[0169] As used herein, the term immunogenic refers to a potential to induce an immune response to a substance. An immune response may be induced when an immune system of an organism or a certain type of immune cell is exposed to an immunogenic substance. The term non-immunogenic refers to a lack of or absence of an immune response above a detectable threshold to a substance. No immune response is detected when an immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic substance. In some embodiments, a non-immunogenic circular polyribonucleotide as provided herein, does not induce an immune response above a pre-determined threshold when measured by an immunogenicity assay. In some embodiments, no innate immune response is detected when an immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic circular polyribonucleotide as provided herein. In some embodiments, no adaptive immune response is detected when an immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic circular polyribonucleotide as provided herein.

[0170] As used herein, the term circularization efficiency refers to a measurement of resultant circular polyribonucleotide as compared to its linear starting material.

[0171] As used herein, the term translation efficiency refers to a rate or amount of protein or peptide production from a ribonucleotide transcript. In some embodiments, translation efficiency can be expressed as amount of protein or peptide produced per given amount of transcript that codes for the protein or peptide.

[0172] The term nucleotide refers to a ribonucleotide, a deoxyribonucleotide, a modified form thereof, or an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs. Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2, or CN, wherein R is an alkyl moiety as defined herein. Nucleotide analogs are also meant to include nucleotides with bases such as inosine, queuosine, xanthine; sugars such as 2-methyl ribose; non-natural phosphodiester linkages such as methylphosphonate, phosphorothioate and peptide linkages. Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.

[0173] The term nucleic acid and polynucleotide are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, or up to about 10,000 or more bases, composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be produced enzymatically or synthetically (e.g., as described in U.S. Pat. No. 5,948,902 and the references cited therein), which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally occurring nucleic acids are comprised of nucleotides including guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U respectively).

[0174] The terms ribonucleic acid and RNA as used herein mean a polymer composed of ribonucleotides.

[0175] The terms deoxyribonucleic acid and DNA as used herein mean a polymer composed of deoxyribonucleotides.

[0176] Isolated or purified generally refers to isolation of a substance (for example, in some embodiments, a compound, a polynucleotide, a protein, a polypeptide, a polynucleotide composition, or a polypeptide composition) such that the substance comprises a significant percent (e.g., greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, usually up to about 90%-100%) of the sample in which it resides. In certain embodiments, a substantially purified component comprises at least 50%, 80%-85%, or 90%-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density. Generally, a substance is purified when it exists in a sample in an amount, relative to other components of the sample, that is more than as it is found naturally.

[0177] The terms duplexed, double-stranded, or hybridized as used herein refer to nucleic acids formed by hybridization of two single strands of nucleic acids containing complementary sequences. In most cases, genomic DNA is double-stranded. Sequences can be fully complementary or partially complementary.

[0178] As used herein, unstructured with regard to RNA refers to an RNA sequence that is not predicted by the RNAFold software or similar predictive tools to form a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule. In some embodiments, unstructured RNA can be functionally characterized using nuclease protection assays.

[0179] As used herein, structured with regard to RNA refers to an RNA sequence that is predicted by the RNAFold software or similar predictive tools to form a structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule.

[0180] As used herein, two duplex forming regions, homology arms, or homology regions, complement, or are complementary, to one another when the two regions share a sufficient level of sequence identity to one another's reverse complement to act as substrates for a hybridization reaction. As used herein, polynucleotide sequences have homology when they are either identical or share sequence identity to a reverse complement or complementary sequence. The percent sequence identity between a duplex forming region and a counterpart duplex forming region's reverse complement can be any percent of sequence identity that allows for hybridization to occur. In some embodiments, an internal duplex forming region of an inventive polynucleotide is capable of forming a duplex with another internal duplex forming region and does not form a duplex with an external duplex forming region.

[0181] Linear nucleic acid molecules are said to have a 5-terminus (5 end) and a 3-terminus (3 end) because nucleic acid phosphodiester linkages occur at the 5 carbon and 3 carbon of the sugar moieties of the substituent mononucleotides. The end nucleotide of a polynucleotide at which a new linkage would be to a 5 carbon is its 5 terminal nucleotide. The end nucleotide of a polynucleotide at which a new linkage would be to a 3 carbon is its 3 terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3- or 5-terminus.

[0182] Transcription means the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. The invention is not limited with respect to the RNA polymerase that is used for transcription. For example, in some embodiments, a T7-type RNA polymerase can be used.

[0183] Translation means the formation of a polypeptide molecule by a ribosome based upon an RNA template.

[0184] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms a, an, and the include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a cell includes combinations of two or more cells, or entire cultures of cells; reference to a polynucleotide includes, as a practical matter, many copies of that polynucleotide. Unless specifically stated or obvious from context, as used herein, the term or is understood to be inclusive. Unless defined herein and below in the reminder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0185] Unless specifically stated or obvious from context, as used herein, the term about, is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4% 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%0, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term about.

[0186] As used herein, the term encode refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.

[0187] By co-administering is meant administering a therapeutic agent provided herein in conjunction with one or more additional therapeutic agents sufficiently close in time such that the therapeutic agent provided herein can enhance the effect of the one or more additional therapeutic agents, or vice versa.

[0188] The terms treat, and prevent as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. The treatment or prevention provided by the method disclosed herein can include treatment or prevention of one or more conditions or symptoms of the disease. Also, for purposes herein, prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.

[0189] As used herein, autoimmunity is defined as persistent and progressive immune reactions to non-infectious self-antigens, as distinct from infectious non self-antigens from bacterial, viral, fungal, or parasitic organisms which invade and persist within mammals and humans. Autoimmune conditions include scleroderma, Grave's disease, Crohn's disease, Sjorgen's disease, multiple sclerosis, Hashimoto's disease, psoriasis, myasthenia gravis, autoimmune polyendocrinopathy syndromes, Type I diabetes mellitus (TIDM), autoimmune gastritis, autoimmune uveoretinitis, polymyositis, colitis, and thyroiditis, as well as in the generalized autoimmune diseases typified by human Lupus. Autoantigen or self-antigen as used herein refers to an antigen or epitope which is native to the mammal and which is immunogenic in said mammal.

[0190] As used herein, the term expression sequence can refer to a nucleic acid sequence that encodes a product, e.g., a peptide or polypeptide, regulatory nucleic acid, or non-coding nucleic acid. An exemplary expression sequence that codes for a peptide or polypeptide can comprise a plurality of nucleotide triads, each of which can code for an amino acid and is termed as a codon.

[0191] As used herein, a spacer refers to a region of a polynucleotide sequence ranging from 1 nucleotide to hundreds or thousands of nucleotides separating two other elements along a polynucleotide sequence. The sequences can be defined or can be random. A spacer is typically non-coding. In some embodiments, spacers include duplex forming regions.

[0192] As used herein, an internal ribosome entry site or IRES refers to an RNA sequence or structural element ranging in size from 10 nt to 1000 nt or more, capable of initiating translation of a polypeptide in the absence of a typical RNA cap structure. An IRES is typically about 500 nt to about 700 nt in length.

[0193] As used herein, an miRNA site refers to a stretch of nucleotides within a polynucleotide that is capable of forming a duplex with at least 8 nucleotides of a natural miRNA sequence.

[0194] As used herein, an endonuclease site refers to a stretch of nucleotides within a polynucleotide that is capable of being recognized and cleaved by an endonuclease protein.

[0195] As used herein, bicistronic RNA refers to a polynucleotide that includes two expression sequences coding for two distinct proteins. These expression sequences are often separated by a cleavable peptide such as a 2A site or an IRES sequence. They can also be separated by a ribosomal skipping element or a protease cleavage.

[0196] As used herein, the term ribosomal skipping element refers to a nucleotide sequence encoding a short peptide sequence capable of causing generation of two peptide chains from translation of one RNA molecule. While wishing not to be bound by theory, it is hypothesized that the ribosomal skipping elements function by: (1) terminating translation of the first peptide chain and re-initiating translation of the second peptide chain; or (2) cleavage of a peptide bond in the peptide sequence encoded by the ribosomal skipping element by an intrinsic protease activity of the encoded peptide, or by another protease in the environment (e.g., cytosol).

[0197] As used herein, the term co-formulate refers to a nanoparticle formulation comprising two or more nucleic acids or a nucleic acid and other active drug substance. Typically, the ratios are equimolar or defined in the ratiometric amount of the two or more nucleic acids or the nucleic acid and other active drug substance.

[0198] As used herein, transfer vehicle includes any of the standard pharmaceutical carriers, diluents, excipients, and the like, which are generally intended for use in connection with the administration of biologically active agents, including nucleic acids.

[0199] As used herein, the phrase lipid nanoparticle refers to a transfer vehicle comprising one or more lipids (e.g., in some embodiments, cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0200] As used herein, the phrase cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.

[0201] As used herein, the phrase non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.

[0202] As used herein, the phrase anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0203] As used herein, the phrase ionizable lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH 4 and a neutral charge at other pHs such as physiological pH 7.

[0204] In some embodiments, a lipid, e.g., an ionizable lipid, disclosed herein comprises one or more cleavable groups. The terms cleave and cleavable are used herein to mean that one or more chemical bonds (e.g., one or more of covalent bonds, hydrogen-bonds, van der Waals' forces and/or ionic interactions) between atoms in or adjacent to the subject functional group are broken (e.g., hydrolyzed) or are capable of being broken upon exposure to selected conditions (e.g., upon exposure to enzymatic conditions). In certain embodiments, the cleavable group is a disulfide functional group, and in particular embodiments is a disulfide group that is capable of being cleaved upon exposure to selected biological conditions (e.g., intracellular conditions). In certain embodiments, the cleavable group is an ester functional group that is capable of being cleaved upon exposure to selected biological conditions. For example, the disulfide groups may be cleaved enzymatically or by a hydrolysis, oxidation or reduction reaction. Upon cleavage of such disulfide functional group, the one or more functional moieties or groups (e.g., one or more of a head-group and/or a tail-group) that are bound thereto may be liberated. Exemplary cleavable groups may include, but are not limited to, disulfide groups, ester groups, ether groups, and any derivatives thereof (e.g., alkyl and aryl esters). In certain embodiments, the cleavable group is not an ester group or an ether group. In some embodiments, a cleavable group is bound (e.g., bound by one or more of hydrogen-bonds, van der Waals' forces, ionic interactions and covalent bonds) to one or more functional moieties or groups (e.g., at least one head-group and at least one tail-group). In certain embodiments, at least one of the functional moieties or groups is hydrophilic (e.g., a hydrophilic head-group comprising one or more of imidazole, guanidinium, amino, imine, enamine, optionally-substituted alkyl amino and pyridyl).

[0205] As used herein, the term hydrophilic is used to indicate in qualitative terms that a functional group is water-preferring, and typically such groups are water-soluble. For example, disclosed herein are compounds that comprise a cleavable disulfide (SS) functional group bound to one or more hydrophilic groups (e.g., a hydrophilic head-group), wherein such hydrophilic groups comprise or are selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino (e.g., an alkyl amino such as dimethylamino) and pyridyl.

[0206] In certain embodiments, at least one of the functional groups of moieties that comprise the compounds disclosed herein is hydrophobic in nature (e.g., a hydrophobic tail-group comprising a naturally occurring lipid such as cholesterol). As used herein, the term hydrophobic is used to indicate in qualitative terms that a functional group is water-avoiding, and typically such groups are not water soluble. For example, disclosed herein are compounds that comprise a cleavable functional group (e.g., a disulfide (SS) group) bound to one or more hydrophobic groups, wherein such hydrophobic groups comprise one or more naturally occurring lipids such as cholesterol, and/or an optionally substituted, variably saturated or unsaturated C.sub.6-C.sub.20 alkyl and/or an optionally substituted, variably saturated or unsaturated C.sub.6-C.sub.20 acyl.

[0207] Compound described herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including .sup.1H, .sup.2H (D or deuterium), and .sup.3H (T or tritium); C may be in any isotopic form, including .sup.12C, .sup.13C, and .sup.14C; O may be in any isotopic form, including .sup.16O and .sup.18O; F may be in any isotopic form, including .sup.18F and .sup.19F; and the like.

[0208] When describing the invention, which may include compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing such compounds and methods of using such compounds and compositions, the following terms, if present, have the following meanings unless otherwise indicated. It should also be understood that when described herein any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope as set out below. Unless otherwise stated, the term substituted is to be defined as set out below. It should be further understood that the terms groups and radicals can be considered interchangeable when used herein.

[0209] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, C.sub.1-6 alkyl is intended to encompass, C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.1-6, C.sub.1-5, C.sub.1-4, C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-5, C.sub.2-4, C.sub.2-3, C.sub.3-6, C.sub.3-5, C.sub.3-4, C.sub.4-6, C.sub.4-5, and C.sub.5-6 alkyl.

[0210] In certain embodiments, the compounds disclosed herein comprise, for example, at least one hydrophilic head-group and at least one hydrophobic tail-group, each bound to at least one cleavable group, thereby rendering such compounds amphiphilic. As used herein to describe a compound or composition, the term amphiphilic means the ability to dissolve in both polar (e.g., water) and non-polar (e.g., lipid) environments. For example, in certain embodiments, the compounds disclosed herein comprise at least one lipophilic tail-group (e.g., cholesterol or a C.sub.6-C.sub.20 alkyl) and at least one hydrophilic head-group (e.g., imidazole), each bound to a cleavable group (e.g., disulfide).

[0211] It should be noted that the terms head-group and tail-group as used describe the compounds of the present invention, and in particular functional groups that comprise such compounds, are used for ease of reference to describe the orientation of one or more functional groups relative to other functional groups. For example, in certain embodiments a hydrophilic head-group (e.g., guanidinium) is bound (e.g., by one or more of hydrogen-bonds, van der Waals' forces, ionic interactions and covalent bonds) to a cleavable functional group (e.g., a disulfide group), which in turn is bound to a hydrophobic tail-group (e.g., cholesterol).

[0212] As used herein, the term alkyl refers to both straight and branched chain C.sub.1-C.sub.40 hydrocarbons (e.g., C.sub.6-C.sub.20 hydrocarbons), and include both saturated and unsaturated hydrocarbons. In certain embodiments, the alkyl may comprise one or more cyclic alkyls and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with substituents (e.g., one or more of alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide). In certain embodiments, a contemplated alkyl includes (9Z,12Z)-octadeca-9,12-dien. The use of designations such as, for example, C.sub.6-C.sub.20 is intended to refer to an alkyl (e.g., straight or branched chain and inclusive of alkenes and alkyls) having the recited range carbon atoms. In some embodiments, an alkyl group has 1 to 10 carbon atoms (C.sub.1-10 alkyl). In some embodiments, an alkyl group has 1 to 9 carbon atoms (C.sub.1-9 alkyl). In some embodiments, an alkyl group has 1 to 8 carbon atoms (C.sub.1-8 alkyl). In some embodiments, an alkyl group has 1 to 7 carbon atoms (C.sub.1-7 alkyl). In some embodiments, an alkyl group has 1 to 6 carbon atoms (C.sub.1-6 alkyl). In some embodiments, an alkyl group has 1 to 5 carbon atoms (C.sub.1-5 alkyl). In some embodiments, an alkyl group has 1 to 4 carbon atoms (C.sub.1-4 alkyl). In some embodiments, an alkyl group has 1 to 3 carbon atoms (C.sub.1-3 alkyl). In some embodiments, an alkyl group has 1 to 2 carbon atoms (C.sub.1-2 alkyl). In some embodiments, an alkyl group has 1 carbon atom (C.sub.1 alkyl). Examples of C.sub.1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.

[0213] As used herein, alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) (C.sub.2-20 alkenyl). In certain embodiments, alkenyl does not contain any triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (C.sub.2-10 alkenyl). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (C.sub.2-9 alkenyl). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (C.sub.2-8 alkenyl). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (C.sub.2-7 alkenyl). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (C.sub.2-6 alkenyl). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (C.sub.2-5 alkenyl). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (C.sub.2-4 alkenyl). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (C.sub.2-3 alkenyl). In some embodiments, an alkenyl group has 2 carbon atoms (C.sub.2 alkenyl). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C.sub.2-4 alkenyl groups include ethenyl (C.sub.2), 1-propenyl (C.sub.3), 2-propenyl (C.sub.3), 1-butenyl (C.sub.4), 2-butenyl (C.sub.4), butadienyl (C.sub.4), and the like. Examples of C.sub.2-6 alkenyl groups include the aforementioned C.sub.2-4 alkenyl groups as well as pentenyl (C.sub.5), pentadienyl (C.sub.5), hexenyl (C.sub.6), and the like. Additional examples of alkenyl include heptenyl (C.sub.7), octenyl (C.sub.8), octatrienyl (C.sub.8), and the like.

[0214] As used herein, alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds) (C.sub.2-20 alkynyl). In certain embodiments, alkynyl does not contain any double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms (C.sub.2-10 alkynyl). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (C.sub.2-9 alkynyl). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (C.sub.2-8 alkynyl). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (C.sub.2-7 alkynyl). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (C.sub.2-6 alkynyl). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (C.sub.2-5 alkynyl). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (C.sub.2-4 alkynyl). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (C.sub.2-3 alkynyl). In some embodiments, an alkynyl group has 2 carbon atoms (C.sub.2 alkynyl). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C.sub.2-4 alkynyl groups include, without limitation, ethynyl (C.sub.2), 1-propynyl (C.sub.3), 2-propynyl (C.sub.3), 1-butynyl (C.sub.4), 2-butynyl (C.sub.4), and the like. Examples of C.sub.2-6 alkenyl groups include the aforementioned C.sub.2-4 alkynyl groups as well as pentynyl (C.sub.5), hexynyl (C.sub.6), and the like. Additional examples of alkynyl include heptynyl (C.sub.7), octynyl (C.sub.8), and the like.

[0215] As used herein, alkylene, alkenylene, and alkynylene, refer to a divalent radical of an alkyl, alkenyl, and alkynyl group respectively. When a range or number of carbons is provided for a particular alkylene, alkenylene, or alkynylene, group, it is understood that the range or number refers to the range or number of carbons in the linear carbon divalent chain. Alkylene, alkenylene, and alkynylene, groups may be substituted or unsubstituted with one or more substituents as described herein.

[0216] As used herein, the term aryl refers to aromatic groups (e.g., monocyclic, bicyclic and tricyclic structures) containing six to ten carbons in the ring portion. The aryl groups may be optionally substituted through available carbon atoms and in certain embodiments may include one or more heteroatoms such as oxygen, nitrogen or sulfur. In some embodiments, an aryl group has six ring carbon atoms (C.sub.6 aryl; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (C.sub.10 aryl; e.g., naphthyl such as 1-naphthyl and 2-naphthyl).

[0217] As used herein, heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (5-10 membered heteroaryl). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

[0218] The term cycloalkyl refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as C.sub.4-8cycloalkyl, derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclopentanes, cyclobutanes and cyclopropanes.

[0219] As used herein, heterocyclyl or heterocyclic refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (3-10 membered heterocyclyl). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (monocyclic heterocyclyl) or a fused, bridged or spiro ring system such as a bicyclic system (bicyclic heterocyclyl), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. The terms heterocycle, heterocyclyl, heterocyclyl ring, heterocyclic group, heterocyclic moiety, and heterocyclic radical, may be used interchangeably.

[0220] As used herein, cyano refers to CN.

[0221] The terms halo and halogen as used herein refer to an atom selected from fluorine (fluoro, F), chlorine (chloro, Cl), bromine (bromo, Br), and iodine (iodo, I). In certain embodiments, the halo group is either fluoro or chloro.

[0222] The term alkoxy, as used herein, refers to an alkyl group which is attached to another moiety via an oxygen atom (O(alkyl)). Non-limiting examples include e.g., methoxy, ethoxy, propoxy, and butoxy.

[0223] As used herein, oxo refers to C?O.

[0224] In general, the term substituted, whether preceded by the term optionally or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a substituted group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.

[0225] As used herein, pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N.sup.+(C.sub.1-4alkyl).sub.4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0226] In typical embodiments, the present invention is intended to encompass the compounds disclosed herein, and the pharmaceutically acceptable salts, pharmaceutically acceptable esters, tautomeric forms, polymorphs, and prodrugs of such compounds. In some embodiments, the present invention includes a pharmaceutically acceptable addition salt, a pharmaceutically acceptable ester, a solvate (e.g., hydrate) of an addition salt, a tautomeric form, a polymorph, an enantiomer, a mixture of enantiomers, a stereoisomer or mixture of stereoisomers (pure or as a racemic or non-racemic mixture) of a compound described herein.

[0227] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0228] In certain embodiments the compounds and the transfer vehicles of which such compounds are a component (e.g., lipid nanoparticles) exhibit an enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, also provided herein are methods of transfecting one or more target cells. Such methods generally comprise the step of contacting the one or more target cells with the compounds and/or pharmaceutical compositions disclosed herein such that the one or more target cells are transfected with the circular RNA encapsulated therein. As used herein, the terms transfect or transfection refer to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and/or polynucleotides) into a cell, or preferably into a target cell. The term transfection efficiency refers to the relative amount of such encapsulated material (e.g., polynucleotides) up-taken by, introduced into and/or expressed by the target cell which is subject to transfection. In some embodiments, transfection efficiency may be estimated by the amount of a reporter polynucleotide product produced by the target cells following transfection. In some embodiments, a transfer vehicle has high transfection efficiency. In some embodiments, a transfer vehicle has at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% transfection efficiency.

[0229] As used herein, the term liposome generally refers to a vesicle composed of lipids (e.g., amphiphilic lipids) arranged in one or more spherical bilayer or bilayers. In certain embodiments, the liposome is a lipid nanoparticle (e.g., a lipid nanoparticle comprising one or more of the ionizable lipid compounds disclosed herein). Such liposomes may be unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the encapsulated circRNA to be delivered to one or more target cells, tissues and organs. In certain embodiments, the compositions described herein comprise one or more lipid nanoparticles. Examples of suitable lipids (e.g., ionizable lipids) that may be used to form the liposomes and lipid nanoparticles contemplated include one or more of the compounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004 and/or HGT4005). Such liposomes and lipid nanoparticles may also comprise additional ionizable lipids such as C12-200, DLin-KC2-DMA, and/or HGT5001, helper lipids, structural lipids, PEG-modified lipids, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE, HGT5000, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

[0230] As used herein, the phrases non-cationic lipid, non-cationic helper lipid, and helper lipid are used interchangeably and refer to any neutral, zwitterionic or anionic lipid.

[0231] As used herein, the phrase anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[0232] As used herein, the phrase biodegradable lipid or degradable lipid refers to any of a number of lipid species that are broken down in a host environment on the order of minutes, hours, or days ideally making them less toxic and unlikely to accumulate in a host over time. Common modifications to lipids include ester bonds, and disulfide bonds among others to increase the biodegradability of a lipid.

[0233] As used herein, the phrase biodegradable PEG lipid or degradable PEG lipid refers to any of a number of lipid species where the PEG molecules are cleaved from the lipid in a host environment on the order of minutes, hours, or days ideally making them less immunogenic. Common modifications to PEG lipids include ester bonds, and disulfide bonds among others to increase the biodegradability of a lipid.

[0234] In certain embodiments of the present invention, the transfer vehicles (e.g., lipid nanoparticles) are prepared to encapsulate one or more materials or therapeutic agents (e.g., circRNA). The process of incorporating a desired therapeutic agent (e.g., circRNA) into a transfer vehicle is referred to herein as or loading or encapsulating (Lasic, et al., FEBS Lett., 312: 255-258, 1992). The transfer vehicle-loaded or -encapsulated materials (e.g., circRNA) may be completely or partially located in the interior space of the transfer vehicle, within a bilayer membrane of the transfer vehicle, or associated with the exterior surface of the transfer vehicle.

[0235] As used herein, the term structural lipid refers to sterols and also to lipids containing sterol moieties.

[0236] As defined herein, sterols are a subgroup of steroids consisting of steroid alcohols.

[0237] As used herein, the term structural lipid refers to sterols and also to lipids containing sterol moieties.

[0238] As used herein, the term PEG means any polyethylene glycol or other polyalkylene ether polymer.

[0239] As generally defined herein, a PEG-OH lipid (also referred to herein as hydroxy-PEGylated lipid) is a PEGylated lipid having one or more hydroxyl (OH) groups on the lipid.

[0240] As used herein, a phospholipid is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains.

[0241] All nucleotide sequences disclosed herein can represent an RNA sequence or a corresponding DNA sequence. It is understood that deoxythymidine (dT or T) in a DNA is transcribed into a uridine (U) in an RNA. As such, T and U are used interchangeably herein in nucleotide sequences.

[0242] The recitations sequence identity or, for example, comprising a sequence 50% identical to, as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a percentage of sequence identity may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0243] The term antibody (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain may comprise a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise three constant domains, CH1, CH2 and CH3. Each light chain can comprise a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region can comprise one constant domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL may comprise three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as antibody conjugates), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain variable fragments (scFv), camelized antibodies, affybodies, Fab fragments, F(ab).sub.2 fragments, disulfide-linked variable fragments (sdFv), anti-idiotypic (anti-id) antibodies (including, e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as antibody mimetics), and antigen-binding fragments of any of the above. In some embodiments, antibodies described herein refer to polyclonal antibody populations.

[0244] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4. Isotype refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. The term antibody includes, by way of example, both naturally occurring and non-naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or nonhuman Abs; wholly synthetic Abs; and single chain Abs. A nonhuman Ab may be humanized by recombinant methods to reduce its immunogenicity in humans. Where not expressly stated, and unless the context indicates otherwise, the term antibody also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab.

[0245] An antigen binding molecule, antigen binding portion, or antibody fragment refers to any molecule that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived. An antigen binding molecule may include the antigenic complementarity determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab, F(ab).sub.2, Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules. Peptibodies (i.e. Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen. In some embodiments, the antigen binding molecule binds to BCMA. In further embodiments, the antigen binding molecule is an antibody fragment, including one or more of the complementarity determining regions (CDRs) thereof, that specifically binds to the antigen. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of avimers.

[0246] As used herein, the term variable region or variable domain is used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0247] The terms VL and VL domain are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof.

[0248] The terms VH and VH domain are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.

[0249] A number of definitions of the CDRs are commonly in use: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software. The contact definition is based on an analysis of the available complex crystal structures. The term Kabat numbering and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen-binding molecule thereof. In certain aspects, the CDRs of an antibody may be determined according to the Kabat numbering system (see, e.g., Kabat E A & Wu T T (1971) Ann NY Acad Sci 190: 382-391 and Kabat E A et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally may include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme. In certain aspects, the CDRs of an antibody may be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al, (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al, (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-HI loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme.

[0250] As used herein, the terms constant region and constant domain are interchangeable and have a meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which may exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0251] Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y may generally be represented by the dissociation constant (KD or Kd). Affinity may be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA or Ka). The KD is calculated from the quotient of koff/kon, whereas KA is calculated from the quotient of kon/koff. kon refers to the association rate constant of, e.g., an antibody to an antigen, and koff refers to the dissociation of, e.g., an antibody to an antigen. The kon and koff may be determined by techniques known to one of ordinary skill in the art, such as BIACORE? or KinExA.

[0252] As used herein, a conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues within a CDR(s) or within a framework region(s) of an antibody or antigen-binding molecule thereof may be replaced with an amino acid residue with a similar side chain.

[0253] As, used herein, the term heterologous means from any source other than naturally occurring sequences.

[0254] As used herein, an epitope is a term in the art and refers to a localized region of an antigen to which an antibody may specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In some embodiments, the epitope to which an antibody binds may be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody: antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H W et al.; U.S. Patent Publication No. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323).

[0255] As used herein, an antigen binding molecule, an antibody, or an antigen binding molecule thereof cross-competes with a reference antibody or an antigen binding molecule thereof if the interaction between an antigen and the first binding molecule, an antibody, or an antigen binding molecule thereof blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, or an antigen binding molecule thereof to interact with the antigen. Cross competition may be complete, e.g., binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind the antigen, or it may be partial, e.g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind the antigen. In some embodiments, an antigen binding molecule that cross-competes with a reference antigen binding molecule binds the same or an overlapping epitope as the reference antigen binding molecule. In other embodiments, the antigen binding molecule that cross-competes with a reference antigen binding molecule binds a different epitope as the reference antigen binding molecule. Numerous types of competitive binding assays may be used to determine if one antigen binding molecule competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA); solid phase direct or indirect enzyme immunoassay (EIA); sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid phase direct labeled assay, solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

[0256] As used herein, the terms immunospecifically binds, immunospecifically recognizes, specifically binds, and specifically recognizes are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope or immune complex) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIACORE?, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the KA when the molecules bind to another antigen.

[0257] An antigen refers to any molecule that provokes an immune response or is capable of being bound by an antibody or an antigen binding molecule. The immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, may serve as an antigen. An antigen may be endogenously expressed, i.e. expressed by genomic DNA, or may be recombinantly expressed. An antigen may be specific to a certain tissue, such as a cancer cell, or it may be broadly expressed. In addition, fragments of larger molecules may act as antigens. In some embodiments, antigens are tumor antigens.

[0258] The term autologous refers to any material derived from the same individual to which it is later to be re-introduced. For example, the engineered autologous cell therapy (eACT?) method described herein involves collection of lymphocytes from a patient, which are then engineered to express, e.g., a CAR construct, and then administered back to the same patient.

[0259] The term allogeneic refers to any material derived from one individual which is then introduced to another individual of the same species, e.g., allogeneic T cell transplantation.

[0260] A cancer refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A cancer or cancer tissue may include a tumor. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, squamous cell cancer, T cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and combinations of said cancers. In some embodiments, the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Kaposi's sarcoma, sarcoma of soft tissue, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (for example adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the renal pelvis, CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma). The particular cancer may be responsive to chemo- or radiation therapy or the cancer may be refractory. A refractory cancer refers to a cancer that is not amenable to surgical intervention and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.

[0261] An anti-tumor effect as used herein, refers to a biological effect that may present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor. An anti-tumor effect may also refer to the prevention of the occurrence of a tumor, e.g., a vaccine.

[0262] A cytokine, as used herein, refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. Cytokine as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. A cytokine may be endogenously expressed by a cell or administered to a subject. Cytokines may be released by immune cells, including macrophages, B cells, T cells, neutrophils, dendritic cells, eosinophils and mast cells to propagate an immune response. Cytokines may induce various responses in the recipient cell. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and pro-inflammatory cytokines may promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, IL-23, IL-27, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), TGF-beta, IL-35, and perform. Examples of acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0263] The term lymphocyte as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the innate immune system. NK cells reject tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They were termed natural killers because they do not require activation in order to kill cells. T cells play a major role in cell-mediated-immunity (no antibody involvement). T cell receptors (TCR) differentiate T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is the primary site for T cell maturation. There are numerous types of T cells, including: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T-killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem memory cells (TSCM), like naive cells, are CD45RO?, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory cells (TCM) express L-selectin and CCR7, they secrete IL-2, but not IFN? or IL-4, and (iii) effector memory cells (TEM), however, do not express L-selectin or CCR7 but produce effector cytokines like IFN? and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ or CD4+ FoxP3+ regulatory T cells), natural killer T cells (NKT) and gamma delta T cells. B-cells, on the other hand, play a principal role in humoral immunity (with antibody involvement). B-cells make antibodies, are capable of acting as antigen-presenting cells (APCs) and turn into memory B-cells and plasma cells, both short-lived and long-lived, after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow.

[0264] The term genetically engineered or engineered refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some embodiments, the cell that is modified is a lymphocyte, e.g., a T cell, which may either be obtained from a patient or a donor. The cell may be modified to express an exogenous construct, such as, e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is incorporated into the cell's genome.

[0265] An immune response refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0266] A costimulatory signal, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to a T cell response, such as, but not limited to, proliferation and/or upregulation or down regulation of key molecules.

[0267] A costimulatory ligand, as used herein, includes a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A costimulatory ligand induces a signal that is in addition to the primary signal provided by a stimulatory molecule, for instance, by binding of a T cell receptor (TCR)/CD3 complex with a major histocompatibility complex (MHC) molecule loaded with peptide. A co-stimulatory ligand may include, but is not limited to, 3/TR6, 4-IBB ligand, agonist or antibody that binds Toll-like receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), ligand that specifically binds with B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death (PD) LI. A co-stimulatory ligand includes, without limitation, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligand that specifically binds with CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0268] A costimulatory molecule is a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, 4-1BB/CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD 18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha; beta; delta; epsilon; gamma; zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1-1a, CD1-1b, CD1-1c, CD1-1d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSFi4), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD1 1a/CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG/Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0269] The recitations sequence identity or, for example, comprising a sequence 50% identical to, as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a percentage of sequence identity may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where, in the case of polypeptides, the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0270] As used herein, a vaccine refers to a composition for generating immunity for the prophylaxis and/or treatment of diseases. Accordingly, vaccines are medicaments which comprise antigens and are intended to be used in humans or animals for generating specific defense and protective substances upon administration to the human or animal.

[0271] As used herein, a neoantigen refers to a class of tumor antigens which arises from tumor-specific mutations in an expressed protein.

2. Vectors, Precursor RNA, and Circular RNA

[0272] Also provided herein are circular RNAs, precursor RNAs that can circularize into the circular RNAs, and vectors (e.g., DNA vectors) that can be transcribed into the precursor RNAs or the circular RNAs.

[0273] In certain aspects, provided herein are circular RNA polynucleotides comprising a post splicing 3 group I intron fragment, optionally a first spacer, an Internal Ribosome Entry Site (IRES), an expression sequence, optionally a second spacer, and a post splicing 5 group I intron fragment. In some embodiments, these regions are in that order. In some embodiments, the circular RNA is made by a method provided herein or from a vector provided herein.

[0274] In certain embodiments, transcription of a vector provided herein (e.g., comprising a 5 duplex forming region, a 3 group I intron fragment, optionally a first spacer, an Internal Ribosome Entry Site (IRES), a first expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, optionally a second spacer, a 5 group I intron fragment, and a 3 duplex forming region) results in the formation of a precursor linear RNA polynucleotide capable of circularizing. In some embodiments, this precursor linear RNA polynucleotide circularizes when incubated in the presence of guanosine nucleotide or nucleoside (e.g., GTP) and divalent cation (e.g., Mg2+).

[0275] In some embodiments, the vectors and precursor RNA polynucleotides provided herein comprise a first (5) duplex forming region and a second (3) duplex forming region. In certain embodiments, the first and second duplex forming regions may form perfect or imperfect duplexes. Thus, in certain embodiments at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the first and second duplex forming regions may be base paired with one another. In some embodiments, the duplex forming regions are predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base pairing with unintended sequences in the RNA (e.g., non-duplex forming region sequences). In some embodiments, including such duplex forming regions on the ends of the precursor RNA strand, and adjacent or very close to the group I intron fragment, bring the group I intron fragments in close proximity to each other, increasing splicing efficiency. In some embodiments, the duplex forming regions are 3 to 100 nucleotides in length (e.g., 3-75 nucleotides in length, 3-50 nucleotides in length, 20-50 nucleotides in length, 35-50 nucleotides in length, 5-25 nucleotides in length, 9-19 nucleotides in length). In some embodiments, the duplex forming regions are about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, the duplex forming regions have a length of about 9 to about 50 nucleotides. In one embodiment, the duplex forming regions have a length of about 9 to about 19 nucleotides. In some embodiments, the duplex forming regions have a length of about 20 to about 40 nucleotides. In certain embodiments, the duplex forming regions have a length of about 30 nucleotides.

[0276] Two types of spacers have been designed for improving precursor RNA circularization and/or gene expression from circular RNA. The first type of spacer is external spacer, i.e., present in a precursor RNA but removed upon circularization. While not wishing to be bound by theory, it is contemplated that an external spacer may improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other neighboring sequence elements from interfering with its folding and function. The second type of spacer is internal spacer, i.e., present in a precursor RNA and retained in a resulting circular RNA. While not wishing to be bound by theory, it is contemplated that an internal spacer may improve ribozyme-mediated circularization by maintaining the structure of the ribozyme itself and preventing other neighboring sequence elements, particularly the neighboring IRES and coding region, from interfering with its folding and function. It is also contemplated that an internal spacer may improve protein expression from the IRES by preventing neighboring sequence elements, particularly the intron elements, from hybridizing with sequences within the IRES and inhibiting its ability to fold into its most preferred and active conformation.

[0277] In certain embodiments, the vectors, precursor RNA and circular RNA provided herein comprise a first (5) and/or a second (3) spacer. In some embodiments, including a spacer between the 3 group I intron fragment and the IRES may conserve secondary structures in those regions by preventing them from interacting, thus increasing splicing efficiency. In some embodiments, the first (between 3 group I intron fragment and IRES) and second (between the expression sequences and 5 group I intron fragment) spacers comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex forming regions. In some embodiments, such spacer base pairing brings the group I intron fragments in close proximity to each other, further increasing splicing efficiency. Additionally, in some embodiments, the combination of base pairing between the first and second duplex forming regions, and separately, base pairing between the first and second spacers, promotes the formation of a splicing bubble containing the group I intron fragments flanked by adjacent regions of base pairing. Typical spacers are contiguous sequences with one or more of the following qualities: 1) predicted to avoid interfering with proximal structures, for example, the IRES, expression sequences, or intron; 2) is at least 7 nt long and no longer than 100 nt; 3) is located after and adjacent to the 3 intron fragment and/or before and adjacent to the 5 intron fragment; and 4) contains one or more of the following: a) an unstructured region at least 5 nt long, b) a region of base pairing at least 5 nt long to a distal sequence, including another spacer, and c) a structured region at least 7 nt long limited in scope to the sequence of the spacer. Spacers may have several regions, including an unstructured region, a base pairing region, a hairpin/structured region, and combinations thereof. In an embodiment, the spacer has a structured region with high GC content. In an embodiment, a region within a spacer base pairs with another region within the same spacer. In an embodiment, a region within a spacer base pairs with a region within another spacer. In an embodiment, a spacer comprises one or more hairpin structures. In an embodiment, a spacer comprises one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In an embodiment, there is an additional spacer between the 3 group I intron fragment and the IRES. In an embodiment, this additional spacer prevents the structured regions of the IRES from interfering with the folding of the 3 group I intron fragment or reduces the extent to which this occurs. In some embodiments, the 5 spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5 spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5 spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and/or 25 and 35 nucleotides in length. In certain embodiments, the 5 spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 5 spacer sequence is a polyA sequence. In another embodiment, the 5 spacer sequence is a polyAC sequence. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C/T or C/U) content.

[0278] In certain embodiments, a 3 group I intron fragment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 3 proximal fragment of a natural group I intron including the 3 splice site dinucleotide and optionally the adjacent exon sequence at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nt in length) and at most the length of the exon. Typically, a 5 group I intron fragment is a contiguous sequence at least 75% homologous (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous) to a 5 proximal fragment of a natural group I intron including the 5 splice site dinucleotide and optionally the adjacent exon sequence at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nt in length) and at most the length of the exon. As described by Umekage et al. (2012), external portions of the 3 group I intron fragment and 5 group I intron fragment are removed in circularization, causing the circular RNA provided herein to comprise only the portion of the 3 group I intron fragment formed by the optional exon sequence of at least 1 nt in length and 5 group I intron fragment formed by the optional exon sequence of at least 1 nt in length, if such sequences were present on the non-circularized precursor RNA. The part of the 3 group I intron fragment that is retained by a circular RNA is referred to herein as the post splicing 3 group I intron fragment. The part of the 5 group I intron fragment that is retained by a circular RNA is referred to herein as the post splicing 5 group I intron fragment.

[0279] In certain embodiments, the vectors, precursor RNA and circular RNA provided herein comprise an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA (e.g., open reading frames that form the expression sequences). The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22 150-161.

[0280] A multitude of IRES sequences are available and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al., J. Virol. (1989) 63: 1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125-15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like.

[0281] In some embodiments, an IRES is an IRES sequence of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G.

[0282] For driving protein expression, the circular RNA comprises an IRES operably linked to a protein coding sequence. Exemplary IRES sequences are provided in Table 17 below. In some embodiments, the circular RNA disclosed herein comprises an IRES sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequence in Table 17. In some embodiments, the circular RNA disclosed herein comprises an IRES sequence in Table 17. Modifications of IRES and accessory sequences are disclosed herein to increase or reduce IRES activities, for example, by truncating the 5 and/or 3 ends of the IRES, adding a spacer 5 to the IRES, modifying the 6 nucleotides 5 to the translation initiation site (Kozak sequence), modification of alternative translation initiation sites, and creating chimeric/hybrid IRES sequences. In some embodiments, the IRES sequence in the circular RNA disclosed herein comprises one or more of these modifications relative to a native IRES (e.g., a native IRES disclosed in Table 17).

[0283] A multitude of IRES sequences are available and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63: 1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125-15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and the like.

[0284] In some embodiments, the IRES is an IRES sequence of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G.

[0285] In some embodiments, the polynucleotides herein comprise more than one expression sequence. In some embodiments, the circular RNA is a bicistronic RNA. The sequences encoding the two or more polypeptides can be separated by a ribosomal skipping element or a nucleotide sequence encoding a protease cleavage site. In certain embodiments, the ribosomai skipping element encodes thosea-asigna virus 2A peptide (T2A), porcine teschovirus-1 2 A peptide (P2A), foot-and-mouth disease virus 2 A peptide (F2A), equine rhinitis A vims 2A peptide (E2A), cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A), or flacherie vims of B. mori 2A peptide (BmIFV 2A).

[0286] In certain embodiments, the vectors provided herein comprise a 3 UTR. In some embodiments, the 3 UTR is from human beta globin, human alpha globin Xenopus beta globin, Xenopus alpha globin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNF?, dengue virus, hantavirus small mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic ringspot virus, woodchuck hepatitis virus post translationally regulated element, sindbis virus, turnip crinkle virus, tobacco etch virus, or Venezuelan equine encephalitis virus.

[0287] In some embodiments, the vectors provided herein comprise a 5 UTR. In some embodiments, the 5 UTR is from human beta globin, Xenopus laevis beta globin, human alpha globin, Xenopus laevis alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or the adenovirus tripartite leader.

[0288] In some embodiments, a vector provided herein comprises a polyA region external of the 3 and/or 5 group I intron fragments. In some embodiments the polyA region is at least 15, 30, or 60 nucleotides long. In some embodiments, one or both polyA regions is 15-50 nucleotides long. In some embodiments, one or both polyA regions is 20-25 nucleotides long. The polyA sequence is removed upon circularization. Thus, an oligonucleotide hybridizing with the polyA sequence, such as a deoxythymine oligonucleotide (oligo(dT)) conjugated to a solid surface (e.g., a resin), can be used to separate circular RNA from its precursor RNA. Other sequences can also be disposed 5 to the 3 group I intron fragment or 3 to the 5 group I intron fragment and a complementary sequence can similarly be used for circular RNA purification.

[0289] In some embodiments, the DNA (e.g., vector), linear RNA (e.g., precursor RNA), and/or circular RNA polynucleotide provided herein is between 300 and 15000, 300 and 14000, 300 and 13000, 300 and 12000, 300 and 11000, 300 and 10000, 400 and 9000, 500 and 8000, 600 and 7000, 700 and 6000, 800 and 5000, 900 and 5000, 1000 and 5000, 1100 and 5000, 1200 and 5000, 1300 and 5000, 1400 and 5000, and/or 1500 and 5000 nucleotides in length. In some embodiments, the polynucleotide is at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, 10000 nt, 11000 nt, 12000 nt, 13000 nt, 14000 nt, or 15000 nt in length. In some embodiments, the polynucleotide is no more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, 10000 nt, 11000 nt, 12000 nt, 13000 nt, 14000 nt, 15000 nt, or 16000 nt in length. In some embodiments, the length of a DNA, linear RNA, and/or circular RNA polynucleotide provided herein is about 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, 10000 nt, 11000 nt, 12000 nt, 13000 nt, 14000 nt, or 15000 nt.

[0290] In some embodiments, provided herein is a vector. In certain embodiments, the vector comprises, in the following order, a) a 5 duplex forming region, b) a 3 group I intron fragment, c) optionally, a first spacer sequence, d) an IRES, e) a first expression sequence, f) a polynucleotide sequence encoding a cleavage site, g) a second expression sequence, h) optionally, a second spacer sequence, i) a 5 group I intron fragment, and g) a 3 duplex forming region. In some embodiments, the vector comprises a transcriptional promoter upstream of the 5 duplex forming region.

[0291] In some embodiments, provided herein is a vector. In certain embodiments, the vector comprises, in the following order, a) a 5 duplex forming region, b) a 3 group I intron fragment, c) optionally, a first spacer sequence, d) a first IRES, e) a first expression sequence, f) a second IRES, g) a second expression sequence, h) optionally, a second spacer sequence, i) a 5 group I intron fragment, and g) a 3 duplex forming region. In some embodiments, the vector comprises a transcriptional promoter upstream of the 5 duplex forming region.

[0292] In some embodiments, provided herein is a precursor RNA. In certain embodiments, the precursor RNA is a linear RNA produced by in vitro transcription of a vector provided herein. In some embodiments, the precursor RNA comprises, in the following order, a) optionally, a 5 duplex forming region, b) a 3 group I intron fragment, c) optionally, a first spacer sequence, d) an IRES, e) a first expression sequence, f) a polynucleotide sequence encoding a cleavage site, g) a second expression sequence, h) optionally, a second spacer sequence, i) a 5 group I intron fragment, and j) optionally, a 3 duplex forming region. In some embodiments, the precursor RNA comprises, in the following order, a) a 5 duplex forming region, b) a 3 group I intron fragment, c) optionally, a first spacer sequence, d) a first IRES, e) a first expression sequence, f) a second IRES, g) a second expression sequence, h) optionally, a second spacer sequence, i) a 5 group I intron fragment, and j) a 3 duplex forming region. The precursor RNA can be unmodified, partially modified or completely modified.

[0293] In certain embodiments, provided herein is a circular RNA. In certain embodiments, the circular RNA is a circular RNA produced by a vector provided herein. In some embodiments, the circular RNA is circular RNA produced by circularization of a precursor RNA provided herein. In certain embodiments, transcription of a vector provided herein results in the formation of a precursor linear RNA capable of circularizing. In some embodiments, this precursor linear RNA polynucleotide circularizes when incubated in the presence of guanosine nucleotide or nucleoside (e.g., GTP) and divalent cation (e.g., Mg.sup.2+). In some embodiments, the circular RNA comprises, in the following sequence, a) a first spacer sequence, b) an IRES, c) a first expression sequence, d) a polynucleotide sequence encoding a cleavage site, e) a second expression sequence, and f) a second spacer sequence. In some embodiments, the circular RNA comprises, in the following sequence, a) a post splicing 3 group I intron fragment, b) a first spacer sequence, c) an IRES, d) a first expression sequence, e) a polynucleotide sequence encoding a cleavage site, f) a second expression sequence, and g) a second spacer sequence, h) a post splicing 5 group I intron fragment. In some embodiments, the circular RNA comprises, in the following sequence, a) a first spacer sequence, b) a first IRES, c) a first expression sequence, d) a second IRES, e) a second expression sequence, and f) a second spacer sequence. In some embodiments, the circular RNA further comprises the portion of the 3 group I intron fragment that is 3 of the 3 splice site. In some embodiments, the circular RNA further comprises the portion of the 5 group I intron fragment that is 5 of the 5 splice site. In some embodiments, the circular RNA is at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000 nucleotides in size. The circular RNA can be unmodified, partially modified or completely modified.

[0294] In some embodiments, the vectors and precursor RNA polynucleotides provided herein comprise a first (5) duplex forming region and a second (3) duplex forming region. In certain embodiments, the first and second homology regions may form perfect or imperfect duplexes. Thus, in certain embodiments at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the first and second duplex forming regions may be base paired with one another. In some embodiments, the duplex forming regions are predicted to have less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%) base pairing with unintended sequences in the RNA (e.g., non-duplex forming region sequences). In some embodiments, including such duplex forming regions on the ends of the precursor RNA strand, and adjacent or very close to the group I intron fragment, bring the group I intron fragments in close proximity to each other, increasing splicing efficiency. In some embodiments, the duplex forming regions are 3 to 100 nucleotides in length (e.g., 3-75 nucleotides in length, 3-50 nucleotides in length, 20-50 nucleotides in length, 35-50 nucleotides in length, 5-25 nucleotides in length, 9-19 nucleotides in length). In some embodiments, the duplex forming regions are about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, the duplex forming regions have a length of about 9 to about 50 nucleotides. In one embodiment, the duplex forming regions have a length of about 9 to about 19 nucleotides. In some embodiments, the duplex forming regions have a length of about 20 to about 40 nucleotides. In certain embodiments, the duplex forming regions have a length of about 30 nucleotides.

[0295] In some embodiments, the circular RNA provided herein has higher functional stability than mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein has higher functional stability than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

[0296] In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, functional half-life can be assessed through the detection of functional protein synthesis.

[0297] In certain embodiments, the vectors, precursor RNA and circular RNA provided herein comprise a first (5) and/or a second (3) spacer. In some embodiments, including a spacer between the 3 group I intron fragment and the IRES may conserve secondary structures in those regions by preventing them from interacting, thus increasing splicing efficiency. In some embodiments, the first (between 3 group I intron fragment and IRES) and second (between the two expression sequences and 5 group I intron fragment) spacers comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex forming regions. In other embodiments, the first (between 3 group I intron fragment and IRES) and second (between the one of the expression sequences and 5 group I intron fragment) spacers comprise additional base pairing regions that are predicted to base pair with each other and not to the first and second duplex forming regions. In some embodiments, such spacer base pairing brings the group I intron fragments in close proximity to each other, further increasing splicing efficiency. Additionally, in some embodiments, the combination of base pairing between the first and second duplex forming regions, and separately, base pairing between the first and second spacers, promotes the formation of a splicing bubble containing the group I intron fragments flanked by adjacent regions of base pairing. Typical spacers are contiguous sequences with one or more of the following qualities: 1) predicted to avoid interfering with proximal structures, for example, the IRES, expression sequence, or intron; 2) is at least 7 nt long and no longer than 100 nt; 3) is located after and adjacent to the 3 intron fragment and/or before and adjacent to the 5 intron fragment; and 4) contains one or more of the following: a) an unstructured region at least 5 nt long, b) a region of base pairing at least 5 nt long to a distal sequence, including another spacer, and c) a structured region at least 7 nt long limited in scope to the sequence of the spacer. Spacers may have several regions, including an unstructured region, a base pairing region, a hairpin/structured region, and combinations thereof. In an embodiment, the spacer has a structured region with high GC content. In an embodiment, a region within a spacer base pairs with another region within the same spacer. In an embodiment, a region within a spacer base pairs with a region within another spacer. In an embodiment, a spacer comprises one or more hairpin structures. In an embodiment, a spacer comprises one or more hairpin structures with a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In an embodiment, there is an additional spacer between the 3 group I intron fragment and the IRES. In an embodiment, this additional spacer prevents the structured regions of the IRES from interfering with the folding of the 3 group I intron fragment or reduces the extent to which this occurs. In some embodiments, the 5 spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5 spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5 spacer sequence is between 5 and 50, 10 and 50, 20 and 50, 20 and 40, and/or 25 and 35 nucleotides in length. In certain embodiments, the 5 spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 5 spacer sequence is a polyA sequence. In another embodiment, the 5 spacer sequence is a polyAC sequence. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C/T or C/U) content.

[0298] In certain embodiments, a 3 group I intron fragment is a contiguous sequence at least 75% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to a 3 proximal fragment of a natural group I intron including the 3 splice site dinucleotide and optionally the adjacent exon sequence at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nt in length) and at most the length of the exon. Typically, a 5 group I intron fragment is a contiguous sequence at least 75% identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to a 5 proximal fragment of a natural group I intron including the 5 splice site dinucleotide and optionally the adjacent exon sequence at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nt in length) and at most the length of the exon. As described by Umekage et al. (2012), external portions of the 3 group I intron fragment and 5 group I intron fragment are removed in circularization, causing the circular RNA provided herein to comprise only the portion of the 3 group I intron fragment formed by the optional exon sequence of at least 1 nt in length and 5 group I intron fragment formed by the optional exon sequence of at least 1 nt in length, if such sequences were present on the non-circularized precursor RNA. The part of the 3 group I intron fragment that is retained by a circular RNA is referred to herein as the post splicing 3 group I intron fragment. The part of the 5 group I intron fragment that is retained by a circular RNA is referred to herein as the post splicing 5 group I intron fragment.

[0299] In some embodiments, the circular RNA provided herein may have a higher magnitude of expression than equivalent linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells. In some embodiments, the circular RNA provided herein has a higher magnitude of expression than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

[0300] In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a functional half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, functional half-life can be assessed through the detection of functional protein synthesis.

[0301] In some embodiments, the circular RNA polynucleotide provided herein has a half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments, the circular RNA polynucleotide provided herein has a half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNA polynucleotide provided herein has a half-life greater than (e.g., at least 1.5-fold greater than, at least 2-fold greater than) that of an equivalent linear RNA polynucleotide encoding the same protein. In some embodiments, the circular RNA polynucleotide, or pharmaceutical composition thereof, has a functional half-life in a human cell greater than or equal to that of a pre-determined threshold value. In some embodiments the functional half-life is determined by a functional protein assay. For example in some embodiments, the functional half-life is determined by an in vitro luciferase assay, wherein the activity of Gaussia luciferase (GLuc) is measured in the media of human cells (e.g. HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, the functional half-life is determined by an in vivo assay, wherein levels of a protein encoded by the expression sequence of the circular RNA polynucleotide are measured in patient serum or tissue samples every 1, 2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In some embodiments, the pre-determined threshold value is the functional half-life of a reference linear RNA polynucleotide comprising the same expression sequence as the circular RNA polynucleotide.

[0302] In some embodiments, the circular RNA provided herein may have a higher magnitude of expression than equivalent linear mRNA, e.g., a higher magnitude of expression 24 hours after administration of RNA to cells. In some embodiments, the circular RNA provided herein has a higher magnitude of expression than mRNA comprising the same expression sequence, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

[0303] In some embodiments, the circular RNA provided herein may be less immunogenic than an equivalent mRNA when exposed to an immune system of an organism or a certain type of immune cell. In some embodiments, the circular RNA provided herein is associated with modulated production of cytokines when exposed to an immune system of an organism or a certain type of immune cell. For example, in some embodiments, the circular RNA provided herein is associated with reduced production of TNF?, RIG-I, IL-2, IL-6, IFN?, and/or a type 1 interferon, e.g., IFN-?1, when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein is associated with less TNF?, RIG-I, IL-2, IL-6, IFN?, and/or type 1 interferon, e.g., IFN-?1, transcript induction when exposed to an immune system of an organism or a certain type of immune cell as compared to mRNA comprising the same expression sequence. In some embodiments, the circular RNA provided herein is less immunogenic than mRNA comprising the same expression sequences. In some embodiments, the circular RNA provided herein is less immunogenic than mRNA comprising the same expression sequences, 5moU modifications, an optimized UTR, a cap, and/or a polyA tail.

[0304] In some embodiments, the compositions and methods described herein provide RNA (e.g., circRNA) with higher stability or functional stability than an equivalent linear RNA without the need for nucleoside modifications. In some embodiments, methods for producing RNA lacking nucleoside modifications produce higher percentages of full length transcripts than methods for producing RNA containing nucleoside modifications due to reduced abortive transcription. In some embodiments, the compositions and methods described herein are capable of producing large (e.g., 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, or 15 kb) RNA constructs without the added abortive transcription associated with RNA containing nucleoside modifications.

[0305] In certain embodiments, the circular RNA provided herein can be transfected into a cell as is, or can be transfected in DNA vector form and transcribed in the cell. Transcription of circular RNA from a transfected DNA vector can be via added polymerases or polymerases encoded by nucleic acids transfected into the cell, or preferably via endogenous polymerases.

[0306] In certain embodiments, a circular RNA polynucleotide provided herein comprises modified RNA nucleotides and/or modified nucleosides. In some embodiments, the modified nucleoside is m.sup.5C (5-methylcytidine). In another embodiment, the modified nucleoside is m.sup.5U (5-methyluridine). In another embodiment, the modified nucleoside is m.sup.6A (N.sup.6-methyladenosine). In another embodiment, the modified nucleoside is s.sup.2U (2-thiouridine). In another embodiment, the modified nucleoside is ? (pseudouridine). In another embodiment, the modified nucleoside is Um (2-O-methyluridine). In other embodiments, the modified nucleoside is m.sup.1A (1-methyladenosine); m.sup.2A (2-methyladenosine); Am (2-O-methyladenosine); ms.sup.2 m.sup.6A (2-methylthio-N.sup.6-methyladenosine); i.sup.6A (N.sup.6-isopentenyladenosine); ms.sup.2i6A (2-methylthio-N.sup.6 isopentenyladenosine); io.sup.6A (N.sup.6-(cis-hydroxyisopentenyl)adenosine); ms.sup.2io.sup.6A (2-methylthio-N.sup.6-(cis-hydroxyisopentenyl)adenosine); g.sup.6A (N.sup.6-glycinylcarbamoyladenosine); t.sup.6A (N.sup.6-threonylcarbamoyladenosine); ms.sup.2t.sup.6A (2-methylthio-N.sup.6-threonyl carbamoyladenosine); m.sup.6t.sup.6A (N.sup.6-methyl-N.sup.6-threonylcarbamoyladenosine); hn.sup.6A(N.sup.6-hydroxynorvalylcarbamoyladenosine); ms.sup.2hn.sup.6A (2-methylthio-N.sup.6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2-O-ribosyladenosine (phosphate)); I (inosine); m.sup.1I (1-methylinosine); m.sup.1Im (1,2-O-dimethylinosine); m.sup.3C (3-methylcytidine); Cm (2-O-methylcytidine); s.sup.2C (2-thiocytidine); ac.sup.4C (N.sup.4-acetylcytidine); f.sup.5C (5-formylcytidine); m.sup.5Cm (5,2-O-dimethylcytidine); ac.sup.4Cm (N.sup.4-acetyl-2-O-methylcytidine); k.sup.2C (lysidine); m.sup.1G (1-methylguanosine); m.sup.2G (N.sup.2-methylguanosine); m.sup.7G (7-methylguanosine); Gm (2-O-methylguanosine); m.sup.2 2G (N.sup.2,N.sup.2-dimethylguanosine); m.sup.2Gm (N.sup.2,2-O-dimethylguanosine); m.sup.2.sub.2Gm (N.sup.2,N.sup.2,2-O-trimethylguanosine); Gr(p) (2-O-ribosylguanosine(phosphate)); yW (wybutosine); o.sub.2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQ.sub.0 (7-cyano-7-deazaguanosine); preQ.sub.1 (7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine); m.sup.5Um (5,2-O-dimethyluridine); s.sup.4U (4-thiouridine); m.sup.5s.sup.2U (5-methyl-2-thiouridine); s.sup.2Um (2-thio-2-O-methyluridine); acp.sup.3U (3-(3-amino-3-carboxypropyl)uridine); ho.sup.5U (5-hydroxyuridine); mo.sup.5U (5-methoxyuridine); cmo.sup.5U (uridine 5-oxyacetic acid); mcmo.sup.5U (uridine 5-oxyacetic acid methyl ester); chm.sup.5U (5-(carboxyhydroxymethyl)uridine)); mchm.sup.5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm.sup.5U (5-methoxycarbonylmethyluridine); mcm.sup.5Um (5-methoxycarbonylmethyl-2-O-methyluridine); mcm.sup.5s.sup.2U (5-methoxycarbonylmethyl-2-thiouridine); nm.sup.5S2U (5-aminomethyl-2-thiouridine); mnm.sup.5U (5-methylaminomethyluridine); mnm.sup.5s.sup.2U (5-methylaminomethyl-2-thiouridine); mnm.sup.5se.sup.2U (5-methylaminomethyl-2-selenouridine); ncm.sup.5U (5-carbamoylmethyluridine); ncm.sup.5Um (5-carbamoylmethyl-2-O-methyluridine); cmnm.sup.5U (5-carboxymethylaminomethyluridine); cmnm.sup.5Um (5-carboxymethylaminomethyl-2-O-methyluridine); cmnm.sup.5s2U (5-carboxymethylaminomethyl-2-thiouridine); m.sup.6.sub.2A (N.sup.6,N.sup.6-dimethyladenosine); Im (2-O-methylinosine); m.sup.4C (N.sup.4-methylcytidine); m.sup.4Cm (N.sup.4,2-O-dimethylcytidine); hm.sup.5C (5-hydroxymethylcytidine); m.sup.3U (3-methyluridine); cm.sup.5U (5-carboxymethyluridine); m.sup.6Am (N.sup.6,2-O-dimethyladenosine); m.sup.6.sub.2Am (N.sup.6,N.sup.6,O-2-trimethyladenosine); m.sup.2,7G (N.sup.2,7-dimethylguanosine); m.sup.2,2.sup.7G (N.sup.2,N.sup.2,7-trimethylguanosine); m.sup.3Um (3,2-O-dimethyluridine); m.sup.5D (5-methyldihydrouridine); f.sup.5Cm (5-formyl-2-O-methylcytidine); m.sup.1Gm (1,2-O-dimethylguanosine); m.sup.1Am (1,2-O-dimethyladenosine); ?m.sup.5U (5-taurinomethyluridine); ?m.sup.5s2U (5-taurinomethyl-2-thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac.sup.6A (N.sup.6-acetyladenosine).

[0307] In some embodiments, the modified nucleoside may include a compound selected from the group of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-m ethoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In another embodiment, the modifications are independently selected from the group consisting of 5-methylcytosine, pseudouridine and 1-methylpseudouridine.

[0308] In some embodiments, the modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methyl-pseudouridine, N6-methyladenosine, and/or pseudouridine. In some embodiments, such modified nucleosides provide additional stability and resistance to immune activation.

[0309] In particular embodiments, polynucleotides may be codon-optimized. A codon optimized sequence may be one in which codons in a polynucleotide encoding a polypeptide have been substituted in order to increase the expression, stability and/or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, and/or (x) systematic variation of codon sets for each amino acid. In some embodiments, a codon optimized polynucleotide may minimize ribozyme collisions and/or limit structural interference between the expression sequence and the IRES.

[0310] In certain embodiments circular RNA provided herein is produced inside a cell. In some embodiments, precursor RNA is transcribed using a DNA template (e.g., in some embodiments, using a vector provided herein) in the cytoplasm by a bacteriophage RNA polymerase, or in the nucleus by host RNA polymerase II and then circularized.

[0311] In certain embodiments, the circular RNA provided herein is injected into an animal (e.g., a human), such that a polypeptide encoded by the circular RNA molecule is expressed inside the animal.

3. Payload

[0312] In some embodiments, the first expression sequence encodes a therapeutic protein. In some embodiments, the second expression sequence encodes a therapeutic protein. In some embodiments, one or both of the therapeutic proteins are selected from the proteins listed in the following table.

TABLE-US-00001 Target cell/ Payload Sequence organ Preferred delivery formulation CD19 CAR Any of sequences 309-314 T cells [00001] (50 mol %) DSPC (10 mol %) Beta-sitosterol (28.5% mol %) Cholesterol (10 mol %) PEG DMG (1.5 mol %) BCMA CAR MALPVTALLLPLALLL HAARPDIVLTQSPASLA VSLGERATINCRASESV SVIGAHLIHWYQQKPG QPPKLLIYLASNLETGV PARFSGSGSGTDFTLTIS SLQAEDAAIYYCLQSRI FPRTFGQGTKLEIKGST SGSGKPGSGEGSTKGQ VQLVQSGSELKKPGAS T cells [00002] VKVSCKASGYTFTDYSI (50 mol %) NWVRQAPGQGLEWMG DSPC (10 mol %) WINTETREPAYAYDFR Beta-sitosterol (28.5% mol %) GRFVFSLDTSVSTAYLQ Cholesterol (10 mol %) ISSLKAEDTAVYYCAR PEG DMG (1.5 mol %) DYSYAMDYWGQGTLV TVSSAAATTTPAPRPPT PAPTIASQPLSLRPEACR PAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLL SLVITLYCKRGRKKLLY IFKQPFRPVQTTQEED GCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQ NQLYNELNLGRREEYD VLDKRRGRDPEMGGKP RRKNPQEGLYNELQKD KMAEAYSEIGMKGERR RGKGHDGLYQGLSTAT KDTYDALHMQALPPR MAGE- A4 TCR TCR alpha chain: KNQVEQSPQSLIILEGK NCTLQCNYTVSPFSNLR WYKQDTGRGPVSLTIM TFSENTKSNGRYTATLD ADTKQSSLHITASQLSD SASYICVVNHSGGSYIP TFGRGTSLIVHPYIQKP DPAVYQLRDSKSSDKS VCLFTDFDSQTNVSQSK T cells [00003] DSDVYITDKTVLDMRS (50 mol %) MDFKSNSAVAWSNKS DSPC (10 mol %) DFACANAFNNSIIPEDT Beta-sitosterol (28.5% mol %) FFPSPESS Cholesterol (10 mol %) TCR beta chain: PEG DMG (1.5 mol %) DVKVTQSSRYLVKRTG EKVFLECVQDMDHEN MFWYRQDPGLGLRLIY FSYDVKMKEKGDIPEG YSVSREKKERFSLILES ASTNQTSMYLCASSFL MTSGDPYEQYFGPGTR LTVTEDLKNVFPPEVA VFEPSEAEISHTQKATL VCLATGFYPDHVELSW WVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSS RLRVSATFWQNPRNHF RCQVQFYGLSENDEWT QDRAKPVTQIVSAEAW GRAD NY- ESO TCR TCRalpha extracellular sequence MQEVTQIPAALSVPEGE NLVLNCSFTDSAIYNLQ WFRQDPGKGLTSLLLIQ SSQREQTSGRLNASLDK SSGRSTLYIAASQPGDS ATYLCAVRPTSGGSYIP TFGRGTSLIVHPY TCRbeta extracellular T cells [00004] sequence (50 mol %) MGVTQTPKFQVLKTGQ DSPC (10 mol %) SMTLQCAQDMNHEYM Beta-sitosterol (28.5% mol %) SWYRQDPGMGLRLIHY Cholesterol (10 mol %) SVGAGITDQGEVPNGY PEG DMG (1.5 mol %) NVSRSTTEDFPLRLLSA APSQTSVYFCASSYVG NTGELFFGEGSRLTVL EPO APPRLICDSRVLERYLL Kidney EAKEAENITTGCAEHCS or bone LNENITVPDTKVNFYA marrow WKRMEVGQQAVEVW QGLALLSEAVLRGQAL LVNSSQPWEPLQLHVD KAVSGLRSLTTLLRALG AQKEAISPPDAASAAPL RTITADTFRKLFRVYSN FLRGKLKLYTGEACRT GDR PAH MSTAVLENPGLGRKLS DFGQETSYIEDNCNQN GAISLIFSLKEEVGALA KVLRLFEENDVNLTHIE SRPSRLKKDEYEFFTHL DKRSLPALTNIIKILRHD IGATVHELSRDKKKDT VPWFPRTIQELDRFANQ ILSYGAELDADHPGFKD PVYRARRKQFADIAYN YRHGQPIPRVEYMEEE Hepatic cells [00005] KKTWGTVFKTLKSLYK (50 mol %) THACYEYNHIFPLLEKY DSPC (10 mol %) CGFHEDNIPQLEDVSQF Cholesterol (38.5% mol %) LQTCTGFRLRPVAGLLS PEG-DMG (1.5%) SRDFLGGLAFRVFHCT OR QYIRHGSKPMYTPEPDI MC3 (50 mol %) CHELLGHVPLFSDRSFA DSPC (10 mol %) QFSQEIGLASLGAPDEY Cholesterol (38.5% mol %) IEKLATIYWFTVEFGLC PEG-DMG (1.5%) KQGDSIKAYGAGLLSSF GELQYCLSEKPKLLPLE LEKTAIQNYTVTEFQPL YYVAESFNDAKEKVRN FAATIPRPFSVRYDPYT QRIEVLDNTQQLKILAD SINSEIGILCSALQKIK CPS1 LSVKAQTAHIVLEDGT KMKGYSFGHPSSVAGE VVFNTGLGGYPEAITDP AYKGQILTMANPIIGNG GAPDTTALDELGLSKY LESNGIKVSGLLVLDYS KDYNHWLATKSLGQW LQEEKVPAIYGVDTRM LTKIIRDKGTMLGKIEF EGQPVDFVDPNKQNLI AEVSTKDVKVYGKGNP Hepatic cells [00006] TKVVAVDCGIKNNVIR (50 mol %) LLVKRGAEVHLVPWN DSPC (10 mol %) HDFTKMEYDGILIAGGP Cholesterol (38.5% mol %) GNPALAEPLIQNVRKIL PEG-DMG (1.5%) ESDRKEPLFGISTGNLIT OR GLAAGAKTYKMSMAN MC3 (50 mol %) RGQNQPVLNITNKQAFI DSPC (10 mol %) TAQNHGYALDNTLPAG Cholesterol (38.5% mol %) WKPLFVNVNDQTNEGI PEG-DMG (1.5%) MHESKPFFAVQFHPEV TPGPIDTEYLFDSFFSLI KKGKATTITSVLPKPAL VASRVEVSKVLILGSGG LSIGQAGEFDYSGSQAV KAMKEENVKTVLMNP NIASVQTNEVGLKQAD TVYFLPITPQFVTEVIKA EQPDGLILGMGGQTAL NCGVELFKRGVLKEYG VKVLGTSVESIMATED RQLFSDKLNEINEKIAPS FAVESIEDALKAADTIG YPVMIRSAYALGGLGS GICPNRETLMDLSTKAF AMTNQILVEKSVTGWK EIEYEVVRDADDNCVT VCNMENVDAMGVHTG DSVVVAPAQTLSNAEF QMLRRTSINVVRHLGIV GECNIQFALHPTSMEYC IIEVNARLSRSSALASK ATGYPLAFIAAKIALGIP LPEIKNVVSGKTSACFE PSLDYMVTKIPRWDLD RFHGTSSRIGSSMKSVG EVMAIGRTFEESFQKAL RMCHPSIEGFTPRLPMN KEWPSNLDLRKELSEPS STRIYAIAKAIDDNMSL DEIEKLTYIDKWFLYK MRDILNMEKTLKGLNS ESMTEETLKRAKEIGFS DKQISKCLGLTEAQTRE LRLKKNIHPWVKQIDTL AAEYPSVTNYLYVTYN GQEHDVNFDDHGMMV LGCGPYHIGSSVEFDW CAVSSIRTLRQLGKKTV VVNCNPETVSTDFDEC DKLYFEELSLERILDIYH QEACGGCIISVGGQIPN NLAVPLYKNGVKIMGT SPLQIDRAEDRSIFSAVL DELKVAQAPWKAVNT LNEALEFAKSVDYPCLL RPSYVLSGSAMNVVFS EDEMKKFLEEATRVSQ EHPVVLTKFVEGAREV EMDAVGKDGRVISHAI SEHVEDAGVHSGDATL MLPTQTISQGAIEKVKD ATRKIAKAFAISGPFNV QFLVKGNDVLVIECNL RASRSFPFVSKTLGVDF IDVATKVMIGENVDEK HLPTLDHPIIPADYVAIK APMFSWPRLRDADPILR CEMASTGEVACFGEGI HTAFLKAMLSTGFKIPQ KGILIGIQQSFRPRFLGV AEQLHNEGFKLFATEA TSDWLNANNVPATPVA WPSQEGQNPSLSSIRKLI RDGSIDLVINLPNNNTK FVHDNYVIRRTAVDSGI PLLTNFQVTKLFAEAV QKSRKVDSKSLFHYRQ YSAGKAA Cas9 MKRNYILGLDIGITSVG YGIIDYETRDVIDAGVR LFKEANVENNEGRRSK RGARRLKRRRRHRIQR VKKLLFDYNLLTDHSE LSGINPYEARVKGLSQK LSEEEFSAALLHLAKRR GVHNVNEVEEDTGNEL STKEQISRNSKALEEKY VAELQLERLKKDGEVR Immune cells [00007] GSINRFKTSDYVKEAK (50 mol %) QLLKVQKAYHQLDQSF DSPC (10 mol %) IDTYIDLLETRRTYYEG Beta-sitosterol (28.5% mol %) PGEGSPFGWKDIKEWY Cholesterol (10 mol %) EMLMGHCTYFPEELRS PEG DMG (1.5 mol %) VKYAYNADLYNALND LNNLVITRDENEKLEYY EKFQIIENVFKQKKKPT LKQIAKEILVNEEDIKG YRVTSTGKPEFTNLKV YHDIKDITARKEIIENAE LLDQIAKILTIYQSSEDI QEELTNLNSELTQEEIE QISNLKGYTGTHNLSLK AINLILDELWHTNDNQI AIFNRLKLVPKKVDLSQ QKEIPTTLVDDFILSPVV KRSFIQSIKVINAIIKKY GLPNDIIIELAREKNSKD AQKMINEMQKRNRQT NERIEEIIRTTGKENAKY LIEKIKLHDMQEGKCLY SLEAIPLEDLLNNPFNY EVDHIIPRSVSFDNSFNN KVLVKQEENSKKGNRT PFQYLSSSDSKISYETFK KHILNLAKGKGRISKTK KEYLLEERDINRFSVQK DFINRNLVDTRYATRG LMNLLRSYFRVNNLDV KVKSINGGFTSFLRRK WKFKKERNKGYKHHA EDALIIANADFIFKEWK KLDKAKKVMENQMFE EKQAESMPEIETEQEYK EIFITPHQIKHIKDFKDY KYSHRVDKKPNRELIN DTLYSTRKDDKGNTLI VNNLNGLYDKDNDKL KKLINKSPEKLLMYHH DPQTYQKLKLIMEQYG DEKNPLYKYYEETGNY LTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDY PNSRNKVVKLSLKPYR FDVYLDNGVYKFVTVK NLDVIKKENYYEVNSK CYEEAKKLKKISNQAEF IASFYNNDLIKINGELY RVIGVNNDLLNRIEVN MIDITYREYLENMNDK RPPRIIKTIASKTQSIKK YSTDILGNLYEVKSKK HPQIIKKG ADAM TS13 AAGGILHLELLVAVGP DVFQAHQEDTERYVLT NLNIGAELLRDPSLGAQ FRVHLVKMVILTEPEG APNITANLTSSLLSVCG WSQTINPEDDTDPGHA DLVLYITRFDLELPDGN RQVRGVTQLGGACSPT WSCLITEDTGFDLGVTI AHEIGHSFGLEHDGAPG SGCGPSGHVMASDGAA Hepatic cells [00008] PRAGLAWSPCSRRQLL (50 mol %) SLLSAGRARCVWDPPR DSPC (10 mol %) PQPGSAGHPPDAQPGL Cholesterol (38.5% mol %) YYSANEQCRVAFGPKA PEG-DMG (1.5%) VACTFAREHLDMCQAL OR SCHTDPLDQSSCSRLLV MC3 (50 mol %) PLLDGTECGVEKWCSK DSPC (10 mol %) GRCRSLVELTPIAAVHG Cholesterol (38.5% mol %) RWSSWGPRSPCSRSCG PEG-DMG (1.5%) GGVVTRRRQCNNPRPA FGGRACVGADLQAEM CNTQACEKTQLEFMSQ QCARTDGQPLRSSPGG ASFYHWGAAVPHSQG DALCRHMCRAIGESFIM KRGDSFLDGTRCMPSG PREDGTLSLCVSGSCRT FGCDGRMDSQQVWDR CQVCGGDNSTCSPRKG SFTAGRAREYVTFLTVT PNLTSVYIANHRPLFTH LAVRIGGRYVVAGKMS ISPNTTYPSLLEDGRVE YRVALTEDRLPRLEEIRI WGPLQEDADIQVYRRY GEEYGNLTRPDITFTYF QPKPRQAWVWAAVRG PCSVSCGAGLRWVNYS CLDQARKELVETVQCQ GSQQPPAWPEACVLEP CPPYWAVGDFGPCSAS CGGGLRERPVRCVEAQ GSLLKTLPPARCRAGA QQPAVALETCNPQPCP ARWEVSEPSSCTSAGG AGLALENETCVPGADG LEAPVTEGPGSVDEKLP APEPCVGMSCPPGWGH LDATSAGEKAPSPWGSI RTGAQAAHVWTPAAG SCSVSCGRGLMELRFLC MDSALRVPVQEELCGL ASKPGSRREVCQAVPC PARWQYKLAACSVSCG RGVVRRILYCARAHGE DDGEEILLDTQCQGLPR PEPQEACSLEPCPPRWK VMSLGPCSASCGLGTA RRSVACVQLDQGQDVE VDEAACAALVRPEASV PCLIADCTYRWHVGTW MECSVSCGDGIQRRRD TCLGPQAQAPVPADFC QHLPKPVTVRGCWAGP CVGQGTPSLVPHEEAA APGRTTATPAGASLEW SQARGLLFSPAPQPRRL LPGPQENSVQSSACGR QHLEPTGTIDMRGPGQ ADCAVAIGRPLGEVVT LRVLESSLNCSAGDML LLWGRLTWRKMCRKL LDMTFSSKTNTLVVRQ RCGRPGGGVLLRYGSQ LAPETFYRECDMQLFG PWGEIVSPSLSPATSNA GGCRLFINVAPHARIAI HALATNMGAGTEGAN ASYILIRDTHSLRTTAFH GQQVLYWESESSQAEM EFSEGFLKAQASLRGQ YWTLQSWVPEMQDPQ SWKGKEGT FOXP3 MPNPRPGKPSAPSLALG PSPGASPSWRAAPKAS DLLGARGPGGTFQGRD LRGGAHASSSSLNPMPP SQLQLPTLPLVMVAPSG ARLGPLPHLQALLQDR PHFMHQLSTVDAHART PVLQVHPLESPAMISLT PPTTATGVFSLKARPGL PPGINVASLEWVSREPA Immune cells [00009] LLCTFPNPSAPRKDSTL (50 mol %) SAVPQSSYPLLANGVC DSPC (10 mol %) KWPGCEKVFEEPEDFL Beta-sitosterol (28.5% mol %) KHCQADHLLDEKGRA Cholesterol (10 mol %) QCLLQREMVQSLEQQL PEG DMG (1.5 mol %) VLEKEKLSAMQAHLAG KMALTKASSVASSDKG SCCIVAAGSQGPVVPA WSGPREAPDSLFAVRR HLWGSHGNSTFPEFLH NMDYFKFHNMRPPFTY ATLIRWAILEAPEKQRT LNEIYHWFTRMFAFFR NHPATWKNAIRHNLSL HKCFVRVESEKGAVWT VDELEFRKKRSQRPSRC SNPTPGP IL-10 SPGQGTQSENSCTHFPG NLPNMLRDLRDAFSRV KTFFQMKDQLDNLLLK ESLLEDFKGYLGCQALS EMIQFYLEEVMPQAEN QDPDIKAHVNSLGENL KTLRLRLRRCHRFLPCE NKSKAVEQVKNAFNKL QEKGIYKAMSEFDIFIN YIEAYMTMKIRN Immune cells [00010] (50 mol %) DSPC (10 mol %) Beta-sitosterol (28.5% mol %) Cholesterol (10 mol %) PEG DMG (1.5 mol %) IL-2 APTSSSTKKTQLQLEHL LLDLQMILNGINNYKNP KLTRMLTFKFYMPKKA TELKHLQCLEEELKPLE EVLNLAQSKNFHLRPR DLISNINVIVLELKGSET TFMCEY ADETATIVEFL NRWITFCQSIISTLT Immune cells [00011] (50 mol %) DSPC (10 mol %) Beta-sitosterol (28.5% mol %) Cholesterol (10 mol %) PEG DMG (1.5 mol %)

[0313] In some embodiments, the at least one of the expression sequences encodes a therapeutic protein. In some embodiments, the first or second expression sequence encodes a cytokine, e.g., IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-?, IFN-?, IL-10, TGF-beta, IL-4, or IL-35, or a functional fragment thereof. In some embodiments, the first or second expression sequence encodes an immune checkpoint inhibitor. In some embodiments, the first or second expression sequence encodes an agonist (e.g., a TNFR family member such as CD137L, OX40L, ICOSL, LIGHT, or CD70). In some embodiments, the first or second expression sequence encodes a chimeric antigen receptor. In some embodiments, the first or second expression sequence encodes an inhibitory receptor agonist (e.g., PDL1, PDL2, Galectin-9, VISTA, B7H4, or MHCII) or inhibitory receptor (e.g., PD1, CTLA4, TIGIT, LAG3, or TIM3). In some embodiments, the first or second expression sequence encodes an inhibitory receptor antagonist. In some embodiments, the first or second expression sequence encodes one or more TCR chains (alpha and beta chains or gamma and delta chains). In some embodiments, the first or second expression sequence encodes a secreted T cell or immune cell engager (e.g., a bispecific antibody such as BiTE, targeting, e.g., CD3, CD137, or CD28 and a tumor-expressed protein e.g., CD19, CD20, or BCMA etc.). In some embodiments, the first or second expression sequence encodes a transcription factor (e.g., FOXP3, HELIOS, TOX1, or TOX2). In some embodiments, the first or second expression sequence encodes an immunosuppressive enzyme (e.g., IDO or CD39/CD73). In some embodiments, the first or second expression sequence encodes a GvHD (e.g., anti-HLA-A2 CAR-Tregs).

[0314] In some embodiments, the first and second expression sequences encode the alpha and beta chains of a T cell receptor (TCR). In some embodiments, the first and second expression sequences encode the gamma and delta chains of a TCR. The invention includes methods of treating a subject suffering from cancer comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide encoding a TCR alpha chain and a TCR beta chain or a TCR gamma chain and a TCR delta chain.

[0315] In some embodiments, the first and second expression sequences encode a chimeric antigen receptor (CAR) and an antagonist of PD1 or PDL1. In some embodiments, the first and second expression sequences encode a chimeric antigen receptor (CAR) and a cytokine. In some embodiments, the cytokine is IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-?, IFN-?, IL-10, TGF-beta, IL-4, or IL-35, or a functional fragment thereof. The invention includes methods of treating a subject suffering from cancer comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide encoding a CAR and an antagonist of PD1 or PDL1. The invention includes methods of treating a subject suffering from cancer comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide encoding a CAR and a cytokine.

[0316] In some embodiments, the first and second expression sequences encode a transcription factor and a cytokine. In some embodiments, the transcription factor is FOXP3, STAT5B, or HELIOS and the cytokine is IL10, IL12, or TGF beta. The invention includes methods of treating a subject suffering from an autoimmune disorder comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide encoding a transcription factor, e.g., FOXP3, and a cytokine.

[0317] In some embodiments, the first and second expression sequences encode a transcription factor and a CAR. In some embodiments, the transcription factor is FOXP3, STAT5B, or HELIOS. The invention includes methods of treating a subject suffering from an autoimmune disorder comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide encoding a transcription factor, e.g., FOXP3, and a CAR.

[0318] In some embodiments, the first and second expression sequences encode a cytokine and an antigen. In some embodiments, the cytokine is IFN?. In some embodiments, the antigen is a neoantigen. The invention includes methods of treating a subject suffering from cancer comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide encoding a cytokine, e.g., IFN?, and a tumor antigen or fragment thereof.

[0319] In some embodiments, the first expression sequence encodes a first chimeric antigen receptor (CAR) and the second expression sequence encodes a second CAR. In some embodiments, the first CAR is specific for a first antigen and contains a costimulatory domain and an intracellular signaling domain, and the second CAR is specific for a second antigen and contains a costimulatory domain and a intracellular signaling domain. In some embodiments, expressing CARs targeting multiple tumor antigens provides a more effective therapy against a tumor with heterogeneous antigen expression. The invention includes methods of treating a subject suffering from cancer comprising administering a therapeutically effective amount of a composition comprising a circular RNA polynucleotide encoding a first CAR and a second CAR.

[0320] In some embodiments, the first expression sequence encodes a first cytokine and the second expression sequence encodes a second cytokine. In some embodiments, the first and second cytokines are in the group IL-10, TGF?, and IL-35. In some embodiments, the first and second cytokines are in the group IFN?, IL-2, IL-7, IL-15, and IL-18.

[0321] In some embodiments, a polynucleotide encodes a protein that is made up of subunits that are encoded by more than one gene. For example, the protein may be a heterodimer, wherein each chain or subunit of the protein is encoded by a separate gene. It is possible that more than one circRNA molecule is delivered in the transfer vehicle and each circRNA encodes a separate subunit of the protein. Alternatively, a single circRNA may be engineered to encode more than one subunit. In certain embodiments, separate circRNA molecules encoding the individual subunits may be administered in separate transfer vehicles.

3.1 Cytokines

[0322] Descriptions and/or amino acid sequences of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-270, IFN?, and/or TGF?1 are provided herein and at the www.uniprot.org database at accession numbers: P60568 (IL-2), P29459 (IL-12A), P29460 (IL-12B), P13232 (IL-7), P22301 (IL-10), P40933 (IL-15), Q14116 (IL-18), Q14213 (IL-270), P01579 (IFN?), and/or P01137 (TGF?1).

3.2 PD-1 and PD-L1 Antagonists

[0323] In some embodiments, a PD-1 inhibitor is pembrolizumab, pidilizumab, or nivolumab. In some embodiments, Nivolumab is described in International Patent Publication No. WO2006/121168. In some embodiments, Pembrolizumab is described in WO2009/114335. In some embodiments, Pidilizumab is described in International Patent Publication No. WO2009/101611. Additional anti-PD1 antibodies are described in U.S. Pat. No. 8,609,089, U.S. Patent Publication Nos. US 2010028330 and US 20120114649, and International Patent Publication Nos. WO2010/027827 and WO2011/066342.

[0324] In some embodiments, a PD-L1 inhibitor is atezolizumab, avelumab, durvalumab, BMS-936559, or CK-301.

[0325] Descriptions and/or amino acid sequences of heavy and light chains of PD-1, and/or PD-L1 antibodies are provided herein and at the www.drugbank.ca database at accession numbers: DB09037 (Pembrolizumab), DB09035 (Nivolumab), DB15383 (Pidilizumab), DB11595 (Atezolizumab), DB 11945 (Avelumab), and DB11714 (Durvalumab).

3.3 Chimeric Antigen Receptors

[0326] Chimeric antigen receptors (CARs or CAR-Ts) are genetically-engineered receptors. These engineered receptors may be inserted into and expressed by immune cells, including T cells via circular RNA as described herein. With a CAR, a single receptor may be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR may target and kill the tumor cell. In some embodiments, the CAR encoded by the polynucleotide comprises (i) an antigen-binding molecule that specifically binds to a target antigen, (ii) a hinge domain, a transmembrane domain, and an intracellular domain, and (iii) an activating domain.

[0327] In some embodiments, an orientation of the CARs in accordance with the disclosure comprises an antigen binding domain (such as scFv) in tandem with a costimulatory domain and an activating domain. The costimulatory domain may comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple costimulatory domains may be utilized in tandem.

Antigen Binding Domain

[0328] CARs may be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen. In some embodiments, the antigen binding molecule is an antibody fragment thereof, e.g., one or more single chain antibody fragment (scFv). An scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. See, for example, U.S. Pat. Nos. 7,741,465, and 6,319,494 as well as Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. An scFv retains the parent antibody's ability to specifically interact with target antigen. scFvs are useful in chimeric antigen receptors because they may be engineered to be expressed as part of a single chain along with the other CAR components. Id. See also Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161: 2791-2797. It will be appreciated that the antigen binding molecule is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to the antigen of interest. Bispecific and multispecific CARs are contemplated within the scope of the invention, with specificity to more than one target of interest.

[0329] In some embodiments, the antigen binding molecule comprises a single chain, wherein the heavy chain variable region and the light chain variable region are connected by a linker. In some embodiments, the VH is located at the N terminus of the linker and the VL is located at the C terminus of the linker. In other embodiments, the VL is located at the N terminus of the linker and the VH is located at the C terminus of the linker. In some embodiments, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids.

[0330] In some embodiments, the antigen binding molecule comprises a nanobody. In some embodiments, the antigen binding molecule comprises a DARPin. In some embodiments, the antigen binding molecule comprises an anticalin or other synthetic protein capable of specific binding to target protein.

[0331] In some embodiments, the CAR comprises an antigen binding domain specific for an antigen selected from the group CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GaINAca-Ser/Thr)), prostate-specific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), Interleukin-13 receptor subunit alpha-2, mesothelin, Interleukin 11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), Protease Serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y) antigen, CD24, Platelet-derived growth factor receptor beta (PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, Folate receptor alpha, HER2, HER3, Mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), Prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), Ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type-A receptor 2 (EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight-melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), Polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), Hepatitis A virus cellular receptor 1 (HAVCR1), adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), Olfactory receptor 51E2 (OR51E2), TCR Gamma Alternate Reading Frame Protein (TARP), Wilms tumor protein (WTi), Cancer/testis antigen 1 (NY-ESO-1), Cancer/testis antigen 2 (LAGE-1a), MAGE family members (including MAGE-A1, MAGE-A3 and MAGE-A4), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X Antigen Family, Member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, surviving, telomerase, prostate carcinoma tumor antigen-1, melanoma antigen recognized by T cells 1, Rat sarcoma (Ras) mutant, human Telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-Acetyl glucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), Androgen receptor, Cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Ras Homolog Family Member C (RhoC), Tyrosinase-related protein 2 (TRP-2), Cytochrome P450 1B1 (CYPIB1), CCCTC-Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), Paired box protein Pax-5 (PAX5), proacrosin binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), Receptor for Advanced Glycation Endproducts (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), intestinal carboxyl esterase, heat shock protein 70-2 mutated (mut hsp70-2), CD79a, CD79b, CD72, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), Glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, ?v?? integrin, ?v?6 integrin, alphafetoprotein (AFP), B7-H6, ca-125, CA9, CD44, CD44v7/8, CD52, E-cadherin, EMA (epithelial membrane antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), kinase insert domain receptor (KDR), k-light chain, L1 cell adhesion molecule, MUC18, NKG2D, oncofetal antigen (h5T4), tumor/testis-antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT10, MART-1, immunoglobulin lambda-like polypeptide 1 (IGLL1), Hepatitis B Surface Antigen Binding Protein (HBsAg), viral capsid antigen (VCA), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen, cytomegalovirus (CMV) antigen, large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, haemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen, parainfluenza type 2 antigen, parainfluenza type 3 antigen, parainfluenza type 4 antigen, Human Metapneumovirus (HMPV) antigen, hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-cell lympotrophic virus (HTLV-1) antigen, Merkel cell polyoma virus small T antigen, Merkel cell polyoma virus large T antigen, Kaposi sarcoma-associated herpesvirus (KSHV) lytic nuclear antigen and KSHV latent nuclear antigen. In some embodiments, an antigen binding domain comprises SEQ ID NO: 321 and/or 322.

Hinge/Spacer Domain

[0332] In some embodiments, a CAR of the instant disclosure comprises a hinge or spacer domain. In some embodiments, the hinge/spacer domain may comprise a truncated hinge/spacer domain (THD) the THD domain is a truncated version of a complete hinge/spacer domain (CHD). In some embodiments, an extracellular domain is from or derived from (e.g., comprises all or a fragment of) ErbB2, glycophorin A (GpA), CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8[T CD11a (IT GAL), CD11b (IT GAM), CD11c (ITGAX), CD11d (IT GAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAMI), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CD11a/CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, a Toll ligand receptor, and fragments or combinations thereof. A hinge or spacer domain may be derived either from a natural or from a synthetic source.

[0333] In some embodiments, a hinge or spacer domain is positioned between an antigen binding molecule (e.g., an scFv) and a transmembrane domain. In this orientation, the hinge/spacer domain provides distance between the antigen binding molecule and the surface of a cell membrane on which the CAR is expressed. In some embodiments, a hinge or spacer domain is from or derived from an immunoglobulin. In some embodiments, a hinge or spacer domain is selected from the hinge/spacer regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM, or a fragment thereof. In some embodiments, a hinge or spacer domain comprises, is from, or is derived from the hinge/spacer region of CD8 alpha. In some embodiments, a hinge or spacer domain comprises, is from, or is derived from the hinge/spacer region of CD28. In some embodiments, a hinge or spacer domain comprises a fragment of the hinge/spacer region of CD8 alpha or a fragment of the hinge/spacer region of CD28, wherein the fragment is anything less than the whole hinge/spacer region. In some embodiments, the fragment of the CD8 alpha hinge/spacer region or the fragment of the CD28 hinge/spacer region comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus or C-Terminus, or both, of the CD8 alpha hinge/spacer region, or of the CD28 hinge/spacer region.

Transmembrane Domain

[0334] The CAR of the present disclosure may further comprise a transmembrane domain and/or an intracellular signaling domain. The transmembrane domain may be designed to be fused to the extracellular domain of the CAR. It may similarly be fused to the intracellular domain of the CAR. In some embodiments, the transmembrane domain that naturally is associated with one of the domains in a CAR is used. In some instances, the transmembrane domain may be selected or modified (e.g., by an amino acid substitution) to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0335] Transmembrane regions may be derived from (i.e. comprise) a receptor tyrosine kinase (e.g., ErbB2), glycophorin A (GpA), 4-1BB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BFAME (SEAMF8), BTEA, CD100 (SEMA4D), CD103, CD160 (BY55), CD 18, CD 19, CD 19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAMI, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IE-2R beta, IE-2R gamma, IE-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, a ligand that specifically binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof.

[0336] In some embodiments, suitable intracellular signaling domain include, but are not limited to, activating Macrophage/Myeloid cell receptors CSFR1, MYD88, CD14, TIE2, TLR4, CR3, CD64, TREM2, DAP10, DAP12, CD169, DECTIN1, CD206, CD47, CD163, CD36, MARCO, TIM4, MERTK, F4/80, CD91, C1QR, LOX-1, CD68, SRA, BAI-1, ABCA7, CD36, CD31, Lactoferrin, or a fragment, truncation, or combination thereof.

[0337] In some embodiments, a receptor tyrosine kinase may be derived from (e.g., comprise) Insulin receptor (InsR), Insulin-like growth factor I receptor (IGF1R), Insulin receptor-related receptor (IRR), platelet derived growth factor receptor alpha (PDGFRa), platelet derived growth factor receptor beta (PDGFRfi). KIT proto-oncogene receptor tyrosine kinase (Kit), colony stimulating factor 1 receptor (CSFR), fms related tyrosine kinase 3 (FLT3), fins related tyrosine kinase 1 (VEGFR-1), kinase insert domain receptor (VEGFR-2), fms related tyrosine kinase 4 (VEGFR-3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), protein tyrosine kinase 7 (CCK4), neurotrophic receptor tyrosine kinase 1 (trkA), neurotrophic receptor tyrosine kinase 2 (trkB), neurotrophic receptor tyrosine kinase 3 (trkC), receptor tyrosine kinase like orphan receptor 1 (ROR1), receptor tyrosine kinase like orphan receptor 2 (ROR2), muscle associated receptor tyrosine kinase (MuSK), MET proto-oncogene, receptor tyrosine kinase (MET), macrophage stimulating 1 receptor (Ron), AXL receptor tyrosine kinase (Axl), TYR03 protein tyrosine kinase (Tyro3), MER proto-oncogene, tyrosine kinase (Mer), tyrosine kinase with immunoglobulin like and EGF like domains 1 (TIE1), TEK receptor tyrosine kinase (TIE2), EPH receptor A1 (EphA1), EPH receptor A2 (EphA2), (EPH receptor A3) EphA3, EPH receptor A4 (EphA4), EPH receptor A5 (EphA5), EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8 (EphA8), EPH receptor A10 (EphAlO), EPH receptor B1 (EphB1), EPH receptor B2 (EphB2), EPH receptor B3 (EphB3), EPH receptor B4 (EphB4), EPH receptor B6 (EphB6), ret proto oncogene (Ret), receptor-like tyrosine kinase (RYK), discoidin domain receptor tyrosine kinase 1 (DDR1), discoidin domain receptor tyrosine kinase 2 (DDR2), c-ros oncogene 1, receptor tyrosine kinase (ROS), apoptosis associated tyrosine kinase (Lmr1), lemur tyrosine kinase 2 (Lmr2), lemur tyrosine kinase 3 (Lmr3), leukocyte receptor tyrosine kinase (LTK), ALK receptor tyrosine kinase (ALK), or serine/threonine/tyrosine kinase 1 (STYK1).

Costimulatory Domain

[0338] In certain embodiments, the CAR comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises 4-1BB (CD137), CD28, or both, and/or an intracellular T cell signaling domain. In a preferred embodiment, the costimulatory domain is human CD28, human 4-1BB, or both, and the intracellular T cell signaling domain is human CD3 zeta (?). The 4-1BB, CD28, CD3 zeta, or any of these may comprise less than the whole 4-1BB, CD28 or CD3 zeta, respectively. Chimeric antigen receptors may incorporate costimulatory (signaling) domains to increase their potency. See U.S. Pat. Nos. 7,741,465, and 6,319,494, as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Amur. Rev. Pharmacol. Toxicol. 56:59-83 (2016).

[0339] In some embodiments, a costimulatory domain comprises the amino acid sequence of SEQ ID NO: 318 or 320.

Intracellular Signaling Domain

[0340] The intracellular (signaling) domain of the engineered T cells disclosed herein may provide signaling to an activating domain, which then activates at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0341] In some embodiments, suitable intracellular signaling domain include (e.g., comprise), but are not limited to 4-1BB/CD137, activating NK cell receptors, an immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD 19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAMI, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE, ITGAL, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand that specifically binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Lyl08), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a/CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof.

[0342] CD3 is an element of the T cell receptor on native T cells, and has been shown to be an important intracellular activating element in CARs. In some embodiments, the CD3 is CD3 zeta. In some embodiments, the activating domain comprises an amino acid sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the polypeptide sequence of SEQ ID NO: 319.

3.4 T Cell Receptors

[0343] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature, and links to the IMGT public database of TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly, each chain may comprise variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region is often considered as part of the joining region. Each variable region may comprise three CDRs (Complementarity Determining Regions) embedded in a framework sequence, one being the hypervariable region named CDR3. There are several types of alpha chain variable (V?) regions and several types of beta chain variable (V?) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The V? types are referred to in IMGT nomenclature by a unique TRAV number. Thus TRAV21 defines a TCR V? region having unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence which is partly defined by an amino acid sequence which is preserved from TCR to TCR but which also includes an amino acid sequence which varies from TCR to TCR. In the same way, TRBV5-1 defines a TCR V? region having unique framework and CDR1 and CDR2 sequences, but with only a partly defined CDR3 sequence.

[0344] The joining regions of the TCR are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRAC and TRBC nomenclature.

[0345] The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJ regions are often considered together as the joining region.

[0346] The unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the TCR field. For example, they can be found in the IMGT public database. The T cell Receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 also discloses sequences defined by the IMGT nomenclature, but because of its publication date and consequent time-lag, the information therein sometimes needs to be confirmed by reference to the IMGT database.

[0347] Native TCRs exist in heterodimeric ?? or ?? forms. However, recombinant TCRs consisting of ac or RR homodimers have previously been shown to bind to peptide MHC molecules. Therefore, the TCR of the invention may be a heterodimeric ?? TCR or may be an ac or RR homodimeric TCR.

[0348] For use in adoptive therapy, an ?? heterodimeric TCR may, for example, be transfected as full length chains having both cytoplasmic and transmembrane domains. In certain embodiments TCRs of the invention may have an introduced disulfide bond between residues of the respective constant domains, as described, for example, in WO 2006/000830.

[0349] TCRs of the invention, particularly alpha-beta heterodimeric TCRs, may comprise an alpha chain TRAC constant domain sequence and/or a beta chain TRBC1 or TRBC2 constant domain sequence. The alpha and beta chain constant domain sequences may be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and/or beta chain constant domain sequence(s) may also be modified by substitution of cysteine residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulfide bond between the alpha and beta constant domains of the TCR.

[0350] Binding affinity (inversely proportional to the equilibrium constant K.sub.D) and binding half-life (expressed as T?) can be determined by any appropriate method. It will be appreciated that doubling the affinity of a TCR results in halving the K.sub.D. T? is calculated as ln 2 divided by the off-rate (koff). So doubling of T? results in a halving in koff K.sub.D and koff values for TCRs are usually measured for soluble forms of the TCR, i.e. those forms which are truncated to remove cytoplasmic and transmembrane domain residues. Therefore it is to be understood that a given TCR has an improved binding affinity for, and/or a binding half-life for the parental TCR if a soluble form of that TCR has the said characteristics. Preferably the binding affinity or binding half-life of a given TCR is measured several times, for example 3 or more times, using the same assay protocol, and an average of the results is taken.

[0351] Since the TCRs of the invention have utility in adoptive therapy, the invention includes a non-naturally occurring and/or purified and/or or engineered cell, especially a T-cell, presenting a TCR of the invention. There are a number of methods suitable for the transfection of T cells with nucleic acid (such as DNA, cDNA or RNA) encoding the TCRs of the invention (see for example Robbins et al., (2008) J Immunol. 180: 6116-6131). T cells expressing the TCRs of the invention will be suitable for use in adoptive therapy-based treatment of cancers such as those of the pancreas and liver. As will be known to those skilled in the art, there are a number of suitable methods by which adoptive therapy can be carried out (see for example Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

[0352] As is well-known in the art TCRs of the invention may be subject to post-translational modifications when expressed by transfected cells. Glycosylation is one such modification, which may comprise the covalent attachment of oligosaccharide moieties to defined amino acids in the TCR chain. For example, asparagine residues, or serine/threonine residues are well-known locations for oligosaccharide attachment. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation and the availability of certain enzymes. Furthermore, glycosylation status (i.e oligosaccharide type, covalent linkage and total number of attachments) can influence protein function. Therefore, when producing recombinant proteins, controlling glycosylation is often desirable. Glycosylation of transfected TCRs may be controlled by mutations of the transfected gene (Kuball J et al. (2009), J Exp Med 206(2):463-475). Such mutations are also encompassed in this invention.

[0353] A TCR may be specific for an antigen in the group MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, Lage-2, SP17, and TRP2-Int2, (MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, pi5(58), CEA, NY-ESO (LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, pi85erbB2, pi80erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, ?-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS.

3.5 Transcription factors

[0354] Regulatory T cells (Treg) are important in maintaining homeostasis, controlling the magnitude and duration of the inflammatory response, and in preventing autoimmune and allergic responses.

[0355] In general, Tregs are thought to be mainly involved in suppressing immune responses, functioning in part as a self-check for the immune system to prevent excessive reactions. In particular, Tregs are involved in maintaining tolerance to self-antigens, harmless agents such as pollen or food, and abrogating autoimmune disease.

[0356] Tregs are found throughout the body including, without limitation, the gut, skin, lung, and liver. Additionally, Treg cells may also be found in certain compartments of the body that are not directly exposed to the external environment such as the spleen, lymph nodes, and even adipose tissue. Each of these Treg cell populations is known or suspected to have one or more unique features and additional information may be found in Lehtimaki and Lahesmaa, Regulatory T cells control immune responses through their non-redundant tissue specific features, 2013, FRONTIERS IN IMMUNOL., 4(294): 1-10, the disclosure of which is hereby incorporated in its entirety.

[0357] Typically, Tregs are known to require TGF-0 and IL-2 for proper activation and development. Tregs, expressing abundant amounts of the IL-2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. Tregs are known to produce both IL-10 and TGF-0, both potent immune suppressive cytokines. Additionally, Tregs are known to inhibit the ability of antigen presenting cells (APCs) to stimulate T cells. One proposed mechanism for APC inhibition is via CTLA-4, which is expressed by Foxp3+ Tregs. It is thought that CTLA-4 may bind to B7 molecules on APCs and either block these molecules or remove them by causing internalization resulting in reduced availability of B7 and an inability to provide adequate co-stimulation for immune responses. Additional discussion regarding the origin, differentiation and function of Tregs may be found in Dhamne et al., Peripheral and thymic Foxp3+ regulatory T cells in search of origin, distinction, and function, 2013, Frontiers in Immunol., 4 (253): 1-11, the disclosure of which is hereby incorporated in its entirety.

[0358] Descriptions and/or amino acid sequences of FOXP3, STAT5B, and/or HELIOS are provided herein and at the www.uniprot.org database at accession numbers: Q9BZS1 (FOXP3), P51692 (STAT5b), and/or Q9UKS7 (HELIOS).

Foxp3

[0359] In some embodiments, a transcription factor is the Forkhead box P3 transcription factor (Foxp3). Foxp3 has been shown to be a key regulator in the differentiation and activity of Tregs. In fact, loss-of-function mutations in the Foxp3 gene have been shown to lead to the lethal IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, X-linked). Patients with IPEX suffer from severe autoimmune responses, persistent eczema, and colitis. Treg cells expressing Foxp3 play a key role in limiting inflammatory responses in the intestine (Josefowicz, S. Z. et al. Nature, 2012, 482, 395-U1510).

STAT

[0360] Members of the signal transducer and activator of transcription (STAT) protein family are intracellular transcription factors that mediate many aspects of cellular immunity, proliferation, apoptosis and differentiation. They are primarily activated by membrane receptor-associated Janus kinases (JAK). Dysregulation of this pathway is frequently observed in primary tumors and leads to increased angiogenesis, enhanced survival of tumors and immunosuppression. Gene knockout studies have provided evidence that STAT proteins are involved in the development and function of the immune system and play a role in maintaining immune tolerance and tumor surveillance.

[0361] There are seven mammalian STAT family members that have been identified: STAT1, STAT2, STAT3, STAT4, STAT5 (including STAT5A and STAT5B), and STATE.

[0362] Extracellular binding of cytokines or growth factors induce activation of receptor-associated Janus kinases, which phosphorylate a specific tyrosine residue within the STAT protein promoting dimerization via their SH2 domains. The phosphorylated dimer is then actively transported to the nucleus via an importin a/0 ternary complex. Originally, STAT proteins were described as latent cytoplasmic transcription factors as phosphorylation was thought to be required for nuclear retention. However, unphosphorylated STAT proteins also shuttle between the cytosol and nucleus, and play a role in gene expression. Once STAT reaches the nucleus, it binds to a consensus DNA-recognition motif called gamma-activated sites (GAS) in the promoter region of cytokine-inducible genes and activates transcription. The STAT protein can be dephosphorylated by nuclear phosphatases, which leads to inactivation of STAT and subsequent transport out of the nucleus by an exportin-RanGTP complex.

[0363] In some embodiments, a STAT protein of the present disclosure may be a STAT protein that comprises a modification that modulates its expression level or activity. In some embodiments such modifications include, among other things, mutations that effect STAT dimerization, STAT protein binding to signaling partners, STAT protein localization or STAT protein degradation. In some embodiments, a STAT protein of the present disclosure is constitutively active. In some embodiments, a STAT protein of the present disclosure is constitutively active due to constitutive dimerization. In some embodiments, a STAT protein of the present disclosure is constitutively active due to constitutive phosphorylation as described in Onishi, M. et al., Mol. Cell. Biol. July 1998 vol. 18 no. 7 3871-3879 the entirety of which is herein incorporated by reference.

3.6 Vaccines

[0364] In an embodiment, one or more expression sequences encodes an antigen, e.g., a tumor antigen, or a fragment thereof. In some embodiments, expression of such a sequence produces an immunogenic composition, e.g., a vaccine composition capable of raising a specific T-cell response. In some embodiments, an antigen is a neoantigen.

4. Cleavage Site

[0365] In some embodiments, two or more expression sequences in a polynucleotide construct may be separated by one or more cleavage site sequences.

[0366] A cleavage site may be any sequence which enables the two or more polypeptides to become separated. A cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual polypeptides without the need for any external cleavage activity.

[0367] A cleavage site may be a furin cleavage site.

[0368] Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor. Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg) and is enriched in the Golgi apparatus.

[0369] A cleavage site may encode a self-cleaving peptide.

[0370] A cleavage site may operate by ribosome skipping such as the skipping of a glycyl-propyl bond at the C-terminus of a 2A self-cleaving peptide. In some embodiments, steric hinderance causes ribosome skipping. In some embodiments, a 2A self-cleaving peptide contains the sequence GDVEXNPGP (SEQ ID NO: 324), wherein X is E or S. In some embodiments, the protein encoded upstream of the 2A self-cleaving peptide is attached to the 2A self-cleaving peptide except the C-terminal proline post translation. In some embodiments, the protein encoded downstream of the 2A self-cleaving peptide is attached to a proline at its N-terminus post translation.

[0371] A self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus. The primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A cleaving at its own C-terminus. In apthoviruses, such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating cleavage at its own C-terminus (Donelly et al. (2001)).

[0372] 2A-like sequences have been found in picornaviruses other than aptho- or cardioviruses, picornavirus-like insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al. (2001)). The cleavage site may comprise one of these 2A-like sequences, such as those listed in Table 8.

[0373] In some embodiments, a self-cleaving peptide is F2A. In some embodiments, a self-cleaving peptide is derived from foot-and-mouth disease virus. In some embodiments, a self-cleaving peptide is E2A. In some embodiments, a self-cleaving peptide is derived from equine rhinitis A virus. In some embodiments, a self-cleaving peptide is P2A. In some embodiments, a self-cleaving peptide is derived from porcine teschovirus-1. In some embodiments, a self-cleaving peptide is T2A. In some embodiments, a self-cleaving peptide is derived from thosea asigna virus. In some embodiments, a self-cleaving peptide has a sequence listed in Table 8.

[0374] In an embodiment, expression sequences encoding therapeutic proteins separated by a cleavage site have the same level of protein expression.

[0375] In some embodiments, a self-cleaving peptide is described in Liu, Z., Chen, O., Wall, J. B. J. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017).

5. Polynucleotides Containing a Second IRES

[0376] In some embodiments, the ratios of expression of the therapeutic proteins encoded by the first and second expression sequences can be controlled or influenced by the IRES used in the circRNA and whether a cleavage site or a second IRES separates the first and second expression sequences. When equal expression of the proteins encoded by the first and second expression sequences is desired, the circRNA may encode a cleavage site, e.g., a 2A self-cleaving peptide, between the first expression sequence and the second expression sequence. When greater expression of the protein encoded by the first expression sequence is desired, the circRNA may encode a first IRES and a second IRES, wherein the first IRES is associated with greater expression than the second IRES, or wherein the second IRES is an intergenic region (IGR) IRES. When greater expression of the protein encoded by the second expression sequence is desired, the circRNA may encode a first IRES and a second IRES, wherein the second IRES is associated with greater expression than the first IRES.

[0377] In some embodiments, an RNA polynucleotide contains a first IRES and a second IRES as described herein. In some embodiments, a DNA vector encodes a first IRES and a second IRES as described herein.

[0378] In an embodiment, the first IRES and the second IRES have the same sequence. In an embodiment, the first IRES and the second IRES have different sequences. In an embodiment, the first IRES is an IRES having a sequence as listed in Table 1 (SEQ ID NO: 1-72). In some embodiments, the first IRES is a Salivirus IRES. In some embodiments, the first IRES is a Salivirus SZ1 IRES. In an embodiment, the second IRES is an IRES having a sequence as listed in Table 1 (SEQ ID NO: 1-72). In some embodiments, the second IRES is a Salivirus IRES. In some embodiments, the first IRES is a Salivirus SZ1 IRES.

[0379] In some embodiments, the first IRES is associated with greater expression than the second IRES (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater expression when compared using constructs containing a single IRES). In some embodiments, the second IRES is associated with greater expression than the first IRES (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater expression when compared using constructs containing a single IRES). In some embodiments, the second IRES is an intergenic region (IGR) IRES.

[0380] Expressing 2 proteins with a single circRNA polynucleotide has advantages over expression using multiple polynucleotides. In some embodiments, expression of 2 proteins from an inventive circRNA polynucleotide leads to more consistent ratios of expression than expression from multiple polynucleotides. In some embodiments, expression of 2 proteins from an inventive circRNA polynucleotide leads to transient expression, which may be desirable over the lasting expression of DNA.

6. Production of Polynucleotides

[0381] The vectors provided herein can be made using standard techniques of molecular biology. For example, the various elements of the vectors provided herein can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells, or by deriving the polynucleotides from a vector known to include the same.

[0382] The various elements of the vectors provided herein can also be produced synthetically, rather than cloned, based on the known sequences. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223: 1299; and Jay et al., J. Biol. Chem. (1984) 259:6311.

[0383] Thus, particular nucleotide sequences can be obtained from vectors harboring the desired sequences or synthesized completely, or in part, using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate. One method of obtaining nucleotide sequences encoding the desired vector elements is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Additionally, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide directed mutagenesis of preexisting nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239: 1534-1536), and enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033) can be used.

[0384] The precursor RNA provided herein can be generated by incubating a vector provided herein under conditions permissive of transcription of the precursor RNA encoded by the vector. For example, in some embodiments a precursor RNA is synthesized by incubating a vector provided herein that comprises an RNA polymerase promoter upstream of its 5 duplex forming region and/or expression sequences with a compatible RNA polymerase enzyme under conditions permissive of in vitro transcription. In some embodiments, the vector is incubated inside of a cell by a bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase II.

[0385] In certain embodiments, provided herein is a method of generating precursor RNA by performing in vitro transcription using a vector provided herein as a template (e.g., a vector provided herein with a RNA polymerase promoter positioned upstream of the 5 duplex forming region).

[0386] In certain embodiments, the resulting precursor RNA can be used to generate circular RNA (e.g., a circular RNA polynucleotide provided herein) by incubating it in the presence of magnesium ions and guanosine nucleotide or nucleoside at a temperature at which RNA circularization occurs (e.g., between 20? C. and 60? C.).

[0387] Thus, in certain embodiments provided herein is a method of making circular RNA. In certain embodiments, the method comprises synthesizing precursor RNA by transcription (e.g., run-off transcription) using a vector provided herein (e.g., a post splicing 3 group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence, a polynucleotide sequence encoding a cleavage site, a second expression sequence, and a 5 group I intron fragment) as a template, and incubating the resulting precursor RNA in the presence of divalent cations (e.g., magnesium ions) and GTP such that it circularizes to form circular RNA. In some embodiments, the precursor RNA disclosed herein is capable of circularizing in the absence of magnesium ions and GTP and/or without the step of incubation with magnesium ions and GTP. It has been discovered that circular RNA has reduced immunogenicity relative to a corresponding mRNA, at least partially because the mRNA contains an immunogenic 5 cap. When transcribing a DNA vector from certain promoters (e.g., a T7 promoter) to produce a precursor RNA, it is understood that the 5 end of the precursor RNA is G. To reduce the immunogenicity of a circular RNA composition that contains a low level of contaminant linear mRNA, an excess of GMP relative to GTP can be provided during transcription such that most transcripts contain a 5 GMP, which cannot be capped. Therefore, in some embodiments, transcription is carried out in the presence of an excess of GMP. In some embodiments, transcription is carried out where the ratio of GMP concentration to GTP concentration is within the range of about 3:1 to about 15:1, for example, about 3:1 to about 10:1, about 3:1 to about 5:1, about 3:1, about 4:1, or about 5:1.

[0388] In some embodiments, a composition comprising circular RNA has been purified. Circular RNA may be purified by any known method commonly used in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some embodiments, purification comprises one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some embodiments, purification comprises the following steps in order: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some embodiments, purification comprises reverse phase HPLC. In some embodiments, a purified composition contains less double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes and/or nicked RNA than unpurified RNA. In some embodiments, a purified composition is less immunogenic than an unpurified composition. In some embodiments, immune cells exposed to a purified composition produce less TNF?, RIG-I, IL-2, IL-6, IFN?, and/or a type 1 interferon, e.g., IFN-?1, than immune cells exposed to an unpurified composition.

7. Nanoparticles

[0389] In certain aspects, provided herein are pharmaceutical compositions comprising the circular RNA provided herein. In certain embodiments, such pharmaceutical compositions are formulated with nanoparticles to facilitate delivery.

[0390] In certain embodiments, the circular RNA provided herein may be delivered and/or targeted to a cell in a transfer vehicle, e.g., a nanoparticle, or a composition comprising a nanoparticle. In some embodiments, the circular RNA may also be delivered to a subject in a transfer vehicle or a composition comprising a transfer vehicle. In some embodiments, the transfer vehicle is a nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle, a solid lipid nanoparticle, a polymeric core-shell nanoparticle, or a biodegradable nanoparticle. In some embodiments, the transfer vehicle comprises or is coated with one or more cationic lipids, non-cationic lipids, ionizable lipids, PEG-modified lipids, polyglutamic acid polymers, Hyaluronic acid polymers, poly n-amino esters, poly beta amino peptides, or positively charged peptides.

[0391] In one embodiment, the transfer vehicle may be selected and/or prepared to optimize delivery of the circRNA to a target cell. For example, if the target cell is a hepatocyte, the properties of the transfer vehicle (e.g., size, charge and/or pH) may be optimized to effectively deliver such transfer vehicle to the target cell, reduce immune clearance and/or promote retention in that target cell.

[0392] The use of transfer vehicles to facilitate the delivery of nucleic acids to target cells is contemplated by the present invention. Liposomes (e.g., liposomal lipid nanoparticles) are generally useful in a variety of applications in research, industry, and medicine, particularly for their use as transfer vehicles of diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16: 307-321, 1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and are usually characterized as microscopic vesicles having an interior aqueous space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).

[0393] In the context of the present invention, a transfer vehicle typically serves to transport the circRNA to the target cell. For the purposes of the present invention, the transfer vehicles are prepared to contain or encapsulate the desired nucleic acids. The process of incorporation of a desired entity (e.g., a nucleic acid) into a liposome is often referred to as loading (Lasic, et al., FEBS Lett., 312: 255-258, 1992). The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The purpose of incorporating a circRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in an embodiment of the present invention, the selected transfer vehicle is capable of enhancing the stability of the circRNA contained therein. The liposome can allow the encapsulated circRNA to reach the target cell, or alternatively limit the delivery of such circRNA to other sites or cells where the presence of the administered circRNA may be useless or undesirable. Furthermore, incorporating the circRNA into a transfer vehicle, such as, for example, a cationic liposome, also facilitates the delivery of such circRNA into a target cell. In some embodiments, a transfer vehicle disclosed herein may serve to promote endosomal or lysosomal release of, for example, contents that are encapsulated in the transfer vehicle (e.g., lipid nanoparticle).

[0394] Ideally, transfer vehicles are prepared to encapsulate one or more desired circRNA such that the compositions demonstrate a high transfection efficiency and enhanced stability. While liposomes can facilitate introduction of nucleic acids into target cells, the addition of polycations (e.g., poly L-lysine and protamine), as a copolymer can in some instances markedly enhance the transfection efficiency of several types of cationic liposomes by 2-28 fold in a number of cell lines both in vitro and in vivo. (See N J. Caplen, et al., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther. 1997; 4, 891.)

[0395] In certain embodiments disclosed herein are ionizable lipids that may be used as a component of a transfer vehicle to facilitate or enhance the delivery and release of circular RNA to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells). In certain embodiments, an ionizable lipid comprises one or more cleavable functional groups (e.g., a disulfide) that allow, for example, a hydrophilic functional head-group to dissociate from a lipophilic functional tail-group of the compound (e.g., upon exposure to oxidative, reducing or acidic conditions), thereby facilitating a phase transition in the lipid bilayer of the one or more target cells.

[0396] In some embodiments, an ionizable lipid is a lipid as described in international patent application PCT/US2018/058555.

[0397] In some of embodiments, a cationic lipid has the following formula:

##STR00012##

wherein: [0398] R.sub.1 and R.sub.2 are either the same or different and independently optionally substituted C.sub.10-C.sub.24 alkyl, optionally substituted C.sub.10-C.sub.24 alkenyl, optionally substituted C.sub.10-C.sub.24 alkynyl, or optionally substituted C.sub.10-C.sub.24 acyl; [0399] R.sub.3 and R.sub.4 are either the same or different and independently optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, or optionally substituted C.sub.2-C.sub.6 alkynyl or R.sub.3 and R.sub.4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; [0400] R.sub.5 is either absent or present and when present is hydrogen or C.sub.1-C.sub.6 alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and [0401] Y and Z are either the same or different and independently O, S, or NH.

[0402] In one embodiment, R.sub.1 and R.sub.2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid.

[0403] In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0404] In various other embodiments, a cationic lipid has the following structure:

##STR00013##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0405] R.sub.1 and R.sub.2 are each independently selected from the group consisting of H and C.sub.1-C.sub.3 alkyls; and [0406] R.sub.3 and R.sub.4 are each independently an alkyl group having from about 10 to about 20 carbon atoms, wherein at least one of R.sub.3 and R.sub.4 comprises at least two sites of unsaturation.

[0407] In some embodiments, R.sub.3 and R.sub.4 are each independently selected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In an embodiment, R.sub.3 and R.sub.4 and are both linoleyl. In some embodiments, R.sub.3 and/or R.sub.4 may comprise at least three sites of unsaturation (e.g., R.sub.3 and/or R.sub.4 may be, for example, dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl).

[0408] In some embodiments, a cationic lipid has the following structure:

##STR00014##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0409] R.sub.1 and R.sub.2 are each independently selected from H and C.sub.1-C.sub.3 alkyls; [0410] R.sub.3 and R.sub.4 are each independently an alkyl group having from about 10 to about 20 carbon atoms, wherein at least one of R.sub.3 and R.sub.4 comprises at least two sites of unsaturation.

[0411] In one embodiment, R.sub.3 and R.sub.4 are the same, for example, in some embodiments R.sub.3 and R.sub.4 are both linoleyl (C.sub.18-alkyl). In another embodiment, R.sub.3 and R.sub.4 are different, for example, in some embodiments, R.sub.3 is tetradectrienyl (C.sub.14-alkyl) and R.sub.4 is linoleyl (Cis-alkyl). In a preferred embodiment, the cationic lipid(s) of the present invention are symmetrical, i.e., R.sub.3 and R.sub.4 are the same. In another preferred embodiment, both R.sub.3 and R.sub.4 comprise at least two sites of unsaturation. In some embodiments, R.sub.3 and R.sub.4 are each independently selected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In an embodiment, R.sub.3 and R.sub.4 are both linoleyl. In some embodiments, R.sub.3 and/or R.sub.4 comprise at least three sites of unsaturation and are each independently selected from dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0412] In various embodiments, a cationic lipid has the formula:

##STR00015##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0413] X.sub.aa is a D- or L-amino acid residue having the formula NR.sup.NCR.sup.1R.sup.2C(C?O), or a peptide or a peptide of amino acid residues having the formula {NR.sup.NCR.sup.1R.sup.2C(C?O)}.sub.n, wherein n is an integer from 2 to 20; [0414] R.sup.1 is independently, for each occurrence, a non-hydrogen or a substituted or unsubstituted side chain of an amino acid; [0415] R.sup.2 and R.sup.N are independently, for each occurrence, hydrogen, an organic group consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, or any combination of the foregoing, and having from 1 to 20 carbon atoms, C.sub.(1-5)alkyl, cycloalkyl, cycloalkylalkyl, C.sub.(1-5)alkenyl, C.sub.(1-5)alkynyl, C.sub.(1-5)alkanoyl, C.sub.(1-5)alkanoyloxy, C.sub.(1-5)alkoxy, C.sub.(1-5)alkoxy-C.sub.(1-5)alkyl, C.sub.(1-5)alkoxy-C.sub.(1-5)alkoxy, C.sub.(1-5)alkyl-amino-C.sub.(1-5)alkyl-, C.sub.(1-5)dialkyl-amino-C.sub.(1-5)alkyl-, nitro-C.sub.(1-5)alkyl, cyano-C.sub.(1-5)alkyl, aryl-C.sub.(1-5)alkyl, 4-biphenyl-C.sub.(1-5)alkyl, carboxyl, or hydroxyl; [0416] Z is NH, O, S, CH.sub.2S, CH.sub.2S(O), or an organic linker consisting of 1-40 atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfur atoms (preferably, Z is NH or O); [0417] R.sup.x and R.sup.y are, independently, (i) a lipophilic tail derived from a lipid (which can be naturally occurring or synthetic), e.g., a phospholipid, a glycolipid, a triacylglycerol, a glycerophospholipid, a sphingolipid, a ceramide, a sphingomyelin, a cerebroside, or a ganglioside, wherein the tail optionally includes a steroid; (ii) an amino acid terminal group selected from hydrogen, hydroxyl, amino, and an organic protecting group; or (iii) a substituted or unsubstituted C.sub.(3-22)alkyl, C.sub.(6-12)cycloalkyl, C.sub.(6-12)cycloalkyl-C.sub.(3-22)alkyl, C.sub.(3-22)alkenyl, C.sub.(3-22)alkynyl, C.sub.(3-22)alkoxy, or C.sub.(6-12)-alkoxy C.sub.(3-22)alkyl;

[0418] In some embodiments, one of R.sup.x and R.sup.Y is a lipophilic tail as defined above and the other is an amino acid terminal group. In some embodiments, both R.sup.x and R.sup.Y are lipophilic tails.

[0419] In some embodiments, at least one of R.sup.x and R.sup.y is interrupted by one or more biodegradable groups (e.g., OC(O), C(O)O, SC(O), C(O)S, OC(S), C(S)O, SS, C(O)(NR.sup.5), N(R.sup.5)C(O), C(S)(NR.sup.5), N(R.sup.5)C(O), N(R.sup.5)C(O)N(R.sup.5), OC(O)O, OSi(R.sup.5).sub.2O, C(O)(CR.sup.3R.sub.4)C(O)O, OC(O)(CR.sup.3R.sub.4)C(O), or

##STR00016##

[0420] In some embodiments, R.sup.11 is a C.sub.2-C.sub.8alkyl or alkenyl.

[0421] In some embodiments, each occurrence of R.sup.5 is, independently, H or alkyl.

[0422] In some embodiments, each occurrence of R.sup.3 and R.sup.4 are, independently H, halogen, OH, alkyl, alkoxy, NH.sub.2, alkylamino, or dialkylamino; or R.sub.3 and R.sub.4, together with the carbon atom to which they are directly attached, form a cycloalkyl group. In some particular embodiments, each occurrence of R.sup.3 and R.sup.4 are, independently H or C.sub.1-C.sub.4alkyl.

[0423] In some embodiments, R.sup.x and R.sup.y each, independently, have one or more carbon-carbon double bonds.

[0424] In some embodiments, the cationic lipid is one of the following:

##STR00017##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0425] R.sub.1 and R.sub.2 are each independently alkyl, alkenyl, or alkynyl, each of which can optionally substituted; [0426] R.sub.3 and R.sub.4 are each independently a C.sub.1-C.sub.6 alkyl, or R.sub.3 and R.sub.4 are taken together to form an optionally substituted heterocyclic ring.

[0427] A representative useful dilinoleyl amino lipid has the formula:

##STR00018##

wherein n is 0, 1, 2, 3, or 4.

[0428] In one embodiment, a cationic lipid is DLin-K-DMA. In one embodiment, a cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).

[0429] In one embodiment, a cationic lipid has the following structure:

##STR00019##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0430] R.sub.1 and R.sub.2 are each independently for each occurrence optionally substituted C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.10-C.sub.30 alkenyl, optionally substituted C.sub.10-C.sub.30 alkynyl or optionally substituted C.sub.10-C.sub.30 acyl; [0431] R.sub.3 is H, optionally substituted C.sub.2-C.sub.10 alkyl, optionally substituted C.sub.2-C.sub.10 alkenyl, optionally substituted C.sub.2-C.sub.10 alkylyl, alkylhetrocycle, alkylpbosphate, alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonate, alkylamine, hydroxyalkyl, ?-aminoalkyl, ?-(substituted)aminoalkyl, ?-phosphoalkyl, ?-thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl, or heterocycle, or a linker ligand, for example, in some embodiments, R.sub.3 is (CH.sub.3).sub.2N(CH.sub.2).sub.n, wherein n is 1, 2, 3 or 4; [0432] E is O, S, N(Q), C(O), OC(O), C(O)O, N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O).sub.2N(Q), S(O).sub.2, N(Q)S(O).sub.2, SS, O?N, aryl, heteroaryl, cyclic or heterocycle, for example C(O)O, wherein - is a point of connection to R.sub.3; and [0433] Q is H, alkyl, ?-aminoalkyl, ?-(substituted)aminoalkyl, ?-phosphoalkyl or ?-thiophosphoalkyl.

[0434] In one specific embodiment, the cationic lipid of Embodiments 1, 2, 3, 4 or 5 has the following structure

##STR00020##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0435] E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O), N(Q), S(O).sub.2, N(Q)S(O, SS, O?N, aryl, heteroaryl, cyclic or heterocycle; [0436] Q is H, alkyl, ?-aminoalkyl, ?-(substituted)amninoalky, ?-phosphoalkyl or ?-thiophosphoalkyl; [0437] R.sub.1 and R.sub.2 and R.sub.x are each independently for each occurrence H, optionally substituted C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.10-C.sub.30 alkyl, optionally substituted C.sub.1-C.sub.36 alkenyl, optionally substituted C.sub.10-C.sub.30 alkynyl, optionally substituted C.sub.10-C.sub.30 acyl, or linker-ligand, provided that at least one of R.sub.1, R.sub.2 and R.sub.x is no H; [0438] R.sub.3 is H, optionally substituted C.sub.1-C.sub.10 alkyl, optionally substituted C.sub.2-C.sub.10 alkenyl, optionally substituted C.sub.2-C.sub.10 alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate, alkylphosphonate, allkylamine, hydroxyalkyl, ?-aminoalkyl, ?-(substituted)aminoalkyl, ?-phosphoalkyl, ?-thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl, or heterocycle, or linker-ligand; and [0439] n is 0, 1, 2, or 3.

[0440] In one embodiment, the cationic lipid of Embodiments 1, 2, 3, 4 or 5 has the structure of Formula I:

##STR00021##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0441] one of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O, and the other of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O or a direct bond; [0442] R.sup.a is H or C.sub.1-C.sub.12 alkyl; [0443] R.sup.1a and R.sup.1b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.1a is H or C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0444] R.sup.2a and R.sup.2b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.2a is H or C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.2b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0445] R.sup.3a and R.sup.3b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.3a is H or C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.3b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0446] R.sup.4a and R.sup.4b are, at each occurrence, independently either (a) H or C.sub.1-C.sub.12 alkyl, or (b) R.sup.4a is H or C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0447] R.sup.5 and R.sup.6 are each independently methyl or cycloalkyl; [0448] R.sup.7 is, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; [0449] R.sup.8 and R.sup.9 are each independently unsubstituted C.sub.1-C.sub.12 alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; [0450] a and d are each independently an integer from 0 to 24; [0451] b and c are each independently an integer from 1 to 24; [0452] e is 1 or 2; and [0453] x is 0, 1 or 2.

[0454] In some embodiments of Formula I, L.sup.1 and L.sup.2 are independently O(C?O) or (C?O)O.

[0455] In certain embodiments of Formula I, at least one of R.sup.1a, R.sup.2a, R.sup.3a, or R.sup.4a is C.sub.1-C.sub.12 alkyl, or at least one of L.sup.1 or L.sup.2 is O(C?O) or (C?O)O. In other embodiments, R.sup.1a and R.sup.1b are not isopropyl when a is 6 or n-butyl when a is 8.

[0456] In still further embodiments of Formula I, at least one of R.sup.1a, R.sup.2a, R.sup.3a or R.sup.4a is C.sub.1-C.sub.12 alkyl, or at least one of L.sup.1 or L.sup.2 is O(C?O) or (C?O)O; and [0457] R.sup.1a and R.sup.1b are not isopropyl when a is 6 or n-butyl when a is 8.

[0458] In other embodiments of Formula I, R.sup.8 and R.sup.9 are each independently unsubstituted C.sub.1-C.sub.12 alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;

[0459] In certain embodiments of Formula I, any one of L.sup.1 or L.sup.2 may be O(C?O) or a carbon-carbon double bond. L.sup.1 and L.sup.2 may each be O(C?O) or may each be a carbon-carbon double bond.

[0460] In some embodiments of Formula I, one of L.sup.1 or L.sup.2 is O(C?O). In other embodiments, both L.sup.1 and L.sup.2 are O(C?O).

[0461] In some embodiments of Formula I, one of L.sup.1 or L.sup.2 is (C?O)O. In other embodiments, both L and L.sup.2 are (C?O)O.

[0462] In some other embodiments of Formula I, one of L.sup.1 or L.sup.2 is a carbon-carbon double bond. In other embodiments, both L.sup.1 and L.sup.2 are a carbon-carbon double bond.

[0463] In still other embodiments of Formula I, one of L.sup.1 or L.sup.2 is O(C?O) and the other of L.sup.1 or L.sup.2 is (C?O)O. In more embodiments, one of L.sup.1 or L.sup.2 is O(C?O) and the other of L.sup.1 or L.sup.2 is a carbon-carbon double bond. In yet more embodiments, one of L.sup.1 or L.sup.2 is (C?O)O and the other of L.sup.1 or L.sup.2 is a carbon-carbon double bond.

[0464] It is understood that carbon-carbon double bond, as used throughout the specification, refers to one of the following structures:

##STR00022##

wherein R.sup.a and R.sup.b are, at each occurrence, independently H or a substituent. For example, in some embodiments R.sup.a and R.sup.b are, at each occurrence, independently H, C.sub.1-C.sub.12 alkyl or cycloalkyl, for example H or C.sub.1-C.sub.12 alkyl.

[0465] In other embodiments, the lipid compounds of Formula I have the following Formula (Ia):

##STR00023##

[0466] In other embodiments, the lipid compounds of Formula I have the following Formula (Ib):

##STR00024##

[0467] In yet other embodiments, the lipid compounds of Formula I have the following Formula (Ic):

##STR00025##

[0468] In certain embodiments of the lipid compound of Formula I, a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0469] In some other embodiments of Formula I, b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0470] In some more embodiments of Formula I, c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0471] In some certain other embodiments of Formula I, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16. In some other various embodiments of Formula I, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0472] The sum of a and b and the sum of c and d in Formula I are factors which may be varied to obtain a lipid of formula I having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a, b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.

[0473] In some embodiments of Formula I, e is 1. In other embodiments, e is 2.

[0474] The substituents at R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a of Formula I are not particularly limited. In certain embodiments R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a are H at each occurrence. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.12 alkyl. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.8 alkyl. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.6 alkyl. In some of the foregoing embodiments, the C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0475] In certain embodiments of Formula I, R.sup.1a, R.sup.1b, R.sup.4a and R.sup.4b are C.sub.1-C.sub.12 alkyl at each occurrence.

[0476] In further embodiments of Formula I, at least one of R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b is H or R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b are H at each occurrence.

[0477] In certain embodiments of Formula I, R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0478] The substituents at R.sup.5 and R.sup.6 of Formula I are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R.sup.5 or R.sup.6 is methyl. In certain other embodiments one or both of R.sup.5 or R.sup.6 is cycloalkyl for example cyclohexyl. In these embodiments the cycloalkyl may be substituted or not substituted. In certain other embodiments the cycloalkyl is substituted with C.sub.1-C.sub.12 alkyl, for example tert-butyl.

[0479] The substituents at R.sup.7 are not particularly limited in the foregoing embodiments of Formula I. In certain embodiments at least one R.sup.7 is H. In some other embodiments, R.sup.7 is H at each occurrence. In certain other embodiments R.sup.7 is C.sub.1-C.sub.12 alkyl.

[0480] In certain other of the foregoing embodiments of Formula I, one of R.sup.8 or R.sup.9 is methyl. In other embodiments, both R.sup.8 and R.sup.9 are methyl.

[0481] In some different embodiments of Formula I, R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.

[0482] In some embodiments of Embodiment 3, the first and second cationic lipids are each, independently selected from a lipid of Formula I.

[0483] In various different embodiments, the lipid of Formula I has one of the structures set forth in Table 1 below.

TABLE-US-00002 TABLE 1 Representative Lipids of Formula I No. Structure pKa I-1 [00026] I-2 [00027] 5.64 I-3 [00028] 7.15 I-4 [00029] 6.43 I-5 [00030] 6.28 I-6 [00031] 6.12 I-7 [00032] I-8 [00033] I-9 [00034] I-10 [00035] I-11 [00036] 6.36 I-12 [00037] I-13 [00038] 6.51 I-14 [00039] I-15 [00040] 6.30 I-16 [00041] 6.63 I-17 [00042] I-18 [00043] I-19 [00044] 6.72 I-20 [00045] 6.44 I-21 [00046] 6.28 I-22 [00047] 6.53 I-23 [00048] 6.24 I-24 [00049] 6.28 I-25 [00050] 6.20 I-33 [00051] 6.27 I-34 [00052] I-35 [00053] 6.21 I-36 [00054] I-37 [00055] I-38 [00056] 6.24 I-39 [00057] 6.82 I-40 [00058] 6.38 I-41 [00059] 5.91

[0484] In some embodiments, the cationic lipid of Embodiments 1, 2, 3, 4 or 5 has a structure of Formula II:

##STR00060##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0485] one of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O, and the other of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O or a direct bond; [0486] G.sup.1 is C.sub.1-C.sub.2 alkylene, (C?O), O(C?O), SC(?O), NR.sup.aC(?O) or a direct bond; [0487] G.sup.2 is C(?O), (C?O)O, C(?O)S, C(?O)NR.sup.a or a direct bond; [0488] G.sup.3 is C.sub.1-C.sub.6 alkylene; [0489] R.sup.a is H or C.sub.1-C.sub.12 alkyl; [0490] R.sup.1a and R.sup.1b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a is H or C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent Rib and the carbon atom to which it is bound to form a carbon-carbon double bond; [0491] R.sup.2a and R.sup.2b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a is H or C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.2b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0492] R.sup.3a and R.sup.3b are, at each occurrence, independently either (a): H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.3b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0493] R.sup.4a and R.sup.4b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.4a is H or C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0494] R.sup.5 and R.sup.6 are each independently H or methyl; [0495] R.sup.7 is C.sub.4-C.sub.20 alkyl; [0496] R.sup.8 and R.sup.9 are each independently C.sub.1-C.sub.12 alkyl; or R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; [0497] a, b, c and d are each independently an integer from 1 to 24; and [0498] x is 0, 1 or 2.

[0499] In some embodiments of Formula (II), L.sup.1 and L.sup.2 are each independently O(C?O), (C?O)O or a direct bond. In other embodiments, G.sup.1 and G.sup.2 are each independently (C?O) or a direct bond. In some different embodiments, L.sup.1 and L.sup.2 are each independently O(C?O), (C?O)O or a direct bond; and G.sup.1 and G.sup.2 are each independently (C?O) or a direct bond.

[0500] In some different embodiments of Formula (II), L.sup.1 and L.sup.2 are each independently C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.a, NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a, NR.sup.aC(?O)O, NR.sup.aS(O).sub.xNR.sup.a, NR.sup.aS(O).sub.x or S(O).sub.xNR.sup.a.

[0501] In other of the foregoing embodiments of Formula (II), the lipid compound has one of the following Formulae (IIA) or (IIB):

##STR00061##

[0502] In some embodiments of Formula (II), the lipid compound has Formula (IIA). In other embodiments, the lipid compound has Formula (IIB).

[0503] In any of the foregoing embodiments of Formula (II), one of L.sup.1 or L.sup.2 is O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 are O(C?O).

[0504] In some different embodiments of Formula (11), one of L.sup.1 or L.sup.2 is (C?O)O. For example, in some embodiments each of L.sup.1 and L.sup.2 is (C?O)O.

[0505] In different embodiments of Formula (II), one of L.sup.1 or L.sup.2 is a direct bond. As used herein, a direct bond means the group (e.g., L.sup.1 or L.sup.2) is absent. For example, in some embodiments each of L.sup.1 and L.sup.2 is a direct bond.

[0506] In other different embodiments of Formula (II), for at least one occurrence of R.sup.1a and R.sup.1b, R.sup.1a is H or C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0507] In still other different embodiments of Formula (II), for at least one occurrence of R.sup.4a and R.sup.4b, R.sup.4a is H or C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0508] In more embodiments of Formula (II), for at least one occurrence of R.sup.2a and R.sup.2b, R.sup.2a is H or C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.2b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0509] In other different embodiments of Formula (II), for at least one occurrence of R.sup.3a and R.sup.3b, R.sup.3a is H or C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.3b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0510] In various other embodiments of Formula (II), the lipid compound has one of the following Formulae (TIC) or (IID):

##STR00062##

wherein e, f, g and h are each independently an integer from 1 to 12.

[0511] In some embodiments of Formula (II), the lipid compound has Formula (IIC). In other embodiments, the lipid compound has Formula (IID).

[0512] In various embodiments of Formulae (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[0513] In certain embodiments of Formula (II), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0514] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0515] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0516] In some certain embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0517] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.

[0518] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

[0519] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

[0520] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.

[0521] In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.

[0522] The sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a, b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[0523] The substituents at R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a of Formula (II) are not particularly limited. In some embodiments, at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is H. In certain embodiments R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a are H at each occurrence. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.12 alkyl. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.8 alkyl. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.6 alkyl. In some of the foregoing embodiments, the C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0524] In certain embodiments of Formula (II), R.sup.1a, R.sup.1b, R.sup.4a and R.sup.4b are C.sub.1-C.sub.12 alkyl at each occurrence.

[0525] In further embodiments of Formula (II), at least one of R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b is H or R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b are H at each occurrence.

[0526] In certain embodiments of Formula (II), R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0527] The substituents at R.sup.5 and R.sup.6 of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments one of R.sup.5 or R.sup.6 is methyl. In other embodiments each of R.sup.5 or R.sup.6 is methyl.

[0528] The substituents at R.sup.7 of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments R.sup.7 is C.sub.6-C.sub.16 alkyl. In some other embodiments, R.sup.7 is C.sub.6-C.sub.9 alkyl. In some of these embodiments, R.sup.7 is substituted with (C?O)OR.sup.b, O(C?O)R.sup.b, C(?O)R.sup.b, OR.sup.b, S(O).sub.xR.sup.b, SSR.sup.b, C(?O)SR.sup.b, SC(?O)R.sup.b, NR.sup.aR.sup.b, NR.sup.aC(?O)R.sup.b, C(?O)NR.sup.aR.sup.b, NR.sup.aC(?O)NR.sup.aR.sup.b, OC(?O)NR.sup.aR.sup.b, NR.sup.aC(?O)OR.sup.b, NR.sup.aS(O)NR.sup.aR.sup.b, NR.sup.aS(O).sub.xR.sup.b or S(O).sub.xNR.sup.aR.sup.b, wherein: R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.b is C.sub.1-C.sub.15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R.sup.7 is substituted with (C?O)OR.sup.b or O(C?O)R.sup.b.

[0529] In some of the foregoing embodiments of Formula (II), R.sup.b is branched C.sub.1-C.sub.16 alkyl. For example, in some embodiments R.sup.b has one of the following structures:

##STR00063##

[0530] In certain other of the foregoing embodiments of Formula (II), one of R.sup.8 or R.sup.9 is methyl. In other embodiments, both R.sup.8 and R.sup.9 are methyl. In some different embodiments of Formula (II), R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R.sup.8 and R.sup.9, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.

[0531] In certain embodiments of Embodiment 3, the first and second cationic lipids are each, independently selected from a lipid of Formula II.

[0532] In still other embodiments of the foregoing lipids of Formula (II), G.sup.3 is C.sub.2-C.sub.4 alkylene, for example C.sub.3 alkylene. In various different embodiments, the lipid compound has one of the structures set forth in Table 2 below

TABLE-US-00003 TABLE 2 Representative Lipids of Formula (II) No. Structure pKa II-1 [00064] 5.64 II-2 [00065] II-3 [00066] II-4 [00067] II-5 [00068] 6.27 II-6 [00069] 6.14 II-7 [00070] 5.93 II-8 [00071] 5.35 II-9 [00072] 6.27 II-10 [00073] 6.16 II-11 [00074] 6.13 II-12 [00075] 6.21 II-13 [00076] 6.22 II-14 [00077] 6.33 II-15 [00078] 6.32 II-16 [00079] 6.37 II-17 [00080] 6.27 II-18 [00081] II-19 [00082] II-20 [00083] II-21 [00084] II-22 [00085] II-23 [00086] II-24 [00087] 6.14 II-25 [00088] II-26 [00089] II-27 [00090] II-28 [00091] II-29 [00092] II-30 [00093] II-31 [00094] II-32 [00095] II-33 [00096] II-34 [00097] II-35 [00098] 5.97 II-36 [00099] 6.13 II-37 [00100] 5.61 II-38 [00101] 6.45 II-39 [00102] 6.45 II-40 [00103] 6.57 II-41 [00104] II-42 [00105] II-43 [00106] II-44 [00107] II-45 [00108] II-46 [00109]

[0533] In some other embodiments, the cationic lipid of Embodiments 1, 2, 3, 4 or 5 has a structure of Formula III:

##STR00110##

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: [0534] one of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O, and the other of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O or a direct bond; [0535] G.sup.1 and G.sup.2 are each independently unsubstituted C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; [0536] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24 alkenylene, C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.8 cycloalkenylene; [0537] R.sup.a is H or C.sub.1-C.sub.12 alkyl; [0538] R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkenyl; [0539] R.sup.3 is H, OR.sup.5, CN, C(?O)OR.sup.4, OC(?O)R.sup.4 or NR.sup.5C(?O)R.sup.4; [0540] R.sup.4 is C.sub.1-C.sub.12 alkyl; [0541] R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and [0542] x is 0, 1 or 2.

[0543] In some of the foregoing embodiments of Formula (III), the lipid has one of the following Formulae (IIIA) or (IIIB):

##STR00111##

wherein: [0544] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; [0545] R.sup.6 is, at each occurrence, independently H, OH or C.sub.1-C.sub.24 alkyl; [0546] n is an integer ranging from 1 to 15.

[0547] In some of the foregoing embodiments of Formula (III), the lipid has Formula (IIIA), and in other embodiments, the lipid has Formula (IIIB).

[0548] In other embodiments of Formula (III), the lipid has one of the following Formulae (IIIC) or (IIID):

##STR00112##

wherein y and z are each independently integers ranging from 1 to 12.

[0549] In any of the foregoing embodiments of Formula (III), one of L.sup.1 or L.sup.2 is O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 are O(C?O). In some different embodiments of any of the foregoing, L.sup.1 and L.sup.2 are each independently (C?O)O or O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 is (C?O)O.

[0550] In some different embodiments of Formula (III), the lipid has one of the following Formulae (IIIE) or (IIIF):

##STR00113##

[0551] In some of the foregoing embodiments of Formula (III), the lipid has one of the following Formulae (IIIG), (IIIH), (IIII), or (IIIJ):

##STR00114##

[0552] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0553] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0554] In some of the foregoing embodiments of Formula (III), R.sup.6 is H. In other of the foregoing embodiments, R.sup.6 is C.sub.1-C.sub.24 alkyl. In other embodiments, R.sup.6 is OH.

[0555] In some embodiments of Formula (III), G.sup.3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G.sup.3 is linear C.sub.1-C.sub.24 alkylene or linear C.sub.1-C.sub.24 alkenylene.

[0556] In some other foregoing embodiments of Formula (III), R.sup.1 or R.sup.2, or both, is C.sub.6-C.sub.24 alkenyl. For example, in some embodiments, R.sup.1 and R.sup.2 each, independently have the following structure:

##STR00115##

wherein: [0557] R.sup.7a and R.sup.7b are, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; and [0558] a is an integer from 2 to 12, [0559] wherein R.sup.7a, R.sup.7b and a are each selected such that R.sup.1 and R.sup.2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0560] In some of the foregoing embodiments of Formula (III), at least one occurrence of R.sup.7a is H. For example, in some embodiments, R.sup.7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R.sup.7b is C.sub.1-C.sub.8 alkyl. For example, in some embodiments, C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0561] In different embodiments of Formula (III), R.sup.1 or R.sup.2, or both, has one of the following structures:

##STR00116##

[0562] In some of the foregoing embodiments of Formula (III), R.sup.3 is OH, CN, C(?O)OR.sup.4, OC(?O)R.sup.4 or NHC(?O)R.sup.4. In some embodiments, R.sup.4 is methyl or ethyl.

[0563] In some specific embodiments of Embodiment 3, the first and second cationic lipids are each, independently selected from a lipid of Formula III.

[0564] In various different embodiments, a cationic lipid of any one of the disclosed embodiments (e.g., the cationic lipid, the first cationic lipid, the second cationic lipid) of Formula (III) has one of the structures set forth in Table 3 below.

TABLE-US-00004 TABLE 3 Representative Compounds of Formula (III) No. Structure pKa III-1 [00117] 5.89 III-2 [00118] 6.05 III-3 [00119] 6.09 III-4 [00120] 5.60 III-5 [00121] 5.59 III-6 [00122] 5.42 III-7 [00123] 6.11 III-8 [00124] 5.84 III-9 [00125] III-10 [00126] III-11 [00127] III-12 [00128] III-13 [00129] III-14 [00130] III-15 [00131] 6.14 III-16 [00132] 6.31 III-17 [00133] 6.28 III-18 [00134] III-19 [00135] III-20 [00136] 6.36 III-21 [00137] III-22 [00138] 6.10 III-23 [00139] 5.98 III-24 [00140] III-25 [00141] 6.22 III-26 [00142] 5.84 III-27 [00143] 5.77 III-28 [00144] III-29 [00145] III-30 [00146] 6.09 III-31 [00147] III-32 [00148] III-33 [00149] III-34 [00150] III-35 [00151] III-36 [00152] III-37 [00153] III-38 [00154] III-39 [00155] III-40 [00156] III-41 [00157] III-42 [00158] III-43 [00159] III-44 [00160] III-45 [00161] III-46 [00162] III-47 [00163] III-48 [00164] III-49 [00165]

[0565] In one embodiment, the cationic lipid ofany one of Embodiments 1, 2, 3, 4 or 5 has a structure of Formula (IV):

##STR00166##

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: [0566] one of G.sup.1 or G.sup.2 is, at each occurrence, O(C?O), (C?O)O, C(?O), O, S(O).sub.y, SS, C(?O)S, SC(?O), N(R.sup.a)C(?O), C(?O)N(R.sup.a), N(R.sup.a)C(?O)N(R.sup.a), OC(?O)N(R.sup.a) or N(R)C(?O)O, and the other of G.sup.1 or G.sup.2 is, at each occurrence, O(C?O), (C?O)O, C(?O), O, S(O).sub.y, SS, C(?O)S, SC(?O), N(R.sup.a)C(?O), C(?O)N(R.sup.a), N(R.sup.a)C(?O)N(R.sup.a), OC(?O)N(R.sup.a) or N(R.sup.a)C(?O)O or a direct bond; [0567] L is, at each occurrence, ?(C?O), wherein ? represents a covalent bond to X; [0568] X is CR.sup.a; [0569] Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; [0570] R.sup.a is, at each occurrence, independently H, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 hydroxylalkyl, C.sub.1-C.sub.12 aminoalkyl, C.sub.1-C.sub.12 alkylaminylalkyl, C.sub.1-C.sub.12 alkoxyalkyl, C.sub.1-C.sub.12 alkoxycarbonyl, C.sub.1-C.sub.12 alkylcarbonyloxy, C.sub.1-C.sub.12 alkylcarbonyloxyalkyl or C.sub.1-C.sub.12 alkylcarbonyl; [0571] R is, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; [0572] R.sup.1 and R.sup.2 have, at each occurrence, the following structure, respectively:

##STR00167## [0573] a.sup.1 and a.sup.2 are, at each occurrence, independently an integer from 3 to 12; [0574] b.sup.1 and b.sup.2 are, at each occurrence, independently 0 or 1; [0575] c.sup.1 and c.sup.2 are, at each occurrence, independently an integer from 5 to 10; [0576] d.sup.1 and d.sup.2 are, at each occurrence, independently an integer from 5 to 10; [0577] y is, at each occurrence, independently an integer from 0 to 2; and [0578] n is an integer from 1 to 6, [0579] wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.

[0580] In some embodiments of Formula (TV), G.sup.1 and G.sup.2 are each independently O(C?O) or (C?O)O.

[0581] In other embodiments of Formula (IV), X is CH.

[0582] In different embodiments of Formula (IV), the sum of a.sup.1+b.sup.1+c.sup.1 or the sum of a.sup.2+b.sup.2+c.sup.2 is an integer from 12 to 26.

[0583] In still other embodiments of Formula (IV), a.sup.1 and a.sup.2 are independently an integer from 3 to 10. For example, in some embodiments a.sup.1 and a.sup.2 are independently an integer from 4 to 9.

[0584] In various embodiments of Formula (IV), b.sup.1 and b.sup.2 are 0. In different embodiments, b.sup.1 and b.sup.2 are 1.

[0585] In more embodiments of Formula (IV), c.sup.1, c.sup.2, d.sup.1 and d.sup.2 are independently an integer from 6 to 8.

[0586] In other embodiments of Formula (IV), c.sup.1 and c.sup.2 are, at each occurrence, independently an integer from 6 to 10, and d.sup.1 and d.sup.2 are, at each occurrence, independently an integer from 6 to 10.

[0587] In other embodiments of Formula (IV), c.sup.1 and c.sup.2 are, at each occurrence, independently an integer from 5 to 9, and d.sup.1 and d.sup.2 are, at each occurrence, independently an integer from 5 to 9.

[0588] In more embodiments of Formula (IV), Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1. In other embodiments, Z is alkyl.

[0589] In various embodiments of the foregoing Formula (IV), R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond. In certain embodiments, each R is H. In other embodiments at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0590] In other embodiments of the compound of Formula (IV), R.sup.1 and R.sup.2 independently have one of the following structures:

##STR00168##

[0591] In certain embodiments of Formula (IV), the compound has one of the following structures:

##STR00169## ##STR00170## ##STR00171## ##STR00172##

[0592] In still different embodiments the cationic lipid of Embodiments 1, 2, 3, 4 or 5 has the structure of Formula

##STR00173##

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: [0593] one of G.sup.1 or G.sup.2 is, at each occurrence, O(C?O), (C?O)O, C(?O), O, S(O).sub.y, SS, C(?O)S, SC(?O), N(R.sup.a)C(?O), C(?O)N(R.sup.a), N(R.sup.a)C(?O)N(R.sup.a), OC(?O)N(R.sup.a) or N(R.sup.a)C(?O)O, and the other of G.sup.1 or G.sup.2 is, at each occurrence, O(C?O), (C?O)O, C(?O), O, S(O).sub.y, SS, C(?O)S, SC(?O), N(R.sup.a)C(?O), C(?O)N(R.sup.a), N(R.sup.a)C(?O)N(R.sup.a), OC(?O)N(R.sup.a) or N(R.sup.a)C(?O)O or a direct bond; [0594] L is, at each occurrence, ?O(C?O), wherein ? represents a covalent bond to X; [0595] X is CR.sup.a; [0596] Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; [0597] R.sup.a is, at each occurrence, independently H, C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 hydroxylalkyl, C.sub.1-C.sub.12 aminoalkyl, C.sub.1-C.sub.12 alkylaminylalkyl, C.sub.1-C.sub.12 alkoxyalkyl, C.sub.1-C.sub.12 alkoxycarbonyl, C.sub.1-C.sub.12 alkylcarbonyloxy, C.sub.1-C.sub.12 alkylcarbonyloxyalkyl or C.sub.1-C.sub.12 alkylcarbonyl; [0598] R is, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; [0599] R.sup.1 and R.sup.2 have, at each occurrence, the following structure, respectively:

##STR00174## [0600] R is, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; [0601] a.sup.1 and a.sup.2 are, at each occurrence, independently an integer from 3 to 12; [0602] b.sup.1 and b.sup.2 are, at each occurrence, independently 0 or 1; [0603] c.sup.1 and c.sup.2 are, at each occurrence, independently an integer from 2 to 12; [0604] d.sup.1 and d.sup.2 are, at each occurrence, independently an integer from 2 to 12; [0605] y is, at each occurrence, independently an integer from 0 to 2; and [0606] n is an integer from 1 to 6, [0607] wherein a.sup.1, a.sup.2, c.sup.1, c.sup.2, d.sup.1 and d.sup.2 are selected such that the sum of a.sup.1+c.sup.1+d.sup.1 is an integer from 18 to 30, and the sum of a.sup.2+c.sup.2+d.sup.2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.

[0608] In certain embodiments of Formula (V), G.sup.1 and G.sup.2 are each independently O(C?O) or (C?O)O.

[0609] In other embodiments of Formula (V), X is CH.

[0610] In some embodiments of Formula (V), the sum of a.sup.1+c.sup.1+d.sup.1 is an integer from 20 to 30, and the sum of a.sup.2+c.sup.2+d.sup.2 is an integer from 18 to 30. In other embodiments, the sum of a.sup.1+c.sup.1+d.sup.1 is an integer from 20 to 30, and the sum of a.sup.2+c.sup.2+d.sup.2 is an integer from 20 to 30. In more embodiments of Formula (V), the sum of a.sup.1+b.sup.1+c.sup.1 or the sum of a.sup.2+b.sup.2+c.sup.2 is an integer from 12 to 26. In other embodiments, a.sup.1, a.sup.2, c.sup.1, c.sup.2, d.sup.1 and d.sup.2 are selected such that the sum of a.sup.1+c.sup.1+d.sup.1 is an integer from 18 to 28, and the sum of a.sup.2+c.sup.2+d.sup.2 is an integer from 18 to 28,

[0611] In still other embodiments of Formula (V), at and a.sup.2 are independently an integer from 3 to 10, for example an integer from 4 to 9.

[0612] In yet other embodiments of Formula (V), b.sup.1 and b.sup.2 are 0. In different embodiments b.sup.1 and b.sup.2 are 1.

[0613] In certain other embodiments of Formula (V), c.sup.1, c.sup.2, d.sup.1 and d.sup.2 are independently an integer from 6 to 8.

[0614] In different other embodiments of Formula (V), Z is alkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1.

[0615] In more embodiments of Formula (V), Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1. In other embodiments, Z is alkyl.

[0616] In other different embodiments of Formula (V), R is, at each occurrence, independently either: (a) H or methyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond. For example in some embodiments each R is H. In other embodiments at least one R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0617] In more embodiments, each R is H.

[0618] In certain embodiments of Formula (V), the sum of a.sup.1+c.sup.1+d.sup.1 is an integer from 20 to 25, and the sum of a.sup.2+c.sup.2+d.sup.2 is an integer from 20 to 25.

[0619] In other embodiments of Formula (V), R.sup.1 and R.sup.2 independently have one of the following structures:

##STR00175##

[0620] In more embodiments of Formula (V), the compound has one of the following structures:

##STR00176## ##STR00177## ##STR00178##

[0621] In any of the foregoing embodiments of Formula (IV) or (V), n is 1. In other of the foregoing embodiments of Formula (IV) or (V), n is greater than 1.

[0622] In more of any of the foregoing embodiments of Formula (IV) or (V), Z is a mono- or polyvalent moiety comprising at least one polar functional group. In some embodiments, Z is a monovalent moiety comprising at least one polar functional group. In other embodiments, Z is a polyvalent moiety comprising at least one polar functional group.

[0623] In more of any of the foregoing embodiments of Formula (IV) or (V), the polar functional group is a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylaminyl, heterocyclyl or heteroaryl functional group.

[0624] In any of the foregoing embodiments of Formula (IV) or (V), Z is hydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl, alkylaminylalkyl, heterocyclyl or heterocyclylalkyl.

[0625] In some other embodiments of Formula (IV) or (V), Z has the following structure:

##STR00179##

wherein: [0626] R.sup.5 and R.sup.6 are independently H or C.sub.1-C.sub.6 alkyl; [0627] R.sup.7 and R.sup.8 are independently H or C.sub.1-C.sub.6 alkyl or R.sup.7 and R.sup.8, together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring; and [0628] x is an integer from 0 to 6.

[0629] In still different embodiments of Formula (IV) or (V), Z has the following structure:

##STR00180##

wherein: [0630] R.sup.5 and R.sup.6 are independently H or C.sub.1-C.sub.6 alkyl; [0631] R.sup.7 and R.sup.8 are independently H or C.sub.1-C.sub.6 alkyl or R.sup.7 and R.sup.8, together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring; and [0632] x is an integer from 0 to 6.

[0633] In still different embodiments of formula (IV) or (V), Z has the following structure:

##STR00181##

wherein: [0634] R.sup.5 and R.sup.6 are independently H or C.sub.1-C.sub.6 alkyl; [0635] R.sup.7 and R.sup.8 are independently H or C.sub.1-C.sub.6 alkyl or R.sup.7 and R.sup.8, together with the nitrogen atom to which they are attached, join to form a 3-7 membered heterocyclic ring; and [0636] x is an integer from 0 to 6.

[0637] In some other embodiments of Formula (IV) or (V), Z is hydroxylalkyl, cyanoalkyl or an alkyl substituted with one or more ester or amide groups.

[0638] For example, in any of the foregoing embodiments of Formula (IV) or (V), Z has one of the following structures:

##STR00182##

[0639] In other embodiments of Formula (IV) or (V), Z-L has one of the following structures:

##STR00183## ##STR00184## ##STR00185## ##STR00186## ##STR00187## ##STR00188##

[0640] In other embodiments, Z-L has one of the following structures:

##STR00189##

[0641] In still other embodiments, X is CH and Z-L has one of the following structures:

##STR00190##

[0642] In various different embodiments, a cationic lipid of any one Embodiments 1, 2, 3, 4 or 5 has one of the structures set forth in Table 4 below.

TABLE-US-00005 TABLE 4 Representative Compounds of Formula (IV) or (V) No. Structure IV-1 [00191] IV-2 [00192] IV-3 [00193]

[0643] In one embodiment, the cationic lipid is a compound having the following structure (VI):

##STR00194##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0644] L.sup.1 and L.sup.2 are each independently O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a, NR.sup.aC(?O)O or a direct bond; [0645] G.sup.1 is C.sub.1-C.sub.2 alkylene, (C?O), O(C?O), SC(?O), NR.sup.aC(?O) or a direct bond; [0646] G.sup.2 is C(?O), (C?O)O, C(?O)S, C(?O)NR.sup.a or a direct bond; [0647] G3 is C.sub.1-C.sub.6 alkylene; [0648] R.sup.a is H or C.sub.1-C.sub.12 alkyl; [0649] R.sup.1a and R.sup.1b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.1a is H or C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0650] R.sup.2a and R.sup.2b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.2a is H or C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.2b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0651] R.sup.3a and R.sup.3b are, at each occurrence, independently either (a): H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.3a is H or C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.3b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0652] R.sup.4a and R.sup.4b are, at each occurrence, independently either: (a) H or C.sub.1-C.sub.12 alkyl; or (b) R.sup.4a is H or C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond; [0653] R.sup.5 and R.sup.6 are each independently H or methyl; [0654] R.sup.7 is H or C.sub.1-C.sub.20 alkyl; [0655] R.sup.8 is OH, N(R.sub.9)(C?O)R.sup.10, (C?O)NR.sup.9R.sup.10, NR.sup.9R.sup.10, (C?O)OR.sup.11 or O(C?O)R.sup.11, provided that G.sup.3 is C.sub.4-C.sub.6 alkylene when R.sup.8 is NR.sup.9R.sup.10, [0656] R.sup.9 and R.sup.10 are each independently H or C.sub.1-C.sub.12 alkyl; [0657] R.sup.1 is aralkyl; [0658] a, b, c and d are each independently an integer from 1 to 24; and [0659] x is 0, 1 or 2,
wherein each alkyl, alkylene and aralkyl is optionally substituted.

[0660] In some embodiments of structure (VI), L and L.sup.2 are each independently O(C?O), (C?O)O or a direct bond. In other embodiments, G.sup.1 and G.sup.2 are each independently (C?O) or a direct bond. In some different embodiments, L.sup.1 and L.sup.2 are each independently O(C?O), (C?O)O or a direct bond; and G.sup.1 and G.sup.2 are each independently (C?O) or a direct bond.

[0661] In some different embodiments of structure (VI), L.sup.1 and L.sup.2 are each independently C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.a, NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a, NR.sup.aC(?O)O, NR.sup.aS(O).sub.xNR.sup.a, NR.sup.aS(O).sub.x or S(O).sub.xNR.sup.a.

[0662] In other of the foregoing embodiments of structure (VI), the compound has one of the following structures (VIA) or (VIB):

##STR00195##

[0663] In some embodiments, the compound has structure (VIA). In other embodiments, the compound has structure (VIB).

[0664] In any of the foregoing embodiments of structure (VI), one of L.sup.1 or L.sup.2 is O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 are O(C?O).

[0665] In some different embodiments of any of the foregoing, one of L.sup.1 or L.sup.2 is (C?O)O. For example, in some embodiments each of Lt and L.sup.2 is (C?O)O.

[0666] In different embodiments of structure (VI), one of L.sup.1 or L.sup.2 is a direct bond. As used herein, a direct bond means the group (e.g., L.sup.1 or L.sup.2) is absent. For example, in some embodiments each of L.sup.1 and L.sup.2 is a direct bond.

[0667] In other different embodiments of the foregoing, for at least one occurrence of R.sup.1a and R.sup.1b, R.sup.1a is H or C.sub.1-C.sub.12 alkyl, and R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent Rib and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0668] In still other different embodiments of structure (VI), for at least one occurrence of R.sup.4a and R.sup.4b, R.sup.4a is H or C.sub.1-C.sub.12 alkyl, and R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0669] In more embodiments of structure (VI), for at least one occurrence of R.sup.2a and R.sup.2b, R.sup.2a is H or C.sub.1-C.sub.12 alkyl, and R.sup.2b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.2b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0670] In other different embodiments of any of the foregoing, for at least one occurrence of R.sup.3a and R.sup.3b, R.sup.3a is H or C.sub.1-C.sub.12 alkyl, and R.sup.3b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.3b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0671] It is understood that carbon-carbon double bond refers to one of the following structures:

##STR00196##

wherein R.sub.c and R.sup.d are, at each occurrence, independently H or a substituent. For example, in some embodiments R.sup.c and R.sup.d are, at each occurrence, independently H, C.sub.1-C.sub.12 alkyl or cycloalkyl, for example H or C.sub.1-C.sub.12 alkyl.

[0672] In various other embodiments, the compound has one of the following structures (VIC) or (VID):

##STR00197##

wherein e, f, g and h are each independently an integer from 1 to 12.

[0673] In some embodiments, the compound has structure (VIC). In other embodiments, the compound has structure (VID).

[0674] In various embodiments of the compounds of structures (VIC) or (VID), e, f, g and h are each independently an integer from 4 to 10.

[0675] In other different embodiments,

##STR00198##

or both, independently has one of the following structures:

##STR00199##

[0676] In certain embodiments of the foregoing, a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0677] In some embodiments of structure (VI), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0678] In some embodiments of structure (VI), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0679] In some certain embodiments of structure (VI), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16. In some embodiments of structure (VI), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12. In some embodiments of structure (VI), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

[0680] In some embodiments of structure (VI), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12. In some embodiments of structure (VI), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12. In some other various embodiments of structure (VI), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments a and d are the same and b and c are the same.

[0681] The sum of a and b and the sum of c and d are factors which may be varied to obtain a lipid having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a, b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[0682] The substituents at R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a are not particularly limited. In some embodiments, at least one of R.sup.a, R.sup.2a, R.sup.3a and R.sup.4a is H. In certain embodiments R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a are H at each occurrence. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.12 alkyl. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.8 alkyl. In certain other embodiments at least one of R.sup.1a, R.sup.2a, R.sup.3a and R.sup.4a is C.sub.1-C.sub.6 alkyl. In some of the foregoing embodiments, the C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0683] In certain embodiments of the foregoing, R.sup.1a, R.sup.1b, R.sup.4a and R.sup.4b are C.sub.1-C.sub.12 alkyl at each occurrence.

[0684] In further embodiments of the foregoing, at least one of R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b is H or R.sup.1b, R.sup.2b, R.sup.3b and R.sup.4b are H at each occurrence.

[0685] In certain embodiments of the foregoing, R.sup.1b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.1b and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R.sup.4b together with the carbon atom to which it is bound is taken together with an adjacent R.sup.4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0686] The substituents at R.sup.5 and R.sup.6 are not particularly limited in the foregoing embodiments. In certain embodiments one of R.sup.5 or R.sub.6 is methyl. In other embodiments each of R.sup.5 or R.sup.6 is methyl.

[0687] The substituents at R.sup.7 are not particularly limited in the foregoing embodiments. In certain embodiments R.sup.7 is C.sub.6-C.sub.16 alkyl. In some other embodiments, R.sup.7 is C.sub.6-C.sub.9 alkyl. In some of these embodiments, R.sup.7 is substituted with (C?O)OR.sup.b, O(C?O)R.sup.b, C(?O)R.sup.b, OR.sup.b, S(O).sub.xR.sup.b, SSR.sup.b, C(?O)SR.sup.b, SC(?O)R.sup.b, NR.sup.aR.sup.b, NR.sup.aC(?O)R.sup.b, C(?O)NR.sup.aR.sup.b, NR.sup.aC(?O)NR.sup.aR.sup.b, OC(?O)NR.sup.aR.sup.b, NR.sup.aC(?O)OR.sup.b, NR.sup.aS(O).sub.xNR.sup.aR.sup.b, NR.sup.aS(O).sub.xR.sup.b or S(O).sub.xNR.sup.aR.sup.b, wherein: R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.b is C.sub.1-C.sub.15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R.sup.7 is substituted with (C?O)OR.sup.b or O(C?O)R.sup.b.

[0688] In various of the foregoing embodiments of structure (VI), R.sup.b is branched C.sub.3-C.sub.15 alkyl. For example, in some embodiments R.sup.b has one of the following structures:

##STR00200##

[0689] In certain embodiments, R.sup.8 is OH.

[0690] In other embodiments of structure (VI), R.sup.8 is N(R.sup.9)(C?O)R.sup.10. In some other embodiments, R.sup.8 is (C?O)NR.sup.9R.sup.10. In still more embodiments, R.sup.8 is NR.sup.9R.sup.10. In some of the foregoing embodiments, R.sup.9 and R.sup.10 are each independently H or C.sub.1-C.sub.8 alkyl, for example H or C.sub.1-C.sub.3 alkyl. In more specific of these embodiments, the C.sub.1-C.sub.8 alkyl or C.sub.1-C.sub.3 alkyl is unsubstituted or substituted with hydroxyl. In other of these embodiments, R.sup.9 and R.sup.10 are each methyl.

[0691] In yet more embodiments of structure (VI), R.sup.8 is (C?O)OR.sup.11. In some of these embodiments R.sup.11 is benzyl.

[0692] In yet more specific embodiments of structure (VI), R.sup.8 has one of the following structures: [0693] OH;

##STR00201##

[0694] In still other embodiments of the foregoing compounds, G is C.sub.2-C.sub.5 alkylene, for example C.sub.2-C.sub.4 alkylene, C.sub.3 alkylene or C.sub.4 alkylene. In some of these embodiments, R.sup.8 is OH. In other embodiments, G.sup.2 is absent and R.sup.7 is C.sub.1-C.sub.2 alkylene, such as methyl.

[0695] In various different embodiments, the compound has one of the structures set forth in Table 5 below.

TABLE-US-00006 TABLE 5 Representative cationic lipids of structure (VI) No. Structure VI- 1 [00202] VI- 2 [00203] VI- 3 [00204] VI- 4 [00205] VI- 5 [00206] VI- 6 [00207] VI- 7 [00208] VI- 8 [00209] VI- 9 [00210] VI- 10 [00211] VI- 11 [00212] VI- 12 [00213] VI- 13 [00214] VI- 14 [00215] VI- 15 [00216] VI- 16 [00217] VI- 17 [00218] VI- 18 [00219] VI- 19 [00220] VI- 20 [00221] VI- 21 [00222] VI- 22 [00223] VI- 23 [00224] VI- 24 [00225] VI- 25 [00226] VI- 26 [00227] VI- 27 [00228] VI- 28 [00229] VI- 29 [00230] VI- 30 [00231] VI- 31 [00232] VI- 32 [00233] VI- 33 [00234] VI- 34 [00235] VI- 35 [00236] VI- 36 [00237] VI- 37 [00238]

[0696] In one embodiment, the cationic lipid is a compound having the following structure (VII):

##STR00239##

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: [0697] X and X are each independently N or CR; [0698] Y and Y are each independently absent, O(C?O), (C?O)O or NR, provided that: [0699] a) Y is absent when X is N; [0700] b) Y is absent when X is N; [0701] c) Y is O(C?O), (C?O)O or NR when X is CR; and [0702] d) Y is O(C?O), (C?O)O or NR when X is CR, [0703] L.sup.1 and L.sup.1 are each independently O(C?O)R.sup.1, (C?O)OR.sup.1, C(?O)R.sup.1, OR.sup.1, S(O)R.sup.1, SSR.sup.1, C(?O)SR.sup.1, SC(?O)R.sup.1, NR.sup.aC(?O)R.sup.1, C(?O)NR.sup.bR.sup.c, NR.sup.aC(?O)NR.sup.bR.sup.c, OC(?O)NR.sup.bR.sup.c or NR.sup.aC(?O)OR.sup.1; [0704] L.sup.2 and L.sup.2 are each independently O(C?O)R.sup.2, (C?O)OR.sup.2, C(?O)R.sup.2, OR.sup.2, S(O).sub.zR.sup.2, SSR.sup.2, C(?O)SR.sup.2, SC(?O)R.sup.2, NR.sup.dC(?O)R.sup.2, C(?O)NR.sup.eR.sup.f, NR.sup.dC(?O)NR.sup.eR.sup.f, OC(?O)NR.sup.eR.sup.f; NR.sup.dC(?O)OR.sup.2 or a direct bond to R.sup.2; [0705] G.sup.1, G.sup.1, G.sup.2 and G.sup.2 are each independently C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; [0706] G.sup.3 is C.sub.2-C.sub.24 heteroalkylene or C.sub.2-C.sub.24 heteroalkenylene; [0707] R.sup.a, R.sup.b, R.sup.d and R.sup.e are, at each occurrence, independently H, C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0708] R.sup.c and R.sup.f are, at each occurrence, independently C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0709] R is, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; [0710] R.sup.1 and R.sup.2 are, at each occurrence, independently branched C.sub.6-C.sub.24 alkyl or branched C.sub.6-C.sub.24 alkenyl; [0711] z is 0, 1 or 2, and
wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.

[0712] In other different embodiments of structure (VII): [0713] X and X are each independently N or CR; [0714] Y and Y are each independently absent or NR, provided that: [0715] a) Y is absent when X is N; [0716] b) Y is absent when X is N; [0717] c) Y is NR when X is CR; and [0718] d) Y is NR when X is CR, [0719] L.sup.1 and L.sup.1 are each independently O(C?O)R.sup.1, (C?O)OR.sup.1, C(?O)R.sup.1, OR.sup.1, S(O)R.sup.1, SSR.sup.1, C(?O)SR.sup.1, SC(?O)R.sup.1, NR.sup.aC(?O)R.sup.1, C(?O)NR.sup.bR.sup.c, NR.sup.aC(?O)NR.sup.bR.sup.c, OC(?O)NR.sup.bR.sup.c or NR.sup.aC(?O)OR.sup.1; [0720] L.sup.2 and L.sup.2 are each independently O(C?O)R.sup.2, (C?O)OR.sup.2, C(O)R.sup.2, OR.sup.2, S(O).sub.2R.sup.2, SSR.sup.2, C(?O)SR.sup.2, SC(?O)R.sup.2, NR.sup.dC(?O)R.sup.2, C(?O)NR.sup.eR.sup.f, NR.sup.dC(?O)NR.sup.eR.sup.f, OC(?O)NR.sup.eR.sup.f; NR.sup.dC(?O)OR.sup.2 or a direct bond to R.sup.2; [0721] G.sup.1, G.sup.1, G.sup.2 and G.sup.2 are each independently C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; [0722] G.sup.3 is C.sub.2-C.sub.24 alkyleneoxide or C.sub.2-C.sub.24 alkenyleneoxide; [0723] R.sup.a, R.sup.b, R.sup.d and R.sup.e are, at each occurrence, independently H, C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0724] R.sup.c and R.sup.f are, at each occurrence, independently C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0725] R is, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; [0726] R.sup.1 and R.sup.2 are, at each occurrence, independently branched C.sub.6-C.sub.24 alkyl or branched C.sub.6-C.sub.24 alkenyl; [0727] z is 0, 1 or 2, and
wherein each alkyl, alkenyl, alkylene, alkenylene, alkyleneoxide and alkenyleneoxide is independently substituted or unsubstituted unless otherwise specified.

[0728] In some embodiments of structure (VII), G.sup.3 is C.sub.2-C.sub.24 alkyleneoxide or C.sub.2-C.sub.24 alkenyleneoxide. In certain embodiments, G.sup.3 is unsubstituted. In other embodiments, G.sup.3 is substituted, for example substituted with hydroxyl. In more specific embodiments G.sup.3 is C.sub.2-C.sub.12 alkyleneoxide, for example, in some embodiments G.sup.3 is C.sub.3-C.sub.7 alkyleneoxide or in other embodiments G.sup.3 is C.sub.3-C.sub.12 alkyleneoxide.

[0729] In other embodiments of structure (VII), G.sup.3 is C.sub.2-C.sub.24 alkyleneaminyl or C.sub.2-C.sub.24 alkenyleneaminyl, for example C.sub.6-C.sub.12 alkyleneaminyl. In some of these embodiments, G.sup.3 is unsubstituted. In other of these embodiments, G.sup.3 is substituted with C.sub.1-C.sub.6 alkyl.

[0730] In some embodiments of structure (VII), X and X are each N, and Y and Y are each absent. In other embodiments, X and X are each CR, and Y and Y are each NR. In some of these embodiments, R is H.

[0731] In certain embodiments of structure (VII), X and X are each CR, and Y and Y are each independently O(C?O) or (C?O)O.

[0732] In some of the foregoing embodiments of structure (VII), the compound has one of the following structures (VIIA), (VIIB), (VIIC), (VIID), (VIIE), (VIIF), (VIIG) or (VIIH):

##STR00240##

wherein R.sup.d is, at each occurrence, independently H or optionally substituted C.sub.1-C.sub.6 alkyl. For example, in some embodiments R.sup.d is H. In other embodiments, R.sup.d is C.sub.1-C.sub.6 alkyl, such as methyl. In other embodiments, R.sup.d is substituted C.sub.1-C.sub.6 alkyl, such as C.sub.1-C.sub.6 alkyl substituted with O(C?O)R, (C?O)OR, NRC(?O)R or C(?O)N(R).sub.2, wherein R is, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl.

[0733] In some of the foregoing embodiments of structure (VII), L.sup.1 and L.sup.1 are each independently O(C?O)R.sup.1, (C?O)OR.sup.1 or C(?O)NR.sup.bR.sup.c, and L.sup.2 and L.sup.2 are each independently O(C?O)R.sup.2, (C?O)OR.sup.2 or C(?O)NR.sup.eR.sup.f. For example, in some embodiments L.sup.1 and L.sup.1 are each (C?O)OR.sup.1, and L.sup.2 and L.sup.2 are each (C?O)OR.sup.2. In other embodiments L.sup.1 and L.sup.1 are each (C?O)OR.sup.1, and L.sup.2 and L.sup.2 are each C(?O)NR.sup.cR.sup.f. In other embodiments L.sup.1 and L.sup.1 are each C(?O)NR.sup.bR.sup.c, and L.sup.2 and L.sup.2 are each C(?O)NR.sup.eR.sup.f.

[0734] In some embodiments of the foregoing, G.sup.1, G.sup.1, G.sup.2 and G.sup.2 are each independently C.sub.2-C.sub.8 alkylene, for example C.sub.4-C.sub.8 alkylene.

[0735] In some of the foregoing embodiments of structure (VII), R.sup.1 or R.sup.2, are each, at each occurrence, independently branched C.sub.6-C.sub.24 alkyl. For example, in some embodiments, R.sup.1 and R.sup.2 at each occurrence, independently have the following structure:

##STR00241##

wherein: [0736] R.sup.7a and R.sup.7b are, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; and [0737] a is an integer from 2 to 12,
wherein R.sup.7a, R.sup.7b and a are each selected such that R.sup.1 and R.sup.2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0738] In some of the foregoing embodiments of structure (VII), at least one occurrence of R.sup.7a is H. For example, in some embodiments, R.sup.7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R.sup.7b is C.sub.1-C.sub.8 alkyl. For example, in some embodiments, C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0739] In different embodiments of structure (VII), R.sup.1 or R.sup.2, or both, at each occurrence independently has one of the following structures:

##STR00242##

[0740] In some of the foregoing embodiments of structure (VII), R.sup.b, R.sup.c, R.sup.e and R.sup.f, when present, are each independently C.sub.3-C.sub.12 alkyl. For example, in some embodiments R.sup.b, R.sup.c, R.sup.e and R.sup.f, when present, are n-hexyl and in other embodiments R.sup.b, R.sup.c, R.sup.e and R.sup.f, when present, are n-octyl.

[0741] In various different embodiments of structure (VII), the cationic lipid has one of the structures set forth in Table 6 below.

TABLE-US-00007 TABLE 6 Representative cationic lipids of structure (VII) No. Structure VII- 1 [00243] VII- 2 [00244] VII- 3 [00245] VII- 4 [00246] VII- 5 [00247] VII- 6 [00248] VII- 7 [00249] VII- 8 [00250] VII- 9 [00251] VII- 10 [00252] VII- 11 [00253]

[0742] In one embodiment, the cationic lipid is a compound having the following structure (VIII):

##STR00254##

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: [0743] X is N, and Y is absent; or X is CR, and Y is NR; [0744] L.sup.1 is O(C?O)R.sup.1, (C?O)OR.sup.1, C(?O)R.sup.1, OR.sup.1, S(O)R.sup.1, SSR.sup.1, C(?O)SR.sup.1, SC(?O)R.sup.1, NR.sup.aC(?O)R.sup.1, C(?O)NR.sup.bR.sup.c, NR.sup.aC(?O)NR.sup.bR.sup.c, OC(?O)NR.sup.bR.sup.c or NR.sup.aC(?O)OR.sup.1; [0745] L.sup.2 is O(C?O)R.sup.2, (C?O)OR.sup.2, C(O)R.sup.2, OR.sup.2, S(O)R.sup.2, SSR.sup.2, C(?O)SR.sup.2, SC(?O)R.sup.2, NR.sup.dC(?O)R.sup.2, C(?O)NR.sup.eR.sup.f, NR.sup.dC(?O)NR.sup.eR.sup.f, OC(?O)NR.sup.eR.sup.f; NR.sup.dC(?O)OR.sup.2 or a direct bond to R.sup.2; [0746] L.sup.3 is O(C?O)R.sup.3 or (C?O)OR.sup.3; [0747] G.sup.1 and G.sup.2 are each independently C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; [0748] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, C.sub.1-C.sub.24 heteroalkylene or C.sub.2-C.sub.24 heteroalkenylene; [0749] R.sup.a, R.sup.b, R.sup.d and R.sup.e are each independently H or C.sub.1-C.sub.12 alkyl or C.sub.1-C.sub.12 alkenyl; [0750] R.sup.c and R.sup.f are each independently C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0751] each R is independently H or C.sub.1-C.sub.12 alkyl; [0752] R.sup.1, R.sup.2 and R.sup.3 are each independently C.sub.1-C.sub.24 alkyl or C.sub.2-C.sub.24 alkenyl; and [0753] x is 0, 1 or 2, and [0754] wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.

[0755] In more embodiments of structure (I): [0756] X is N, and Y is absent, or X is CR, and Y is NR; [0757] L.sup.1 is O(C?O)R.sup.1, (C?O)OR.sup.1, C(?O)R.sup.1, OR.sup.1, S(O)R.sup.1, SSR.sup.1, C(?O)SR.sup.1, SC(?O)R.sup.1, NR.sup.aC(?O)R.sup.1, C(?O)NR.sup.bR.sup.c, NR.sup.aC(?O)NR.sup.bR.sup.c, OC(?O)NR.sup.bR.sup.c or NR.sup.aC(?O)OR.sup.1; [0758] L.sup.2 is O(C?O)R.sup.2, (C?O)OR.sup.2, C(?O)R.sup.2, OR.sup.2, S(O)R.sup.2, SSR.sup.2, C(?O)SR.sup.2, SC(?O)R.sup.2, NR.sup.dC(?O)R.sup.2, C(?O)NR.sup.eR.sup.f, NR.sup.dC(?O)NR.sup.eR.sup.f, OC(?O)NR.sup.eR.sup.f; NR.sup.dC(?O)OR.sup.2 or a direct bond to R.sup.2; [0759] L.sup.3 is O(C?O)R.sup.3 or (C?O)OR.sup.3; [0760] G.sup.1 and G.sup.2 are each independently C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; [0761] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, C.sub.1-C.sub.24 heteroalkylene or C.sub.2-C.sub.24 heteroalkenylene when X is CR, and Y is NR; and G.sup.3 is C.sub.1-C.sub.24 heteroalkylene or C.sub.2-C.sub.24 heteroalkenylene when X is N, and Y is absent; [0762] R.sup.a, R.sup.b, R.sup.d and R.sup.e are each independently H or C.sub.1-C.sub.12 alkyl or C.sub.1-C.sub.12 alkenyl; [0763] R.sup.c and R.sup.f are each independently C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0764] each R is independently H or C.sub.1-C.sub.12 alkyl; [0765] R.sup.1, R.sup.2 and R.sup.3 are each independently C.sub.1-C.sub.24 alkyl or C.sub.2-C.sub.24 alkenyl; and [0766] x is 0, 1 or 2, and [0767] wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.

[0768] In other embodiments of structure (I): [0769] X is N and Y is absent, or X is CR and Y is NR; [0770] L.sup.1 is O(C?O)R.sup.1, (C?O)OR.sup.1, C(?O)R.sup.1, OR.sup.1, S(O)R.sup.1, SSR.sup.1, C(?O)SR.sup.1, SC(?O)R.sup.1, NR.sup.aC(?O)R.sup.1, C(?O)NR.sup.bR.sup.c, NR.sup.aC(?O)NR.sup.bR.sup.c, OC(?O)NR.sup.bR.sup.c or NR.sup.aC(?O)OR.sup.1; [0771] L.sup.2 is O(C?O)R.sup.2, (C?O)OR.sup.2, C(?O)R.sup.2, OR.sup.2, S(O)R.sup.2, SSR.sup.2, C(?O)SR.sup.2, SC(?O)R.sup.2, NR.sup.dC(?O)R.sup.2, C(?O)NR.sup.eR.sup.f, NR.sup.dC(?O)NR.sup.eR.sup.f, OC(?O)NR.sup.eR.sup.f; NR.sup.dC(?O)OR.sup.2 or a direct bond to R.sup.2; [0772] L.sup.3 is O(C?O)R.sup.3 or (C?O)OR.sup.3; [0773] G.sup.1 and G.sup.2 are each independently C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; [0774] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, C.sub.1-C.sub.24 heteroalkylene or C.sub.2-C.sub.24 heteroalkenylene; [0775] R.sup.a, R.sup.b, R.sup.d and R.sup.e are each independently H or C.sub.1-C.sub.12 alkyl or C.sub.1-C.sub.12 alkenyl; [0776] R.sup.c and R.sup.f are each independently C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0777] each R is independently H or C.sub.1-C.sub.12 alkyl; [0778] R.sup.1, R.sup.2 and R.sup.3 are each independently branched C.sub.6-C.sub.24 alkyl or branched C.sub.6-C.sub.24 alkenyl, and [0779] x is 0, 1 or 2, and
wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.

[0780] In certain embodiments of structure (VIII), G.sup.3 is unsubstituted. In more specific embodiments G.sup.3 is C.sub.2-C.sub.12 alkylene, for example, in some embodiments G.sup.3 is C.sub.3-C.sub.7 alkylene or in other embodiments G.sup.3 is C.sub.3-C.sub.12 alkylene. In some embodiments, G.sup.3 is C.sub.2 or C.sub.3 alkylene.

[0781] In other embodiments of structure (VIII), G.sup.3 is C.sub.1-C.sub.12 heteroalkylene, for example C.sub.1-C.sub.12 aminylalkylene.

[0782] In certain embodiments of structure (VIII), X is N and Y is absent. In other embodiments, X is CR and Y is NR, for example in some of these embodiments R is H.

[0783] In some of the foregoing embodiments of structure (VIII), the compound has one of the following structures (VIIIA), (VIIIB), (VIIIC) or (VIIID):

##STR00255##

[0784] In some of the foregoing embodiments of structure (VIII), L.sup.1 is O(C?O)R.sup.1, (C?O)OR.sup.1 or C(?O)NR.sup.bR.sup.c, and L.sup.2 is O(C?O)R.sup.2, (C?O)OR.sup.2 or C(?O)NR.sup.eR.sup.f. In other specific embodiments, L.sup.1 is (C?O)OR.sup.1 and L.sup.2 is (C?O)OR.sup.2. Tn any of the foregoing embodiments, L.sup.3 is (C?O)OR.sup.3.

[0785] In some of the foregoing embodiments of structure (VIII), G.sup.1 and G.sup.2 are each independently C.sub.2-C.sub.12 alkylene, for example C.sub.4-C.sub.10 alkylene.

[0786] In some of the foregoing embodiments of structure (VIII), R.sup.1, R.sup.2 and R.sup.3 are each, independently branched C.sub.6-C.sub.24 alkyl. For example, in some embodiments, R.sup.1, R.sup.2 and R.sup.3 each, independently have the following structure:

##STR00256##

wherein: [0787] R.sup.7a and R.sup.7b are, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl, and [0788] a is an integer from 2 to 12,
wherein R.sup.7a, R.sup.7b and a are each selected such that R.sup.1 and R.sup.2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0789] In some of the foregoing embodiments of structure (VIII), at least one occurrence of R.sup.7a is H. For example, in some embodiments, R.sup.7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R.sup.7b is C.sub.1-C.sub.8 alkyl. For example, in some embodiments, C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0790] In some of the foregoing embodiments of structure (VIII), X is CR, Y is NR and R.sup.3 is C.sub.1-C.sub.12 alkyl, such as ethyl, propyl or butyl. In some of these embodiments, R.sup.1 and R.sup.2 are each independently branched C.sub.6-C.sub.24 alkyl.

[0791] In different embodiments of structure (Viii), R.sup.1, R.sup.2 and R.sup.3 each, independently have one or the following structures:

##STR00257##

[0792] In certain embodiments of structure (VIII), R.sup.1 and R.sup.2 and R.sup.3 are each, independently, branched C.sub.6-C.sub.24 alkyl and R.sup.3 is C.sub.1-C.sub.24 alkyl or C.sub.2-C.sub.24 alkenyl.

[0793] In some of the foregoing embodiments of structure (VIII), R.sup.b, R.sup.c, R.sup.e and R.sup.f are each independently C.sub.3-C.sub.12 alkyl. For example, in some embodiments R.sup.b, R.sup.c, R.sup.e and R.sup.f are n-hexyl and in other embodiments R.sup.b, R.sup.c, R.sup.e and R.sup.f are n-octyl.

[0794] In various different embodiments of structure (VIII), the compound has one of the structures set forth in Table 7 below.

TABLE-US-00008 TABLE 7 Representative cationic lipids of structure (VIII) No. Structure VIII-1 [00258] VIII-2 [00259] VIII-3 [00260] VIII-4 [00261] VIII-5 [00262] VIII-6 [00263] VIII-7 [00264] VIII-8 [00265] VIII-9 [00266] VIII-10 [00267] VIII-11 [00268] VIII-12 [00269]

[0795] In one embodiment, the cationic lipid is a compound having the following structure (IX):

##STR00270##

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: [0796] L.sup.1 is O(C?O)R.sup.1, (C?O)OR.sup.1, C(?O)R.sup.1, OR.sup.1, S(O)R.sup.1, SSR.sup.1, C(?O)SR.sup.1, SC(?O)R.sup.1, NR.sup.aC(?O)R.sup.1, C(?O)NR.sup.bR.sup.c, NR.sup.aC(?O)NR.sup.bR.sup.c, OC(?O)NR.sup.bR.sup.c or NR.sup.aC(?O)OR.sup.1. [0797] L.sup.2 is O(C?O)R.sup.2, (C?O)OR.sup.2, C(?O)R.sup.2, OR.sup.2, S(O)R.sup.2, SSR.sup.2, C(?O)SR.sup.2, SC(?O)R.sup.2, NR.sup.dC(?O)R.sup.2, C(?O)NR.sup.eR.sup.f, NR.sup.dC(?O)NR.sup.eR.sup.f, OC(?O)NR.sup.eR.sup.f, NR.sup.dC(?O)OR.sup.2 or a direct bond to R.sup.2; [0798] G.sup.1 and G.sup.2 are each independently C.sub.2-C.sub.12 alkylene or C.sub.2-C.sub.12 alkenylene; [0799] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, C.sub.3-C.sub.8 cycloalkylene or C.sub.3-C.sub.8 cycloalkenylene, [0800] R.sup.a, R.sup.b, R.sup.d and R.sup.e are each independently H or C.sub.1-C.sub.12 alkyl or C.sub.1-C.sub.12 alkenyl; [0801] R.sup.c and R.sup.f are each independently C.sub.1-C.sub.12 alkyl or C.sub.2-C.sub.12 alkenyl; [0802] R.sup.1 and R.sup.2 are each independently branched C.sub.6-C.sub.24 alkyl or branched C.sub.6-C.sub.24 alkenyl; [0803] R.sup.3 is N(R.sup.4)R.sup.5; [0804] R.sup.4 is C.sub.1-C.sub.12 alkyl; [0805] R.sup.5 is substituted C.sub.1-C.sub.12 alkyl; and [0806] x is 0, 1 or 2, and
wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted unless otherwise specified.

[0807] In certain embodiments of structure (XI), G.sup.3 is unsubstituted. In more specific embodiments G.sup.3 is C.sub.2-C.sub.12 alkylene, for example, in some embodiments G.sup.3 is C.sub.3-C.sub.7 alkylene or in other embodiments G.sup.3 is C.sub.3-C.sub.12 alkylene. In some embodiments, G.sup.3 is C.sub.2 or C.sub.3 alkylene.

[0808] In some of the foregoing embodiments of structure (IX), the compound has the following structure (IX A):

##STR00271##

wherein y and z are each independently integers ranging from 2 to 12, for example an integer from 2 to 6, from 4 to 10, or for example 4 or 5. In certain embodiments, y and z are each the same and selected from 4, 5, 6, 7, 8 and 9.

[0809] In some of the foregoing embodiments of structure (IX), L.sup.1 is O(C?O)R.sup.1, (C?O)OR.sup.1 or C(?O)NR.sup.bR.sup.c, and L.sup.2 is O(C?O)R.sup.2, (C?O)OR.sup.2 or C(?O)NR.sup.eR.sup.f. For example, in some embodiments L.sup.1 and L.sup.2 are (C?O)OR.sup.1 and (C?O)OR.sup.2, respectively. In other embodiments L.sup.1 is (C?O)OR.sup.1 and L.sup.2 is C(?O)NR.sup.eR.sup.f. In other embodiments L.sup.1 is C(?O)NR.sup.bR.sup.c and L.sup.2 is C(?O)NR.sup.eR.sup.f.

[0810] In other embodiments of the foregoing, the compound has one of the structures or

##STR00272##

[0811] In some of the foregoing embodiments, the compound has structure (IXB), in other embodiments, the compound has structure (IXC) and in still other embodiments the compound has the structure (IXD). In other embodiments, the compound has structure (IXE).

[0812] In some different embodiments of the foregoing, the compound has one of the following structures (IXF), (IXG), (IXH) or (IXJ):

##STR00273##

wherein y and z are each independently integers ranging from 2 to 12, for example an integer from 2 to 6, for example 4.

[0813] In some of the foregoing embodiments of structure (IX), y and z are each independently an integer ranging from 2 to 10, 2 to 8, from 4 to 10 or from 4 to 7. For example, in some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, z is 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, y and z are the same, while in other embodiments y and z are different.

[0814] In some of the foregoing embodiments of structure (IX), R.sup.1 or R.sup.2, or both is branched C.sub.6-C.sub.24 alkyl. For example, in some embodiments, R.sup.1 and R.sup.2 each, independently have the following

##STR00274##

wherein: [0815] R.sup.7a and R.sup.7b are, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; and [0816] a is an integer from 2 to 12,
wherein R.sup.7a, R.sup.7b and a are each selected such that R.sup.1 and R.sup.2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0817] In some of the foregoing embodiments of structure (IX), at least one occurrence of R.sup.7a is H. For example, in some embodiments, R.sup.7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R.sup.7b is C.sub.1-C.sub.8 alkyl. For example, in some embodiments, C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0818] In different embodiments of structure (IX), R.sup.1 or R.sup.2, or both, has one of the following structures:

##STR00275##

[0819] In some of the foregoing embodiments of structure (IX), R.sup.b, R.sup.c, R.sup.e and R.sup.f are each independently C.sub.3-C.sub.12 alkyl. For example, in some embodiments R.sup.b, R.sup.c, R.sup.e and R.sup.f are n-hexyl and in other embodiments R.sup.b, R.sup.c, R.sup.e and R.sup.f are n-octyl.

[0820] In any of the foregoing embodiments of structure (IX), R.sup.4 is substituted 6 or unsubstituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl or n-nonyl. For example, in some embodiments R.sup.4 is unsubstituted. In other R.sup.4 is substituted with one or more substituents selected from the group consisting of OR.sup.g, NR.sup.gC(?O)R.sup.h, C(?O)NR.sup.gR.sup.h, C(?O)R.sup.h, OC(?O)R.sup.h, C(?O)OR.sup.h and OR.sup.iOH, wherein: [0821] R.sup.g is, at each occurrence independently H or C.sub.1-C.sub.6 alkyl; [0822] R.sup.h is at each occurrence independently C.sub.1-C.sub.6 alkyl; and [0823] R.sup.i is, at each occurrence independently C.sub.1-C.sub.6 alkylene.

[0824] In other of the foregoing embodiments of structure (IX), R.sup.5 is substituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl or n-nonyl. In some embodiments, R.sup.5 is substituted ethyl or substituted propyl. In other different embodiments, R.sup.5 is substituted with hydroxyl. In still more embodiments, R.sup.5 is substituted with one or more substituents selected from the group consisting of OR.sup.g, NR.sup.gC(?O)R.sup.h, C(?O)NR.sup.gR.sup.h, C(?O)R.sup.h, OC(?O)R.sup.h, C(O)OR.sup.h and OR.sup.iOH, wherein: [0825] R.sup.g is, at each occurrence independently H or C.sub.1-C.sub.6 alkyl; [0826] R.sup.h is at each occurrence independently C.sub.1-C.sub.6 alkyl; and [0827] R.sup.i is, at each occurrence independently C.sub.1-C.sub.6 alkylene.

[0828] In other embodiments of structure (IX), R.sup.4 is unsubstituted methyl, and R.sup.5 is substituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl or n-nonyl. In some of these embodiments, R.sup.5 is substituted with hydroxyl.

[0829] In some other specific embodiments of structure (IX), R.sup.3 has one of the following structures:

##STR00276## ##STR00277##

[0830] In various different embodiments of structure (IX), the cationic lipid has one of the structures set forth in Table 8 below.

TABLE-US-00009 TABLE 8 Representative cationic lipids of structure (IX) No. Structure IX-1 [00278] IX-2 [00279] IX-3 [00280] IX-4 [00281] IX-5 [00282] IX-6 [00283] IX-7 [00284] IX-8 [00285] IX-9 [00286] IX-10 [00287] IX-11 [00288] IX-12 [00289] IX-13 [00290] IX-14 [00291] IX-15 [00292] IX-16 [00293] IX-17 [00294] IX-18 [00295]

[0831] In one embodiment, the cationic lipid is a compound having the following structure (X):

##STR00296##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0832] G.sup.1 is OH, NR.sup.3R.sup.4, (C?O)NR.sup.5 or NR.sup.3(C?O)R.sup.5; [0833] G.sup.2 is CH.sub.2 or (C?O); [0834] R is, at each occurrence, independently H or OH; [0835] R.sup.1 and R.sup.2 are each independently branched, saturated or unsaturated C.sub.12-C.sub.36 alkyl; [0836] R.sup.3 and R.sup.4 are each independently H or straight or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl; [0837] R.sup.5 is straight or branched, saturated or unsaturated C.sub.1-C.sub.6 alkyl; and [0838] n is an integer from 2 to 6.

[0839] In some embodiments, R.sup.1 and R.sup.2 are each independently branched, saturated or unsaturated C.sub.12-C.sub.30 alkyl, C.sub.12-C.sub.20 alkyl, or C.sub.15-C.sub.20 alkyl. In some specific embodiments, R.sup.1 and R.sup.2 are each saturated. In certain embodiments, at least one of R.sup.1 and R.sup.2 is unsaturated.

[0840] In some of the foregoing embodiments of structure (X), R.sup.1 and R.sup.2 have the following structure:

##STR00297##

[0841] In some of the foregoing embodiments of structure (X), the compound has the following structure

##STR00298##

wherein: [0842] R.sup.6 and R.sup.7 are, at each occurrence, independently H or straight or branched, saturated or unsaturated C.sub.1-C.sub.14 alkyl; [0843] a and b are each independently an integer ranging from 1 to 15, [0844] provided that R.sup.6 and a, and R.sup.7 and b, are each independently selected such that R.sup.1 and R.sup.2, respectively, are each independently branched, saturated or unsaturated C.sub.12-C.sub.36 alkyl.

[0845] In some of the foregoing embodiments, the compound has the following structure (XB):

##STR00299##

wherein: [0846] R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are each independently straight or branched, saturated or unsaturated C.sub.4-C.sub.12 alkyl, provided that R.sup.8 and R.sup.9, and R.sup.10 and R.sup.11, are each independently selected such that R.sup.1 and R.sup.2, respectively, are each independently branched, saturated or unsaturated C.sub.12-C.sub.36 alkyl. In some embodiments of (XB), R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are each independently straight or branched, saturated or unsaturated C.sub.6-C.sub.10 alkyl. In certain embodiments of (XB), at least one of R.sup.8, R.sup.9, R.sup.10 and R.sup.11 is unsaturated. In other certain specific embodiments of (XB), each of R.sup.8, R.sup.9, R.sup.10 and R.sup.11 is saturated.

[0847] In some of the foregoing embodiments, the compound has structure (XA), and in other embodiments, the compound has structure (XB).

[0848] In some of the foregoing embodiments, G.sup.1 is OH, and in some embodiments G.sup.1 is NR.sup.3R.sup.4. For example, in some embodiments, G.sup.1 is NH.sub.2, NHCH.sub.3 or N(CH.sub.3).sub.2. In certain embodiments, G.sup.1 is (C?O)NR.sup.5. In certain other embodiments, G.sup.1 is NR.sup.3(C?O)R.sup.5. For example, in some embodiments G.sup.1 is NH(C?O)CH.sub.3 or NH(C?O)CH.sub.2CH.sub.2CH.sub.3.

[0849] In some of the foregoing embodiments of structure (X), G.sup.2 is CH.sub.2. In some different embodiments, G.sup.2 is (C?O).

[0850] In some of the foregoing embodiments of structure (X), n is an integer ranging from 2 to 6, for example, in some embodiments n is 2, 3, 4, 5 or 6. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0851] In certain of the foregoing embodiments of structure (X), at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 is unsubstituted. For example, in some embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are each unsubstituted. In some embodiments, R.sup.3 is substituted. In other embodiments R.sup.4 is substituted. In still more embodiments, R.sup.5 is substituted. In certain specific embodiments, each of R.sup.3 and R.sup.4 are substituted. In some embodiments, a substituent on R.sup.3, R.sup.4 or R.sup.5 is hydroxyl. In certain embodiments, R.sup.3 and R.sup.4 are each substituted with hydroxyl.

[0852] In some of the foregoing embodiments of structure (X), at least one R is OH. In other embodiments, each R is H.

[0853] In various different embodiments of structure (X), the compound has one of the structures set forth in Table 9 below.

TABLE-US-00010 TABLE 9 Representative cationic lipids of structure (X) No. Structure X-1 [00300] X-2 [00301] X-3 [00302] X-4 [00303] X-5 [00304] X-6 [00305] X-7 [00306] X-8 [00307] X-9 [00308] X-10 [00309] X-11 [00310] X-12 [00311] X-13 [00312] X-14 [00313] X-15 [00314] X-16 [00315] X-17 [00316]

[0854] In any of Embodiments 1, 2, 3, 4 or 5, the LNPs further comprise a neutral lipid. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1. In certain embodiments, the neutral lipid is present in any of the foregoing LNPs in a concentration ranging from 5 to 10 mol percent, from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. In certain specific embodiments, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent. In some embodiments, the molar ratio of cationic lipid to the neutral lipid ranges from about 4.1:1.0 to about 4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, or from about 4.7:1.0 to 4.8:1.0. In some embodiments, the molar ratio of total cationic lipid to the neutral lipid ranges from about 4.1:1.0 to about 4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, or from about 4.7:1.0 to 4.8:1.0.

[0855] Exemplary neutral lipids for use in any of Embodiments 1, 2, 3, 4 or 5 include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-Icarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DIVIPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC). In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0856] In various embodiments of Embodiments 1, 2, 3, 4 or 5, any of the disclosed lipid nanoparticles comprise a steroid or steroid analogue. In certain embodiments, the steroid or steroid analogue is cholesterol. In some embodiments, the steroid is present in a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In certain specific embodiments, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent.

[0857] In certain embodiments, the molar ratio of cationic lipid to the steroid ranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In some of these embodiments, the molar ratio of cationic lipid to cholesterol ranges from about 5:1 to 1:1. In certain embodiments, the steroid is present in a concentration ranging from 32 to 40 mol percent of the steroid.

[0858] In certain embodiments, the molar ratio of total cationic to the steroid ranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In some of these embodiments, the molar ratio of total cationic lipid to cholesterol ranges from about 5:1 to 1:1. In certain embodiments, the steroid is present in a concentration ranging from 32 to 40 mol percent of the steroid.

[0859] In some embodiments of Embodiments 1, 2, 3 4 or 5, the LNPs further comprise a polymer conjugated lipid. In various other embodiments of Embodiments 1, 2, 3 4 or 5, the polymer conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2,3-di(tetradecanoyloxy)propyl-1-O-(?-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(?-methoxy(polyethoxy)ethyl)carbamate.

[0860] In various embodiments, the polymer conjugated lipid is present in a concentration ranging from 1.0 to 2.5 molar percent. In certain specific embodiments, the polymer conjugated lipid is present in a concentration of about 1.7 molar percent. In some embodiments, the polymer conjugated lipid is present in a concentration of about 1.5 molar percent.

[0861] In certain embodiments, the molar ratio of cationic lipid to the polymer conjugated lipid ranges from about 35:1 to about 25:1. In some embodiments, the molar ratio of cationic lipid to polymer conjugated lipid ranges from about 100:1 to about 20:1.

[0862] In certain embodiments, the molar ratio of total cationic lipid (i.e., the sum of the first and second cationic lipid) to the polymer conjugated lipid ranges from about 35:1 to about 25:1. In some embodiments, the molar ratio of total cationic lipid to polymer conjugated lipid ranges from about 100:1 to about 20:1.

[0863] In some embodiments of Embodiments 1, 2, 3 4 or 5, the pegylated lipid, when present, has the following Formula (XI):

##STR00317##

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: [0864] R.sup.12 and R.sup.13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and [0865] w has a mean value ranging from 30 to 60.

[0866] In some embodiments, R.sup.12 and R.sup.13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average w ranges from 42 to 55, for example, the average w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some specific embodiments, the average w is about 49.

[0867] In some embodiments, the pegylated lipid has the following Formula (XIa):

##STR00318##

wherein the average w is about 49.

[0868] In some embodiments of Embodiments 1, 2, 3 4 or 5, the nucleic acid is selected from antisense and messenger RNA. For example, messenger RNA may be used to induce an immune response (e.g., as a vaccine), for example by translation of immunogenic proteins.

[0869] In other embodiments of Embodiments 1, 2, 3 4 or 5, the nucleic acid is mRNA, and the mRNA to lipid ratio in the LNP (i.e., N/P, were N represents the moles of cationic lipid and P represents the moles of phosphate present as part of the nucleic

[0870] In an embodiment, the transfer vehicle comprises a lipid or an ionizable lipid described in US patent publication number 20190314524.

[0871] Some embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein as structures listed in Table 10, that provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo.

[0872] In one embodiment, an ionizable lipid has the following structure (XII):

##STR00319##

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: [0873] one of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O) NR.sup.aC(?O) C(?O)NR.sup.a, NR.sup.aC(?)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O, and the other of L.sup.1 or L.sup.2 is O(C?O), (C?O), C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O) NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O or a direct bond; [0874] G.sup.1 and G.sup.2 are each independently unsubstituted C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; [0875] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24 alkenylene, C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.8 cycloalkenylene; [0876] R.sup.a is H or C.sub.1-C.sub.12 alkyl; [0877] R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkenyl; [0878] R.sup.3 is H, OR.sup.5, CN, C(?O)OR.sup.4, OC(?O)R.sup.4 or NR.sup.5C(?O)R.sup.4; [0879] R.sup.4 is C.sub.1-C.sub.12 alkyl; [0880] R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and [0881] x is 0, 1 or 2.

[0882] In some embodiments, an ionizable lipid has one of the following structures (XIIA) or (XIIB):

##STR00320##

wherein: [0883] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; [0884] R.sub.6 is, at each occurrence, independently H, OH or C.sub.1-C.sub.24 alkyl; and [0885] n is an integer ranging from 1 to 15.

[0886] In some embodiments, the ionizable lipid has structure (XIIA), and in other embodiments, the ionizable lipid has structure (XIIB).

[0887] In other embodiments, an ionizable lipid has one of the following structures (XIIC) or (XIID):

##STR00321##

wherein y and z are each independently integers ranging from 1 to 12.

[0888] In some embodiments, one of L.sup.1 or L.sup.2 is O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 are O(C?O). In some different embodiments of any of the foregoing, L.sup.1 and L.sup.2 are each independently (C?O)O or O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 is (C?O).

[0889] In some embodiments, an ionizable lipid has one of the following structures (XIIE) or (XIIF):

##STR00322##

[0890] In some embodiments, an ionizable lipid has one of the following structures (XIIG), (XIIH), (XIII), or (XIIJ):

##STR00323##

[0891] In some embodiments, n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0892] In some embodiments, y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0893] In some embodiments, R.sub.6 is H. In other embodiments, R.sub.6 is C.sub.1-C.sub.24 alkyl. In other embodiments, R.sub.6 is OH.

[0894] In some embodiments, G.sup.3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G.sup.3 is linear C.sub.1-C.sub.24 alkylene or linear C.sub.1-C.sub.24 alkenylene.

[0895] In some embodiments, R.sup.1 or R.sup.2, or both, is C.sub.6-C.sub.24 alkenyl. For example, in some embodiments, R.sup.1 and R.sup.2 each, independently have the following structure:

##STR00324##

wherein: [0896] R.sup.7a and R.sup.7b are, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; and [0897] a is an integer from 2 to 12, [0898] wherein R.sup.7a, R.sup.7b and a are each selected such that R.sup.1 and R.sup.2 each independently comprise from 6 to 20 carbon atoms.

[0899] In some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0900] In some embodiments, at least one occurrence of R.sup.7a is H. For example, in some embodiments, R.sup.7a is H at each occurrence. In other different embodiments, at least one occurrence of R.sup.7b is C.sub.1-C.sub.8 alkyl. For example, in some embodiments, C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0901] In different embodiments, R.sup.1 or R.sup.2, or both, has one of the following structures:

##STR00325##

[0902] In some embodiments, R.sub.3 is OH, CN, C(?O)OR.sup.4, OC(?O)R.sup.4 or NHC(?O)R.sup.4. In some embodiments, R.sup.4 is methyl or ethyl.

[0903] In some embodiments, an ionizable lipid is a compound of Formula (1):

##STR00326##

wherein: [0904] each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and [0905] L.sub.1 and L.sub.3 are each independently OC(O)* or C(O)O*, wherein * indicates the attachment point to R.sub.1 or R.sub.3; [0906] R.sub.1 and R.sub.3 are each independently a linear or branched C.sub.9-C.sub.20 alkyl or C.sub.9-C.sub.20 alkenyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl.

[0907] In some embodiments, R.sub.1 and R.sub.3 are the same. In some embodiments, R.sub.1 and R.sub.3 are different.

[0908] In some embodiments, R.sub.1 and R.sub.3 are each independently a branched saturated C.sub.9-C.sub.20 alkyl. In some embodiments, one of R.sub.1 and R.sub.3 is a branched saturated C.sub.9-C.sub.20 alkyl, and the other is an unbranched saturated C.sub.9-C.sub.20 alkyl. In some embodiments, R.sub.1 and R.sub.3 are each independently selected from a group consisting of

##STR00327##

[0909] In various embodiments, R.sub.2 is selected from a group consisting of:

##STR00328## ##STR00329##

[0910] In some embodiments, R.sub.2 may be as described in International Pat. Pub. No. WO2019/152848 A1, which is incorporated herein by reference in its entirety.

[0911] In some embodiments, an ionizable lipid is a compound of Formula (1-1) or Formula (1-2):

##STR00330##

wherein n, R.sub.1, R.sub.2, and R.sub.3 are as defined in Formula (1).

[0912] Preparation methods for the above compounds and compositions are described herein below and/or known in the art.

[0913] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include, e.g., hydroxyl, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxyl include, e.g., trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include, e.g., t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include, e.g., C(O)R (where R is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include, e.g., alkyl, aryl, or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in, e.g., Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin, or a 2-chlorotrityl-chloride resin.

[0914] It will also be appreciated by those skilled in the art, although such protected derivatives of compounds of this invention may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as prodrugs. All prodrugs of compounds of this invention are included within the scope of the invention.

[0915] Furthermore, all compounds of the invention which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds of the invention can also be converted to their free base or acid form by standard techniques.

[0916] The following reaction scheme illustrates an exemplary method to make compounds of Formula (1):

##STR00331##

A1 are purchased or prepared according to methods known in the art. Reaction of A1 with diol A2 under appropriate condensation conditions (e.g., DCC) yields ester/alcohol A3, which can then be oxidized (e.g., with PCC) to aldehyde A4. Reaction of A4 with amine A5 under reductive amination conditions yields a compound of Formula (1).

[0917] The following reaction scheme illustrates a second exemplary method to make compounds of Formula (1), wherein R.sub.1 and R.sub.3 are the same:

##STR00332##

[0918] Modifications to the above reaction scheme, such as using protecting groups, may yield compounds wherein R.sub.1 and R.sub.3 are different. The use of protecting groups, as well as other modification methods, to the above reaction scheme will be readily apparent to one of ordinary skill in the art.

[0919] It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make other compounds of Formula (1) not specifically illustrated herein by using the appropriate starting materials and modifying the parameters of the synthesis. In general, starting materials may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this invention.

[0920] In some embodiments, an ionizable lipid is a compound of Formula (2):

##STR00333##

wherein each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0921] In some embodiments, as used in Formula (2), R.sub.1 and R.sub.2 are as defined in Formula (1).

[0922] In some embodiments, as used in Formula (2), R.sub.1 and R.sub.2 are each independently selected from a group consisting of:

##STR00334## ##STR00335## ##STR00336##

[0923] In some embodiments, R.sub.1 and/or R.sub.2 as used in Formula (2) may be as described in International Pat. Pub. No. WO2015/095340 A1, which is incorporated herein by reference in its entirety. In some embodiments, R.sub.1 as used in Formula (2) may be as described in International Pat. Pub. No. WO2019/152557 A1, which is incorporated herein by reference in its entirety.

[0924] In some embodiments, as used in Formula (2), R.sub.3 is selected from a group consisting of

##STR00337##

[0925] In some embodiments, an ionizable lipid is a compound of Formula (3)

##STR00338##

wherein X is selected from O, S, or OC(O)*, wherein * indicates the attachment point to R.sub.1.

[0926] In some embodiments, an ionizable lipid is a compound of Formula (3-1):

##STR00339##

[0927] In some embodiments, an ionizable lipid is a compound of Formula (3-2):

##STR00340##

[0928] In some embodiments an ionizable lipid is a compound of Formula (3-3):

##STR00341##

[0929] In some embodiments, as used in Formula (3-1), (3-2), or (3-3), each R.sub.1 is independently a branched saturated C.sub.9-C.sub.20 alkyl. In some embodiments, each R.sub.1 is independently selected from a group consisting of:

##STR00342##

[0930] In some embodiments, each R.sub.1 in Formula (3-1), (3-2), or (3-3) are the same.

[0931] In some embodiments, as used in Formula (3-1), (3-2), or (3-3), R.sub.2 is selected from a group consisting of:

##STR00343## ##STR00344##

[0932] In some embodiments, R.sub.2 as used in Formula (3-1), (3-2), or (3-3) may be as described in International Pat. Pub. No. WO2019/152848A1, which is incorporated herein by reference in its entirety.

[0933] In some embodiments, an ionizable lipid is a compound of Formula (5):

##STR00345##

wherein: [0934] each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and [0935] R.sub.2 is as defined in Formula (1).

[0936] In some embodiments, as used in Formula (5), R.sub.4 and R.sub.5 are defined as R.sub.1 and R.sub.3, respectively, in Formula (1). In some embodiments, as used in Formula (5), R.sub.4 and R may be as described in International Pat. Pub. No. WO2019/191780 A1, which is incorporated herein by reference in its entirety.

[0937] In some embodiments, an ionizable lipid of the disclosure is selected from Table 10a. In some embodiments, the ionizable lipid is Lipid 26 in Table 10a. In some embodiments, the ionizable lipid is Lipid 27 in Table 10a. In some embodiments, the ionizable lipid is Lipid 53 in Table 10a. In some embodiments, the ionizable lipid is Lipid 54 in Table 10a.

[0938] In some embodiments, an ionizable lipid of the disclosure is selected from the group consisting of:

##STR00346##

TABLE-US-00011 TABLE 10a Ionizable lipid number Structure 1 [00347] 2 [00348] 3 [00349] 4 [00350] 5 [00351] 6 [00352] 7 [00353] 8 [00354] 9 [00355] 10 [00356] 11 [00357] 12 [00358] 13 [00359] 14 [00360] 15 [00361] 16 [00362] 17 [00363] 18 [00364] 19 [00365] 20 [00366] 21 [00367] 22 [00368] 23 [00369] 24 [00370] 25 [00371] 26 [00372] 27 [00373] 28 [00374] 29 [00375] 30 [00376] 31 [00377] 32 [00378] 33 [00379] 34 [00380] 35 [00381] 36 [00382] 37 [00383] 38 [00384] 39 [00385] 40 [00386] 41 [00387] 42 [00388] 43 [00389] 44 [00390] 45 [00391] 46 [00392] 47 [00393] 48 [00394] 49 [00395] 50 [00396] 51 [00397] 52 [00398] 53 [00399] 54 [00400] 55 [00401] 56 [00402] 57 [00403] 58 [00404] 59 [00405] 60 [00406] 61 [00407] 62 [00408] 63 [00409] 64 [00410] 65 [00411] 66 [00412] 67 [00413] 68 [00414] 69 [00415] 70 [00416] 71 [00417] 72 [00418] 73 [00419] 74 [00420] 75 [00421] 76 [00422] 77 [00423] 78 [00424] 79 [00425] 80 [00426] 81 [00427] 82 [00428] 83 [00429] 84 [00430] 85 [00431] 86 [00432] 87 [00433] 88 [00434] 89 [00435] 90 [00436] 91 [00437] 92 [00438] 93 [00439] 94 [00440] 95 [00441] 96 [00442] 97 [00443] 98 [00444] 99 [00445] 100 [00446] 101 [00447] 102 [00448] 103 [00449] 104 [00450] 105 [00451] 106 [00452] 107 [00453] 108 [00454] 109 [00455] 110 [00456] 111 [00457] 112 [00458] 113 [00459] 114 [00460] 115 [00461] 116 [00462] 117 [00463] 118 [00464] 119 [00465] 120 [00466] 121 [00467] 122 [00468] 123 [00469] 124 [00470] 125 [00471] 126 [00472] 127 [00473] 128 [00474] 129 [00475] 130 [00476] 131 [00477] 132 [00478] 133 [00479] 134 [00480] 135 [00481] 136 [00482]

[0939] In some embodiments, the ionizable lipid has a beta-hydroxyl amine head group. In some embodiments, the ionizable lipid has a gamma-hydroxyl amine head group.

[0940] In some embodiments, an ionizable lipid of the disclosure is a lipid selected from Table 10b. In some embodiments, an ionizable lipid of the disclosure is Lipid 15 from Table 10b. In an embodiment, the ionizable lipid is described in US patent publication number US20170210697A1. In an embodiment, the ionizable lipid is described in US patent publication number US20170119904A1.

TABLE-US-00012 TABLE 10b Ionizable lipid number Structure 1 [00483] 2 [00484] 3 [00485] 4 [00486] 5 [00487] 6 [00488] 7 [00489] 8 [00490] 9 [00491] 10 [00492] 11 [00493] 12 [00494] 13 [00495] 14 [00496] 15 [00497] 16 [00498] 17 [00499] 18 [00500] 19 [00501] 20 [00502] 21 [00503] 22 [00504] 23 [00505] 24 [00506] 25 [00507] 26 [00508] 27 [00509] 28 [00510] 29 [00511] 30 [00512] 31 [00513] 32 [00514] 33 [00515] 34 [00516] 35 [00517] 36 [00518] 37 [00519] 38 [00520]

[0941] In some embodiments, an ionizable lipid has one of the structures set forth in Table 10 below.

TABLE-US-00013 TABLE 10 Number Structure 1 [00521] 2 [00522] 3 [00523] 4 [00524] 5 [00525] 6 [00526] 7 [00527] 8 [00528] 9 [00529] 10 [00530] 11 [00531] 12 [00532] 13 [00533] 14 [00534] 15 [00535] 16 [00536] 17 [00537] 18 [00538] 19 [00539] 20 [00540] 21 [00541] 22 [00542] 23 [00543] 24 [00544] 25 [00545] 26 [00546] 27 [00547] 28 [00548] 29 [00549] 30 [00550] 31 [00551] 32 [00552] 33 [00553] 34 [00554] 35 [00555] 36 [00556] 37 [00557] 38 [00558] 39 [00559] 40 [00560] 41 [00561] 42 [00562] 43 [00563] 44 [00564] 45 [00565] 46 [00566] 47 [00567] 48 [00568] 49 [00569]

[0942] In some embodiments, the ionizable lipid has one of the structures set forth in Table 11 below. In some embodiments, the ionizable lipid as set forth in Table 11 is as described in international patent application PCT/US2010/061058.

TABLE-US-00014 TABLE 11 [00570] [00571] [00572] [00573] [00574] [00575] [00576] [00577] [00578] [00579] [00580] [00581] [00582] [00583] [00584] [00585] [00586] [00587] [00588] [00589] [00590] [00591] [00592] [00593] [00594] [00595] [00596] [00597] [00598] [00599] [00600] [00601] [00602] [00603] [00604] [00605] [00606] [00607] [00608] [00609] [00610] [00611] [00612] [00613] [00614] [00615] [00616] [00617] [00618] [00619] [00620] [00621] [00622] [00623] [00624] [00625] [00626] [00627] [00628] [00629] [00630] [00631] [00632] [00633] [00634] [00635] [00636] [00637] [00638] [00639] [00640] [00641] [00642] [00643] [00644] [00645] [00646] [00647] [00648] [00649] [00650] [00651] [00652] [00653] [00654] [00655] [00656] [00657] [00658] [00659] [00660] [00661] [00662] [00663] [00664] [00665] [00666] [00667] [00668] [00669] [00670] [00671] [00672] [00673] [00674] [00675] [00676] [00677] [00678] [00679] [00680] [00681] [00682] [00683] [00684] [00685] [00686] [00687] [00688] [00689] [00690] [00691] [00692] [00693] [00694] [00695] [00696] [00697] [00698] [00699] [00700] [00701] [00702] [00703] [00704] [00705] [00706] [00707] [00708] [00709] [00710] [00711] [00712] [00713] [00714] [00715] [00716] [00717] [00718] [00719] [00720] [00721] [00722] [00723] [00724] [00725] [00726] [00727] [00728] [00729] [00730] [00731] [00732] [00733] [00734] [00735] [00736] [00737] [00738] [00739] [00740] [00741] [00742] [00743] [00744] [00745] [00746] [00747] [00748] [00749] [00750] [00751] [00752] [00753] [00754] [00755] [00756] [00757] [00758] [00759] [00760] [00761] [00762] [00763] [00764] [00765] [00766] [00767] [00768] [00769] [00770] [00771] [00772] [00773] [00774] [00775] [00776] [00777] [00778] [00779] [00780] [00781] [00782] [00783] [00784] [00785] [00786] [00787] [00788] [00789] [00790] [00791] [00792] [00793] [00794] [00795] [00796] [00797] [00798] [00799] [00800] [00801] [00802] [00803] [00804] [00805] [00806] [00807] [00808] [00809] [00810] [00811] [00812] [00813] [00814] [00815] [00816] [00817] [00818] [00819] [00820] [00821] [00822] [00823] [00824] [00825] [00826] [00827] [00828] [00829] [00830] [00831] [00832] [00833] [00834] [00835] [00836] [00837] [00838] [00839] [00840] [00841] [00842] [00843] [00844] [00845] [00846] [00847] [00848] [00849] [00850] [00851] [00852] [00853] [00854] [00855] [00856] [00857] [00858] [00859] [00860] [00861] [00862] [00863] [00864] [00865] [00866] [00867] [00868] [00869] [00870] [00871] [00872] [00873] [00874] [00875] [00876] [00877] [00878] [00879] [00880] [00881] [00882] [00883] [00884] [00885] [00886] [00887] [00888] [00889] [00890] [00891] [00892] [00893] [00894] [00895] [00896] [00897] [00898] [00899] [00900] [00901] [00902] [00903] [00904] [00905] [00906] [00907] [00908] [00909] [00910] [00911] [00912] [00913] [00914] [00915] [00916] [00917] [00918] [00919] [00920] [00921] [00922] [00923] [00924] [00925] [00926] [00927] [00928] [00929] [00930] [00931] [00932] [00933] [00934] [00935] [00936] [00937] [00938] [00939] [00940] [00941] [00942] [00943] [00944] [00945] [00946] [00947] [00948] [00949] [00950] [00951] [00952] [00953] [00954] [00955] [00956] [00957] [00958] [00959] [00960] [00961] [00962] [00963] [00964] [00965] [00966] [00967] [00968] [00969] [00970] [00971] [00972] [00973] [00974] [00975] [00976] [00977] [00978] [00979] [00980] [00981] [00982] [00983] [00984] [00985] [00986] [00987] [00988] [00989] [00990] [00991] [00992] [00993] [00994] [00995] [00996] [00997] [00998] [00999] [01000] [01001] [01002] [01003] [01004] [01005] [01006] [01007] [01008] [01009] [01010] [01011] [01012] [01013] [01014] [01015] [01016] [01017] [01018] [01019] [01020] [01021] [01022] [01023] [01024] [01025] [01026] [01027] [01028] [01029] [01030] [01031] [01032] [01033] [01034] [01035] [01036] [01037] [01038] [01039] [01040] [01041] [01042] [01043] [01044] [01045] [01046] [01047] [01048] [01049] [01050] [01051] [01052] [01053] [01054] [01055] [01056] [01057] [01058] [01059] [01060] [01061] [01062] [01063] [01064] [01065] [01066] [01067] [01068] [01069] [01070] [01071] [01072] [01073] [01074] [01075] [01076] [01077] [01078] [01079] [01080] [01081] [01082] [01083] [01084] [01085] [01086] [01087] [01088] [01089] [01090] [01091] [01092] [01093] [01094] [01095] [01096] [01097] [01098] [01099] [01100] [01101] [01102] [01103] [01104] [01105] [01106] [01107] [01108] [01109] [01110] [01111]

[0943] In some embodiments, the transfer vehicle comprises Lipid A, Lipid B, Lipid C, and/or Lipid D. In some embodiments, inclusion of Lipid A, Lipid B, Lipid C, and/or Lipid D improves encapsulation and/or endosomal escape. In some embodiments, Lipid A, Lipid B, Lipid C, and/or Lipid D are described in international patent application PCT/US2017/028981.

[0944] In some embodiments, an ionizable lipid is Lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca9,12-dienoate, also called 3-((4,44bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:

##STR01112##

[0945] Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86), incorporated by reference in its entirety.

[0946] In some embodiments, an ionizable lipid is Lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate). Lipid B can be depicted as:

##STR01113##

[0947] Lipid B may be synthesized according to WO2014/136086 (e.g., pp. 107-09), incorporated by reference in its entirety.

[0948] In some embodiments, an ionizable lipid is Lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9Z,12Z,12Z)-bis(octadeca-9,12-dienoate). Lipid C can be depicted as:

##STR01114##

[0949] In some embodiments, an ionizable lipid is Lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octylundecanoate. Lipid D can be depicted as:

##STR01115##

[0950] Lipid C and Lipid D may be synthesized according to WO2015/095340, incorporated by reference in its entirety.

[0951] In some embodiments, an ionizable lipid is described in US patent publication number 20190321489. In some embodiments, an ionizable lipid is described in international patent publication WO 2010/053572, incorporated herein by reference. In some embodiments, an ionizable lipid is C12-200, described at paragraph [00225] of WO 2010/053572.

[0952] Several ionizable lipids have been described in the literature, many of which are commercially available. In certain embodiments, such ionizable lipids are included in the transfer vehicles described herein. In some embodiments, the ionizable lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or DOTMA is used. (Felgner et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone or can be combined with a neutral lipid, dioleoylphosphatidylethanolamine or DOPE or other cationic or non-cationic lipids into a lipid nanoparticle. Other suitable cationic lipids include, for example, ionizable cationic lipids as described in U.S. provisional patent application 61/617,468, filed Mar. 29, 2012 (incorporated herein by reference), such as, e.g., (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), C12-200 (described in WO 2010/053572), 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLinKC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), 2-(2,2-di((9Z,2Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA), (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), (15Z,18 Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002), 5-carboxyspermylglycine-dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane (DODAP), 1,2-Dioleoyl-3-Trimethylammonium-Propane or (DOTAP). Contemplated ionizable lipids also include 1,2-distcaryloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5-(cholest-5-en-3-beta-oxy)-3-oxapentoxy)-3-dimethyl-1-(cis,cis-9,1-2-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N-Dilinoleylcarbamyl-3-dimethylamninopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA) or GL67, or mixtures thereof (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). The use of cholesterol-based ionizable lipids to formulate the transfer vehicles (e.g., lipid nanoparticles) is also contemplated by the present invention. Such cholesterol-based ionizable lipids can be used, either alone or in combination with other lipids. Suitable cholesterol-based ionizable lipids include, for example, DC-Cholesterol (N,N-dimethyl-N-ethylcarboxamidocholesterol), and 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al., Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335).

[0953] Also contemplated are cationic lipids such as dialkylamino-based, imidazole-based, and guanidinium-based lipids. For example, also contemplated is the use of the ionizable lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate (ICE), as disclosed in International Application No. PCT/US2010/058457, incorporated herein by reference.

[0954] Also contemplated are ionizable lipids such as the dialkylamino-based, imidazole-based, and guanidinium-based lipids. For example, certain embodiments are directed to a composition comprising one or more imidazole-based ionizable lipids, for example, the imidazole cholesterol ester or ICE lipid, (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (XIII) below. In an embodiment, a transfer vehicle for delivery of circRNA may comprise one or more imidazole-based ionizable lipids, for example, the imidazole cholesterol ester or ICE lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (XIII).

##STR01116##

[0955] Without wishing to be bound by a particular theory, it is believed that the fusogenicity of the imidazole-based cationic lipid ICE is related to the endosomal disruption which is facilitated by the imidazole group, which has a lower pKa relative to traditional ionizable lipids. The endosomal disruption in turn promotes osmotic swelling and the disruption of the liposomal membrane, followed by the transfection or intracellular release of the nucleic acid(s) contents loaded therein into the target cell.

[0956] The imidazole-based ionizable lipids are also characterized by their reduced toxicity relative to other ionizable lipids.

[0957] In some embodiments, an ionizable lipid is described by US patent publication number 20190314284. In certain embodiments, the an ionizable lipid is described by structure 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., HGT4001, HGT4002, HGT4003, HGT4004 and/or HGT4005). In certain embodiments, the one or more cleavable functional groups (e.g., a disulfide) allow, for example, a hydrophilic functional head-group to dissociate from a lipophilic functional tail-group of the compound (e.g., upon exposure to oxidative, reducing or acidic conditions), thereby facilitating a phase transition in the lipid bilayer of the one or more target cells. For example, when a transfer vehicle (e.g., a lipid nanoparticle) comprises one or more of the lipids of structures 3-10, the phase transition in the lipid bilayer of the one or more target cells facilitates the delivery of the circRNA into the one or more target cells.

[0958] In certain embodiments, the ionizable lipid is described by structure (XIV),

##STR01117##

wherein: [0959] R.sub.1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino (e.g., an alkyl amino such as dimethylamino) and pyridyl; [0960] R.sub.2 is selected from the group consisting of structure XV and structure XVI;

##STR01118##

##STR01119##

wherein R.sub.3 and R.sub.4 are each independently selected from the group consisting of an optionally substituted, variably saturated or unsaturated C.sub.6-C.sub.20 alkyl and an optionally substituted, variably saturated or unsaturated C.sub.6-C.sub.20 acyl; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more). In certain embodiments, R.sub.3 and R.sub.4 are each an optionally substituted, polyunsaturated Cis alkyl, while in other embodiments R.sub.3 and R.sub.4 are each an unsubstituted, polyunsaturated C.sub.18 alkyl. In certain embodiments, one or more of R.sub.3 and R.sub.4 are (9Z,12Z)-octadeca-9,12-dien.

[0961] Also disclosed herein are pharmaceutical compositions that comprise the compound of structure XIV, wherein R.sub.1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino (e.g., an alkyl amino such as dimethylamino) and pyridyl; wherein R.sub.2 is structure XV; and wherein n is zero or any positive integer. Further disclosed herein are pharmaceutical compositions comprising the compound of structure XIV, wherein R.sub.1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino (e.g., an alkyl amino such as dimethylamino) and pyridyl; wherein R.sub.2 is structure XVI; wherein R.sub.3 and R.sub.4 are each independently selected from the group consisting of an optionally substituted, variably saturated or unsaturated C.sub.6-C.sub.20 alkyl and an optionally substituted, variably saturated or unsaturated C.sub.6-C.sub.20 acyl; and wherein n is zero or any positive integer. In certain embodiments. R.sub.3 and R.sub.4 are each an optionally substituted, polyunsaturated C.sub.18 alkyl, while in other embodiments R.sub.3 and R.sub.4 are each an unsubstituted, polyunsaturated C.sub.18 alkyl (e.g., octadeca-9,12-dien).

[0962] In certain embodiments, the R.sub.1 group or head-group is a polar or hydrophilic group (e.g., one or more of the imidazole, guanidinium and amino groups) and is bound to the R.sub.2 lipid group by way of the disulfide (SS) cleavable linker group, for example as depicted in structure XIV. Other contemplated cleavable linker groups may include compositions that comprise one or more disulfide (SS) linker group bound (e.g., covalently bound) to, for example an alkyl group (e.g., C.sub.1 to C.sub.10 alkyl). In certain embodiments, the R1 group is covalently bound to the cleavable linker group by way of a C.sub.1-C.sub.20 alkyl group (e.g., where n is one to twenty), or alternatively may be directly bound to the cleavable linker group (e.g., where n is zero). In certain embodiments, the disulfide linker group is cleavable in vitro and/or in vivo (e.g., enzymatically cleavable or cleavable upon exposure to acidic or reducing conditions).

[0963] In certain embodiments, the inventions relate to the compound 5-(((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-TH-cyclopenta[a]phenanthren-3-yl)disulfanyl)methyl)-1H-imidazole, having structure XVII (referred to herein as HGT4001).

##STR01120##

[0964] In certain embodiments, the inventions relate to the compound 1-(2-(((3S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12, 13, 14, 15, 16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)ethyl)guanidine, having structure XVIII (referred to herein as HGT4002).

##STR01121##

[0965] In certain embodiments, the inventions relate to the compound 2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine, having structure XIX (referred to herein as HGT4003).

##STR01122##

[0966] In other embodiments, the inventions relate to the compound 5-(((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)methyl)-1H-imidazole having the structure of structure XX (referred to herein as HGT4004).

##STR01123##

[0967] In still other embodiments, the inventions relate to the compound 1-(((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)methyl)guanidine having structure XXI (referred to herein as HGT4005).

##STR01124##

[0968] In certain embodiments, the compounds described as structures 3-10 are ionizable lipids.

[0969] The compounds, and in particular the imidazole-based compounds described as structures 3-8 (e.g., HGT4001 and HGT4004), are characterized by their reduced toxicity, in particular relative to traditional ionizable lipids. In some embodiments, the transfer vehicles described herein comprise one or more imidazole-based ionizable lipid compounds such that the relative concentration of other more toxic ionizable lipids in such pharmaceutical or liposomal composition may be reduced or otherwise eliminated.

[0970] The ionizable lipids include those disclosed in international patent application PCT/US2019/025246, and US patent publications 2017/0190661 and 2017/0114010, incorporated herein by reference in their entirety. The ionizable lipids may include a lipid selected from the following tables 12, 13, 14, or 15.

TABLE-US-00015 TABLE 12 [01125] ATX-001 [01126] ATX-002 [01127] ATX-003 [01128] ATX-004 [01129] ATX-005 [01130] ATX-006 [01131] ATX-007 [01132] ATX-008 [01133] ATX-009 [01134] ATX-010 [01135] ATX-011 [01136] ATX-012 [01137] ATX-013 [01138] ATX-014 [01139] ATX-015 [01140] ATX-016 [01141] ATX-017 [01142] ATX-018 [01143] ATX-019 [01144] ATX-020 [01145] ATX-021 [01146] ATX-022 [01147] ATX-023 [01148] ATX-024 [01149] ATX-025 [01150] ATX-026 [01151] ATX-027 [01152] ATX-028 [01153] ATX-029 [01154] ATX-030 [01155] ATX-031 [01156] ATX-032

TABLE-US-00016 TABLE 13 [01157] ATX-B-1 [01158] ATX-B-2 [01159] ATX-B-3 [01160] ATX-B-4 [01161] ATX-B-5 [01162] ATX-B-6 [01163] ATX-B-7 [01164] ATX-B-8 [01165] ATX-B-9 [01166] ATX-B-10 [01167] ATX-B-11 [01168] ATX-B-12

TABLE-US-00017 TABLE 14 Compound ATX-# [01169] 0063 [01170] 0130 [01171] 0131 [01172] 0044 [01173] 0111 [01174] 0132 [01175] 0134 [01176] 0133 [01177] 0064 [01178] 0061 [01179] 0100 [01180] 0117 [01181] 0114 [01182] 0115 [01183] 0101 [01184] 0106 [01185] 0116 [01186] 0043 [01187] 0086 [01188] 0058 [01189] 0081 [01190] 0123 [01191] 0122 [01192] 0057 [01193] 0088 [01194] 0087 [01195] 0124 [01196] 0128 [01197] 0127 [01198] 0126 [01199] 0129 [01200] 0082 [01201] 0085 [01202] 0083 [01203] 0121 [01204] 0091 [01205] 0102 [01206] 0098 [01207] 0092 [01208] 0084 [01209] 0095 [01210] 0125 [01211] 0094 [01212] 0109 [01213] 0110 [01214] 0118 [01215] 0108 [01216] 0107 [01217] 0093 [01218] 0097 [01219] 0096

TABLE-US-00018 TABLE 15 [01220] 11 [01221] 13

[0971] In some embodiments, an ionizable lipid is as described in international patent application PCT/US2019/015913. In some embodiments, an ionizable lipid is chosen from the following:

##STR01222## ##STR01223## ##STR01224## ##STR01225## ##STR01226## ##STR01227## ##STR01228## ##STR01229## ##STR01230## ##STR01231## ##STR01232## ##STR01233## ##STR01234## ##STR01235## ##STR01236## ##STR01237## ##STR01238## ##STR01239## ##STR01240## ##STR01241##

##STR01242## ##STR01243## ##STR01244## ##STR01245## ##STR01246## ##STR01247## ##STR01248## ##STR01249## ##STR01250## ##STR01251## ##STR01252## ##STR01253## ##STR01254## ##STR01255## ##STR01256## ##STR01257## ##STR01258## ##STR01259##

##STR01260## ##STR01261## ##STR01262## ##STR01263## ##STR01264## ##STR01265## ##STR01266## ##STR01267## ##STR01268## ##STR01269## ##STR01270## ##STR01271## ##STR01272## ##STR01273## ##STR01274## ##STR01275## ##STR01276## ##STR01277## ##STR01278## ##STR01279## ##STR01280## ##STR01281##

Amine Lipids

[0972] In certain embodiments, transfer vehicle compositions for the delivery of circular RNA comprise an amine lipid. In certain embodiments, an ionizable lipid is an amine lipid. In some embodiments, an amine lipid is described in international patent application PCT/US2018/053569.

[0973] In some embodiments, the amine lipid is Lipid E, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate.

[0974] Lipid E can be depicted as:

##STR01282##

[0975] Lipid E may be synthesized according to WO2015/095340 (e.g., pp. 84-86). In certain embodiments, the amine lipid is an equivalent to Lipid E.

[0976] In certain embodiments, an amine lipid is an analog of Lipid E. In certain embodiments, a Lipid E analog is an acetal analog of Lipid E. In particular transfer vehicle compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In additional embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is chosen from a C4, C5, C6, C7, C9, C10, C11 and C12 acetal analog.

[0977] Amine lipids and other biodegradable lipids suitable for use in the transfer vehicles, e.g., lipid nanoparticles, described herein are biodegradable in vivo. The amine lipids described herein have low toxicity (e.g., are tolerated in animal models without adverse effect in amounts of greater than or equal to 10 mg/kg). In certain embodiments, transfer vehicles composing an amine lipid include those where at least 75% of the amine lipid is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days.

[0978] Biodegradable lipids include, for example, the biodegradable lipids of WO2017/173054, WO2015/095340, and WO2014/136086.

[0979] Lipid clearance may be measured by methods known by persons of skill in the art. See, for example, Maier, M. A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78.

[0980] Transfer vehicle compositions comprising an amine lipid can lead to an increased clearance rate. In some embodiments, the clearance rate is a lipid clearance rate, for example the rate at which a lipid is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, for example the rate at which an circRNA is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which transfer vehicles are cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which transfer vehicles are cleared from a tissue, such as liver tissue or spleen tissue. In certain embodiments, a high rate of clearance leads to a safety profile with no substantial adverse effects. The amine lipids and biodegradable lipids may reduce transfer vehicle accumulation in circulation and in tissues. In some embodiments, a reduction in transfer vehicle accumulation in circulation and in tissues leads to a safety profile with no substantial adverse effects.

[0981] Lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipid, such as an amine lipid, may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood, where pH is approximately 7.35, the lipid, such as an amine lipid, may not be protonated and thus bear no charge.

[0982] The ability of a lipid to bear a charge is related to its intrinsic pKa. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. For example, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.5. Lipids with a pKa ranging from about 5.1 to about 7.4 are effective for delivery of cargo in vivo, e.g., to the liver. Further, it has been found that lipids with a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g., into tumors. See, e.g., WO2014/136086.

Lipids Containing a Disulfide Bond

[0983] In some embodiments, the ionizable lipid is described in U.S. Pat. No. 9,708,628.

[0984] The present invention provides a lipid represented by structure (XXII):

##STR01283##

[0985] In structure (XXII), X.sup.a and X.sup.b are each independently X.sup.1 or X.sup.2 shown below.

##STR01284##

[0986] R.sup.4 in X.sup.1 is an alkyl group having 1-6 carbon atoms, which may be linear, branched or cyclic. The alkyl group preferably has a carbon number of 1-3. Specific examples of the alkyl group having 1-6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, t-pentyl group, 1,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, cyclohexyl group and the like. R.sup.4 is preferably a methyl group, an ethyl group, a propyl group or an isopropyl group, most preferably a methyl group.

[0987] The s in X.sup.2 is 1 or 2. When s is 1, X.sup.2 is a pyrrolidinium group, and when s is 2, X.sup.2 is a piperidinium group. s is preferably 2. While the binding direction of X.sup.2 is not limited, a nitrogen atom in X.sup.2 preferably binds to R.sup.1a and R.sup.1b.

[0988] X.sup.a may be the same as or different from X.sup.b, and X.sup.a is preferably the same group as X.sup.b.

[0989] n.sup.a and n.sup.b are each independently 0 or 1, preferably 1. When n.sup.a is 1, R.sup.3a binds to X.sup.a via Y.sup.a and R.sup.2a, and when n.sup.a is 0, a structure of R.sup.3aX.sup.aR.sup.1aS is taken. Similarly, when n.sup.b is 1, R.sup.3b binds to X.sup.b via Y.sup.b and R.sup.2b, and when n.sup.b is 0, a structure of R.sup.3bX.sup.bR.sup.1bS is taken.

[0990] n.sup.a may be the same as or different from n.sup.b, and n.sup.a is preferably the same as n.sup.b.

[0991] R.sup.1a and R.sup.1b are each independently an alkylene group having 1-6 carbon atoms, which may be linear or branched, preferably linear. Specific examples of the alkylene group having 1-6 carbon atoms include methylene group, ethylene group, trimethylene group, isopropylene group, tetramethylene group, isobutylene group, pentamethylene group, neopentylene group and the like. R.sup.1a and R.sup.1b are each preferably a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, most preferably an ethylene group.

[0992] R.sup.1a may be the same as or different from R.sup.1b, and R.sup.1a is preferably the same group as R.sup.1b.

[0993] R.sup.2a and R.sup.2b are each independently an alkylene group having 1-6 carbon atoms, which may be linear or branched, preferably linear. Examples of the alkylene group having 1-6 carbon atoms include those recited as the examples of the alkylene group having 1-6 carbon atoms for R.sup.1a or R.sup.1b. R.sup.2a and R.sup.2b are each preferably a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group.

[0994] When X.sup.a and X.sup.b are each X.sup.1, R.sup.2a and R.sup.2b are preferably trimethylene groups. When X.sup.a and X.sup.b are each X.sup.2, R.sup.2a and R.sup.2b are preferably ethylene groups.

[0995] R.sup.2a may be the same as or different from R.sup.2b, and R.sup.2a is preferably the same group as R.sup.2b.

[0996] Y.sup.a and Y.sup.b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably an ester bond, an amide bond or a carbamate bond, most preferably an ester bond. While the binding direction of Y.sup.a and Y.sup.b is not limited, when Y.sup.a is an ester bond, a structure of R.sup.3aCOOR.sup.2a is preferable, and when Y.sup.b is an ester bond, a structure of R.sup.3bCOOR.sup.2b is preferable.

[0997] Y.sup.a may be the same as or different from Y.sup.b, and Y.sup.a is preferably the same group as Y.sup.b.

[0998] R.sup.3a and R.sup.3b are each independently a sterol residue, a liposoluble vitamin residue or an aliphatic hydrocarbon group having 12-22 carbon atoms, preferably a liposoluble vitamin residue or an aliphatic hydrocarbon group having 12-22 carbon atoms, most preferably a liposoluble vitamin residue.

[0999] Examples of the sterol residue include a cholesteryl group (cholesterol residue), a cholestaryl group (cholestanol residue), a stigmasteryl group (stigmasterol residue), a ?-sitosteryl group (?-sitosterol residue), a lanosteryl group (lanosterol residue), and an ergosteryl group (ergosterol residue) and the like. The sterol residue is preferably a cholesteryl group or a cholestaryl group.

[1000] As the liposoluble vitamin residue, a residue derived from liposoluble vitamin, as well as a residue derived from a derivative obtained by appropriately converting a hydroxyl group, aldehyde or carboxylic acid, which is a functional group in liposoluble vitamin, to other reactive functional group can be used. As for liposoluble vitamin having a hydroxyl group, for example, the hydroxyl group can be converted to a carboxylic acid by reacting with succinic acid anhydride, glutaric acid anhydride and the like. Examples of the liposoluble vitamin include retinoic acid, retinol, retinal, ergosterol, 7-dehydrocholesterol, calciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol and the like. Preferable examples of the liposoluble vitamin include retinoic acid and tocopherol.

[1001] The aliphatic hydrocarbon group having 12-22 carbon atoms may be linear or branched, preferably linear. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the aliphatic hydrocarbon group generally contains 1-6, preferably 1-3, more preferably 1-2 unsaturated bonds. While the unsaturated bond includes a carbon-carbon double bond and a carbon-carbon triple bond, it is preferably a carbon-carbon double bond. The aliphatic hydrocarbon group has a carbon number of preferably 12-18, most preferably 13-17. While the aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group and the like, it is preferably an alkyl group or an alkenyl group. Specific examples of the aliphatic hydrocarbon group having 12-22 carbon atoms include dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group, docosyl group, dodecenyl group, tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosenyl group, docosenyl group, decadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group and the like. The aliphatic hydrocarbon group having 12-22 carbon atoms is preferably tridecyl group, tetradecyl group, heptadecyl group, octadecyl group, heptadecadienyl group or octadecadienyl group, particularly preferably tridecyl group, heptadecyl group or heptadecadienyl group.

[1002] In one embodiment, an aliphatic hydrocarbon group having 12-22 carbon atoms, which is derived from fatty acid, aliphatic alcohol, or aliphatic amine is used. When R.sup.3a (or R.sup.3b) is derived from fatty acid, Y.sup.a (or Y.sup.b) is an ester bond or an amide bond, and fatty acid-derived carbonyl carbon is included in Y.sup.a (or Y.sup.b). For example, when linoleic acid is used, R.sup.3a (or R.sup.3b) is a heptadecadienyl group.

[1003] R.sup.3a may be the same as or different from R.sup.3b, and R.sup.3a is preferably the same group as R.sup.3b.

[1004] In one embodiment, X.sup.a is the same as X.sup.b, n.sup.a is the same as n.sup.b, R.sup.1a is the same as R.sup.1b, R.sup.2a is the same as R.sup.2b, R.sup.3a is the same as R.sup.3b, and Y.sup.a is the same as Y.sup.b.

[1005] In one embodiment, [1006] X.sup.a and X.sup.b are each independently X1, [1007] R.sup.4 is an alkyl group having 1-3 carbon atoms, n.sup.a and n.sup.b are each 1, [1008] R.sup.1a and R.sup.1b are each independently an alkylene group having 1-6 carbon atoms, [1009] R.sup.2a and R.sup.2b are each independently an alkylene group having 1-6 carbon atoms, [1010] Y.sup.a and Y.sup.b are each an ester bond or an amide bond, and [1011] R.sup.3a and R.sup.3b are each independently an aliphatic hydrocarbon group having 12-22 carbon atoms.

[1012] In one embodiment, [1013] X.sup.a and X.sup.b are each X1, [1014] R.sup.4 is an alkyl group having 1-3 carbon atoms, n.sup.a and n.sup.b are each 1, [1015] R.sup.1a and R.sup.1b are each an alkylene group having 1-6 carbon atoms, [1016] R.sup.2a and R.sup.2b are each an alkylene group having 1-6 carbon atoms, [1017] Y.sup.a and Y.sup.b are each an ester bond or an amide bond, [1018] R.sup.3a and R.sup.3b are each an aliphatic hydrocarbon group having 12-22 carbon atoms, [1019] X.sup.a is the same as X.sup.b, [1020] R.sup.1a is the same as R.sup.1b, [1021] R.sup.2a is the same as R.sup.2b, and [1022] R.sup.3a is the same as R.sup.3b.

[1023] In one embodiment, [1024] X.sup.a and X.sup.b are each X.sup.1, [1025] R.sup.4 is a methyl group, n.sup.a and n.sup.b are each 1, [1026] R.sup.1a and R.sup.1b are each an ethylene group, [1027] R.sup.2a and R.sup.2b are each a trimethylene group, [1028] Y.sup.a and Y.sup.b are each COO, and [1029] R.sup.3a and R.sup.3b are each independently an alkyl group or alkenyl group having 13-17 carbon atoms.

[1030] In one embodiment, [1031] X.sup.a and X.sup.b are each X.sup.1, [1032] R.sup.4 is a methyl group, n.sup.a and n.sup.b are each 1, [1033] R.sup.1a and R.sup.1b are each an ethylene group, [1034] R.sup.2a and R.sup.2b are each a trimethylene group, [1035] Y.sup.a and Y.sup.b are each COO, [1036] R.sup.3a and R.sup.3b are each an alkyl group or alkenyl group having 13-17 carbon atoms, and [1037] R.sup.3a is the same as R.sup.3b.

[1038] In one embodiment, [1039] X.sup.a and X.sup.b are each independently X.sup.1, [1040] R.sup.4 is an alkyl group having 1-3 carbon atoms, n.sup.a and n.sup.b are each 1, [1041] R.sup.1a and R.sup.1b are each independently an alkylene group having 1-6 carbon atoms, [1042] R.sup.2a and R.sup.2b are each independently an alkylene group having 1-6 carbon atoms, [1043] Y.sup.a and Y.sup.b are each an ester bond or an amide bond, and [1044] R.sup.3a and R.sup.3b are each independently a liposoluble vitamin residue (e.g., retinoic acid residue, tocopherol residue).

[1045] In one embodiment, [1046] X.sup.a and X.sup.b are each X.sup.1, [1047] R.sup.4 is an alkyl group having 1-3 carbon atoms, n.sup.a and n.sup.b are each 1, [1048] R.sup.1a and R.sup.1b are each an alkylene group having 1-6 carbon atoms, [1049] R.sup.2a and R.sup.2b are each an alkylene group having 1-6 carbon atoms, [1050] Y.sup.a and Y.sup.b are each an ester bond or an amide bond, [1051] R.sup.3a and R.sup.3b are each a liposoluble vitamin residue (e.g., retinoic acid residue, tocopherol residue), [1052] X.sup.a is the same as X.sup.b, [1053] R.sup.1a is the same as R.sup.1b, [1054] R.sup.2a is the same as R.sup.2b, and [1055] R.sup.3a is the same as R.sup.3b.

[1056] In one embodiment, [1057] X.sup.a and X.sup.b are each X.sup.1, [1058] R.sup.4 is a methyl group, n.sup.a and n.sup.b are each 1, [1059] R.sup.1a and R.sup.1b are each an ethylene group, [1060] R.sup.2a and R.sup.2b are each a trimethylene group, [1061] Y.sup.a and Y.sup.b are each COO, and [1062] R.sup.3a and R.sup.3b are each independently a liposoluble vitamin residue (e.g., retinoic acid residue, tocopherol residue).

[1063] In one embodiment, [1064] X.sup.a and X.sup.b are each X.sup.1, [1065] R.sup.4 is a methyl group, n.sup.a and n.sup.b are each 1, [1066] R.sup.1a and R.sup.1b are each an ethylene group, [1067] R.sup.2a and R.sup.2b are each a trimethylene group, [1068] Y.sup.a and Y.sup.b are each COO, [1069] R.sup.3a and R.sup.3b are each a liposoluble vitamin residue (e.g., retinoic acid residue, tocopherol residue), and [1070] R.sup.3a is the same as R.sup.3b.

[1071] In one embodiment, [1072] X.sup.a and X.sup.b are each independently X.sup.2, [1073] t is 2, [1074] R.sup.1a and R.sup.1b are each independently an alkylene group having 1-6 carbon atoms, [1075] R.sup.2a and R.sup.2b are each independently an alkylene group having 1-6 carbon atoms, [1076] Y.sup.a and Y.sup.b are each an ester bond, and [1077] R.sup.3a and R.sup.3b are each independently a liposoluble vitamin residue (e.g., retinoic acid residue, tocopherol residue) or an aliphatic hydrocarbon group having 12-22 carbon atoms (e.g., alkyl group having 12-22 carbon atoms).

[1078] In one embodiment, [1079] X.sup.a and X.sup.b are each independently X.sup.2, [1080] t is 2, [1081] R.sup.1a and R.sup.1b are each independently an alkylene group having 1-6 carbon atoms, [1082] R.sup.2a and R.sup.2b are each independently an alkylene group having 1-6 carbon atoms, [1083] Y.sup.a and Y.sup.b are each an ester bond, [1084] R.sup.3a and R.sup.3b are each independently a liposoluble vitamin residue (e.g., retinoic acid residue, tocopherol residue) or an aliphatic hydrocarbon group having 12-22 carbon atoms (e.g., alkyl group having 12-22 carbon atoms), [1085] X.sup.a is the same as X.sup.b, [1086] R.sup.1a is the same as Rib, [1087] R.sup.2a is the same as R.sup.2b, and [1088] R.sup.3a is the same as R.sup.3b.

[1089] In one embodiment, [1090] X.sup.a and X.sup.b are each independently X.sup.2, [1091] t is 2, [1092] R.sup.1a and R.sup.1b are each an ethylene group, [1093] R.sup.2a and R.sup.2b are each independently an alkylene group having 1-6 carbon atoms, [1094] Y.sup.a and Y.sup.b are each an ester bond, [1095] R.sup.3a and R.sup.3b are each independently a liposoluble vitamin residue (e.g., retinoic acid residue, tocopherol residue) or an aliphatic hydrocarbon group having 12-22 carbon atoms (e.g., alkyl group having 12-22 carbon atoms), [1096] X.sup.a is the same as X.sup.b, [1097] R.sup.2a is the same as R.sup.2b, and [1098] R.sup.3a is the same as R.sup.3b.

[1099] In some embodiments, an ionizable lipid has one of the structures set forth in Table 15b below.

TABLE-US-00019 TABLE 15b Number Structure 1 [01285] 2 [01286] 3 [01287] 4 [01288] 5 [01289] 6 [01290] 7 [01291] 8 [01292] 9 [01293] 10 [01294] 11 [01295] 12 [01296] 13 [01297] 14 [01298] 15 [01299]

[1100] A lipid of the present invention may have an SS (disulfide) bond. The production method for such a compound includes, for example, a method including producing

R.sup.3a(Y.sup.aR.sup.2a)n.sup.a-X.sup.aR.sup.1aSH, and

R.sup.3b(Y.sup.bR.sup.2b)n.sup.b-X.sup.bR.sup.1bSH, and

subjecting them to oxidation (coupling) to give a compound containing SS, a method including sequentially bonding necessary parts to a compound containing an SS bond to finally obtain the compound of the present invention and the like. Preferred is the latter method.

[1101] A specific example of the latter method is shown below, which is not to be construed as limiting.

[1102] Examples of the starting compound include SS bond-containing two terminal carboxylic acid, two terminal carboxylate, two terminal amine, two terminal isocyanate, two terminal alcohol, two terminal alcohol having a leaving group such as MsO (mesylate group) and the like, a two terminal carbonate having a leaving group such as pNP (p-nitrophenylcarbonate group) and the like.

[1103] For example, when a compound containing X.sup.1 or X.sup.2 for X.sup.a and X.sup.b is produced, two terminal functional groups of compound (1) containing an SS bond are reacted with an NH group in compound (2) having the NH group and one functional group at the terminal, the functional group at the terminal in compound (2) which did not contribute to the reaction is reacted with a functional group in compound (3) containing R.sup.3, whereby the compound of the present invention containing an SS bond, R.sup.1a and R.sup.1b, X.sup.a and X.sup.b, R.sup.2a and R.sup.2b, Y.sup.a and Y.sup.b, and R.sup.3a and R.sup.3b can be obtained.

[1104] In the reaction of compound (1) and compound (2), an alkali catalyst such as potassium carbonate, sodium carbonate, potassium t-butoxide and the like may be used as a catalyst, or the reaction may be performed without a catalyst. Preferably, potassium carbonate or sodium carbonate is used as a catalyst.

[1105] The amount of catalyst is 0.1-100 molar equivalents, preferably, 0.1-20 molar equivalents, more preferably 0.1-5 molar equivalents, relative to compound (1). The amount of compound (2) to be charged is 1-50 molar equivalents, preferably 1-10 molar equivalents, relative to compound (1).

[1106] The solvent to be used for the reaction of compound (1) and compound (2) is not particularly limited as long as it is a solvent or aqueous solution that does not inhibit the reaction. For example, ethyl acetate, dichloromethane, chloroform, benzene, toluene and the like can be mentioned. Among these, toluene and chloroform are preferable.

[1107] The reaction temperature is ?20 to 200? C., preferably 0 to 80? C., more preferably 20 to 50? C., and the reaction time is 1-48 hr, preferably 2-24 hr.

[1108] When the reaction product of compound (1) and compound (2) is reacted with compound (3), an alkali catalyst such as potassium carbonate, sodium carbonate, potassium t-butoxide and the like, or an acid catalyst such as PTS (p-toluenesulfonic acid), MSA (methanesulfonic acid) and the like may be used, like the catalyst used for the reaction of compound (1) and compound (2), or the reaction may be performed without a catalyst.

[1109] In addition, the reaction product of compound (1) and compound (2) may be directly reacted with compound (3) by using a condensing agent such as DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and the like. Alternatively, compound (3) may be treated with a condensing agent to be once converted to an anhydride and the like, after which it is reacted with the reaction product of compound (1) and compound (2).

[1110] The amount of compound (3) to be charged is 1-50 molar equivalents, preferably 1-10 molar equivalents, relative to the reaction product of compound (1) and compound (2).

[1111] The catalyst to be used is appropriately selected according to the functional groups to be reacted.

[1112] The amount of catalyst is 0.05-100 molar equivalents, preferably 0.1-20 molar equivalents, more preferably 0.2-5 molar equivalent, relative to compound (1).

[1113] The solvent to be used for the reaction of the reaction product of compound (1) and compound (2) with compound (3) is not particularly limited as long as it is a solvent or aqueous solution that does not inhibit the reaction. For example, ethyl acetate, dichloromethane, chloroform, benzene, toluene and the like can be mentioned. Among these, toluene and chloroform are preferable.

[1114] The reaction temperature is 0 to 200? C., preferably 0 to 120? C., more preferably 20 to 50? C., and the reaction time is 1 hr-48 hr, preferably 2-24 hr.

[1115] The reaction product obtained by the above-mentioned reaction can be appropriately purified by a general purification method, for example, washing with water, silica gel column chromatography, crystallization, recrystallization, liquid-liquid extraction, reprecipitation, ion exchange column chromatography and the like.

Structure XXIH Lipids

[1116] In some embodiments, an ionizable lipid is described in U.S. Pat. No. 9,765,022.

[1117] The present invention provides a compound represented by structure (XXIII):

##STR01300##

[1118] In structure XXIII, a hydrophilic and optionally positively charged head is

##STR01301##

in which each of R.sub.a, R.sub.a, R.sub.a, and R.sub.a, independently, is H, a C.sub.1-C.sub.20 monovalent aliphatic radical, a C.sub.1-C.sub.20 monovalent heteroaliphatic radical, a monovalent aryl radical, or a monovalent heteroaryl radical, and Z is a C.sub.1-C.sub.20 bivalent aliphatic radical, a C.sub.1-C.sub.20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical; B is a C.sub.1-C.sub.24 monovalent aliphatic radical, a C.sub.1-C.sub.24 monovalent heteroaliphatic radical, a monovalent aryl radical, a monovalent heteroaryl radical, or

##STR01302##

each of R.sub.1 and R.sub.4, independently, is a bond, a C.sub.1-C.sub.10 bivalent aliphatic radical, a C.sub.1-C.sub.10 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical; each of R.sub.2 and R.sub.5, independently, is a bond, a C.sub.1-C.sub.20 bivalent aliphatic radical, a C.sub.1-C.sub.20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical; each of R.sub.3 and R.sub.6, independently, is a C.sub.1-C.sub.20 monovalent aliphatic radical, a C.sub.1-C.sub.20 monovalent heteroaliphatic radical, a monovalent aryl radical, or a monovalent heteroaryl radical; each of

##STR01303##

a hydrophobic tail, and

##STR01304##

also a hydrophobic tail, has 8 to 24 carbon atoms; and each of X, a linker, and Y, also a linker, independently, is

##STR01305##

in which each of m, n, p, q, and t, independently, is 1-6; W is O, S, or NR.sub.c; each of L.sub.1, L.sub.3, L.sub.5, L.sub.7, and L.sub.9, directly linked to R.sub.1, R.sub.2, R.sub.4, or R.sub.5, independently, is a bond, O, S, or NR.sub.d; each of L.sub.2, L.sub.4, L.sub.6, L.sub.8, and L.sub.10, independently, is a bond, O, S, or NR.sub.e; V is OR.sub.f, SR.sub.g, or NR.sub.hR.sub.i; and each of R.sub.b, R.sub.c, R.sub.d, R.sub.e, R.sub.f, R.sub.g, R.sub.h, and R.sub.i, independently, is H, OH, C.sub.1-10 oxyaliphatic radical, C.sub.1-C.sub.10 monovalent aliphatic radical, C.sub.1-C.sub.10 monovalent heteroaliphatic radical, a monovalent aryl radical, or a monovalent heteroaryl radical.

[1119] A subset of the above-described lipid-like compounds include those in which A is

##STR01306##

each of R.sub.a and R.sub.a, independently, being a C.sub.1-C.sub.10 monovalent aliphatic radical, a C.sub.1-C.sub.10 monovalent heteroaliphatic radical, a monovalent aryl radical, or a monovalent heteroaryl radical; and Z being a C.sub.1-C.sub.10 bivalent aliphatic radical, a C.sub.1-C.sub.10 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical.

[1120] Some lipid-like compounds of this invention feature each of R.sub.1 and R.sub.4, independently, being C.sub.1-C.sub.6 (e.g., C.sub.1-C.sub.4) bivalent aliphatic radical or a C.sub.1-C.sub.6 (e.g., C.sub.1-C.sub.4) bivalent heteroaliphatic radical, the total carbon number for R.sub.2 and R.sub.3 being 12-20 (e.g., 14-18), the total carbon number of R.sub.5 and R.sub.6 also being 12-20 (e.g., 14-18), and each of X and Y, independently, is

##STR01307##

[1121] Specific examples of X and Y include

##STR01308##

m being 2-6.

[1122] Still within the scope of this invention is a pharmaceutical composition containing a nanocomplex that is formed of a protein and a bioreducible compound. In this pharmaceutical composition, the nanocomplex has a particle size of 50 to 500 nm; the bioreducible compound contains a disulfide hydrophobic moiety, a hydrophilic moiety, and a linkerjoining the disulfide hydrophobic moiety and the hydrophilic moiety; and the protein binds to the bioreducible compound via a non-covalent interaction, a covalent bond, or both.

[1123] In certain embodiments, the disulfide hydrophobic moiety is a heteroaliphatic radical containing one or more SS groups and 8 to 24 carbon atoms; the hydrophilic moiety is an aliphatic or heteroaliphatic radical containing one or more hydrophilic groups and 1-20 carbon atoms, each of the hydrophilic groups being amino, alkylamino, dialkylamino, trialkylamino, tetraalkylammonium, hydroxyamino, hydroxyl, carboxyl, carboxylate, carbamate, carbamide, carbonate, phosphate, phosphite, sulfate, sulfite, or thiosulfate; and the linker is O, S, Si, C.sub.1-C.sub.6 alkylene,

##STR01309##

or in which the variables are defined above.

[1124] Specific examples of X and Y include O, S, Si, C.sub.1-C.sub.6 alkylene,

##STR01310##

[1125] In some embodiments, a lipid-like compound of this invention, as shown instructure XXIII above, includes (i) a hydrophilic head, A; (ii) a hydrophobic tail, R.sub.2SSR.sub.3; and (iii) a linker, X. Optionally, these compounds contain a second hydrophobic tail, R.sub.5SSR.sub.6 and a second linker, Y.

[1126] The hydrophilic head of structure XXIII contains one or more hydrophilic functional groups, e.g., hydroxyl, carbonyl, carboxyl, amino, sulfhydryl, phosphate, amide, ester, ether, carbamate, carbonate, carbamide, and phosphodiester. These groups can form hydrogen bonds and are optionally positively or negatively charged.

[1127] Examples of the hydrophilic head include:

##STR01311## ##STR01312## ##STR01313## ##STR01314## ##STR01315##

[1128] Other examples include those described in Akinc et al., Nature Biotechnology, 26, 561-69 (2008) and Mahon et al., US Patent Application Publication 2011/0293703.

[1129] The hydrophobic tail of structure XXIII is a saturated or unsaturated, linear or branched, acyclic or cyclic, aromatic or nonaromatic hydrocarbon moiety containing a disulfide bond and 8-24 carbon atoms. One or more of the carbon atoms can be replaced with a heteroatom, such as N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. The tail is optionally substituted with one or more groups described above. The lipid-like compounds containing this disulfide bond can be bioreducible.

[1130] Examples include:

##STR01316##

[1131] A linker of structure XXIII links the hydrophilic head and the hydrophobic tail. The linker can be any chemical group that is hydrophilic or hydrophobic, polar or non-polar, e.g., O, S, Si, amino, alkylene, ester, amide, carbamate, carbamide, carbonate, phosphate, phosphite, sulfate, sulfite, and thiosulfate. Examples include:

##STR01317##

[1132] Shown below are exemplary lipid-like compounds of this invention:

##STR01318## ##STR01319##

[1133] The lipid-like compounds of structure XXIII can be prepared by methods well known the art. See Wang et al., ACS Synthetic Biology, 1, 403-07 (2012); Manoharan, et al., International Patent Application Publication WO 2008/042973; and Zugates et al., U.S. Pat. No. 8,071,082. The route shown below exemplifies synthesis of these lipid-like compounds:

##STR01320##

[1134] Each of L.sub.a, L.sub.a, L, and L can be one of L.sub.1-L.sub.10; each of W.sub.a and W.sub.b, independently, is W or V; and R.sub.a and R.sub.1-R.sub.6 are defined above, as well as L.sub.1-L.sub.10, W, and V.

[1135] In this exemplary synthetic route, an amine compound, i.e., compound D, reacts with bromides E1 and E2 to form compound F, which is then coupled with both G1 and G2 to afford the final product, i.e., compound H. One or both of the double bonds in this compound (shown above) can be reduced to one or two single bonds to obtain different lipid-like compounds of structure XXIII.

[1136] Other lipid-like compounds of this invention can be prepared using other suitable starting materials through the above-described synthetic route and others known in the art. The method set forth above can include an additional step(s) to add or remove suitable protecting groups in order to ultimately allow synthesis of the lipid-like compounds. In addition, various synthetic steps can be performed in an alternate sequence or order to give the desired material. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing applicable lipid-like compounds are known in the art, including, for example, R. Larock, Comprehensive Organic Transformations (2nd Ed., VCH Publishers 1999); P. G. M. Wuts and T. W. Greene, Greene's Protective Groups in Organic Synthesis (4th Ed., John Wiley and Sons 2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons 1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2nd ed., John Wiley and Sons 2009) and subsequent editions thereof. Certain lipid-like compounds may contain a non-aromatic double bond and one or more asymmetric centers. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans-isomeric forms. All such isomeric forms are contemplated.

[1137] As mentioned above, these lipid-like compounds are useful for delivery of pharmaceutical agents. They can be preliminarily screened for their efficacy in delivering pharmaceutical agents by an in vitro assay and then confirmed by animal experiments and clinic trials. Other methods will also be apparent to those of ordinary skill in the art.

[1138] Not to be bound by any theory, the lipid-like compounds of structure XXIII facilitate delivery of pharmaceutical agents by forming complexes, e.g., nanocomplexes and microparticles. The hydrophilic head of such a lipid-like compound, positively or negatively charged, binds to a moiety of a pharmaceutical agent that is oppositely charged and its hydrophobic moiety binds to a hydrophobic moiety of the pharmaceutical agent. Either binding can be covalent or non-covalent.

[1139] The above described complexes can be prepared using procedures described in publications such as Wang et al., ACS Synthetic Biology, 1, 403-07 (2012). Generally, they are obtained by incubating a lipid-like compound and a pharmaceutical agent in a buffer such as a sodium acetate buffer or a phosphate buffered saline (PBS).

Hydrophilic Groups

[1140] In certain embodiments, the selected hydrophilic functional group or moiety may alter or otherwise impart properties to the compound or to the transfer vehicle of which such compound is a component (e.g., by improving the transfection efficiencies of a lipid nanoparticle of which the compound is a component). For example, the incorporation of guanidinium as a hydrophilic head-group in the compounds disclosed herein may promote the fusogenicity of such compounds (or of the transfer vehicle of which such compounds are a component) with the cell membrane of one or more target cells, thereby enhancing, for example, the transfection efficiencies of such compounds. It has been hypothesized that the nitrogen from the hydrophilic guanidinium moiety forms a six-membered ring transition state which grants stability to the interaction and thus allows for cellular uptake of encapsulated materials. (Wender, et al., Adv. Drug Del. Rev. (2008) 60: 452-472.) Similarly, the incorporation of one or more amino groups or moieties into the disclosed compounds (e.g., as a head-group) may further promote disruption of the endosomal/lysosomal membrane of the target cell by exploiting the fusogenicity of such amino groups. This is based not only on the pKa of the amino group of the composition, but also on the ability of the amino group to undergo a hexagonal phase transition and fuse with the target cell surface, i.e. the vesicle membrane. (Koltover, et al. Science (1998) 281: 78-81.) The result is believed to promote the disruption of the vesicle membrane and release of the lipid nanoparticle contents into the target cell.

[1141] Similarly, in certain embodiments the incorporation of, for example, imidazole as a hydrophilic head-group in the compounds disclosed herein may serve to promote endosomal or lysosomal release of, for example, contents that are encapsulated in a transfer vehicle (e.g., lipid nanoparticle) of the invention. Such enhanced release may be achieved by one or both of a proton-sponge mediated disruption mechanism and/or an enhanced fusogenicity mechanism. The proton-sponge mechanism is based on the ability of a compound, and in particular a functional moiety or group of the compound, to buffer the acidification of the endosome. This may be manipulated or otherwise controlled by the pKa of the compound or of one or more of the functional groups comprising such compound (e.g., imidazole). Accordingly, in certain embodiments the fusogenicity of, for example, the imidazole-based compounds disclosed herein (e.g., HGT4001 and HGT4004) are related to the endosomal disruption properties, which are facilitated by such imidazole groups, which have a lower pKa relative to other traditional ionizable lipids. Such endosomal disruption properties in turn promote osmotic swelling and the disruption of the liposomal membrane, followed by the transfection or intracellular release of the polynucleotide materials loaded or encapsulated therein into the target cell. This phenomenon can be applicable to a variety of compounds with desirable pKa profiles in addition to an imidazole moiety. Such embodiments also include multi-nitrogen based functionalities such as polyamines, polypeptide (histidine), and nitrogen-based dendritic structures.

[1142] Exemplary ionizable and/or cationic lipids are described in International PCT patent publications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085, WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740, WO20 12/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO2010/088537, WO2010/054401, WO2010/054406, WO2010/054405, WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO2013/126803, WO2006/007712, WO2011/038160, WO2005/121348, WO2011/066651, WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563, WO2013/089151, WO2017/099823, WO2015/095346, and WO2013/086354, and US patent publications US2016/0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US2016/0376224, US2017/0119904, US2012/0149894, US2015/0057373, US2013/0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/0203446, US2018/0005363, US2014/0308304, US2013/0338210, US2012/0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US2013/0123338, US2015/0064242, US2006/0051405, US2013/0065939, US2006/0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032, US2018/0028664, US2016/0317458, and US2013/0195920, the contents of all of which are incorporated herein by reference in their entirety. International patent application WO 2019/131770 is also incorporated herein by reference in its entirety.

8. PEG Lipids

[1143] The use and inclusion of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) in the liposomal and pharmaceutical compositions described herein is contemplated, preferably in combination with one or more of the compounds and lipids disclosed herein. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, the PEG-modified lipid employed in the compositions and methods of the invention is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (2000 MW PEG) DMG-PEG2000. The addition of PEG-modified lipids to the lipid delivery vehicle may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-polynucleotide composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipid and derivatized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in a liposomal lipid nanoparticle.

[1144] In an embodiment, a PEG-modified lipid is described in International Pat. Appl. No. PCT/US2019/015913, which is incorporated herein by reference in their entirety. In an embodiment, a transfer vehicle comprises one or more PEG-modified lipids.

[1145] Non-limiting examples of PEG-modified lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. In some further embodiments, a PEG-modified lipid may be, e.g., PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE.

[1146] In some still further embodiments, the PEG-modified lipid includes, but is not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[1147] In various embodiments, a PEG-modified lipid may also be referred to as PEGylated lipid or PEG-lipid.

[1148] In one embodiment, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[1149] In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C.sub.14 to about C.sub.22, such as from about C.sub.14 to about C.sub.16. In some embodiments, a PEG moiety, for example a mPEG-NH.sub.2, has a size of about 1000, about 2000, about 5000, about 10,000, about 15,000 or about 20,000 daltons. In one embodiment, the PEG-lipid is PEG2k-DMG.

[1150] In one embodiment, the lipid nanoparticles described herein can comprise a lipid modified with a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.

[1151] PEG-lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Pat. Publ. No. WO2015/130584 A2, which are incorporated herein by reference in their entirety.

[1152] In various embodiments, lipids (e.g., PEG-lipids), described herein may be synthesized as described International Pat. Publ. No. PCT/US2016/000129, which is incorporated by reference in its entirety.

[1153] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[1154] In some embodiments the PEG-modified lipids are a modified form of PEG-DMG. PEG-DMG has the following structure:

##STR01321##

[1155] In some embodiments the PEG-modified lipids are a modified form of PEG-C18, or PEG-1. PEG-1 has the following structure

##STR01322##

[1156] In one embodiment, PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[1157] In some embodiments, the PEG lipid is a compound of Formula (P1):

##STR01323## [1158] or a salt or isomer thereof, wherein: [1159] r is an integer between 1 and 100; [1160] R is C.sub.10-40 alkyl, C.sub.10-40 alkenyl, or C.sub.10-40 alkynyl; and optionally one or more methylene groups of R are independently replaced with C.sub.3-10 carbocyclylene, 4 to 10 membered heterocyclylene, C.sub.6-10 arylene, 4 to 10 membered heteroarylene, N(R.sup.N), O, S, C(O), C(O)N(R.sup.N), NR.sup.NC(O), NR.sup.NC(O)N(R.sup.N), C(O)O, OC(O), OC(O)O, OC(O)N(R.sup.N), NR.sup.NC(O)O, C(O)S, SC(O), C(?NR.sup.N), C(?NR.sup.N)N(R.sup.N), NR.sup.NC(?NR.sup.N), NR.sup.NC(?NR.sup.N)N(R.sup.N), C(S), C(S)N(R.sup.N), NR.sup.NC(S), NR.sup.NC(S)N(R.sup.N), S(O), OS(O), S(O)O, OS(O)O, OS(O).sub.2, S(O).sub.2O, OS(O).sub.2O, N(R.sup.N)S(O), S(O)N(R.sup.N), N(R.sup.N)S(O)N(R.sup.N), OS(O)N(R.sup.N), N(R.sup.N)S(O)O, S(O).sub.2, N(R.sup.N)S(O).sub.2, S(O).sub.2N(R.sup.N), N(R.sup.N)S(O).sub.2N(R.sup.N), OS(O).sub.2N(R.sup.N), or N(R.sup.N)S(O).sub.2O; and each instance of R.sup.N is independently hydrogen, C.sub.1-6 alkyl, or a nitrogen protecting group.

[1161] For example, R is C.sub.17 alkyl. For example, the PEG lipid is a compound of Formula (P1-a):

##STR01324##

or a salt or isomer thereof, wherein r is an integer between 1 and 100.

[1162] For example, the PEG lipid is a compound of the following formula:

##STR01325##

9. Helper Lipids

[1163] In some embodiments, the transfer vehicle (e.g., LNP) described herein comprises one or more non-cationic helper lipids. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is a phospholipid substitute or replacement. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[1164] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[1165] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[1166] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[1167] In some embodiments, the helper lipid is a 1,2-distearoyl-177-glycero-3-phosphocholine (DSPC) analog, a DSPC substitute, oleic acid, or an oleic acid analog.

[1168] In some embodiments, a helper lipid is a non-phosphatidyl choline (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a DSPC substitute.

[1169] In some embodiments, a helper lipid is described in PCT/US2018/053569. Helper lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Such helper lipids are preferably used in combination with one or more of the compounds and lipids disclosed herein. Examples of helper lipids include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoylsn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-paimitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), paimitoyioieoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanol amine (DOPE) dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC) or dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC). Helper lipids function to stabilize and improve processing of the transfer vehicles. Such helper lipids are preferably used in combination with other excipients, for example, one or more of the ionizable lipids disclosed herein. In some embodiments, when used in combination with an ionizable lipid, the helper lipid may comprise a molar ratio of 5% to about 90%, or about 10% to about 70% of the total lipid present in the lipid nanoparticle.

10. Structural Lipids

[1170] In an embodiment, a structural lipid is described in international patent application PCT/US2019/015913.

[1171] The transfer vehicles described herein comprise one or more structural lipids. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, bassicasterol, tomatidine, tomatine, ursolic, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[1172] In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.

[1173] The transfer vehicles described herein comprise one or more structural lipids. Incorporation of structural lipids in a transfer vehicle, e.g., a lipid nanoparticle, may help mitigate aggregation of other lipids in the particle. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[1174] In some embodiments, the structural lipid is a sterol. Structural lipids can include, but are not limited to, sterols (e.g., phytosterols or zoosterols).

[1175] In certain embodiments, the structural lipid is a steroid. For example, sterols can include, but are not limited to, cholesterol, ?-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol.

[1176] In some embodiments, a transfer vehicle includes an effective amount of an immune cell delivery potentiating lipid, e.g., a cholesterol analog or an amino lipid or combination thereof, that, when present in a transfer vehicle, e.g., an lipid nanoparticle, may function by enhancing cellular association and/or uptake, internalization, intracellular trafficking and/or processing, and/or endosomal escape and/or may enhance recognition by and/or binding to immune cells, relative to a transfer vehicle lacking the immune cell delivery potentiating lipid. Accordingly, while not intending to be bound by any particular mechanism or theory, in one embodiment, a structural lipid or other immune cell delivery potentiating lipid of the disclosure binds to C1q or promotes the binding of a transfer vehicle comprising such lipid to C1q. Thus, for in vitro use of the transfer vehicles of the disclosure for delivery of a nucleic acid molecule to an immune cell, culture conditions that include C1q are used (e.g., use of culture media that includes serum or addition of exogenous C1q to serum-free media). For in vivo use of the transfer vehicles of the disclosure, the requirement for C1q is supplied by endogenous C1q.

[1177] In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is a lipid in Table 16: Table 16

TABLE-US-00020 TABLE 16 CMPD No. S- Structure 1 [01326] 22 [01327] 2 [01328] 3 [01329] 4 [01330] 5 [01331] 6 [01332] 7 [01333] 23 [01334] 24 [01335] 25 [01336] 26 [01337] 27 [01338] 28 [01339] 8 [01340] 9 [01341] 10 [01342] 11 [01343] 12 [01344] 13 [01345] 29 [01346] 30 [01347] 31 [01348] 32 [01349] 33 [01350] 34 [01351] 14 [01352] 15 [01353] 16 [01354] 17 [01355] 18 [01356] 19 [01357] 35 [01358] 36 [01359] 37 [01360] 38 [01361] 39 [01362] 40 [01363] 20 [01364] 21 [01365] 150 [01366] 154 [01367] 162 [01368] 41 [01369] 42 [01370] 165 [01371] 169 [01372] 170 [01373] 163 [01374] 164 [01375] 184 [01376] 171 [01377] 172 [01378] 43 [01379] 47 [01380] 44 [01381] 45 [01382] 46 [01383] 175 [01384] 176 [01385] 48 [01386] 49 [01387] 50 [01388] 177 [01389] 178 [01390] 51 [01391] 52 [01392] 53 [01393] 54 [01394] 55 [01395] 56 [01396] 57 [01397] 60 [01398] 61 [01399] 62 [01400] 63 [01401] 64 [01402] 65 [01403] 66 [01404] 58 [01405] 59 [01406] 153 [01407] 67 [01408] 149 [01409] 68 [01410] 69 [01411] 71 [01412] 72 [01413] 70 [01414] 73 [01415] 74 [01416] 75 [01417] 76 [01418] 77 [01419] 78 [01420] 79 [01421] 80 [01422] 81 [01423] 82 [01424] 83 [01425] 84 [01426] 85 [01427] 86 [01428] 87 [01429] 152 [01430] 157 [01431] 88 [01432] 89 [01433] 90 [01434] 91 [01435] 93 [01436] 94 [01437] 95 [01438] 96 [01439] 92 [01440] 97 [01441] 98 [01442] 99 [01443] 100 [01444] 101 [01445] 102 [01446] 103 [01447] 104 [01448] 105 [01449] 180 [01450] 181 [01451] 182 [01452] 106 [01453] 107 [01454] 108 [01455] 109 [01456] 110 [01457] 121 [01458] 111 [01459] 112 [01460] 113 [01461] 114 [01462] 115 [01463] 116 [01464] 117 [01465] 122 [01466] 123 [01467] 124 [01468] 125 [01469] 126 [01470] 127 [01471] 128 [01472] 118 [01473] 119 [01474] 120 [01475] 156 [01476] 158 [01477] 160 [01478] 129 [01479] 130 [01480] 155 [01481] 167 [01482] 168 [01483] 173 [01484] 161 [01485] 166 [01486] 174 [01487] 179 [01488] 131 [01489] 132 [01490] 133 [01491] 134 [01492] 135 [01493] 136 [01494] 137 [01495] 138 [01496] 139 [01497] 140 [01498] 142 [01499] 143 [01500] 144 [01501] 145 [01502] 146 [01503] 147 [01504] 148 [01505] 141 [01506] 159 [01507] 151 [01508] Composition S- No. Structure 183 [01509] Compound 141 [01510] Compound 140 [01511] Compound 143 [01512] Compound 148

11. LNP Formulations

[1178] The formation of a lipid nanoparticle (LNP) described herein may be accomplished by any methods known in the art. For example, as described in U.S. Pat. Pub. No. US2012/0178702 A1, which is incorporated herein by reference in its entirety. Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).

[1179] In one embodiment, the LNP formulation may be prepared by, e.g., the methods described in International Pat. Pub. No. WO 2011/127255 or WO 2008/103276, the contents of each of which are herein incorporated by reference in their entirety.

[1180] In one embodiment, LNP formulations described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be a composition selected from Formulae 1-60 of U.S. Pat. Pub. No. US2005/0222064 A1, the content of which is herein incorporated by reference in its entirety.

[1181] In one embodiment, the lipid nanoparticle may be formulated by the methods described in U.S. Pat. Pub. No. US2013/0156845 A1, and International Pat. Pub. No. WO2013/093648 A2 or WO2012/024526 A2, each of which is herein incorporated by reference in its entirety.

[1182] In one embodiment, the lipid nanoparticles described herein may be made in a sterile environment by the system and/or methods described in U.S. Pat. Pub. No. US2013/0164400 A1, which is incorporated herein by reference in its entirety.

[1183] In one embodiment, the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle described in U.S. Pat. No. 8,492,359, which is incorporated herein by reference in its entirety.

[1184] A nanoparticle composition may optionally comprise one or more coatings. For example, a nanoparticle composition may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[1185] In some embodiments, the lipid nanoparticles described herein may be synthesized using methods comprising microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to, a slit interdigitial micromixer including, but not limited to, those manufactured by Precision Nanosystems (Vancouver, BC, Canada), Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al. (2012) Langmuir. 28:3633-40; Belliveau, N. M. et al. Mol. Ther. Nucleic. Acids. (2012) 1:e37; Chen, D. et al. J. Am. Chem. Soc. (2012) 134(16):6948-51; each of which is herein incorporated by reference in its entirety).

[1186] In some embodiments, methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Pat. Pub. Nos. US2004/0262223 A1 and US2012/0276209 A1, each of which is incorporated herein by reference in their entirety.

[1187] In one embodiment, the lipid nanoparticles may be formulated using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany). In one embodiment, the lipid nanoparticles are created using microfluidic technology (see, Whitesides (2006) Nature. 442: 368-373; and Abraham et al. (2002) Science. 295: 647-651; each of which is herein incorporated by reference in its entirety). As a non-limiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (see, e.g., Abraham et al. (2002) Science. 295: 647651; which is herein incorporated by reference in its entirety).

[1188] In one embodiment, the circRNA of the present invention may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA), Dolomite Microfluidics (Royston, UK), or Precision Nanosystems (Van Couver, BC, Canada). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[1189] In one embodiment, the lipid nanoparticles may have a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm. In one embodiment, the lipid nanoparticles may have a diameter from about 10 to 500 nm. In one embodiment, the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm. Each possibility represents a separate embodiment of the present invention.

[1190] In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm.

[1191] In some embodiments, the lipid nanoparticles described herein can have a diameter from below 0.1 ?m to up to 1 mm such as, but not limited to, less than 0.1 ?m, less than 1.0 ?m, less than 5 ?m, less than 10 ?m, less than 15 ?m, less than 20 ?m, less than 25 ?m, less than 30 ?m, less than 35 ?m, less than 40 ?m, less than 50 ?m, less than 55 ?m, less than 60 ?m, less than 65 ?m, less than 70 ?m, less than 75 ?m, less than 80 ?m, less than 85 ?m, less than 90 ?m, less than 95 ?m, less than 100 ?m, less than 125 ?m, less than 150 ?m, less than 175 ?m, less than 200 ?m, less than 225 ?m, less than 250 ?m, less than 275 ?m, less than 300 ?m, less than 325 ?m, less than 350 ?m, less than 375 ?m, less than 400 ?m, less than 425 ?m, less than 450 ?m, less than 475 ?m, less than 500 ?m, less than 525 ?m, less than 550 ?m, less than 575 ?m, less than 600 ?m, less than 625 ?m, less than 650 ?m, less than 675 ?m, less than 700 ?m, less than 725 ?m, less than 750 ?m, less than 775 ?m, less than 800 ?m, less than 825 ?m, less than 850 ?m, less than 875 ?m, less than 900 ?m, less than 925 ?m, less than 950 ?m, less than 975 ?m.

[1192] In another embodiment, LNPs may have a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and/or from about 70 to about 90 nm. Each possibility represents a separate embodiment of the present invention.

[1193] A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.1 1, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition may be from about 0.10 to about 0.20. Each possibility represents a separate embodiment of the present invention.

[1194] The zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition may be from about ?20 mV to about +20 mV, from about ?20 mV to about +15 mV, from about ?20 mV to about +10 mV, from about ?20 mV to about +5 mV, from about ?20 mV to about 0 mV, from about ?20 mV to about ?5 mV, from about ?20 mV to about ?10 mV, from about ?20 mV to about ?15 mV from about ?20 mV to about +20 mV, from about ?20 mV to about +15 mV, from about ?20 mV to about +10 mV, from about ?20 mV to about +5 mV, from about ?20 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV. Each possibility represents a separate embodiment of the present invention.

[1195] The efficiency of encapsulation of a therapeutic agent describes the amount of therapeutic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic agent in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic agent (e.g., nucleic acids) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic agent may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the lipid nanoparticle has a polydiversity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200 nm.

[1196] The properties of a lipid nanoparticle formulation may be influenced by factors including, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the selection of the non-cationic lipid component, the degree of noncationic lipid saturation, the selection of the structural lipid component, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. As described herein, the purity of a PEG lipid component is also important to an LNP's properties and performance.

12. Methods

[1197] In one embodiment, a lipid nanoparticle formulation may be prepared by the methods described in International Publication Nos. WO2011127255 or WO2008103276, each of which is herein incorporated by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. WO2019131770, which is herein incorporated by reference in its entirety.

[1198] In some embodiments, circular RNA is formulated according to a process described in U.S. patent application Ser. No. 15/809,680. In some embodiments, the present invention provides a process of encapsulating circular RNA in transfer vehicles comprising the steps of forming lipids into pre-formed transfer vehicles (i.e. formed in the absence of RNA) and then combining the pre-formed transfer vehicles with RNA. In some embodiments, the novel formulation process results in an RNA formulation with higher potency (peptide or protein expression) and higher efficacy (improvement of a biologically relevant endpoint) both in vitro and in vivo with potentially better tolerability as compared to the same RNA formulation prepared without the step of preforming the lipid nanoparticles (e.g., combining the lipids directly with the RNA).

[1199] For certain cationic lipid nanoparticle formulations of RNA, in order to achieve high encapsulation of RNA, the RNA in buffer (e.g., citrate buffer) has to be heated. In those processes or methods, the heating is required to occur before the formulation process (i.e. heating the separate components) as heating post-formulation (post-formation of nanoparticles) does not increase the encapsulation efficiency of the RNA in the lipid nanoparticles. In contrast, in some embodiments of the novel processes of the present invention, the order of heating of RNA does not appear to affect the RNA encapsulation percentage. In some embodiments, no heating (i.e. maintaining at ambient temperature) of one or more of the solutions comprising the pre-formed lipid nanoparticles, the solution comprising the RNA and the mixed solution comprising the lipid nanoparticle encapsulated RNA is required to occur before or after the formulation process.

[1200] RNA may be provided in a solution to be mixed with a lipid solution such that the RNA may be encapsulated in lipid nanoparticles. A suitable RNA solution may be any aqueous solution containing RNA to be encapsulated at various concentrations. For example, a suitable RNA solution may contain an RNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml. In some embodiments, a suitable RNA solution may contain an RNA at a concentration in a range from about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7 mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2-0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6 mg/ml.

[1201] Typically, a suitable RNA solution may also contain a buffering agent and/or salt. Generally, buffering agents can include HEPES, Tris, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate or sodium phosphate. In some embodiments, suitable concentration of the buffering agent may be in a range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM.

[1202] Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentration of salts in an RNA solution may be in a range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM.

[1203] In some embodiments, a suitable RNA solution may have a pH in a range from about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5.

[1204] Various methods may be used to prepare an RNA solution suitable for the present invention. In some embodiments, RNA may be directly dissolved in a buffer solution described herein. In some embodiments, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation.

[1205] According to the present invention, a lipid solution contains a mixture of lipids suitable to form transfer vehicles for encapsulation of RNA. In some embodiments, a suitable lipid solution is ethanol based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e. 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol based. In another embodiment, a suitable lipid solution is dimethylsulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide.

[1206] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration in a range from about 0.1-100 mg/ml, 0.5-90 mg/ml, 1.0-80 mg/ml, 1.0-70 mg/ml, 1.0-60 mg/ml, 1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml, 1.0-10 mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or 1.0-5 mg/ml.

13. Targeting

[1207] The present invention also contemplates the discriminatory targeting of target cells and tissues by both passive and active targeting means. The phenomenon of passive targeting exploits the natural distributions patterns of a transfer vehicle in vivo without relying upon the use of additional excipients or means to enhance recognition of the transfer vehicle by target cells. For example, transfer vehicles which are subject to phagocytosis by the cells of the reticulo-endothelial system are likely to accumulate in the liver or spleen, and accordingly may provide a means to passively direct the delivery of the compositions to such target cells.

[1208] Alternatively, the present invention contemplates active targeting, which involves the use of targeting moieties that may be bound (either covalently or non-covalently) to the transfer vehicle to encourage localization of such transfer vehicle at certain target cells or target tissues. For example, targeting may be mediated by the inclusion of one or more endogenous targeting moieties in or on the transfer vehicle to encourage distribution to the target cells or tissues. Recognition of the targeting moiety by the target tissues actively facilitates tissue distribution and cellular uptake of the transfer vehicle and/or its contents in the target cells and tissues (e.g., the inclusion of an apolipoprotein-E targeting ligand in or on the transfer vehicle encourages recognition and binding of the transfer vehicle to endogenous low density lipoprotein receptors expressed by hepatocytes). As provided herein, the composition can comprise a moiety capable of enhancing affinity of the composition to the target cell. Targeting moieties may be linked to the outer bilayer of the lipid particle during formulation or post-formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusogenic polymers such as PEAA, hemagluttinin, other lipopeptides (see U.S. patent application Ser. No. 08/835,281, and 60/083,294, which are incorporated herein by reference) and other features useful for in vivo and/or intracellular delivery. In other some embodiments, the compositions of the present invention demonstrate improved transfection efficacies, and/or demonstrate enhanced selectivity towards target cells or tissues of interest. Contemplated therefore are compositions which comprise one or more moieties (e.g., peptides, aptamers, oligonucleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their nucleic acid contents for the target cells or tissues. Suitable moieties may optionally be bound or linked to the surface of the transfer vehicle. In some embodiments, the targeting moiety may span the surface of a transfer vehicle or be encapsulated within the transfer vehicle. Suitable moieties and are selected based upon their physical, chemical or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features). Cell-specific target sites and their corresponding targeting ligand can vary widely. Suitable targeting moieties are selected such that the unique characteristics of a target cell are exploited, thus allowing the composition to discriminate between target and non-target cells. For example, compositions of the invention may include surface markers (e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition of, or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers). As an example, the use of galactose as a targeting moiety would be expected to direct the compositions of the present invention to parenchymal hepatocytes, or alternatively the use of mannose containing sugar residues as a targeting ligand would be expected to direct the compositions of the present invention to liver endothelial cells (e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor present in hepatocytes). (See Hillery A M, et al. Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists (2002) Taylor & Francis, Inc.) The presentation of such targeting moieties that have been conjugated to moieties present in the transfer vehicle (e.g., a lipid nanoparticle) therefore facilitate recognition and uptake of the compositions of the present invention in target cells and tissues. Examples of suitable targeting moieties include one or more peptides, proteins, aptamers, vitamins and oligonucleotides.

[1209] In particular embodiments, a transfer vehicle comprises a targeting moiety. In some embodiments, the targeting moiety mediates receptor-mediated endocytosis selectively into a specific population of cells. In some embodiments, the targeting moiety is capable of binding to a T cell antigen. In some embodiments, the targeting moiety is capable of binding to a NK, NKT, or macrophage antigen. In some embodiments, the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, PD-1, 4-1BB, and CD2. In some embodiments, the targeting moiety is an single chain Fv (scFv) fragment, nanobody, peptide, peptide-based macrocycle, minibody, heavy chain variable region, light chain variable region or fragment thereof. In some embodiments, the targeting moiety is selected from T-cell receptor motif antibodies, T-cell ? chain antibodies, T-cell ? chain antibodies, T-cell ? chain antibodies, T-cell ? chain antibodies, CCR7 antibodies, CD3 antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8 antibodies, CD11b antibodies, CD11c antibodies, CD16 antibodies, CD19 antibodies, CD20 antibodies, CD21 antibodies, CD22 antibodies, CD25 antibodies, CD28 antibodies, CD34 antibodies, CD35 antibodies, CD40 antibodies, CD45RA antibodies, CD45RO antibodies, CD52 antibodies, CD56 antibodies, CD62L antibodies, CD68 antibodies, CD80 antibodies, CD95 antibodies, CD117 antibodies, CD127 antibodies, CD133 antibodies, CD137 (4-1BB) antibodies, CD163 antibodies, F4/80 antibodies, IL-4R? antibodies, Sca-1 antibodies, CTLA-4 antibodies, GITR antibodies GARP antibodies, LAP antibodies, granzyme B antibodies, LFA-1 antibodies, transferrin receptor antibodies, and fragments thereof. In some embodiments, the targeting moiety is a small molecule binder of an ectoenzyme on lymphocytes. Small molecule binders of ectoenzymes include A2A inhibitors CD73 inhibitors, CD39 or adesines receptors A2aR and A2bR. Potential small molecules include AB928.

[1210] In some embodiments, transfer vehicles are formulated and/or targeted as described in Shobaki N, Sato Y, Harashima H. Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting. Int J Nanomedicine. 2018; 13:8395-8410. Published 2018 Dec. 10. In some embodiments, a transfer vehicle is made up of 3 lipid types. In some embodiments, a transfer vehicle is made up of 4 lipid types. In some embodiments, a transfer vehicle is made up of 5 lipid types. In some embodiments, a transfer vehicle is made up of 6 lipid types.

14. Target Cells

[1211] Where it is desired to deliver a nucleic acid to an immune cell, the immune cell represents the target cell. In some embodiments, the compositions of the invention transfect the target cells on a discriminatory basis (i.e., do not transfect non-target cells). The compositions of the invention may also be prepared to preferentially target a variety of target cells, which include, but are not limited to, T cells, B cells, macrophages, and dentritic cells.

[1212] In some embodiments, the target cells are deficient in a protein or enzyme of interest. For example, where it is desired to deliver a nucleic acid to a hepatocyte, the hepatocyte represents the target cell. In some embodiments, the compositions of the invention transfect the target cells on a discriminatory basis (i.e., do not transfect non-target cells). The compositions of the invention may also be prepared to preferentially target a variety of target cells, which include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

[1213] The compositions of the invention may be prepared to preferentially distribute to target cells such as in the heart, lungs, kidneys, liver, and spleen. In some embodiments, the compositions of the invention distribute into the cells of the liver or spleen to facilitate the delivery and the subsequent expression of the circRNA comprised therein by the cells of the liver (e.g., hepatocytes) or the cells of spleen (e.g., immune cells). The targeted cells may function as a biological reservoir or depot capable of producing, and systemically excreting a functional protein or enzyme. Accordingly, in one embodiment of the invention the transfer vehicle may target hepatocytes or immune cells and/or preferentially distribute to the cells of the liver or spleen upon delivery. In an embodiment, following transfection of the target hepatocytes or immune cells, the circRNA loaded in the vehicle are translated and a functional protein product is produced, excreted and systemically distributed. In other embodiments, cells other than hepatocytes (e.g., lung, spleen, heart, ocular, or cells of the central nervous system) can serve as a depot location for protein production.

[1214] In one embodiment, the compositions of the invention facilitate a subject's endogenous production of one or more functional proteins and/or enzymes. In an embodiment of the present invention, the transfer vehicles comprise circRNA which encode a deficient protein or enzyme. Upon distribution of such compositions to the target tissues and the subsequent transfection of such target cells, the exogenous circRNA loaded into the transfer vehicle (e.g., a lipid nanoparticle) may be translated in vivo to produce a functional protein or enzyme encoded by the exogenously administered circRNA (e.g., a protein or enzyme in which the subject is deficient). Accordingly, the compositions of the present invention exploit a subject's ability to translate exogenously- or recombinantly-prepared circRNA to produce an endogenously-translated protein or enzyme, and thereby produce (and where applicable excrete) a functional protein or enzyme. The expressed or translated proteins or enzymes may also be characterized by the in vivo inclusion of native post-translational modifications which may often be absent in recombinantly-prepared proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme.

[1215] The administration of circRNA encoding a deficient protein or enzyme avoids the need to deliver the nucleic acids to specific organelles within a target cell. Rather, upon transfection of a target cell and delivery of the nucleic acids to the cytoplasm of the target cell, the circRNA contents of a transfer vehicle may be translated and a functional protein or enzyme expressed.

[1216] In some embodiments, a circular RNA comprises one or more miRNA binding sites. In some embodiments, a circular RNA comprises one or more miRNA binding sites recognized by miRNA present in one or more non-target cells or non-target cell types (e.g., Kupffer cells or hepatic cells) and not present in one or more target cells or target cell types (e.g., hepatocytes or T cells). In some embodiments, a circular RNA comprises one or more miRNA binding sites recognized by miRNA present in an increased concentration in one or more non-target cells or non-target cell types (e.g., Kupffer cells or hepatic cells) compared to one or more target cells or target cell types (e.g., hepatocytes or T cells). miRNAs are thought to function by pairing with complementary sequences within RNA molecules, resulting in gene silencing.

[1217] In some embodiments, the compositions of the invention transfect or distribute to target cells on a discriminatory basis (i.e. do not transfect non-target cells). The compositions of the invention may also be prepared to preferentially target a variety of target cells, which include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

15. Pharmaceutical Compositions

[1218] In certain embodiments, provided herein are compositions (e.g., pharmaceutical compositions) comprising a therapeutic agent provided herein. In some embodiments, the therapeutic agent is a circular RNA polynucleotide provided herein. In some embodiments the therapeutic agent is a vector provided herein. In some embodiments, the therapeutic agent is a cell comprising a circular RNA or vector provided herein (e.g., a human cell, such as a human T cell). In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the compositions provided herein comprise a therapeutic agent provided herein in combination with other pharmaceutically active agents or drugs, such as anti-inflammatory drugs or antibodies capable of targeting B cell antigens, e.g., anti-CD20 antibodies, e.g., rituximab.

[1219] With respect to pharmaceutical compositions, the pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active agent(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the therapeutic agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[1220] The choice of carrier will be determined in part by the particular therapeutic agent, as well as by the particular method used to administer the therapeutic agent. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions provided herein.

[1221] In certain embodiments, the pharmaceutical composition comprises a preservative. In certain embodiments, suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. The preservative or mixtures thereof are typically present in an amount of about 0.00010% to about 2% by weight of the total composition.

[1222] In some embodiments, the pharmaceutical composition comprises a buffering agent. In some embodiments, suitable buffering agents may include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents optionally may be used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition.

[1223] In some embodiments, the concentration of therapeutic agent in the pharmaceutical composition can vary, e.g., less than about 1%, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%%, 15, 20%, 25%, 30%, 35%, 40%, 45%, or about 50% or more by weight, and can be selected primarily by fluid volumes, and viscosities, in accordance with the particular mode of administration selected.

[1224] The following formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and intrathecal), and topical administration are merely exemplary and are in no way limiting. More than one route can be used to administer the therapeutic agents provided herein, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[1225] Formulations suitable for oral administration can comprise or consist of (a) liquid solutions, such as an effective amount of the therapeutic agent dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard or soft shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can comprise the therapeutic agent with a flavorant, usually sucrose, acacia or tragacanth. Pastilles can comprise the therapeutic agent with an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.

[1226] Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In some embodiments, the therapeutic agents provided herein can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids including water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol or hexadecyl alcohol, a glycol such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[1227] Oils, which can be used in parenteral formulations in some embodiments, include petroleum, animal oils, vegetable oils, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral oil. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[1228] Suitable soaps for use in certain embodiments of parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alky, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-?-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[1229] In some embodiments, the parenteral formulations will contain, for example, from about 0.5% to about 25% by weight of the therapeutic agent in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having, for example, a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range, for example, from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol, sorbitan, fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules or vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[1230] In certain embodiments, injectable formulations are provided herein. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed, pages 622-630 (1986)).

[1231] In some embodiments, topical formulations are provided herein. Topical formulations, including those that are useful for transdermal drug release, are suitable in the context of certain embodiments provided herein for application to skin. In some embodiments, the therapeutic agent alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa.

[1232] In certain embodiments, the therapeutic agents provided herein can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes can serve to target the therapeutic agents to a particular tissue. Liposomes also can be used to increase the half-life of the therapeutic agents. Many methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[1233] In some embodiments, the therapeutic agents provided herein are formulated in time-released, delayed release, or sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to, cause sensitization of the site to be treated. Such systems can avoid repeated administrations of the therapeutic agent, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain composition embodiments provided herein. In one embodiment, the compositions of the invention are formulated such that they are suitable for extended-release of the circRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice a day, daily or every other day. In an embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, every four weeks, once a month, every six weeks, every eight weeks, every three months, every four months, every six months, every eight months, every nine months or annually.

[1234] In some embodiments, a protein encoded by an inventive polynucleotide is produced by a target cell for sustained amounts of time. For example, the protein may be produced for more than one hour, more than four, more than six, more than 12, more than 24, more than 48 hours, or more than 72 hours after administration. In some embodiments the polypeptide is expressed at a peak level about six hours after administration. In some embodiments the expression of the polypeptide is sustained at least at a therapeutic level. In some embodiments, the polypeptide is expressed at least at a therapeutic level for more than one, more than four, more than six, more than 12, more than 24, more than 48, or more than 72 hours after administration. In some embodiments, the polypeptide is detectable at a therapeutic level in patient tissue (e.g., liver or lung). In some embodiments, the level of detectable polypeptide is from continuous expression from the circRNA composition over periods of time of more than one, more than four, more than six, more than 12, more than 24, more than 48, or more than 72 hours after administration.

[1235] In certain embodiments, a protein encoded by an inventive polynucleotide is produced at levels above normal physiological levels. The level of protein may be increased as compared to a control. In some embodiments, the control is the baseline physiological level of the polypeptide in a normal individual or in a population of normal individuals. In other embodiments, the control is the baseline physiological level of the polypeptide in an individual having a deficiency in the relevant protein or polypeptide or in a population of individuals having a deficiency in the relevant protein or polypeptide. In some embodiments, the control can be the normal level of the relevant protein or polypeptide in the individual to whom the composition is administered. In other embodiments, the control is the expression level of the polypeptide upon other therapeutic intervention, e.g., upon direct injection of the corresponding polypeptide, at one or more comparable time points.

[1236] In certain embodiments, the levels of a protein encoded by an inventive polynucleotide are detectable at 3 days, 4 days, 5 days, or 1 week or more after administration. Increased levels of protein may be observed in a tissue (e.g., liver or lung).

[1237] In some embodiments, the method yields a sustained circulation half-life of a protein encoded by an inventive polynucleotide. For example, the protein may be detected for hours or days longer than the half-life observed via subcutaneous injection of the protein or mRNA encoding the protein. In some embodiments, the half-life of the protein is 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week or more.

[1238] Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems: wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the active composition is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[1239] In some embodiments, the therapeutic agent can be conjugated either directly or indirectly through a linking moiety to a targeting moiety. Methods for conjugating therapeutic agents to targeting moieties is known in the art. See, for instance, Wadwa et al., J, Drug Targeting 3:111 (1995) and U.S. Pat. No. 5,087,616.

[1240] In some embodiments, the therapeutic agents provided herein are formulated into a depot form, such that the manner in which the therapeutic agent is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Pat. No. 4,450,150). Depot forms of therapeutic agents can be, for example, an implantable composition comprising the therapeutic agents and a porous or non-porous material, such as a polymer, wherein the therapeutic agents are encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body and the therapeutic agents are released from the implant at a predetermined rate.

16. Therapeutic Methods

[1241] In certain aspects, provided herein is a method of treating and/or preventing a condition, e.g., an autoimmune disorder or cancer.

[1242] In certain embodiments, the therapeutic agents provided herein are co-administered with one or more additional therapeutic agents (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the therapeutic agent provided herein can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the therapeutic agent provided herein and the one or more additional therapeutic agents can be administered simultaneously.

[1243] In some embodiments, the subject is a mammal. In some embodiments, the mammal referred to herein can be any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, or mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs), or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the mammal is a human.

17. Sequences

[1244]

TABLE-US-00021 TABLE17 IRESsequences. SEQ ID NO IRES Sequence 1 EMCV-A cccccctctccctccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgttt gtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtct tcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtg aaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcag cggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctg caaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctc tcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgat ctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaa ccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaacc 2 EMCV-B ctccccctccccccccttactatactggccgaagccacttggaataaggccggtgtgcgtttgtcta catgctattttctaccgcattaccgtcttatggtaatgtgagggtccagaacctgaccctgtcttcttga cgaacactcctaggggtctttcccctctcgacaaaggagtgtaaggtctgttgaatgtcgtgaagga agcagttcctctggaagcttcttaaagacaaacaacgtctgtagcgaccctttgcaggcagcggaa ccccccacctggtgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaag gcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctca agcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggg gcctcggtgcacgtgctttacacgtgttgagtcgaggtgaaaaaacgtctaggccccccgaacca cggggacgtggttttcctttgaaaaccacgattacaat 3 EMCV-Bf ttgccagtctgctcgatatcgcaggctgggtccgtgactacccactccccctttcaacgtgaaggct acgatagtgccaggggggtactgccgtaagtgccaccccaaacaacaacaacaaaacaaactc cccctccccccccttactatactggccgaagccacttggaataaggccggtgtgcgtttgtctacat gctattttctaccgcattaccgtcttatggtaatgtgagggtccagaacctgaccctgtcttcttgacg aacactcctaggggtctttcccctctcgacaaaggagtgtaaggtctgttgaatgtcgtgaaggaag cagttcctctggaagcttcttaaagacaaacaacgtctgtagcgaccctttgcaggcagcggaacc ccccacctggtgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggc ggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaag cgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggc ctcggtgcacgtgctttacacgtgttgagtcgaggtgaaaaaacgtctaggccccccgaaccacg gggacgtggttttcctttgaaaaccacgattacaat 4 EMCV-Cf ttgccagtctgctcgatatcgcaggctgggtccgtgactacccactccccctttcaacgtgaaggct acgatagtgccaggggggtactgccgtaagtgccaccccaaaacaacaacaaccccccctctc cctccTccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatat gttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgac gagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaa gcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaac cccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaagg cggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaa gcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctgggg cctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacgg ggacgtggttttcctttgaaaaacacgatgataat 5 EMCVpEC9 ccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttat tttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagc attcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcag ttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccc cacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggc acaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgt attcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctc ggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacgggga cgtggttttcctttgaaaaacacgatgataat 6 Picobirnavirus gtaaattaaatgctatttacaaaatttaaacagaaaggagagatgttatgaaccggttttacaaggttt catacatcgaaaatagcactacctggggcagccgacacactaacatcgtctgtttaaccagaagtg ttactgaaaggaggttattta 7 HCVQC64 acctgcccctaataggggcgacactccgccatgaatcactcccctgtgaggaactactgtcttcac gcagaaagcgtctagccatggcgttagtatgagtgtcgtacagcctccaggcccccccctcccgg gagagccatagtggtctgcggaaccggtgagtacaccggaattgccgggaagactgggtcctttc ttggataaacccactctatgcccggacatttgggcgtgcccccgcaagactgctagccgagtagc gttgggttgcgaaaggccttgtggtactgcctgatagggtgcttgcgagtgccccgggaggtctcg tagaccgtgcatc 8 HumanCosavirus ctacaagctttgtgtaaacaaacttttgtttggcttttctcaagcttctctcacatcaggccccaaagat E/D gtcctgaaggtaccccgtgtatctgaggatgagcaccatcgactacccggacctgcaaaattttgc aaacgcatgtggtatcccagccccctcctctcggggagggggctttgctcactcagcacaggatct gatcaggagatccacctccggtgctttacaccggggcgtggatttaaaaattgcccaaggcctggc gcacaacctaggggactaggttttccttatattttaaagctgtcaat 9 HumanCosavirus gtcttaggacgacgcatgtggtatcccagcccccgcctacattggcgggggcttttgaagcacca F gacactggatctgatcaggaggagggtagctgctttacagcccctcttaaaaattgcccaaggtcc ggccacccaacctaggggactaggttttccttttatttttaaattgtcatt 10 HumanCosavirus acatgggggagactgcatgtggcagtcttgaaacgtgtggtttgacgtctaccttatatggcagtgg JMY gtggagtactgcaaagatgtcaccgtgctttacacggtttttgaaccccacaccggctgtttgacgct cgtagggcagcaggtttattttcattaaaattcttactttctagctgcatgagttctattcatgcagacgg agtgatactcccgttccttcttggacaggttgcctccacgccctttgtggatcttaaggtgaccaagtc actggtgttggaggtgaagatagagagtcctcttgggaatgtcatgtggctgtgccaggggttgta gcgatgccattcgtgtgtgcggatttcctctcgtggtgacacgagcctcacaggccaaaagccccg tccgaaaggacccgaatggtggagtgaccctgactcccccctgcatagttttgtgattaggaacttg aggaatttctgtcataaatctctatcacatcaggccccaaagatgtcctgaaggtaccctgtgtatctg aggatgagcaccaccgactacccggacttgcattagcagacacatgtggttgcccagccccacct cttcagaggtggggctttgctcactcagcacaggatctgatcaggagccccgctcgtgtgctttaca ctcgacgcggggttaaaaattgcccaaggcctggcacaacaacctaggggactaggttttcctattt ttgtaaattatgtcaat 11 Rhinovirus gtgacaatcagccagattgttaacggtcaagcacttctgtttccccggtacccttgtatacgcttcacc NAT001 cgaggcgaaaagtgaggttatcgttatccgcaaagtgcctacgagaagcctagtagcacttttgaa gcctatggctggtcgctcaactgtttacccagcagtagacctggcagatgaggctagatgttcccc accagcgatggtgatctagcctgcgtggctgcctgcacactctattgagtgtgaagccagaaagtg gacaaggtgtgaagagcctattgtgctcactttgagtcctccggcccctgaatgtggctaatcctaa ccccgtagctgttgcatgtaatccaacatgtctgcagtcgtaatgggcaactatgggatggaaccaa ctactttgggtgtccgtgtttcttgtttttctttatgcttgcttatggtgacaactgtagttattacatttgtta Cc 12 HRV14 ttaaaacagcggatgggtatcccaccattcgacccattgggtgtagtactctggtactatgtacctttg tacgcctgtttctccccaaccacccttccttaaaattcccacccatgaaacgttagaagcttgacatta aagtacaataggtggcgccatatccaatggtgtctatgtacaagcacttctgtttcccaggagcgag gtataggctgtacccactgccaaaagcctttaaccgttatccgccaaccaactacgtaacagttagt accatcttgttcttgactggacgttcgatcaggtggattttccctccactagtttggtcgatgaggcta ggaattccccacgggtgaccgtgtcctagcctgcgtggcggccaacccagcttatgctgggacgc ccttttaaggacatggtgtgaagactcgcatgtgcttggttgtgagtcctccggcccctgaatgcgg ctaaccttaaccctagagccttatgccacgatccagtggttgtaaggtcgtaatgagcaattccggg acgggaccgactactttgggtgtccgtgtttctcatttttcttcatattgtcttatggtcacagcatatata tacatatactgtgatc 13 HRV89 ttaaaactgggagtgggttgttcccactcactccacccatgcggtgttgtactctgttattacggtaac tttgtacgccagtttttcccacccttccccataatgtaacttagaagtttgtacaatatgaccaataggt gacaatcatccagactgtcaaaggtcaagcacttctgtttccccggtcaatgaggatatgctttaccc aaggcaaaaaccttagagatcgttatccccacactgcctacacagagcccagtaccatttttgatat aattgggttggtcgctccctgcaaacccagcagtagacctggcagatgaggctggacattcccca ctggcgacagtggtccagcctgcgtggctgcctgctcacccttcttgggtgagaagcctaattattg acaaggtgtgaagagccgcgtgtgctcagtgtgcttcctccggcccctgaatgtggctaaccttaa ccctgcagccgttgcccataatccaatgggtttgcggtcgtaatgcgtaagtgcgggatgggacca actactttgggtgtccgtgtttcctgtttttcttttgattgcattttatggtgacaatttatagtgtatagattg tcatc 14 HRVC-02 ttaaaactgggtacaggttgttcccacctgtatcacccacgtggtgtggtgctcttgtattccggtaca cttgcacgccagtttgccacccctcacccgtcgtaacttagaagctaacaactcgaccaacaggcg gtggtaaaccataccacttacggtcaagcactcctgtttccccggtatgcgaggaatagactcctac agggttgaagcctcaagtatcgttatccgcattggtactacgcaaagcttagtagtgccttgaaagtc ccttggttggtcgctccgctagtttcccctagtagacctggcagatgaggcaggacactccccact ggcgacagtggtcctgcctgcgtggctgcctgcgcacccttaggggtgcgaagccaagtgacag acaaggtgtgaagagccccgtgtgctaccaatgagtcctccggcccctgaatgcggctaatccaa ccccacagctattgcacacaagccagtgtgtatgtagtcgtaatgagcaattgtgggacggaaccg actactttgggtgtccgtgtttccttttattcttatcattctgcttatggtgacaatactgtgaaatagtgtt gttacc 15 HRV-A21 taaaactggatccaggttgttcccacctggatctcctattgggagttgtactctattattccggtaatttt gtacgccagttttatcttccccctccccaattgtaacttagaaggttatcaatacgaccaataggtggt agttagccaaactaccaaaggtcaagcacttctgtttccccggtcaaagttgatatgctccaacagg gcaaaaacaactgagatcgttatccgcaaagtgcctacgcaaagcctagtaacacctttgaagattt atggttggtcgttccgctatttcccatagtagacctggcagatgaggctagaaatcccccactggcg acagtgctctagcctgcgtggctgcctgcgcaccccttgggtgcgaagccatacattggacaagg tgtgaagagccccgtgtgctcactttgagtcctccggcccctgaatgtggctaaccttaaccctgca gctagtgcatgtaatccaacatgttgctagtcgtaatgagtaattgcgggacgggaccaactactttg ggtgtccgtgtttcactttttccttttaatattgcttatggtgacaatatatatagctatatatattgacacc 16 SalivirusASH1 ttcccctgcaaccattacgcttactcgcatgtgcattgagtggtgcatgtgttgaacaaacagctaca ctcacatggggggggttttcccgccctacggcttctcgcgaggcccacccctcccctttctcccat aactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtgg ggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagcgaatt ctagtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaact caccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagacc agtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaac cggtggggtaaggttggtgtgcccaacgcccgtactcaggggatacctcaaggcacccaggaat gccagggaggtaccccgcttcacagcgggatctgaccctggggtaaatgtctgcggggggtcttc ttggcccacttctcagtacttttcagg 17 SalivirusFHB acatggggggtctgcggacggcttcggcccacccgcgacaagaatgccgtcatctgtcctcatta cccgtattccttcccttcccccgcaaccaccacgcttactcgcgcacgtgttgagtggcacgtgcgt tgtccaaacagctacacccacacccttcggggcgggtttgtcccgccctcgggttcctcgcggaa cccccccctccctctctctctttctatccgccctcacttcccataactacagtgctttggtaggtgagc accctgaccccccgcggaagctgctaacgtggcaactgtggggatccaggcaggttatcaaagg cacccggtctttccgccttcaggagtatctctgccggtgaattccggtagggctctgcttggtgcca acctcccccaaatgcgcgctgcgggagtgctcttccccaactcatcttagtaacctctcatgtgtgtg cttggtcagcatatctgaggcgacgttccgctgtcccagaccagtccagcaatggacgggccagt gtgcgtagtcgctttccggttttccggcgcatgtttggcgaaacgctgaggtaaggttggtgtgccc aacgcccgtaatttggtgatacctcaagaccacccaggaatgccagggaggtaccccacttcggt gggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctcttttttctggcatg 18 SalivirusNG-J1 tatggcaggcgggcttgtggacggcttcggcccacccacagcaagaatgccatcatctgtcctca cccccaattttcccttttcttcccctgcaaccattacgcttactcgcatgtgcattgagtggtgcatgtgt tgaacaaacagctacactcacatggggggggttttcccgccctacggcctctcgcgaggcccac cccttccctccccttataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctc acgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtat ccctactagtgaattctagcggggctctgcttggtgccaacctcccccaaatgcgcgctggggag tgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttcc gctgtcccagaccagtccagtaatggacgggccagtgcgtgtagtcgtcttccggcttgtccggg gcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgacacctcaa gaccacccaggaatgccagggaggtaccccacctcacggtgggatctgaccctgggctaattgt ctacggtggttcttcttgcttccacttctttcttctgttcacg 19 Human tttgaaaggggtctcctagagagcttggccgtcgggccttataccccgacttgctgagtttctctagg Parechovirus1 agagcccttttcccagccctgaggcggctggtcaataaaagcctcaaacgtaactaacacctaaga agatcatgtaaaccctatgcctggtctccactattcgaaggcaacttgcaataagaagagtgggatc aagacgcttaaagcatagagacagttttttttctaacccacatttgtgtggggtggcagatggcgtg ccataactctaatagtgagataccacgcttgtggaccttatgctcacacagccatcctctagtaagttt gtgagacgtctggtgacgtgtgggaacttattggaaacaacattttgctgcaaagcatcctactgcc agcggaaaaacacctggtaacaggtgcctctggggccaaaagccaaggtttaacagaccctttag gattggttctaaacctgagatgttgtggaagatatttagtacctgctgatctggtagttatgcaaacact agttgtaaggcccatgaaggatgcccagaaggtacccgtaggtaacaagtgacactatggatctg atttggggccagatacctctatcttggtgatctggttaaaaaacatctaatgggccaaacccggggg ggatccccggtttcctcttattctatcaatgccact 20 CrohivirusB gtataagagacaggtgtttgccttgtcttcggactggcatcttgggaccaaccccccttttccccagc catgggttaaatggcaataaaggacgtaacaactttgtaaccattaagctttgtaattttgtaaccact aagctttgtgcacataatgtaaccatcaagcttgttagtcccagcaggaggtttgcatgcttgtagcc gaaatggggctcgaccccccatagtaggatacttgattttgcattccattgtggacctgcaaactcta cacatagaggctttgtcttgcatctaaacacctgagtacagtgtgtacctagaccctatagtacggga ggaccgtttgtttcctcaataaccctacataataggctaggtgggcatgcccaatttgcaagatccca gactgggggtcggtctgggcagggttagatccctgttagctactgcctgatagggtggtgctcaac catgtgtagtttaaattgagctgttcatatacc 21 Yc-3 actgaagatcctacagtaactactgccccaatgaacgccacagatgggtctgctgatgactacctat cttagtgctagttgaggtttgaagtgagccggtttttagaagaaccagtttctgaacattatcatcccc agcatctattctatacgcacaagatagatagtcatcagcagacacatctgtgctactgcttgatagag ttgcggctggtcaacttagattggtataaccagttgagtggcaa 22 RosavirusM-7 tatgcatcactggacggcctaacctcggtcgtggcttcttgccgatttcagcgctaccaggctttctg gtctcgccaggcgttgattagtaggtgcactgtctaagtgaagacagcagtgctctctgtgaaaagt tgatgacactcttcaggtttgtagcgatcactcaaggctagcggatttccccgtgtggtaacacacg cctctaggcccagaaggcacggtgttgacagcaccccttgagtggctggtcttccccaccagcac ctgatttgtggattcttcctagtaacggacaagcatggctgctcttaagcattcagtgcgtccggggc tgaaggatgcccagaaggtacccgcaggtaacgataagctcactgtggatctgatctggggctgc gggctgggtgtctttccacccagccaaaacccgtaaaacggtagtcgcagttaaaaaacgtctag gccccacccccccagggatggggggttcccttaaaccctcacaagttcaac 23 ShanbavirusA tgaaaagggggcgcagggtggtggtggttactaaatacccaccatcgccctgcacttcccttttcc cctgtggctcagggtcacttagccccctctttgggttaccagtagttttctacccctgggcacagggt taactatgcaagacggaacaacaatctcttagtccccctcgccgatagtgggctcgacccccatgt gtaggagtggataagggacggagtgagccgatacggggaagagtgtgcggtcacaccttaattc catgagcgctgcgaagaaggaagctgtgaacaatggcgacctgaaccgtacacatggagctcca caggcatggtactcgttagactacgcagcctggttgggagtgggtataccctgggtgagccgcca gtgaatgggagttcactggttaacacacactgcctgatagggtcagggcctcctgtccccgccgta atgaggtagaccatatgcc 24 PasivirusA gcggctggatattctggccgtgcaactgcttttgaccagtggctctgggtaacttagccaaagtgtc cttctccctttccctattatatgttttatggctttgtctggtcttgtttagtttatatataagatcctttccgcc gatatagacctcgacagtctagtgtaggaggattggtgatattaatttgccccagaagagtgaccgt gacacatagaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatccc attgccatcccaacaagcggagggtatacccactatgtgcacgtctgcagtgggagtctgcagatt tagtcatactgcctgatagggtgtgggcctgcactctggggtactcaggctgtttatataat 25 PasivirusA2 gctggactttctggctgcgcaactgcttttaaccagtggctctgggttacttagccaaaaccccctttc cccgtaccctagtttgtgtgtgtattattattttgttgttgttttgtaaatttttatataagatcctttccgccg atatagacctcgacagtctagtgtaggaggattggtgatattaatatgccccagaagagtgaccgtg acacatagaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatccca ttgccatcccaacaaacggagggtatacccgctatgtgcgcgtctacagtgggaatctgtagattta gtcatactgcctgatagggtgtgggcctgcactctggggtactcaggctgtttatataat 26 EchovirusE14 ttaaaacagcctgtgggttgttcccatccacagggcccactgggcgccagcactctggtattgcgg taccttagtgcgcctgttttatatacccgtcccccaaacgtaacttagacgcatgtcaacgaagacca atagtaagcgcagcacaccagctgtgttccggtcaagcacttctgttaccccggaccgagtatcaa taagctactcacgtggctgaaggagaaaacgttcgttacccgaccaattacttcaagaaacctagta acaccatgaaggttgcgcagtgtttcgctccgcacaaccccagtgtagatcaggtcgatgagtcac cgcattccccacgggtgaccgtggcggtggctgcgctggcggcctgcccatggggaaacccat gggacgcttcaatactgacatggtgcgaagagtctattgagctaattggtagtcctccggcccctga atgcggctaatcctaactgcggagcagatacccacacaccagtgggcagtctgtcgtaacgggca actctgcagcggaaccgactactttgggtgtccgtgtttctctttatccttatactggctgcttatggtg acaattgagagattgttaccatatagctattggattggccatccggtgacaaatagagcaattgtgtat ttgtttgttggtttcgtgccattaaattacaaggttctaaacacccttaatcttattatagcattcaacaca acaaa 27 Human gtacattagatgcgtcatctgcaactttagtcaataaattacctccaatgtcattaccaacattccctac Parechovirus5 cttttcactaacacctaagacaacaagtacctatgcctggtctccactattcgaaggcaacttgcaat aagaagagtggaattaagacgcttaaagcatagagctagttatcttttctaacccacaaagttttgtg gggtggcagatggcgtgccataactctattagtgagataccatgcttgtggatcttatgctcacaca gccatcctctagtaagttgataaggtgtctggtgatatgtgggaactcacatgaaccattaatttaccg taaggtatcctatagccagcggaatcacatctggtgacagatgcctctggggccgaaagccaagg tttaacagaccctataggattggtttcaaaacctgaattgatgtggattgtgtatagtacctgttgatct ggtaacagtgtcaacactagttgtaaggcccacgaaggatgcccagaaggtacccgtaggtaaca agtgacactatggatctgatctggggccagctacctctatcatggtgagttggttaaaaaacgtctag tgggccaaacccaggggggatccctggtttccttttacctaatcaaagccact 28 AichiVirus tttgaaaaggggggggggggcctcggccccctcaccctcttttccggtggtctggtcccggacc accgttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatactcccccc accccccttttgtaactaagtatgtgtgctcgtgatcttgactcccacggaacggaccgatccgttgg tgaacaaacagctaggtccacatcctcccttcccctgggagggcccccgccctcccacatcctcc ccccagcctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgg gtccccccttcatcaagacaccaggtctttcctccttaaggctagccccggcgtgtgaattcacgttg ggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaac ccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccaacctg gtgacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggt gtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcag gtaccccacctccggggggatctgagcctgggctaattgtctacgggtagtttcatttccaatccttt tatgtcggagtc 29 HepatitisAVirus ttcaagaggggtctccggagttttccggaacccctcttggaagtccatggtgaggggacttgatac HA16 ctcaccgccgtttgcctaggctataggctaaatttccctttccctgtccttcccctatttccttttgttttgt ttgtaaatattaattcctgcaggttcagggttctttaatctgtttctctataagaacactcaatttttcacgc tttctgtctcctttcttccagggctctccccttgccctaggctctggccgttgcgcccggcggggtca actccatgattagcatggagctgtaggagtctaaattggggacgcagatgtttgggacgtcgccttg cagtgttaacttggctttcatgaacctctttgatcttccacaaggggtaggctacgggtgaaacctctt aggctaatacttcaatgaagagatgccttggatagggtaacagcggcggatattggtgagttgttaa gacaaaaaccattcaacgccggaggactggctctcatccagtggatgcattgagggaattgattgt cagggctgtctctaggtttaatctcagacctctctgtgcttagggcaaacactatttggccttaaatgg gatcctgtgagagggggtccctccattgacagctggactgttctttggggccttatgtggtgtttgcc tctgaggtactcaggggcatttaggtttttcctcattcttaaataata 30 Phopivirus gggagtaaacctcaccaccgtttgccgtggtttacggctacctatttttggatgtaaatattaattcctg caggttcaggtctcttgaattatgtccacgctagtggcactctcttacccataagtgacgccttagcg gaacctttctacacttgatgtggttaggggttacattatttccctgggccttctttggccctttttcccctg cactatcattctttcttccgggctctcagcatgccaatgttccgaccggtgcgcccgccggggttaa ctccatggttagcatggagctgtaggccctaaaagtgctgacactggaactggactattgaagcat acactgttaactgaaacatgtaactccaatcgatcttctacaaggggtaggctacgggtgaaacccc ttaggttaatactcatattgagagatacttctgataggttaaggttgctggataatggtgagtttaacga caaaaaccattcaacagctgtgggccaacctcatcaggtagatgcttttggagccaagtgcgtagg ggtgtgtgtggaaatgcttcagtggaaggtgccctcccgaaaggtcgtaggggtaatcaggggca gttaggtttccacaattacaatttgaa 31 CVA10 gctcttccgatctgggttgttcccacccacagggcccactgggcgccagcactctgattccacgga atctttgtgcgcctgttttacaacccttcccaatttgtaacgtagaagcaatacacactactgatcaata gtaggcatggcgcgccagtcatgtcatgatcaagcacttctgttcccccggactgagtatcaataga ctgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacctagtagca ccatggaagctgcggagtgtttcgctcagcactttccccgtgtagatcaggtcgatgagtcactgca atccccacgggcgaccgtggcagtggctgcgttggcggcctgcctatggggcaacccataggac gctctaatgtggacatggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcg gctaatcctaactgcggagcacatgccttcaacccaggaggtggtgtgtcgtaacgggtaactctg cagcggaaccgactactttgggtgtccgtgtttccttttatccttatattggctgcttatggtgacaatc acggaattgttgccatatagctattggattggccatccggtgtctaacagagctattgtatacctatttg ttggatttactcccctatcatacaaatctctgaacactttgtgctttatactgaacttaaacacacgaaa 32 EnterovirusC ttaaaacagctctggggttgttcccaccccagaggcccacgtggcggccagtacaccggtaccac ggtacccttgtacgcctgttttatactcccctccccgtaaactagaagcacgaaacacaagttcaata gaagggggtacagaccagtaccaccacgaacaagcacttctgttcccccggtgaggtcacatag actgtccccacggtcaaaagtgactgatccgttatccgctcacgtacttcggaaagcctagtaccac cttggaatctacgatgcgttgcgctcagcactcgaccccggagtgtagcttaggctgatgagtctg gacgttccccactggtgacagtggtccaggctgcgttggcggcctacctgtggtccaaaaccaca ggacgctagtagtgaacaaggtgtgaagagcccactgagctacctgagaatcctccggcccctg aatgcggctaatcccaaccacggagcaggtaatcgcaaaccagcggtcagcctgtcgtaacgcg taagtctgtggcggaaccgactactttgggtgtccgtgtttccttttatttttatggtggctgcttatggt gacaatcatagattgttatcataaagcaaattggattggccatccggagtgagctaaactatctatttc tctgagtgttggattcgtttcacccacattctgaacaatcagcctcattagtgttaccctgttaataaga cgatatcatcacg 33 EnterovirusD ttaaaacagctctggggttgttcccaccccagaggcccacgtggcggctagtactccggtacccc ggtacccttgtacgcctgttttatactccctttcccaagtaactttagaagaaataaactaatgttcaac aggagggggtacaaaccagtaccaccacgaacacacacttctgtttccccggtgaagttgcatag actgtacccacggttgaaagcgatgaatccgttacccgcttaggtacttcgagaagcctagtatcat cttggaatcttcgatgcgttgcgatcagcactctaccccgagtgtagcttgggtcgatgagtctgga caccccacaccggcgacgtggtccaggctgcgttggcggcctacccatggctagcaccatggga cgctagttgtgaacaaggtgcgaagagcctattgagctacctgagagtcctccggcccctgaatgc ggctaatcccaaccacggagcaaatgctcacaatccagtgagtggtttgtcgtaatgcgcaagtct gtggcggaaccgactactttgggtgtccgtgtttccttttatttttattatggctgcttatggtgacaatct gagattgttatcatatagctattggattagccatccggtgatatcttgaaattttgccataactttttcaca aatcctacaacattacactacactttctcttgaataattgagacaactcata 34 EnterovirusJ ttaaaatagcctcagggttgttcccaccctgagggcccacgtggtgtagtactctggtattacggtac ctttgtacgcctattttatacccccttccccaagtaatttagaagcaagcacaaaccagttcagtagta agcagtacaatccagtactgtaatgaacaagtacttctgttaccccggaagggtctatcggtaagct gtacccacggctgaagaatgacctaccgttaaccggctacctacttcgagaagcctagtaatgccg ttgaagttttattgacgttacgctcagcacactaccccgtgtgtagttttggctgatgagtcacggcac tccccacgggcgaccgtggccgtggctgcgttggcggccaaccaaggagtgcaagctccttgga cgtcatattacagacatggtgtgaagagcctattgagctaggtggtagtcctccggcccctgaatgc ggctaatcctaactccggagcatatcggtgcgaaccagcacttggtgtgttgtaatacgtaagtctg gagcggaaccgactactttgggtgtccgtgtttcctgttttaacttttatggctgcttatggtgacaattt aacattgttaccatatagctgttgggttggccatccggattttgttataaaaccatttcctcgtgccttga cctttaacacatttgtgaacttctttaaatcccttttattagtccttaaatactaaga 35 HumanPegivirus aactgttgttgtagcaatgcgcatattgctacttcggtacgcctaattggtaggcgcccggccgacc 2 ggccccgcaagggcctagtaggacgtgtgacaatgccatgagggatcatgacactggggtgag cggaggcagcaccgaagtcgggtgaactcgactcccagtgcgaccacctggcttggtcgttcatg gagggcatgcccacgggaacgctgatcgtgcaaagggatgggtccctgcactggtgccatgcg cggcaccactccgtacagcctgatagggtggcggcgggcccccccagtgtgacgtccgtggag cgcaac 36 GBV-CGT110 tgacgtgggggggttgatTTTccccccccggcactgggtgcaagccccagaaaccgacgcct atctaagtagacgcaatgactcggcgccgactcggcgaccggccaaaaggtggtggatgggtga tgacagggttggtaggtcgtaaatcccggtcatcctggtagccactataggtgggtcttaagagaa ggtcaagattcctcttacgcctgcggcgagaccgcgcacggtccacaggtgttggccctaccggt gtgaataagggcccgacatcaggc 37 GBV-CK1737 gacgtgggggggttgatccccccccTTTggcactgggtgcaagccccagaaaccgacgccta tttaaacagacgttaagaaccggcgccgacccggcgaccggccaaaaggtggtggatgggtgat gccagggttggtaggtcgtaaatcccggtcatcttggtagccactataggtgggtcttaagggttgg ttaaggtccctctggcgcttgtggcgagaaagcgcacggtccacaggtgttggccctaccggtgt gaataagggcccgacgtcaggctcgtcgttaaaccgagcccactacccacctgggcaaacaacg cccacgtacggtccacgtcgcccttcaatgtctctcttgaccaataggcttagccggcgagttgaca aggaccagtgggggctgggcggtaggggaaggacccctgccgctgcccttcccggtggagtg ggaaatgc 38 GBV-CIowa tgacgtgggggggttgatccGccccccccggcactCggtgcaagccccataaaccgacgccta tctaagtagacgcaatgactcggcgccgactcggcgaccggccaaaaggtggtggatgggtggt gacagggttggtaggtcgtaaatcccggtcatcctggtagccactataggtgggtcttaagagaag gtcaagactcctcttgtgcctgcggcgagaccgcgcacggtccacaggtgctggccctaccggtg tgaataagggcccgacgtcaggctcgtcgttaaaccgagcccgtcacccacctgggcaaacgac gcccacgtacggtccacgtcgcccttca 39 PegivirusA1220 tgtagcaatgcgcatattgctacttcggtacgcctaattggtaggcgcccggccgaccggccccgc aagggcctagtaggacgtgtgacaatgccatgcgggatcatgacactggggtgagcggaggca gcaccgaagtcgggtgaactcgactcccagtgcgaccacctggcttggtcgttcatggagggcat gcccacgggaacgctgatcgtgcaaagggatgggtccctgcactggtgccatgcgcggcacca ctccgtacagcctgatagggtggcggcgggcccccccagtgtgacgtccgtggagcgcaac 40 PasivirusA3 attttctggccgtgtagctgcttttgaccagtggctctgggttacttagccaaatcccccttccttcacc cttttaaatttgatggtctgtgttgtttgttttgtcttgtctaaataatatataagatccttcccgccgataca gacctcgacagtctggtgtaggagggttggtgttattaatttgccccagaagagtgaccgtgacac atagaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgcc atcccaacaagcggagggtatacccactatgtgcgcgtttgcagtgggaatctgcaaatttagtcat actgcctgatagggtgtgggcctgcactctggggtactcaggctgttcatataat 41 Sapelovirus cccctccacccttaaggtggttgtatcccacataccccaccctcccttccaaagtggacggacaact ggattttgactaacggcaagtctgaatggtatgatttggatacgtttaaacggcagtagcgtggcga gctatggaaaaatcgcaattgtcgatagccatgttagtgacgcgcttcggcgtgctcctttggtgatt cggcgactggttacaggagagtaggcagtgagctatgggcaaacctctacagtattacttagagg gaatgtgcaattgagacttgacgagcgtctctttgagatgtggcgcatgctcttggcattaccatagt gagcttccaggttgggaaacctggactgggcctatactacctgatagggtcgcggctggccgcct gtaactagtatagtcagttgaaaccccccc 42 RosavirusB gtctctttagtgtctatgcttcagagagcggtgaactgacaccgttgcttcttgcacagcccttcgtgc cggtctttccggttctcgacagcgttgggcatcatggctagttaggctaagatagtggatgatctagt gaacagttttggattgtttggagttttgtagcgatgctagtagtgtgtgtggacctccccacgtggtaa cacgtgccccacaggccaaaagccaaggtgttgaaagcacccctactagtcccagactcacccat ctgggaactcctctcatgaaaaatcttagtaacttttgattcggctattcatcaacctctctagtcaagg gctgaaggatgcccggaaggtacccgcaggtaacgataagctcactgtggatctgatccggggc tttggtgcgaccgtctgtccggcgtagccagagttaaaaaacgtctaggcccttccaccccaaggg attggggtttccccaatcatttgaaagttcact 43 BakunsaVirus ttttgaacgccacctcggagcgatatccggggaccccctcccctttttccttcctaccttcttcccaaa tttccctcttcccttgttattttggtttggatttcctggacatgactcggacggatctatctcatttgctttgt gtctgctccaccagtggcatggtcgaaagatcatcaacactggacgtgtactgtaatggccaaacg tgcccacaggggaaaccatgccggtcgctgtagcggcgggtggacgtggtggacccctctccct gctcataaactttgggtaggtgaagggttcaagcgacgcttgccgtgagggcgcatccggatggt gggaaccaacaaactaggctgtaatggccgacctcaggtggatgagctagggctgctgcaccaa aagggactcgattcgatatcccggcctggtagcctagtgcagtggactcgtagttgggaatctacg actggcctagtacagggtgatagccccgtttcccacgcccacctgttgtagggacacccccccc 44 TremovirusA tttgaaagaggcctccggagtgtccggaggctctctttcgacccaacccatactggggggtgtgtg ggaccgtacctggagtgcacggtatatatgcattcccgcatggcaagggcgtgctaccttgcccct tgacgcatggtatgcgtcatcatttgccttggttaagccccatagaaacgaggcgtcacgtgccga aaatccctttgcgtttcacagaaccatcctaaccatgggtgtagtatgggaatcgtgtatggggatga ttaggatctctcgtagagggataggtgtgccattcaaatccagggagtactctggctctgacattgg gacatttgatgtaaccggacctggttcagtatccgggttgtcctgtattgttacggtgtatccgtcttgg cacactgaaagggtatttttgggtaatcctttcctactgcctgatagggtggcgtgcccggccacga gagattaagggtagcaatttaaac 45 SwinePasivirus1 gcttttgaccagtggctctgggttacttagccaagtccctttctcttattttcactagtttatgttgtgtgtt gtctgttttgttttgtttaaattgtatacaagatccttcccgccgacacagacctcgacagtctggtgta ggagggttggtgatattaatttgccccaaaagagtgaccgtgatacgtggaaaccatgagtacatgt gtatccgtggaggatcgcccgggactggattccatatcccattgccatcccaacaaacggagggt atacccaccacgtgcgcgtttgcagtgggaatctgcaaatttagtcatactgcctgatagggtgtgg gcctgcactttggggtactcaggctgttcatataat 46 PLV-CHN acatggggtatgttgtctgtcctgttttgttgaaacaatatataagatcctttccgccgatatagacctc gacagtctagtgtaggaggattggtgatagtaacttgccccagaagagtgaccgtgacacataga aaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgccatccc aacaaacggagggtatacccactatgtgcgcgtttgcagtgggagcctgcaaatttagtcatactg cctgatagggtgtgggcctgcactctggggtactcaggctgtttatataat 47 PasivirusA tgaaaaagtggttgtgcagctggattttccggctgtgcaactgcttttgaccagtggctctgggttact (longer) tagccaaattcctttcccttatccctattggtttgtgttgtgtgttgtttgttttgttttgtcttaactatataca agatccttcccgccgatacagacctcgacagtctggtgtaggagggttggtgttattaatttgcccca aaagagtgaccgtgacacgtggaaaccatgagtacatgtgtatccgtggaggatcgcccgggact ggattccatatcccattgccatcccaacaaacggagggtatacccaccacgtgcgcgtttgcagtg ggaatctgcaaatttagtcatactgcctgatagggtgtgggcctgcactttggggtactcaggctgtt tatataat 48 Sicinivirus gtgtcattaaggtgtgtttggaagttcgaattagctggtttgtggtgattagtagaccccctggaggta cccaattcggatctgaccagggacccgtgactataccgctccggtaattcgggtttaaaacaatga acgtcaccacacaattacttttctcattttattttcatcattgtcttcctatttaccgattacactcgatttcct tggatgttcctggagatttccctggttacctggaccctcattattgttgttgtttcacccagcgagctgt cccaattgcttattatttgcgcttacaacttcgtcctaatatttttctggttgatcgggttgattgagctcc cgggctatcctgccattcaac 49 HepacivirusK gggaacaatggtccgtccgcggaacgactctagccatgagtctagtacgagtgcgtgccacccat tagcacaaaaaccactgactgagccacacccctcccggaatcctgagtacaggacattcgctcgg acgacgcatgagcctccatgccgagaaaattgggtatacccacgggtaaggggggccacccag cgggaatctgggggctggtcactgactatggtacagcctgatagggtgctgccgcagcgtcagtg gtatgcggctgttcatggaac 50 HepacivirusA acctccgtgctaggcacggtgcgttgtcagcgttttgcgcttgcatgcgctacacgcgtcgtccaac gcggagggaacttcacatcaccatgtgtcactccccctatggagggttccaccccgcttacacgga aatgggttaaccatacccaaagtacgggtatgcgggtcctcctagggcccccccggcaggtcga gggagctggaattcgtgaattcgtgagtacacgaaaatcgcggcttgaacgtctttgaccttcgga gccgaaatttgggcgtgccccacgaaggaaggcgggggcggtgttgggccgccgccccctttat cccacggtctgataggatgcttgcgagggcacctgccggtctcgtagaccataggac 51 BVDV1 gtatacgagaatttgcctaggacctcgtttacaatatgggcaatctaaaattataattaggcctaagg gacaaatcctcctcagcgaaggccgaaaagaggctagccatgcccttagtaggactagcaaaata aggggggtagcaacagtggtgagttcgttggatggctgaagccctgagtacagggtagtcgtcag tggttcgacgcttcggaggacaagcctcgagataccacgtggacgagggcatgcccacagcaca tcttaacctggacgggggtcgttcaggtgaaaacggtttaaccaaccgctacgaatacagcctgat agggtgctgcagaggcccactgtattgctactgaaaatctctgctgtacatggcac 52 BorderDisease gtatacgggagtagctcatgcccgtatacaaaattggatattccaaaactcgattgggttagggagc Virus cctcctagcgacggccgaaccgtgttaaccatacacgtagtaggactagcagacgggaggacta gccatcgtggtgagatccctgagcagtctaaatcctgagtacaggatagtcgtcagtagttcaacg caggcacggttctgccttgagatgctacgtggacgagggcatgcccaagacttgctttaatctcgg cgggggtcgccgaggtgaaaacacctaacggtgttggggttacagcctgatagggtgctgcaga ggcccacgaataggctagtataaaaatctctgctgtacatggcac 53 BVDV2 gtatacgagattagctaaagtactcgtatatggattggacgtcaacaaatttttaattggcaacgtagg gaaccttcccctcagcgaaggccgaaaagaggctagccatgccctttagtaggactagcaaaagt agggggactagcggtagcagtgagttcgttggatggccgaacccctgagtacaggggagtcgtc aatggttcgacactccattagtcgaggagtctcgagatgccatgtggacgagggcatgcccacgg cacatcttaacccatgcgggggttgcatgggtgaaagcgctaatcgtggcgttatggacacagcct gatagggtgtagcagagacctgctattccgctagtaaaaaactctgctgtacatggcac 54 CSFV-PK15C gtatacgaggttagttcattctcgtatgcattattggacaaatcaaaatttcaatttggttcagggcctc cctccagcgacggccgaactgggctagccatgcccatagtaggactagcaaacggagggacta gccgtagtggcgagctccctgggtgttctaagtcctgagtacaggacagtcgtcagtagttcgacg tgagcagaagcccacctcgagatgctatgtggacgagggcatgcccaagacgcaccttaaccct agcgggggtcgctagggtgaaatcacaccacgtgatgggagtccgacctgatagggtgctgcag aggctcactattaggctagtataaaaatctctgctgtacatggcac 55 SF573 aaaaccgaccccagagatcagaaagtcgttgacgcgatcttttattagaggacgttgcgctggcgc Dicistrovirus gagctttaattagcagacgccaaaaataaacaacaaaatgctgatcgcgagacttaattgtcagac gattggccaaatccgatgtgatctttgctgctcccagattgccgaaataggagtagtag 56 HubeiPicorna- ccccaaaaccccccccttaaactcaacactgtagtggattcattttccgttgcaaaacaaaacattac likeVirus tacccgcatttatgtaggctctgtgttttctatgcgaccgttacattaatctctactctgacccactagttt ataaaaccgaagacctgaatgaaacgattttccttcttttcaacctctaacgaacctctgacggcttga gaaacctgaagttagtaattatgtttaaaagaaaggaaagtcaaacgcgatgactcttacatccctat tccataccgttgctccacaatgtgagcgatgcgaggtcgggactgcagtattaggggaacgagct acatggagagttaattatctctcccctcctacgggagtctcatgtgagctgtagaaagcggttggca cctctcgttacctcgcctgtacatgatcc 57 CRPV aaaagcaaaaatgtgatcttgcttgtaaatacaattttgagaggttaataaattacaagtagtgctatttt tgtatttaggttagctatttagctttacgttccaggatgcctagtggcagccccacaatatccaggaag ccctctctgcggtttttcagattaggtagtcgaaaaacctaagaaatttacct 58 SalivirusABN5 tttcctcctttcgaccgccttacggcaggcgggtccgcggacggcttcggcctacccgcgacaag aatgccgtcatctgtccttatcacccatattctttcccttcccccgcaaccatcacgcttactcgcgca cgtgttgagtggcacgtgcgttgtccaaacagttacactcacacccttggggcgggtttgtcccgcc ctcgggttcctcgcggaaccctccctcttctctctccctttctatccgccttcactttccataactacagt gctttggtaggtaagcatcctgaccccccgcggaagctgccaacgtggcaactgtggggatccag gcaggttatcaaaggcacccggtctttccgccttcaggagtatccctgccggtgaattccgacagg gctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcatcttagt aacctctcatgtgtgtgcttggtcagcatatctgaggcgacgttccgctgtcccagaccagtccagc aatggacgggccagtgtgcgtagtcgctttccggtttcccggcgcatgtttggcgaaacgctgagg taaggttggtgtgcccaatgcccgtaatttggtgacacctcaagaccacccaggaatgccaggga ggtaccccacttcggtgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctc ttttttctggcatg 59 SalivirusABN2 tatggcaggcgggcttgtggacggcttcggcccacccacagcaagaatgccatcatctgtcctca cccccatgtttcccctttctttccctgcaaccgttacgcttactcgcaggtgcatttgagtggtgcacgt gttgaataaacagctacactcacatggggggggttttcccgccctgcggcctctcgcgaggccc acccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagct gctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcagg agtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgctgcg ggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgat gttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttttcc ggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgatacct caagaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctgggctaat tgtctacggtggttcttcttgcttccacttctttctactgttc 60 SalivirusA tttcgaccgccttatggcaggcgggcttgtggacggcttcggcccacccacagcaagaatgccat 02394 catctgtcctcacccccatttctcccctccttcccctgcaaccattacgcttactcgcatgtgcattgag tggtgcacgtgttgaacaaacagctacactcacgtggggggggttttcccgcccttcggcctctc gcgaggcccacccttccccttcctcccataactacagtgctttggtaggtaagcatcctgatccccc gcggaagctgctcgcgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttcc gcctccaggagtatccctgctagtgaattctagtggggctctgcttggtgccaacctcccccaaatg cgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatat ctgagacgatgttccgctgtcccagaccagtccagcaatggacgggccagtgtgcgtagtcgtctt ccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactt tggtgacaactcaagaccacccaggaatgccagggaggtaccccgcctcacgggggatctga ccctgggctaattgtctacggtggttcttcttgcttccatttctttcttctgttc 61 SalivirusAGUT tatggcaggcgggcttgtggacggtttcggcccacccacagcaagaatgccatcatctgtcctcac ccccaattttccctttcttcccctgcaatcatcacgcttactcgcatgtgcattgagtggtgcatgtgtt gaacaaacagctacactcacatggggggggttttcccgccctacggcctctcgcgaggcccac ccttcccctccccttataactacagtgctttggcaggtaagcatcctgatcccccgcggaagctgct cacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagc atccccactagtgaattctagtggggctctgcttggtgccaacctcccccaaatgcgcgctgcggg agtgctcttccccaacccatcctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgacgt tccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttgtccg gcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgacacctc aagaccacccaggaatgccagggaggtaccccgcctcacggcgggatctgaccctgggctaatt gtctacggtggttcttcttgcttccacttctttctt 62 SalivirusACH ttctcctgcaaccattacgcttaatcgcatgtgcattgagtggtgcatgtgttgaacaaacagctaca atcacatggggggggttttcccgccccacggcttctcgcgaggcccatccctcccttttctcccat aactacagtgctttggtaggtaagcatcccgatctcccgcggaagctgctcacgtggcaactgtgg ggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagcgaatt ctagtagggctctgcttggtgccaacctctcccaaatgcgcgctgcgggagtgctcttccccaaatc accccagtatcctctcatgtgtgtgcctggtcagcatatctgagacgatgttccgctgtcccagacca gtccagtaatggacgggccagtgtgcgtagtcgtcctccggcttgtccggcgcatgtttggtgaac cggtggggtaaggttggtgtgcccaacgcccgtaatcaggggatacctcaaggcacccaggaat gccagggaggtatcccgcctcacagcgggatctgaccctggggtaaatgtctgcggggggtcct cttggcccaattctcagtaattttcagg 63 SalivirusASZ1 tctgtcctcaccccatcttcccttctttcctgcaccgttacgcttactcgcatgtgcattgagtggtgca cgtgcttgaacaaacagctacactcacatggggggggttttcccgccctgcggcctctcgcgag gcccacccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcgga agctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttc aggagtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgct gcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgaga cgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggctt gtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgat acctcaagaccacccaggaatgccagggaggtaccccgcttcacaggggatctgaccctggg ctaattgtctacggtggttcttcttgcttccacttctttctactgttcatg 64 SalivirusFHB acatggggggtctgcggacggcttcggcccacccgcgacaagaatgccgtcatctgtcctcatta cccgtattccttcccttcccccgcaaccaccacgcttactcgcgcacgtgttgagtggcacgtgcgt tgtccaaacagctacacccacacccttcggggcgggtttgtcccgccctcgggttcctcgcggaa cccccccctccctctctctctttctatccgccctcacttcccataactacagtgctttggtaggtgagc accctgaccccccgcggaagctgctaacgtggcaactgtggggatccaggcaggttatcaaagg cacccggtctttccgccttcaggagtatctctgccggtgaattccggtagggctctgcttggtgcca acctcccccaaatgcgcgctgcgggagtgctcttccccaactcatcttagtaacctctcatgtgtgtg cttggtcagcatatctgaggcgacgttccgctgtcccagaccagtccagcaatggacgggccagt gtgcgtagtcgctttccggttttccggcgcatgtttggcgaaacgctgaggtaaggttggtgtgccc aacgcccgtaatttggtgatacctcaagaccacccaggaatgccagggaggtaccccacttcggt gggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctcttttttctggcatg 65 CVB3 ttaaaacagcctgtgggttgatcccacccacaggcccattgggcgctagcactctggtatcacggt acctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaaca gtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaataga ctgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaaca ccgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgc attccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatggg acgctctaatacagacatggtgcgaagagtctattgagctagttggtagtcctccggcccctgaatg cggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaac tctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtgac aattgagagatcgttaccatatagctattggattggccatccggtgactaatagagctattatatatcc ctttgttgggtttataccacttagcttgaaagaggttaaaacattacaattcattgttaagttgaatacag caaa 66 CVB1 ttaaaacagcctgtgggttgttcccacccacaggcccattgggcgctagcactctggtatcacggta cctttgtgcgcctgttttacatcccctccccaaattgtaatttagaagtttcacacaccgatcattagca agcgtggcacaccagccatgttttgatcaagcacttctgttaccccggactgagtatcaatagaccg ctaacgcggttgaaggagaaaacgttcgttacccggccaactacttcgaaaaacctagtaacacca tggaagttgcggagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgcgttc cccacgggcgaccgtggcggtggctgcgttggcggcctgcctacggggaaacccgtaggacg ctctaatacagacatggtgcgaagagtctattgagctagttggtaatcctccggcccctgaatgcgg ctaatcctaactgcggagcacataccctcaaaccagggggcagtgtgtcgtaacgggcaactctg cagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtgacaatt gacaggttgttaccatatagttattggattggccatccggtgactaacagagcaattatatatctctttg ttgggtttataccacttagcttgaaagaggttaaaacactacatctcatcattaaactaaatacaacaa a 67 Echovirus7 ttaaaacagcctgtgggttgttcccacccacagggcccattgggcgtcagcaccctggtatcacgg tacctttgtgcgcctgttttatatcccttcccccaattgtaacttagaagaaacacacaccgatcaaca gcaagcgtggcacaccagccatgttttggtcaagcacttctgttaccccggactgagtatcaataga ctgctcacgcggttgaaggagaaagcgtccgttatccggccagctacttcgagaaacctagtaac accatggaagttgcggagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccg ctttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatgggggaacccatagg acgctctaatacagacatggtgcgaagagtctattgagctagctggtattcctccggcccctgaatg cggctaatcctaactgtggagcacatgcccctaatccaaggggtagtgtgtcgtaatgagcaattcc gcagcggaaccgactactttgggtgtccgtgtttcctcttattcttgtactggctgcttatggtgacaat tgagagattgttaccatatagctattggattggccatccggtgactaatagagctattgtgtatctcttt gttggatttgtaccacttaatttgaaagaaatcaggacactacgctacattttactattgaacaccgca aa 68 CVB5 ttaaaacagcctgtgggttgtacccacccacagggcccactgggcgctagcactctggtatcacg gtacctttgtgcgcctgttttatgcccccttcccccaattgaaacttagaagttacacacaccgatcaa cagcgggcgtggcataccagccgcgtcttgatcaagcactcctgtttccccggaccgagtatcaat agactgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacctagta gcatcatgaaagttgcgaagcgtttcgctcagcacatccccagtgtagatcaggtcgatgagtcac cgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcctacggggcaacccgt aggacgcttcaatacagacatggtgcgaagagtcgattgagctagttagtagtcctccggcccctg aatccggctaatcctaactgcggagcacataccctcaacccagggggcattgtgtcgtaacgggt aactctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttataatggctgcttatggtg acaattgaaagattgttaccatatagctattggattggccatccggtgtctaacagagctattatatac ctctttgttggatttgtaccacttgatctaaaggaagtcaagacactacaattcatcatacaattgaaca cagcaaa 69 EVA71 ttaaaacagcctgtgggttgcacccactcacagggcccactgggcgcaagcactctggcacttcg gtacctttgtgcgcctgttttatatcccctcccccaatgaaatttagaagcagcaaaccccgatcaata gcaggcataacgctccagttatgtcttgatcaagcacttctgtttccccggactgagtatcaatagac tgctcacgcggttgaaggagaaaacgttcgttatccggctaactacttcggaaagcctagtaacac catggaagttgcggagagtttcgttcagcacttccccagtgtagatcaggtcgatgagtcaccgcat tccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggtaacccatgggac gctctaatacggacatggtgtgaagagtctactgagctagttagtagtcctccggcccctgaatgcg gctaatcccaactgcggagcacacgcccacaagccagtgggtagtgtgtcgtaacgggcaactct gcagcggaaccgactactttgggtgtccgtgtttccttttattcttatgttggctgcttatggtgacaatt aaagagttgttaccatatagctattggattggccatccggtgtgcaacagagcgatcgtttacctattt attggttttgtaccattgacactgaagtctgtgatcacccttaattttatcttaaccctcaacacagccaa ac 70 CVA3 ttaaaacagcctgtgggttgtacccacccacagggcccactgggcgctagcacactggtattacg gtacctttgtgcgcctgttttataccccccccaacctcgaaacttagaagtaaagcaaacccgatca atagcaggtgcggcgcaccagtcgcatcttgatcaagcacttctgtaaccccggaccgagtatcaa tagactgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacccagt agcatcatgaaagttgcagagtgtttcgctcagcactacccccgtgtagatcaggccgatgagtca ccgcacttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatggggcaaccca taggacgctctaatacggacatggtgcgaagagtctattgagctagttagtagtcctccggcccctg aatgcggctaatcctaactgcggagcacatacccttaatccaaagggcagtgtgtcgtaacgggta actctgcagcggaaccgactactttgggtgtccgtgtttccttttaatttttactggctgcttatggtgac aattgaggaattgttgccatatagctattggattggccatccggtgactaacagagctattgtgttcca atttgttggatttaccccgctcacactcacagtcgtaagaacccttcattacgtgttatttctcaactcaa gaaa 71 CVA12 ttaaaacagcctgtgggttgtacccacccacagggcccactgggcgctagcactctggtactacg gtacctttgtgtgcctgttttaagcccctaccccccactcgtaacttagaaggcttctcacactcgatc aatagtaggtgtggcacgccagtcacaccgtgatcaagcacttctgttaccccggtctgagtacca ataagctgctaacgcggctgaaggggaaaacgatcgttatccggctaactacttcgagaaaccca gtaccaccatgaacgttgcagggtgtttcgctcggcacaaccccagtgtagatcaggtcgatgagt caccgtattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggtgaccc atgggacgctctaatactgacatggtgcgaagagtctattgagctagttagtagtcctccggcccct gaatgcggctaatcctaactgcggagcacatacccttaatccaaagggcagtgtgtcgtaacggg caactctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttacattggctgcttatggt gacaattgaaaagttgttaccatatagctattggattggccatccggtgacaaatagagctattgtata tctttttgttggttacgtaccccttaattacaaagtggtttcaactttgaaatacatcctaacactaaattg tagaaa 72 EV24 ttaaaacagcctgtgggttgcacccacccacagggcccacagggcgctagcactctggtatcacg gtacctttgtgcgcctgttttattaccccttccccaattgaaaattagaagcaatgcacaccgatcaac agcaggcgtggcgcaccagtcacgtctcgatcaagcacttctgtttccccggaccgagtatcaata gactgctcacgcggttgaaggagaaagtgttcgttatccggctaaccacttcgagaaacccagtaa caccatgaaagttgcagggtgtttcgctcagcacttccccagtgtagatcaggtcgatgagtcacc gcgttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatgggttaacccatag gacgctctaatacagacatggtgcgaagagtttattgagctggttagtatccctccggcccctgaat gcggctaatcctaactgcggagcacgtgcctccaatccagggggttgcatgtcgtaacgggtaac tctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttatactggctgcttatggtgaca atcgaggaattgttaccatatagctattggattggccatccggtgtctaacagagcgattatatacctc tttgttggatttatgcagctcaataccaccaactttaacacattgaaatatatcttaaagttaaacacag caaa 348 AP1.0 attctcgggctacggccctggagccactccggctcctaaagatttagaagtttgagcacacccgcc cactagggccccccatccaggggggcaacgggcaagcacttctgtttccccggtatgatctgata ggctgtaaccacggctgaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatg atgggtgaaattgaatccgttgatccggtgtctcccccacaccagaaactcatgatgagggttgcca tcccggctacggcgacgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatg atatggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggcta atcctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctggga cggaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctg ggtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttatta aatttacacagagaagaattaca 349 CK1.0 gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccact tgctctgTcacG 350 PV1.0 aacaaaaggctacaccacttgggctacggcccgcgccaccttgtggcgcaaagacattagaaga atagcataccgcccactagggccctgcagccagcagggtaacgggcaagcacttctgtctcccc ggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcg caaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaac gcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcgg cctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccg cctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgta acgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgt atggtgacaagttatagatctcgatctatagcgtttcttgagagatttccaaacatttattcaagtcgta caattcttgtgtttaagcagtacagtgtaacc 351 SV1.0 tctgtcctcaccccatcttcccttctttcctgcaccgttacgcttactcgcatgtgcattgagtggtgca cgtgcttgaacaaacagctacactcacatggggggggttttcccgccctgcggcctctcgcgag gcccacccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcgga agctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttc aggagtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgct gcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgaga cgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggctt gtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgat acctcaagaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctggg ctaattgtctacggtggttcttcttgcttccacttctttctactgttcgccacc 352 Caprine gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac Kobuvirus5?40 agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttttttcacacaactctactgctgacaactcactgactatccact tgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctca caatccttgctcagttcaaagtc 353 Caprine gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac Kobuvirus agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa 5?40/3?122 gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactaaagtc 354 Caprine gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac Kobuvirus agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggggcaa 5?40/3?86_Dista1 gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccact tgctctaaagtc 355 Caprine gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac Kobuvirus agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa 5?40/3?122_ gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg Kozak ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactgccacc 356 Caprine gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac Kobuvirus agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa 5?40/3?86 gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg Proximal ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactttcactgcttttcttctcacaatccttgctca gttcaaagtc 357 Parabovirus tgaaccgttacgcaccactcagttggtgtttggtggcaccaatgatggaacaaaaggctacaccac ttgggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactag ggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgt acccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactg gaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccacc ccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgct cctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatc ctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcgga accgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatct cgatctatagcgtttcttgagagatttccaaacatttattcaagtcgtacaattcttgtgtttaagcagta cagtgtaagg 358 Parabovirus5?48 aacaaaaggctacaccacttgggctacggcccgcgccaccttgtggcgcaaagacattagaaga atagcataccgcccactagggccctgcagccagcagggtaacgggcaagcacttctgtctcccc ggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcg caaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaac gcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcgg cctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccg cctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgta acgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgt atggtgacaagttatagatctcgatctatagcgtttcttgagagatttccaaacatttattcaagtcgta caattcttgtgtttaagcagtacagtgtaagg 359 Parabovirus5?67 tgggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactag ggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgt acccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactg gaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccacc ccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgct cctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatc ctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcgga accgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatct cgatctatagcgtttcttgagagatttccaaacatttattcaagtcgtacaattcttgtgtttaagcagta cagtgtaagg 360 Parabovirus3?60 tgaaccgttacgcaccactcagttggtgtttggtggcaccaatgatggaacaaaaggctacaccac ttgggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactag ggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgt acccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactg gaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccacc ccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgct cctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatc ctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcgga accgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatct cgatctatagcgtttgtaagg 361 Apodemus tttgaaaggggtgcggatatcatggcgtttctcgccatgatatccgcacattgcaaacccatattgca Picornavirus tacccactgggtatgcattatggggaggcccctttcacccctccccccccaattaccttttccccctct agtaaccatacgctttactcagcgtaactactccgggttacgtgatgaagaagaggctacggagatt ctcgggctacggccctggagccactccggctcctaaagatttagaagtttgagcacacccgccca ctagggccccccatccaggggggcaacgggcaagcacttctgtttccccggtatgatctgatagg ctgtaaccacggctgaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatgatg ggtgaaattgaatccgttgatccggtgtctcccccacaccagaaactcatgatgagggttgccatcc cggctacggcgacgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatgata tggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggctaatc ctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctgggacg gaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctgg gtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttattaa atttacacagagaagaattaca 362 Apodemus cccctccccccccaattaccttttccccctctagtaaccatacgctttactcagcgtaactactccggg Picornavirus ttacgtgatgaagaagaggctacggagattctcgggctacggccctggagccactccggctccta 5?105 aagatttagaagtttgagcacacccgcccactagggccccccatccaggggggcaacgggcaa gcacttctgtttccccggtatgatctgataggctgtaaccacggctgaaacagagattatcgttatcc gcttcactacttcgagaagcctagtaatgatgggtgaaattgaatccgttgatccggtgtctccccca caccagaaactcatgatgagggttgccatcccggctacggcgacgtagcgggcatccctgcgct ggcatgaggcctcttaggaggacggatgatatggatcttgtcgtgaagagcctattgagctagtgtc gactcctccgcccccgtgaatgcggctaatcctaaccccggagcaggtgggtccaatccagggc ctggcctgtcgtaatgcgtaagtctgggacggaaccgactactttcgggaaggcgtgtttccatttgt tcattatttgtgtgtttatggtgacaactctgggtaaacgttctattgcgtttattgagagattcccaaca attgaacaaacgagaactacctgttttattaaatttacacagagaagaattaca 363 Apodemus attctcgggctacggccctggagccactccggctcctaaagatttagaagtttgagcacacccgcc Picornavirus cactagggccccccatccaggggggcaacgggcaagcacttctgtttccccggtatgatctgata 5?201 ggctgtaaccacggctgaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatg atgggtgaaattgaatccgttgatccggtgtctcccccacaccagaaactcatgatgagggttgcca tcccggctacggcgacgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatg atatggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggcta atcctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctggga cggaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctg ggtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttatta aatttacacagagaagaattaca 364 Kobuvirus tttcacaccctcttttccggtggtccggacccagaccaccgttactccattcagctacttcggtacctg SZAL6 ttcggaggaattaaacgggcaccctacccaagggttacatgggaccatattcctcctcccctgtaac tttaagttttgtgcccgtattcttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagct agacactttcctcccctccctctgggctgctccggcagtccactccctccccccagcgtaacatgcc ccgctggagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataa tgacaactggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccac atgaatttgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttc cccaagcgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgc gtcgtgccaacctggcgacaggtgacccagtgtgcgtagacttcttccggattcgtccggctcttct ctaggaaacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctagg aatgtgccgcaggtacccctacttcggtagggatctgagcggtagctaattgtctacgggtagtttc atttccatcttctcttcaggtcgacatc 365 Kobuvirus ttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacactttcctcccctccct SZAL65?158 ctgggctgctccggcagtccactccctccccccagcgtaacatgccccgctggagtgatgcacct ggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaactggttgaccccc acactcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatttgcagctggcaac tagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaagcgtatgccttccttc tgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtgccaacctggcgac aggtgacccagtgtgcgtagacttcttccggattcgtccggctcttctctaggaaacatgcgtgtaa ggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtgccgcaggtaccc ctacttcggtagggatctgagcggtagctaattgtctacgggtagtttcatttccatcttctcttcaggt cgacatc 366 Kobuvirus gaattaaacgggcaccctacccaagggttacatgggaccatattcctcctcccctgtaactttaagtt SZAL65?76 ttgtgcccgtattcttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacact ttcctcccctccctctgggctgctccggcagtccactccctccccccagcgtaacatgccccgctg gagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaa ctggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatt tgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaag cgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtg ccaacctggcgacaggtgacccagtgtgcgtagacttcttccggattcgtccggctcttctctagga aacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtg ccgcaggtacccctacttcggtagggatctgagcggtagctaattgtctacgggtagtttcatttcca tcttctcttcaggtcgacatc 367 Kobuvirus tttcacaccctcttttccggtggtccggacccagaccaccgttactccattcagctacttcggtacctg SZAL63?37 ttcggaggaattaaacgggcaccctacccaagggttacatgggaccatattcctcctcccctgtaac tttaagttttgtgcccgtattcttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagct agacactttcctcccctccctctgggctgctccggcagtccactccctccccccagcgtaacatgcc ccgctggagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataa tgacaactggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccac atgaatttgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttc cccaagcgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgc gtcgtgccaacctggcgacaggtgacccagtgtgcgtagacttcttccggattcgtccggctcttct ctaggaaacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctagg aatgtgccgcaggtacccctacttcggtagggatctgagcggtagctaattggacatc 368 SalivirusSZ1 tctgtcctcaccccatcttcccttctttcctgcaccgttacgcttactcgcatgtgcattgagtggtgca cgtgcttgaacaaacagctacactcacatggggggggttttcccgccctgcggcctctcgcgag gcccacccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcgga agctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttc aggagtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgct gcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgaga cgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggctt gtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgat acctcaagaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctggg ctaattgtctacggtggttcttcttgcttccacttctttctactgttcatg 369 CrohivirusB gtataagagacaggtgtttgccttgtcttcggactggcatcttgggaccaaccccccttttccccagc catgggttaaatggcaataaaggacgtaacaactttgtaaccattaagctttgtaattttgtaaccact aagctttgtgcacataatgtaaccatcaagcttgttagtcccagcaggaggtttgcatgcttgtagcc gaaatggggctcgaccccccatagtaggatacttgattttgcattccattgtggacctgcaaactcta cacatagaggctttgtcttgcatctaaacacctgagtacagtgtgtacctagaccctatagtacggga ggaccgtttgtttcctcaataaccctacataataggctaggtgggcatgcccaatttgcaagatccca gactgggggtcggtctgggcagggttagatccctgttagctactgcctgatagggtggtgctcaac catgtgtagtttaaattgagctgttcatatacc 370 CrohivirusB ccccccttttccccagccatgggttaaatggcaataaaggacgtaacaactttgtaaccattaagctt 5?51 tgtaattttgtaaccactaagctttgtgcacataatgtaaccatcaagcttgttagtcccagcaggagg tttgcatgcttgtagccgaaatggggctcgaccccccatagtaggatacttgattttgcattccattgt ggacctgcaaactctacacatagaggctttgtcttgcatctaaacacctgagtacagtgtgtacctag accctatagtacgggaggaccgtttgtttcctcaataaccctacataataggctaggtgggcatgcc caatttgcaagatcccagactgggggtcggtctgggcagggttagatccctgttagctactgcctg atagggtggtgctcaaccatgtgtagtttaaattgagctgttcatatacc 371 CVB3 ttaaaacagcctgtgggttgatcccacccacagggcccattgggcgctagcactctggtatcacgg tacctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaac agtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaatag actgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaac accgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccg cattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatgg gacgctctaatacagacatggtgcgaagagtctattgagctagttggtagtcctccggcccctgaat gcggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaa ctctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtga caattgagagattgttaccatatagctattggattggccatccggtgaccaatagagctattatatatct ctttgttgggtttataccacttagcttgaaagaggttaaaacattacaattcattgttaagttgaatacag caaa 372 CVB33?91 ttaaaacagcctgtgggttgatcccacccacagggcccattgggcgctagcactctggtatcacgg tacctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaac agtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaatag actgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaac accgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccg cattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatgg gacgctctaatacagacatggtgcgaagagtctattgagctagttggtagtcctccggcccctgaat gcggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaa ctctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtga caattgagagattgttaccatatagctattggattggccatccggtgaagcaaa 373 SAFV cacttatttaattcggccttttgtgacaagcccctcggtgaaagaacctctctcttttcgacgtggttgg aattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgttttctgccctgctgg gcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagcc gcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtac gtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgca gcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgt agcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagcccc gtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatgg aaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgc gtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaaaaattgtcac agcttctccaaaccaagtggtcttggttttccaattttgttga 374 SAFV5?46 cctctctcttttcgacgtggttggaattgccatcatttccgacgaaagtgctatcatgcctccccgatta tgtgatgttttctgccctgctgggcggagcattctcgggttgagaaaccttgaatctttttctttggaac cttggttcccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgg gttctatccgtaaaaagggtacgtgctgccccttccttctctggagaattcacacggcggtctttccgt ctctcaacaagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttaga gtgcacatcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagag cctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccct ggttcgatgatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaag aatgtcctgaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgt cactttggtttaaaaattgtcacagcttctccaaaccaagtggtcttggttttccaattttgttga 375 SAFV5?93 gtgctatcatgcctccccgattatgtgatgttttctgccctgctgggcggagcattctcgggttgaga aaccttgaatctttttctttggaaccttggttcccccggtctaagccgcttggaatatgacagggttattt tcttgatcttatttctacttttgcgggttctatccgtaaaaagggtacgtgctgccccttccttctctgga gaattcacacggcggtctttccgtctctcaacaagtgtgaatgcagcatgccggaaacggtgaaga aaacagttttctgtggaaatttagagtgcacatcgaaacagctgtagcgacctcacagtagcagcg gactcccctcttggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtg agcggtgcaaccccagcaccctggttcgatgatcattctctatggaaccagaaaatggttttctcaa gccctccggtagagaagccaagaatgtcctgaaggtaccccgcgtgcgggatctgatcaggaga ccaattggcggtgctttacactgtcactttggtttaaaaattgtcacagcttctccaaaccaagtggtct tggttttccaattttgttga 376 SAFV3?47 cacttatttaattcggccttttgtgacaagcccctcggtgaaagaacctctctcttttcgacgtggttgg aattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgttttctgccctgctgg gcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagcc gcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtac gtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgca gcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgt agcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagcccc gtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatgg aaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgc gtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaatgttga 377 SAFVKozak cacttatttaattcggccttttgtgacaagcccctcggtgaaagaacctctctcttttcgacgtggttgg aattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgttttctgccctgctgg gcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagcc gcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtac gtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgca gcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgt agcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagcccc gtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatgg aaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgc gtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaaaaattgtcac agcttctccaaaccaagtggtcttggttttccaattttgttgaccgcc 378 GLucCKdCTG1 gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactctacGTctgacaactcactgactatcca cttgctctaaagtc 379 GLucCK gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac dCTG1_2 agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttttttcacacaactctacGTcGTacaactcactgactatcc acttgctctaaagtc 380 GLucCK gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac dCTG1_2_3 agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttttttcacacaactctacGTcGTacaactcacGTactat ccacttgctctaaagtc 381 GLucCKdAll gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactctacGTcGTacaactcacGTactaC TcactGTctctaaagtc 382 CKSZ1-L1S gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgacc gggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaa ctgcttcaggtaagtggggtgtgcccaatccctacaaaggttgaaccacccaggaatgccaggga ggtaccccgcttcacagcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccactt ctttctactgttcgccacc 383 CKAichiScan gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat (AV-S) acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgacc gggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaa ctgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgct ccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcatt tccaatccttttatgtcggagtc 384 CKAichiLoop gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat (AV-L1) acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgacc gggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaa ctgcttcaggtaagtggggtgtgcccaatccctacaaaggttgaactgccctaggaatgccaggca ggtaccccacctccgggtgggatctgagcctgggctaattgtctacgggtagttttcctttttcttttca cacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagtt ccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 385 CKSZ1-L2 gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagaca ggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagtgaattctagtagggctct gcttgcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattcttt caccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgt ctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctct cttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatcc ttgctcagttcaaagtc 386 CKAichi gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat TriLoop(AV-L2) acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccaacct ggtgacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtgcgttgtccagaaactgct tcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccgg aggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttt tcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaa gttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 387 CKScan gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat Deletion(?S) acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgacc gggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaa ctgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgct ccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtg 388 CKLoop gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat Deletion(?L1) acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgacc gggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaa ctgcttcaggtaagtggggtgtgcccaatccctacaaaggttgatttcctttttcttttcacacaactct actgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgt ttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 389 CKTriloop gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat Deletion(?L2) acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt cccccttttcccaaccccaaccgccgtatctggtggggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcgcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaa aggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccg gaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgact atccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgctttt cttctcacaatccttgctcagttcaaagtc 413 RhPV gataaaagaacctataatcccttcgcacaccgcgtcacaccgcgctatatgctgctcattaggaatt acggctccttttttgtggatacaatctcttgtatacgatatacttattgttaatttcattgacctttacgcaa tcctgcgtaaatgctggtatagggtgtacttcggatttccgagcctatattggttttgaaaggaccttta agtccctactatactacattgtactagcgtaggccacgtaggcccgtaagatattataactattttatta tattttattcaccccccacattaatcccagttaaagctttataactataagtaagccgtgccgaaacgtt aatcggtcgctagttgcgtaacaactgttagtttaattttccaaaatttatttttcacaatttttagttaaga ttttagcttgccttaagcagtctttatatcttctgtatattattttaaagtttataggagcaaagttcgcttta ctcgcaatagctattttatttattttaggaatattatcacctcgtaattatttaattataacattagctttatct atttata 414 Halastaviarva cttgattctaaccttgccgtatggtgccctaacgggttcatttaatcatgcgatgagggttgctatacc (1xmut) gcatccattctaaggcgattcaatgcttcatttaggaattttgttgacgattaaaaggtacccccacaa aaacaaaaccaatcttacttgattttcgttttaactgaccactgcgatcccaaattttcgccttcttatca aagtatgttgtgttctttgggtgtacaacctgagaacttgtctacaactacatattactcgaggaagaa attcggtttaagccgtgccttctcacgtttagtatatctatctGgacacaccttcttcatcttctaatccc catctagtctcctgatcagagacgtcgttattaacaaataaccccccttgttaataagagacaaagta caatcaagctaagttctcttggagttcctgtaggaacttagccattgtgatagagtcataagtctatgt gcatagacagctctagctcaccatttccttcccaacccatcttttcatcagcttaactctatgaatccga tgcaaaaaccattctaacatcttatggtgctttccaagccaaatgagagctcactcttttgagccgcta tttaatggacaataaacgttttatagtgtacatcatattgtaaaaacaaa 415 Oscivirus cctcggtccctctttccgtcgccgcccacgacgttaaatgcggtgttgtggtgcttaggtgccacac cactgctatttgggtccccctttcccctatatatgtttgtttgtttatttcaatttcttgaggattggcacctc cttatgccaaatctaaatcgtggaggatcccaggctttctggtctttaacagaactccacgtccaggt catagaaactggttggtaggctgcctgagtagtccatttgctagtagtcccttgtgaacagggtggct cccgtttactgctggtattcccggtgtaggtcgccatggtggtaacaccatcctgcattgtgtgtgaa ccagtaccgcaaggatagcaaggtatgaacacttgtggacgaaatggtaagtgatcaattcactttc atggccggaaggtcacgtggcaatcatgccacccaggtaccctcctctgggaggatctgagggt gggctaagcagaccctgccatgtggctgaacttttcccttattgttttactttgtaacatttatagttgtgt tagtgatttgtgtgttgtgcccttgtgagctatatccagtataagttcgcagctagaagttaatccttcg acatcggctgtattggaa 416 CadicivirusB caccaacccttgacctgtaatgtcagtggacagagtgctcctctgttcccggttaccgtgttccagg acacgattgtaatcctgcgcctcaccagcgctgcgtgcacgtctgcataaggaaacgtgccttccc catgtctctatcaattctttggtgagtgaccgccctagttgctcatcctatgggattcttctctcatgggt tctttgtggcatgcgaatgtcaccttaattggaggtctttaattagatatcctttcttcatctttgatatgag tgtcggaatttgattcctagtctctgcaaaacaaccccacttgatgaattcaacttttcaaccgcacaa acataatcaggtttttaaattgaatgtttctaaattctaaatttagtttatttaagtagtttgccatcttgact cgatgtaaaattgtcatacaagtcttcttttcttttctttacactttgaagtttgcacttagcagtcgttctg cacagctttcgagttttgtttgatcgacatcgcaacttccacccacctctctttttctagtgttgaatgcg gctaatcctaacccgagagcaataaacccaggtttattgtcgtaacgcgcaagtcttggacggaac cgactatacacacacctctttaccctttagtacacccttggtacg 417 PSIV(2xmutfor gcaaaatgggtacgtagttaaccactgcgtatcaggattgcaggccacgaagggtatttgcatatct Xba1) ttctatgcggtattacggcttaaaacccgttgtatcttgtTgtttgactgcctgtatcactagtggccatt ttatttaggttagagacccctgatagtaggagagttacaaactctttaaaaattgttgaccccggaaa agatggtgacccctgtaagtagttgatcAagaagatctatgcgctggcatagtaatccagtgtttcc tgttttaggatgacctctgaaagtagatgaccgtggaaagtcacgtagtgccccaataagcacgttt gggcagcgtgcgctatcacaaggcttgatctccgaggagccccttgttttagctggctggaagcca atgatcttaagtagataagtgctgttgcttgtagttcaacagaaagctttgagtacgtctttcttgcgag aaagaacacatgcattcttatgctctcaattctattatttttattttgggcgaaaggaaagctctcacgc gagtacgaatagccaaccctttat 418 PSIVIGR GCTGACTATGTGATCTTATTAAAATTAGGTTAAATTTCGAG GTTAAAAATAGTTTTAATATTGCTATAGTCTTAGAGGTCTT GTATATTTATACTTACCACACAAGATGGACCGGAGCAGCCC TCCAATATCTAGTGTACCCTCG 419 PVMahoney ATGAGTCTGGACATCCCTCACCGGTGACGGTGGTCCAGGCT GCGTTGGCGGCCTACCTATGGCTAACGCCATGGGACGCTAG TTGTGAACAAGGTGTGAAGAGCCTATTGAGCTACATAAGA ATCCTCCGGCCCCTGAATGCGGCTAATCCCAACCTCGGAGC AGGTGGTCACAAACCAGTGATTGGCCTGTCGTAACGCGCA AGTCCGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCC TTTTATTTTATTGTGGCTGCTTATGGTGACAATCACAGATTG TTATCATAAAGCGAATTGGATTGGCC 420 REVA GGGGTCGCCGTCCTACACATTGTTGTGACGTGCGGCCCAGA TTCGAATCTGTAATAAAAGCTTTTTCTTCTATATCCTCAGAT TGGCAGTGAGAGGAGATTTTGTTCGTGGTGTTGGCTGGCCT ACTGGGTGGGGTAGGGATCCGGACTGAATCCGTAGTATTTC GGTACAACATTTGGGGGCTCGTCCGGGATTCCTCCCCATCG GCAGAGGTGCCTACTGTTTCTTCGAACTCCGGCGCCGGTAA GTAAGTACTTGATTTTGGTACCTCGCGAGGGTTTGGGAGGA TCGGAGTGGCGGGACGCTGCCGGGAAGCTCCACCTCCGCT CAGCAGGGGACGCCCTGGTCTGAGCTCTGTGGTATCTGATT GTTGTTGAACCGTCTCTAAGACGGTGATACTATAAGTCGTG GTTTGTGTGTTTGTTTGTTACCTTGTGTTTGTTCGTCACTTGT CGACAGCGCCCTGCGAATTGGTGTACCCACACCGCGCGGCT TGCGAATAATACTTTGGAGAGTCTTTTGCCTCCAGTGTCTTC CGTTTGTACTCGTCCTCCTCTCCCTCTCCGGCCGGGATGGG 421 TropivirusA tgtcgcatgttgccaacatcaaaattctgggagagtcgcgaactccttaacactgccttgcctcgac ggagccgttgttatagtgtcgacgggatacaaacattaaactaaacccacttgcctcgacggaacc ccttaccttttattttattttatagtatgaaagtgaatcttgtatgaatgttcatagaaaactgcaaatgagt accacgtctaacatgagagaatgatactggagaaatccaagtttagaagtcactacgaatcccagc ggaaacaagggaattctgagcttctaataggcgtttaagactatttgcaaaattctggtgcgtaagtg atattttcattgcgtagaacgctggtaaccactccggctagtataagcattgttagtcacttattatgaa actccacactatcctttctggagaagcacacaaacttacatggtaaagctagaccattatcttaagcg gtgagtacactgcaaccttgtaacaatgcttgtatgactactttttgtatatcttgagcaatattgttgag gtggacatgtccaaaggtaatgttgttgggaatggaggggtccattttcccgtgcacgtagtgtact agtattgggtgatagccttgcggcggatcaaccatgtattttaatccgttgactttcac 422 SymapivirusA ttgggaaatccccaatgcttctttcaacaccgcctgactatgcggtggcgcttcggctcaaacaact agtcacttccccctcttaactactacccaagacttctaactacccttacctacttatttgtctaaatttcaa acttttattctcacgcgtcttataaacatcttttctatttgttatggtatgttttgtgatttgtgtggtgtatttc atttaatgggatctagtggaccgtgccccggttgggtatccgctccctttaaatgtttgcaagcactct tgacattataacctatcatttagtttacttgtttgtatgatcgtatttctgaatcgtaacatttatgcaattctt tctcgccgagacttgtctaggagataaagttcctgcatatttagtgttacggttgtataatggagactta gatagcttcacactgaggacgctttttcgctatccttttgacctgattcaggccagtgtggagttaatg attgtatggatgggccctacaatttgtctaagacttggtgatagcctcgcggccgctcgccatttata caactgaatagcggttgaaactctct 423 SakobuvirusA tcacgcgcttttccggtggtcacccaccgttagggagcgccagcgttcgcgcttccgctaccaggt FFUP1(1xmut) gacacactcctttcccctcccccattcccgttcccatcctctggactggtttctcctcacgattgacca gcagctgggagctgttaccagacgttggacagtaagtcccggatgcactatagggctggtggcta gtgcttggtaagcactcaacgccatacctaatgtgtacctcggcttgccctcctggtcgtggtgacc ggctgtttctcttcccttggctcCagacgggctggtgtcctaccaccaccgttgcatgcagacctcc ccctgcgcactcgaacgccctgtcccagcagggttagtatgtgctgtgcagatctgcatgtgacac cccatccactggtagagcaggaagttgccctagctaacgcggcaagtattactttccgctacacgt ccttgagattcctcggacctctggaactagggtgactgtgggcttgggaaaacccaccttggtcctg tactgcctgatagggtcgcggctggccgaccagtggatgtagccagttgttttgggat 424 RosavirusC cagggagatctccatgaataatcttttccaccctctttagcgtctatgctattgaggacgggttggag NFSM6F ccccgttgacccagcgtcagagtgtgtcggtagcaggctttctgctctcgccccatgccggccaca cctcccattagtgatgtgaaggttgtaagttacatgtgaaaaggtttctaataattgagctgaatgtag cgattacctaaggtgagcggattcccccacgtggtaacacgtgcctctcaggccaaaagccaagg tgttaaaagcaccccttaggtaggccactaccccgtggcctcagttctcttagaagattcacttagta gtgtgtgcactggcaactcttaagcagagctagtgagtgggctaaggatgccctgaaggtacccg caggtaacgttaagacactgtggatctgatcaggggctcgagtgctgaagctttacagaggtagct cgagttaaaaaacgtctatgcccctcccccacgggagtgggggttcccccacaccaattttagatt gcact 425 Rosavirus2 ccaggcatggcgttaaacatgcattcccttcccctagtaacctcccttcgccccttccccacgttgta GA7403 ccccctccgagatggctgctaaggcgcttgctgctacagcagtctcgtgtttcgggtgttataagtg ctttcttttccactccactccctgcctatggggagcggaacggccttgtctcggtcgttgcttcttgca gatcttcacccctccaggctttctggactcgccaggggtggagtagtaggcgcactgtctaagtga aggtagcagtgttgttggcgaagagttgtggacctactttgagtttgtagcgatcatccagagctag cggatctccccacgcggtaacgcgtgcctctaggcccaaaaggcacggtgttcacagcacccttt ggatggcgggggtgcccccctccgcacttaaagtagaaaaacagcttagtagtcaaataacatgg ctttcctcaagcattcagtgctacatgggactgaaggatgcccagaaggtacccgcaggcaacga taagctcactgtggatctgatctggggccctgggccaggtgctatacacctggttaaaaccaaatct ggtagtcagggttaaaaaacgtctaagtcccacccccccggggacggggggttcccttaaaccct caactgacacc 426 RhimavirusA cgaattccggacatctcctttcgggggcgagcgtcaccgtgcccctcatggaggcaactgtgcctc taatcggtgacccactgagaaaattttctttctacgtggctaaacaatgcaactttataataacacaaa tttaatgcttaatcttaacaccaaagatttgaacatatgtttggaaagtggcacacttcaaacattgcat agttgctaggggtgaagtccctttaaggggttgcagaggatctttcctctttatgagcggctaggagt atcttcttgatattatgtggtcgtgcaactcacttcccagatgtatgacggtgtactaagcgattggaa ctagtcataacctctttgaattttggtattgcgagtctagcagggggatatttaccgctaaagggtgac acactcgtgagggtggcctttggtgtgtgtatatttattccgcccatcttgcatggggtgctaaaattct aatgctgtgaaataaccattttctgaatacattctctacatttggagtcaaatatgaggaatgccactca ggtacccttgacatgatcttggatctgagagtgggctaattatctaattatttggcgactttctaaaatct tctgtttttagtggtgacaatttatggttataaa 427 Rafivirus gtgtccgggaagcgactcaagcttttgactgagtctctacaccttcatccgtaacatctttaagtttatg LPXYC222841 tgcctatggacctctagtgcactgccatcaccgggggtgtattggactggtttttccacaatccattca tcctgaggaattttggctttgttactaggatggtcccaccacacgcttatctgtgcctattgtgtcaacc atgttcttaagtagttgtgcccgtgggtgagtagataaccacaacaatccgataaagcatctcgcaa ggatgtgagtaatggagtgtatgtgctacagagacccacaacctgaaccaagagagacacagtg aggattgtaaagggggaactctttgaaagggcatgtcccgcaattcctactgactgacaccgggg gttggtgtcggtggattttagcaaatcctgttactgggtgatagccttgtgcacttcacttggttcttgta taagtgctgta 428 Rafivirus tgcgaatttattcgcacagtctcttttcccccatcttgtgtgtgtgatggggtaagccgcagagtaata WHWGGF74766 cctactctgctgcaaacacactcactcttttctatctactttatatcatgtaataataagtagggaacata ttcaattcatattgttcatctcactgaacccgcatgaaggactgcattgcatatcctggacgaagtgac gtggaatatttggacatttatggattggacaccattacgctttgtgcctctacggagatgtaaccataa tcttaagtagtagtaccccagcacaagaggataaagtggcatacacgacaacgggtgttgctcgc accttagtaatgtggatgttcacccttggagcgtgctgaaactctgtgggtaaagacacacattagta caaatgtgggggaactcactgaaagggcatgtcccgtgtactggtgtgccggaaagtgggggtc gctttctggagaacttagtagttcttgttattgggtgatagccttgcggcggatcaactcacagttttaa tccgttgttttgcat 429 Poecivirus actacacaatcgcaacacgcgcaagtttgtagtttgattggcgtgcaaatgtcaaatcaagcatata BCCH-449 acacaatttggtggctgttggtgtttgttataggaattttggttgtgttgaaattgtggatgtgtaggaaa tatgcacaattacgtcagcgtcaggagttttataacctggcgcaacaccaaaatggtcttcgcgcttt aacatcaccagcgaggtgtaaacaaattgaagttgaattagatcgtgtataggccagggaaccatc cctcccaacgccacatcttgtggggaagttgggataatggtgggtctatatgaattggtctgtagac ccacagtgaagagtgaatagtatgcttgcggttccatttgttaatggtctagcatggggggggcgg caaccccgtgaggggttccccactggccaaaagcccaggggttagtcatttcaaccaaggaagct ggtaacctggtgacctgaacttgagtggtgagacccccttgctagagtgtgtaaaccgattgtaagc attttgtttgcttagtatctgtggtataagcagtcaattttgtataggctcaaggctgtggtagttagtag atgcccggaaggttattactgatccggggaccgtgactatacattaggtaaaccggtttaaaaacc 430 MegirivirusA ttcgggacactggatgggcgacttggtggggctgccactctatcttgacctttcgttactgactttcg LY gatctctgactcctccttgtctcttgcgtttggtccacggacggactaattggaatgtttactggctaag cctcgttctgaaataccctagccaatgggttgtagtaggatcctggtgtttccattaaacctcttccga ccatagtagctagagttatggctgtgtaggatgtgggtaagaccgctttttgcgtatctcccacaaga caccggattatggatgtgtccgctggataaggctcgaaacctcccaactgaaggtggtgctgaaat attgcaagcctaggttgtgtagaggcaagtagatgcctgccgcgacattcgtcttccgcccttttgg gttagtagtgtacctacatggacgtggggctgggaatccccaccttgcataacactggttgatagac ctgcggctggtcaagttactatggtataaccagttgaaatggct 431 MegirivirusE gcttggcaacctcatatcgttactctgccgaccagtctgggtcgtgtggccacacaatgggattcgtt ctgttgtgtagagtcacatggcattactgggctgatcggtggggatccgttgccacccctaaaccct tacatttactggactgcttttcttggccccggaatgattcgctcacccgcgatgaggactgttgttctta ttatggcaggattacgcgtctggtccgcgtaaggactaattcctatgtttatacgttactaccttgttct gaacggtgggcgccaccccgcctagtaggatcctggcttatcgtgtagacctctagggaccacatt agctagagtgtaggctgctatggatggagtagtgacccctttttgggtatcactctctaagactccgg aatgtgtcatagtacgctggaaatccttacttgtttttccatgagggggaggtggtgctgaaatattgc aagccacccctcggttaaaacagtttggtgccgcttatgccatattaccgccccttgtagttgggctg tttttgcagctccgggttagtagagtaccatagtggacgcggtgttgggaatcaccgccttggctgc acactgcttgatagagctgcggctggtcaagctaattgtggtataaccagttgatttggcat 432 MegirivirusC ttcccgaccggtctggcaaaccggacggttatcctggttagatgtctgatggttgctggaacgtggt ggctactgctgccaccttctggcttcctttaatgggcatctagctgggttctttgccacaatccatctta ctctcttacccattttctattacccagacttgttgttaactggtaaagttgacctactggcttcgttttgag actattctggtgttggtggacactctttccacaagtagattgtatggagttcatgctcgttttgaaccgg gaatggcacaacccgtagtaggatcttgcctctgccatactaatctgcgcctgttgcttttagactatg ggctgctaaggatgacattggaacccctttttggatattccatgtcaagtcaactgtttcatctggtgta cgctggaaatccttgttccgaggtcttgtctggaggtggtgctgaaatattgcaagccacaggcagt tccttggacttggtgccgctatcagatgctacaccctctatgggcaaatgttgaaccttagtggacgc gtgagatgggaatccacgccggccatagactggctgataagctcgcggctgatcgagttgcaaca gtaatcagttgatttgccact 433 Ludopivirus tagacccccacctagcccttttccccgtcagtggggggcttactcactgggcatctgttaatctggc ctaactagattgacaccactcccttggaacgtaactccacgctaactcactggctctacgcacagac acacggtctttctgctatccccggggaagataccagatggcgaccggctgtcccagcggcctagt agctactcgggttgagtacccaccacggttttgacgcctgctaaaattcaagagacagaggtaggg gtgcttagtgtgtgggggaagttcccacaagcgaggcaaagcattgctccctcgcgtcaccgggt gcaaggtaaattggctggacttccgctctacccttgctactcgccctcttcggagggttcgaagtga cactaggtatacgcatggttgggaaaccatgcctggcctactactgggtgatagcctggcggcgg gtccgtctcttggcttatacccgttgatttgggat 434 Livupivirus tatctacatggggatccaggctgtatggaatgtctgtcttaacaagcactataccagaaagatccac ccaaagtggtgggactgggactgtgaggtgagaaatcccgaaaccagccttctcaagcgtcgga cgatctttctgttttagtgaacaccttgccttttaaatggatgacaacaccccttcagcaaatcgcaatc tgaaatcccaaaagactgtttagccgaactctggtaatcactccggagaagtaggatacgcagccc ctgtggactcttgatttcaggactcaaggtagctagagctggaacttcatggaatgacaaaggaata tatgcacattgtgcgctttcctggccttgtagcccgtcgtgaggatatgtcgttgggaatcgacatctt agtccagtactgcttgatagagtgtcggctggcacagttacctgagaataagtcagttgtacttaaca tgaacaaaaaaaataactaccacaactaccacaatctaccaatacttgaattatgctgaatctcgtac agtaaaaacgttccgtggaaggacaagtattgaagtgcggttacatcatccgatacgcgctggatc cctca 435 AichivirusA cacccatacacccccacccccttttctgtaactcaagtatgtgtgctcgtaatcttgactcccacgga FSS693 atggatcgatccgctggagaacaaactgctagatccacatcctccctccccttgggaggacctcgg tcctcccacatcctccctccagcctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctc acgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccgat gtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgt tccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgct gtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtg attggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgcccta ggaatgccaggcaggtaccccaccttcgggtgggatctgagcctgggctaattgtctacgggtag tttcatttccaattcttttatgctggagtc 436 Aichivirus tactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatactcccccccaccc KVGH cccttttgtaactaagtatgtgtgctcgtgaccttgactcccacggaacggaccgatccgttggtgaa caaacagctaggtccacatcctcctttcccctgggagggtccccgccctcccacatccccccccca gcctgacgtgtcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccc cccttcatcaagacaccaggtctttcctccttaaggctagccccggcgtgtgaactcacgttgggca actagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctg gcccttcactatgtgcctggcaagcacacctgagaaggtgttccgctgtggctgccagcctggtaa caggtgccccagtgtgcgtaaccttcttccgtcttcggacggtggtgattggttaagatttggtgtaa ggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtac cccaccttcgggtgggatctgagcctgggctaattgtctacgggtggtttcatttccaattctttcatgt cggagtc 437 AichivirusDV tactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccaccccc ttttctgcaacttaagtatgtgtgctcgtaatcttgactcccacggaacggatcgatccgctggagaa caaactgctagatccacatcctcccttcccctgggaggaccccggtcctcccacatcctcccccca gcctgacgtaacacaggctgtgtgaagtccccgcgaaagctgctcacgtggcaattgtgggtccc cccttcaccaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggca actagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctg gcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaac aggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaag gttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtacc ccaccttcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgtc ggagtc 438 Murine gtaacttcaagtgtgtgtgctcgtaatcttgactcctgccggaatgccgcccggttcagtgaacaaa Kobuvirus1 cagctaggcaagtccctcccttcccctgtggtcggttctcaccggccaccatccctcccccagcct gacgtgttacaggctgtgcaaagcccccgcgaaagctgctcacgtggcaattgtgggtcccccctt tgtcaagacaccgagtctttctcccttaaggctagcccggtcccacgaacgtggaactggcaacta gtggtgtcactacacgcctccgacctcggacgcggagtgctgttccccaagctgtaaccctgacc caagactgtgctgcctggcaagcaccgtctgggaagatgttccgctgtggctgccaaacctggta acaggtgccccagtgtgtgtagtcttcctccagtctccggactggcagtcttgtgtaaagatgcagt gtaaggttcaagtgccaaatccctggaaggagtgaccctctactgccctaggaatgctgtgcaggt acccccaacttcggttggggatctgagcacaggctaattgtctacgggtagtttcatttcccatcctct cttttttggcatc 439 Porcine tttgaaaaggggggggggggcctcggccccctcaccctcttttccggtggccacccgcccggg KobuvirusK-30 ccaccgttactccactccactccttcgggactggtttggaggaacataacagggcttcccatccctg tttacccttactccactcacccctccccttgaccaaccctatccacaccccactgactgactcctttgg atcttgacctcggaatgcctacttgacctcccacttgcctctcccttttcggattgccggtggtgcctg gcggaaaaagcacaagtgtgttgttggctaccaaactcctacccgacaaaggtgcgtgtccgcgt gctgagtaatgggataggagatgccaataacaggctcgcccatgagtagagcatggactgcggt gcatgtgacttcggtcaccaggggcatagcattgctcacccctgaatcaagtcatcgagatttctct gacctctgaagtgcactgtggttgcgtggctgggaatccacgcttgaccatgtactgcttgatagag tcgcggctggccgactcatgggttaaagtcagttgacaagacac 440 Porcine ccaccgttacttcactccactccctcgggactggtttggaggagcataacagggcttcccatccctg KobuvirusXX ttcaccctcaataccacccaccctttccctcaaccatccctatccacaccccactgactgattcccttg gattttgacctcagaacgcctacttgacctcccacttgcctttcccttctcggattgccggtggtgcct ggcggaaaaagcacaagtgtgttgcaggctaccaaactcctacccgacaaaggtacgtgtccgc gtgctgagtaatgggataggagatgcctacaacaggctcgcccatgagtagagcatggactgcg gtgcatgtgacttcggtcaccacgggcatagcattgctcacccgtgaatcaagtcattgagattcct ctgacctctgaagtgcactgtggttgcgtggctgggaatccacgcttgaccatgtactgcttgatag agtcgcggctggccgactcatgggttaaagtcagttgataagacac 441 Caprine gggggggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat Kobuvirus acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt 12Q108 cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaa gcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgt aagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgacc gggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaa ctgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgct ccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctt tttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctg gttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 442 RabbitKobuvirus gggctataaatatgggcattcctcttcccccttccccttttgaagatgagtgcgcatattcttgactccg cctggattggccgcccaaggcgtgaacaagcagctaggccaccatgacactgcggtggtgtccg aacccgcgggtgccttcacgggcacctgtggtatgtaggactcccaccgtggtcttccctttccccc tcaatctttccccctggttcgactaacgggaccagtgctggaacctgtccggtgaacggtatagca ggcccccccggcagaaacacccggtgcttaccccttaaggctagcccccttccatgaatttggttg gggcaactagtgggtgtacagttggcgtgaaccctccggtctaggagtgctcttgcccaatcctct gtgtgtgccttgcagtagggactggcaatccttcgcgtaggtgatccgctgtgccatgccatcctgg cgacaggaggcccagtgtgcgcaacctacgtcccttctgggtgctgcattgcattacctttggagta agcttggtgtgccgaaaccccagggtttacgtaccactcgtggtgtgaggaatgtgccgcaggtac cccatccttgaggtgggatctgagcggtagctaattgtctagcaccactttcttccttttttctttgctgg tcacg 443 Aalivirus ttgaaagggggtgctcagggtagctccctgagctcttccctccaccctctttcaacgtctggcccac gatacgggccacctttcaatcttaactaactatccctttaatctatttggattttctggtttagaataatttg gaacacataattggattatcttttaggattgtggataggatttgttcgggatatcactcccttcctgtgct aacacatattctaattccctcctttgtctattatctcttggaggtggtgctgaaatattgcaagccacttg agtgtatagatgaagtaggctcaagatgaatgttgtgttactcaaggcaagtgtagctatcactaaga tattggtaacgtgaaacggattaccggtagtagcgtgatcttccgtcttagtgctctagtgactagag gacaacgacatggcatcacatatcttaaccctccagttttggcatccgggacagaatgggctggat atccgctttctttctggggtatgtgatgggtggtattggggtaaccaccttgaccatgacgctcgata agagtgaccgcctgatcattgaaacctctagtataaaattcaggctgaaatc 444 GrusopivirusA tgcctgagtaggattgtgaatttaggtatgagagggttagccaacccattctgaaccataatagatac gtcaatctgaatccatctaaatctatctcttaggcagtggtgctgaaatattgcaagctactagggata gacgtgatctgattcaagaacctatctaatgtggtgatgagaaggctaggtttatccatagtaatccct tgttctgaacaggcaatgcacatgctctagtaggatctcgggctctgcgattggctctaaaccgacc aatccaggtagaggcactaagtgtaggacttgccaaaatgtattacatgctggtaccgactcactag tctggaaactccacactgaaagtgactggggggggccccatcacatttgtgctactgcttgataga gttgcggctggtcaacttggattggtataaccagttgaa 445 GrusopivirusB gccatccgtaggttctggtaaggttccatcaactgttggggcgctagttgctatgaccgcattcacg gacggatgatttatagtatcacccaatccgggcacaacttctttagccacttcttccacattactaagg gctctcttgccgagtttcaacgtctagtccacgacacggaccttcctacttttctatcttcttattttctct actaaattggtatctggtactgaagatatgcggattgtgattttgtgcctgtctaaactaaccctattcta gggttaggtgggtaccatatactaatggtgaacaggattacctatgtatccattagtccctatggatct ggcgacccacaaactcatgttcatagagaggctaagctgagtgctcgccgaataagcattgcttca ggtgccgactattgtctggaaaccactcagtgatagctataggggggggccccgtagcatctgcct tactgcctgatagggtggcggctggtccatgaacatgcagtaaccagttgacttgac 446 Yancheng ccttagggtctggaatgcgtcctttctgggcacttccacaatcctaaggtaattttcaacgccagcga osbecksgrenadier tggagcgatatccaaagaacctttatgttttagttcgtcttgtatgtttataaaatataaaattgggatta anchovy gcaacccacaaaaacatttttgttattctcaccacatgagagggtggttaaacctcttcgtaacctatc picornavirus ttgcttgattggctacttgggctagatttaggacaacctctttagcaagcctaaattactcctcgtactt gcacctggtaacaggcgtacctggaggttacggtggcgctaacttggacttctcgttaattcgtgca agtttaaatgatgcctattttgaatacaagaaagtatgatagtaacttagggcgtgaagttccgcttaa cataaggcagtataagtactaagataaggtgtaagacctaccttaataactgttgtcttttctcatggtc tttccgtgggagcccttgctaggggtaaagttaagtattctcaaataatttttcattcaaactctttctctc tgtttt 447 TurkeyGallivirus ccactcgcacttcctggatagtgcgttagatatgcccgacatatcgcttccaggaccaaagccccc M176 cttttctctttcccaaccagcttcgccactcaagctgtaattccatgtccggtctttccggccttagtatc atggaaatgtggtcgtgctcaaatgaaattgagttgacattgatcaatgaaagttgcactgaactttg ctaaactggctagcgccacctggtgtgtgccgttggtctcctcacatggtaacatgtgccaacggg cccgaaaggctagtgggcaattaccgctccaagggaggggtacccaccccgacctgaacagcg gtaatgaagctcacctcccaggctctgaccccgagaagtttagttatttagtaggtgtaattagtactt gtgattggtcaatttgatagtagtttgaaacgttatggatgaatgagtagaccccctgaaggtacccc attacatgggatctgatcagggccacattctgcgtgtctccccgcacttgtggttaaaaccatgaaa gttcatcccaaacaatcttttcctcttctttttttttagtggtgacaacctactggattggtgattaccaat ctgtactagtgttgtattaagacttgttgtgtggagaaaatggactctttcaagaagatttttg 448 FalcovirusA1 taaaaggggacgcggtgtggcagctttggctgtcatgccgtgttctccttttaccccaaggactagc cttgggggttttccaaattcctttccctgtaggctttacttctctttatctatcttttctgtaactaagttttgc ctattctaaaaatattttagaatgtgtttggatgtaactaagtttgtgcctgccctaaaaatattttagggc ttgtttggataacctcgtcccttgtgttcagtgccgcacaatttgctaggcactgttcacttcctttgtttg tccattatgtatgctaaggtatgaattccatcatatgcttagcctctacatgcataatcttattccctccct ggtgcaaactacgcccccaacatatgtgaatcttttaagcatattcctgaccccacacatatatatgtg ttctcgtgaattcccccaccgtgaggtggtcacttggacgtggtgtgtgtcacacagcatatatatga tgcaggatgttgtttttaagataagcatatgtccttagtgctttgcatcatttcctccacaccccgtgaat gcggctaatcttaaccctgttgggtccgtgggtaaaccaacccattaaccacaggacggaaccga ctactttcgggagtgtgtgtttctttttcttcttttgtcact 449 TremovirusB ttcaaatggcccctgggttgatacccagtggtcatttggacactttggtaaggaggtgtaattatcctt cccatgtggaacctagtgcttaggtttactttatatgttctttgtttgtcctttgtactttctatcgggcaat cttgttgttcaatacaatatgtatttgaactgcctaagataaattcagttttcaaccaacccctctcttgg ggttgtgtctttctttctttcttatatcctcttaagctgacttacttgctaatccgactcctcgtcaacggg agggtaaagcagtatcactagggtattgtgatgtaggagaaaaagtaagtagagatagtgcatgta acgaaagtgacttggtactttaaactctcttaatcccaaagtgtggtattggtcatgttggagtaggct acgggtgaaactccttcacatttagtaatgtgttcacacgctaacgctacggtagatgacagactag gtcttattctcaacgtagggggacgggtgtatgttcatgattagccacatattaaggttttgaggggct gagtcatataagtatgtgcattaatttctggtactggtccctggggactggcccttttctaggttgatttt agtttccccaatttttaaaaactaatgagatttacgac 450 Didelphisaurita tctttggtctggggaactaaaataccagacccgcgtttgcctagcgatataggctttaattgttgtttgt HAV cattgtgcgtttgatatgtgttttaatgtaaataataattctagcaggttctagttcttgatcatgtcctcttt aaggcactcatttcaacttgctatctttcttttcttccttggttctccctacaccaaatgcactggccgct gcgcccggcggggtcaaccacatgattagcatgtggctgtaggtgttgaaggctgggacatgaac atcaatggaatagtgcgcatgcttactggggtccattgaagtagtgggatctttctattggggtaggc tacgggtgaaaccccttaggttaatactcatattgagagataccttggataggttaactgtgctggat atggttgagtttaacgacaaaaagccatcaacagctgtggacagaacctcatccttagattgctcac tatggatatgtgctctgggcgtgtttcttgcatgatggccattggtcaattcatgcctgggccaatgta ggattagccttaaattactttttaaaagtagcctcatttagctggactaatggtggggcgtatgatcctg catttggcctctggggtaatcaggggcatttaggtttccacataatagcaaat 451 HepatovirusG1 gcaaggggtggttttaaccttgcacgcgtttaccgtgcgttaacggttttccatgtttgtatgtcttgttt gtattatgtgttttgtaaatattaattcctgcaggttcagggttctttaatcatgttgggctgtacccacac tcaacttttggccataagtgagtttcttaacgaaccttttaacacaggatgttattagggcccaatatttt ccctgaggccttctttggcctctattttttccccttttctatctccttgtattccgggctcacgtgatgcca atggactgacccatgcgcccgtgggggttaactactggagtagccagtagctgtaggtgctaaaa gtcacgtacgtgtaagactggacgagacctctcagctataactgaaagtagtaagtatgtctgaact tcttgaaggggtaggctacgggtgaaaccccttaggttaatactcatattgagagatacctctgatag gtgaaggtttccggtagaggtgagtttaacgacaaagcctctcaacggatgtgggcccacctcatc agcaagatgctttcatacccaataccgtaggggctgggttgttgagttcagtcccaagcgtccctcc cgcaaggttgtaggggtactcaggggcatttaggtttccacaattaaacaaataca 452 HepatovirusD ctttggatgcccatagtgcgggggtataaataccgcactccctttagctgttccgagggtatcggaa cctatatgtttgttttctgtctgtctgtcagctttatgtgtgctcgtcccctttagggcactcatttcagctt gctttcattcttttcttccccggttctcaccttaccggaggcactggccgttgcgcccggcggggtca acctagtgattagcactaggctgtaggtgtctaaagtggtgacattaagacttggtaactgatttcag cactgttaactgatgttggggatgacttgattgatcttctggaaggggtaggctacgggtgaaaccc cttatcttaataccactatgtagagatagattcagtaggttaagggcagtggataaggttgagttcattt tggacaataaaccttcaacactggtggacccaatctcactgaccagatgctttcttgactgatccttc agaggggtgattcttctgaataggttgccttgacactgatgcctgagacccattgggtcgggcctta aatcatggaactccactggactttcatggcctagcttctgccttagacagactctggggccccacga ccctctgggcccttcggggtactcaggggcatttaggtttttccacaattaaaagagtta 453 HepatovirusH2 gtcatgtttctctttaagaacactcaattttggccataagtgagactcttgtcgaacctttcatgtcagg accatgttagggccattatccttttccctggggcattcttcttgcccctgtttcatctttctatcatctttctt ccgggctctcacaatgccaatggagcgaccgatgcgcacgtcggggttaacccatggattagcc atgggctgtagctgctaaaagttgtgactcctgaagcatactatcaatggtagtagatgtaactgaaa cactgaagcttctctgatcttgaaagaagggtaggctacgggtgaaacccttcaggttaatactcat attgagagatacctttggtaggttaacgttggcggataatgttgagtttaacgacaataaacattcaac gcctgtgggcgaacctcaccaatttcatgctttgaagtgaatgtgcgtagggtctctatcggagatg ctatgtggatggtgccctccctggaaacaggttgtaggggtactcaggtgcacttaggtttccacatt ttaaagatttttc 454 HepatovirusI ggctgcctgtgtctcaggggtaagtactggggccgcgttgaccgtgcggtacggttatgcttttaga ttaggatgtccgtctgtccggcactctcttttgcttaaaatggccttaaatccatgggaggcgtaacca tgggccctttgttacctagacatgattgcattgggggccgtccttggggcttaggccccagccatttc tcttgactcgtctaagagtttacttcatccttttctttactttattttccaggctctcagcatgccgacggct ctgaccactgcgcccggtggggttaactgcatgattagcatgcagctgtaggagttaaaagtgctg acaggccaattctgacgtaagtccactctatattaacttgatcaagtaaggttgattgatctttgtgaga gggtaggctacgggtgaaaccctctaggttaatactcatattgagagatacctccagaaggtgaag gttggcggatattggtgagttcttttaggacaaaaacctttcaacgcctgtgggcccacctcactggc acaatgctttcatccccaattgtgatgggtagtttggactgaaatcaggagtaacctgccctacgagt ttaggggtagttcaggggtatttaggcttccacatttgatagagtttatgagagtgagcc 455 HepatovirusC ttcaaaagccccagcggggtttcattaccccgctgtggcttttggacttccctaggatggggaagta aattaccatcctcgcgtttgccgtgcgttaacggctacttttcttctagctgtagaagtaaaattcagca tgttttatgtttgtttgtcttgtttgttatatacatttttacactcctacaaatgcacatgaagaacagtttgt agagattaacaaacgcttagctgaacctaggtggtgaatctagtagtaagataagtagaggaagct ataccttaagttggttgggccctcgtgtttgctctataaacaaaaccaagtgagtagagtggatgaac agtactaaatccctgagtacagggaacctcacaggtgtgatacacttatgtctatgtgacctggttgg aggttgggcgtgccctatgatactggagtgggagatcttttggggaacccacgttttcacactgcct gatagggtcttgccgagagactcacttgtttcggctgtacttgtaac 456 FipivirusA tgcgggtaaactcccgcatgtgtgaatgaggcgatgtcccaggaactaactgccgatcctggtttta actacgatccgtatttgttactaatgcgatatccccccattgtttgcctccatgttgttttcaacgcttttg gccttgagtgttatcaagtgttttagcgacatagtgggaagctacggctgcgtccccatttttgagtg gcgacccagttttagtggccactctgtccctgaactgcgctataatgtgaatttatgttcacaaaaac ggactgatgtaactgttaatgactaaggaatagtacctcactgaagtatcaagaccccgttcgagcg gtgtacatatatggatggaaaccttgtctgagtcatctcgaatactaatcaatgagggatgtcgagta agcatatcatgaaccacatagaatagtggggtttcggggttagaggctctctgcagcaatgtatctct aacaccatggccgaaatgagagatagagaccacgatgtttgtgtgtaagtaatgatgtgtggaaag aaaattctgaatgttggtatgatatcagtctaaggggagtggctcacctaagagctacccaaacattt cacagcagacaacataacgtactgagagtagttggaaggttccagaaatcagt 457 FipivirusC cgcggttaaacccgcgcaaccttctttcagccgcgtctgagtagcgcggttagtcctgatacacagt ttcctgttgggtactgtgtcttcgggtgaatgctcttgtgtgaatgttttaggctgtttaagggaagcgtt tccccgtgcgctgtgagggtttctcacgctctttcggggtgcagtctcttctgttgttcattaagatgta tggatgcactgttgtgaaggatttgtgaactggggatcgacaccccgtgaggggtgccccagtgtc cataggagtttgctggagaggtgtgttgctgtagtgactatccgtgacctggcattctaaggtgttga ccccaacctgtgagggtctggatcgcagtgttgaagtgctttggagggttcaatggggtttctgtagt ggatattatgtgcttgacgactactggtacgagtgtattgggggtctacatgtgtga 458 FipivirusE ctcttccgatcttgggggttcgcccccatgtctcatttcaactagccgtgtgtctagttaacgcaccgc ctcaccctggtcgttatcgggtcggttcttgcgaccgttagatcgtgagcgtttctgaggatcagttc gtataagttctccggtgtggcgaccgtaaaatcgtcacgtcccatgcaatagatgacgttaaactcg tttgccagttacataaaggaatgttgttacttttaaattgtctgttacatttaacatcttgccagtatgatg ctactgtacactacgggtgtaggaaccttgtagtgtgacgtatcactcatatgtggatgggtgctcca gacctttatggaagctctcagttagtagtgatccttgacttcattgagccctggtaacagtggaagtc aagatgtatatgttgctcaacacacttcggtgctacgaagctgtttgtggaagtactggcgaggttca ttctgaatcatatgtttgtcacatagtcagggagtgccgtcgcttacgacggaccctttttctttataatt acaaatctgtgtctcaagtgttgttggctggttttcttcttctgttttcattgttcatatatatacgtcagagt gaaagactcggtatatacaaaactgatccaga 459 Aquamavirus ttcaaaggtggcgggagagttggcctcacgctgtttagcgtgagagctggctctcctgccccttcc cctgagccggggatcttggctcattcccctcttttctatcctccctcattggactttacggatgacccg gcataaacttgacaaccgatgttggatttcccttgtggctgtgatggaggacataccctcgggtgta gttgtgtgcgtgtcgctctgcgactcgagcttcaaagtggtgctgaaatattgcaagcgtcgttgctc gattaacggagtggtacaatcctatgaacccaagtgcattcatgcgaaagccccggaggggtgag tagcatggactcgaatcagaagagctggagctcgcttggtacggcacgtagcattgctttgcctaa agaccaagggggtatggctataggtgggggcctatagcttgtccagtgctggttgacagactcgtg ctacgcgtctggttcgagtataagtagctgcaactcact 460 AvisivirusA ttcactcgctttccccccctctctataggggggtcttttaattcttattaatttcctactttactatcaaatt tcttctaagtagggactgaggtcacttagccctccctctcctgggctttccagggttatagaggttcta aagctaagccatgtgtcttgagctacacttagtacaaaggtttagtaatgattgtacatgccagtaac cttctagtgcccatggattaaagagtggtaacactctccatggggcccgaaaggctagtgggcata gttggcatcaaggaaggggtccccaccccaacctgaattgctggctagaagctcaccttagaaga agtgctgggtgacaacgtgtccaatcgtgaacgactgatggaaacgtgtggagatggatatgtgg gggttcactgagtagatgccctgaaggagaaatctgatcaggggcccgtgactatacgctaggta aaccgggtataaaaaccatgaaaggtggcccaaaatctcttccttttattttatttctatgttggtgaca gtcaag 461 AvisivirusB caccccctactgccctaacccccaaagttagttatagggtggctccctacccttactccacggggta agccctaacccggttgaatctcaagatcagccttagcgaggactattagtaccgctcaaaccctttg cctgtagtgcccaggggtcacagaggggtgaccctctccctggggcccaaaaggctaggtggca agacagggtccaagtgaggggctactctaagtagccccaagctgaacatcctgtctgaagccacc cttgcagggccaggtttgattggggaaactagacaccagctttgtcctgggattggggggatatcg agttagtccaggaggtgcgagtagatgcccccgaaggtaccccaggcacatctgggatctgatcg ggggcccgtgactatacaataggtaaaccgggttaaaaaacatgaaagcgcctctctctttcctact tcttttattgactggtgacaaaaatagcagt 462 CrohivirusA gttgaagtccatttcttgcttgcccccgatgaatcctgttaaggcctcacggccctaagggtgaaact cggttatcccctcctgtacttcgagaagattagtacaacactatgaaatctacatcttgtgatccggga taaccccaatcccagaaacctgtgatgggcgtcaccacccctcttatggtaacataagggtgtcgc cgcgttggcacaggaccctttgggctggatgtttttagtaatggtgtcgaaggtcctattgagctaca ggagtttcctccgccctggtgaatgcggctaatcttatccctgagcctaaggttgcgatccagcaac ttgatggtcgtaatgcgtaagttgggggcggaaccgactactttccagaaggcgtgtttctttgttttg tctgttactatggtgcatgatatagatattgaatatttgatctttttgagctgtttcttatcttattgctacatc ctttcaggtgttggatttacattttggttaataag 463 KunsagivirusB gattttctggttatcccttttggacttggtaggggcccacgtgcccacccacctctgtgtgtgttgattt ctaatcgatgcctggcagtggcggccacctctccttactggtaaacctccggtgagtgaagttgtca agctacaggtaccgtgcaggatgaaatgcgcacatgtgaacaaactaggagtcatacaccgggtc aaactctggaaacggagtccgggactctgaccttggttgggtgagctcgaggcatcacattgatgg acgcgattcgctatccttccctagtaggaccttgtggtgtacccctggttgggaatccagggctggt cgggtgcagggtgacagcctgttctccacctcaaccattgtaggagaaatcaacccct 464 LimnipivirusA ttctttggatatccatttaacgtgtaccctatacgataattggggtggattctggatgcctagttccagt gattggttaagaactcgtttactacgtatagtatgattagcaaagtgctcgattgatcacgtaatgatct atgtggttaaaaacccagtagtatggtatatactcagtagtgtacactgtgagtacaactcttggcgta gagagaacaattcacccgaatccgtggcgtatccatggaaataagtttacctaattgtatgttacaag gcatatgagacatttatgagatatggtttattttgactaaacgagtgtagaggtggtggagtctatcca acttcaagccatgcaattgttgtgttgattgatatcattgaccatttttgtggattgtgtacacatacaatt tgaaaattaaccccctcaagaataagacatgggaccattcgtggtagataccgtgctcggatgcttg agattagatgggttagactagttttggaatgagattgccgagaaagtcccgctagacatgttttacaa gtcgtggtattccgctagactttttcgcagacacatggaagggtccatgtgttgtgcaattgcagggt gacagcccaactgcagagttttccttactagaataaaaatctgttgtcaatttt 465 LimnipivirusC gtttctgagcactggtaagagcttagacaaacgtttttaaaatttattttctctgcaacttttgtttgtgttt atttttatttgttaattttgcgcctaagcatttgttgcgaagtatttgattcattagtaatattacttattgttta tttagatggtattcaaagtggtgggagtatcgaacccaagcgtcgtatgctatctccttgaacaattttt aatcattgcgaagtgatcattgaaaaggataggtgtttaagaactcaaagagtgttaataatgttggg tgacaggtgtccccatagaatttattaacatgatttggactggttatctagtaagaagaaccatcgaa cgcacgagcgagcattgcttgggggcagttaccctgcgtcgatgtaagtgtgtaccggggggtg cacatgttgattctttatggcctgatagggtgcgtcattcgcgcctagataattagtataatgcgaatg gaataaatttac 466 Orivirus ggtcccaggccaatattcttcgtaaggcttggttccaattttccaccactcgtgtttgggttctggccta tggtacccagaggggcggtttgggggaattaactccccctcccctgtggtcctataccaccccaca cctctgtgggctttctttactatcttcttgttttccgacttttaaacactaggcaggcgcgcctagtcata caccgcccggctggtctttccagctcttgtgggcggtgcgcgctggtccatcgtgcccagcgacat agcaccttgtggacacctccgaacgccctcccctgtatggggtggtgcccaggggtttcagtgtgg tgacacactccctggggcccgaaaggctagtgtgcaacaggtgaggtacagccagctgcccccg tggctggagggaccaagcttgtgaagcacacctcaccttcttggggggggctagtaagtggtga aagcatagtgtccgtgtcgctggccaacactttgggtcaagtccagccactcagtgagtagatgcc caggaggtacccctagtggatctgacttggggcctgttacttaatgcaggttaaaaactatgaaagc tgagtagtgtagcccggctggtggcttctcttccttattcattctattttatggtgacaaacgcaactga agcc 467 HAVFH1 cttgatacctcaccgccgtttgcctaggctataggctaaatttccctttccctgtcctttccctatttcctt ttgttttgtttgtaaatattaattcctgcaggttcagggttctttaatctgtttctctataagaacactcaatt ttcacgctttctgtctcctttcttccagggctctccccttgccctaggctctggccgttgcgcccggcg gggtcaactccatgattagcatggagctgtaggagtctaaattggggacgcagatgtttgggacgt cgccttgcagtgttaacttggctttcatgaacctctttgatcttccacaaggggtaggctacgggtga aacctcttaggctaatacttctatgaagagatgccttggatagggtaacagcggcggatattggtga gttgttaagacaaaaaccattcaacgccgaaggactggctctcatccagtggatgcattgagggaa ttgattgtcagggctgtctctaggtttaatctcagacctctctgtgcttagggcaaacactatttggcct taaatgggatcctgtgagagggggtccctccattgacagctggactgttctttggggccttatgtggt gtttgcctctgaggtactcaggggcatttaggtttttcctcattcttaaacaata 468 HAVHM175 cgccgtttgcctaggctataggctaaattttccctttcccttttccctttcctattccctttgttttgcttgta aatattgatttgtaaatattgattcctgcaggttcagggttcttaaatctgtttctctataagaacactcatt tcacgctttctgtcttctttcttccagggctctccccttgccctaggctctggccgttgcgcccggcgg ggtcaactccatgattagcatggagctgtaggagtctaaattggggacacagatgtttggaacgtca ccttgcagtgttaacttggctttcatgaatctctttgatcttccacaaggggtaggctacgggtgaaac ctcttaggctaatacttctatgaagagatgccttggatagggtaacagcggcggatattggtgagttg ttaagacaaaaaccattcaacgccggaggactgactctcatccagtggatgcattgagtggattga ctgtcggggctgtctttaggcttaattccagacctctctgtgcttggggcaaacatcatttggccttaa atgggattctgtgagaggggatccctccattgccagctggactgttctttggggccttatgtggtgttt gccgctgaggtactcaggggcatttaggtttttcctcattcttaaataata 469 ParechovirusF ggtcggggagatgtgttcatgatcggttaacaccatcatggatcatctctccccgacctctttttgacc cagctatgggttaaatagtacttttcttttctcttttgctttcttttgtgttttgtttgttttgcaacatataaca agcattttatcagtattagtgtctgcaactgtataacaagcaaggtggagcaatcatgcgagtatatct caattgaattgtgacacacaagtgtgcactatgtggaataaatgccattttggccaaacctggttagc cagaccagtagtaggacaatttggcacccttagtgggcgcgacctagatgctagggatgagcaaa cctatttcccctgagtacaggggctctccttcacctctacattttggacctctttttgagtatcctcgata gaaggtgaagtgacggtgtaccggatggttaattgatctcattgctgggtgacagcccgctaggac caggcagcatctttgtatggacctgtacatgtaac 470 ParechovirusD acatggggcaggtgtgctgtgccaagagcaacactacggtggccgagccgatggttcgtcacca cgtagtaggactccgtagtgcttggttacggcggacgtaagtcagttgagtgatgtctaagtggcaa accatgagtacatggtaaccttgtgtggactcgcgggacggaatttcctatcccattgactccttgta gcaaggtgggtatacccaaccacaatggcagcaccctgggtgggaacccaggggcctggatta gtatccagtcacacagcctgatagggtggcggctcagccactgaccagcgtctctaaataattgtg agctgttcatgcacc 471 ParechovirusC cggtcatccccctttccccacagccggtgtgggttctaatcggctcctactaaacacctaagcatca ctgcgcctctatctctcctatccacaggtctaagacgcttggaataagacatgtgggtgcaatagga agattagctagtccaatctctccttccagctacgcttctcccttcgatgagcgtagggggggccccc acctccctcatctctggatagggctcttgctacggggctttcccgtctggaccagcaggcccactg gtgcgcttccattcaagtttagtgtgcattactgtctgaaatattgctttgctaggatctagtgtagcga cctgcatattgccagcggacttccccacatggtaacatgtgcctctgggcccaaaaggcatgtcttt gaccgtatgcagtacaaccccagtataggtcctttctatggcagtatggatctcagtgatgagtctat acagaatatggaagtggttcggatatgtcagcccgaaggatgcccagaaggtacccgcagataa ccttaagagactgtggatctgatctggggcccaccaccttcggggggtagaagctaaccatgcct tgggttaaaaaacgtctaagggctgaccagacccgggggatccgggttttccctatcttgacctact ctaatc 472 LjunganVirus ctcattgcccacacctggttggttcccaggttcatacaataaccatcaataaacttttaacatctaagat 87-012 agtattatcccatactagactggacgaagccgcttggaataagtctagtcttatcttgtatgtgtcctg cactgaacttgtttctgtctctggagtgctctacacttcagtaggggctgtacccgggcggtcccact cttcacaggaatctgcacaggtggctttcacctctggacagtgcattccacacccgctccacggta gaagatgatgtgtgtctttgcttgtgaaaagcttgtgaaaatcgtgtgtaggcgtagcggctacttga gtgccagcggattacccctagtggtaacactagcctctgggcccaaaaggcatgtcatttgaccac tcaggtacacaaccccagtgatgcacacgcttagtaatggcttagtaacaaacattgattgatcattt gaaagctgttaggaggtttaggtatgacgggctgaaggatgccctgaaggtacccataggtaacct taagcgactatggatctgatcaggggcccaccatgtaacacatgggtagaagtcttcggaccttgg gttaaaaaacgtctaggcccgccccccacagggatgtggggtttcccttataaccccaatattgtat a 473 ParechovirusA2 gccgtcgggccttacaccccgacttgctgagtttctctaggagagtccctttcccagccagaggtg gctggtcaaacaataccaaacgtaactaaacatctaagataacatagccctatgcctggtctccacc agttgaaggcatcttgcaataaaatgggtggattaagacgcttaaagcatggagtcaattatcttttct aactagtgatcttcactgggtggcagatggcgtgccataactctattagtgggataccacgctcgtg gatcttatgcccacacagccatcctctagtaagtttgcaaggtgtctgatgaggcgtgggaacttatt ggaaataattacttgctgcgaagcatcctactgccagcggatcaacacctggtaacaggtgcccct ggggccaaaagccacggtttaacagaccctttaggattggttaaaacctgagtaattatggaagata cttagtacctaccaacttggtaacagtgcaaacactagttgtaaggcccacgaaggatgcccagaa ggtacccgcaggtaacaagagacactgtggatctgatctggggccacctacctctatcctggtgag gtggttaaaaaacgtctagtgggccaaacccaggggggatccctggtttccttattttagtgtaaatg tcatt 474 ParechovirusA3 agagtccttttcccagccagaggtggctggttaaataatacctactgtaacaaaacatctaagatgta acaaccacacacctggtctccactggccgaaggcaactagcaataaggcaggtgggttcagacg cttaaagtgtgttgtacatattcttttctaacctgtgttttacacagggtggcagatggcgtgccataac tctaacagtgagataccacgcttgtggaccttatgctcacacagccatcctctagtaagtttgtaagat gtctgatgacgtgtgggaacctgttggagataacagtttgctgcaaagcatcccactgccagcgga tctacatctggtaacagatgcctctggggccaaaagccaaggtttaacagaccctttgggattggtt caaacctgaactgttatggaagacatttagtacctgctgatttggtagtaatgcaaacactagttgtaa ggcccacgaaggatgcccagaaggtacccgtaggtaacaagtggcactatggatctgatctggg gccagctacctctatcttggtgagttggttaaaaaacgtctagtgggccaaacccaggggggatcc tggtttctttttaatttaagtaatcact 475 ParechovirusA8 gggccttataccccgacttgctgagtttctctaggagagtccctttcccagccctgaggcggctgga taataaaggcctcacatgtaacaaacatctaagacaaaataatttgccttgcacctggtccccactag ttgaaggcatctagcaataagatgagtggaacaaggacgcttaaagtgcaatgatagttatcttttct aacccactatttatagtggggtggtggatggcgcaccataattctaatagtgagataccacgcttgtg gaccttatgctcacacagccatcctctagtaagtttgtgagacgtctggtgacgtgtgggaacttact ggaaacaatgctttgccgtaaggctttcattagccagcggaccaccacctggtaacaggtgcctct ggggccaaaagccaaggtttaatagaccctaatggaatggttcaaacctggagcattgtggaaagt acttagtacctgctgatctggtagtaatgcaaacactagttgtacggcccacgaaggatgcccaga aggtacccgtaggtaacaagtgacactatggatctgatctggggccaactacctctatcttggtgag ttggttaaaaaacgtctagtgggccaaacccaggggggatccctggtttccttttattttactttgtcaa t 476 ParechovirusA17 ctctattagtgagataccacgcttgtggaccttatgctcacacagccatcctctagtaagtttgtaaga cgtctggtgacgtgtgggaacttgtgggaatcaatattttgctttaaagcatccattagccagcggat aaaacacctggtaacaggtgcctctggggccaaaagccaaggtttaacagaccctagtggattgg tttcaaaacctgaaatattgtggaacacactcagtacctactgatctggtagtaatgcaagcactagtt gtaaggcccacgaaggatgcccagaaggtacctgtagggaacaagagacactatagatctgatct ggggctggctacctctattttggtgagtcagttaaaaaacgtctagtgggccaaacccagggggga ccctggtttccatttattttacaaaggcact 477 PotamipivirusA cacatggaaagcttttcgcttccatgtttacgcacacactctctttgacaccctgttgtatggtgttaaa ctacaacatttgtctgtctataatcgtttattttgtttaccctatatgtacccaagtatttgattgcttgactc acataagcatcggtaacccatactgttttatgagctactacctctgctgtctacatacattttatatgaat ggtttgagctctgcctcaggatcaaacatggtaacatgttcctttggtcagttagaatcttattgtataat ctaaggtgtctattagtacgtagaaagttgtaacacatatggggcctgatagccgctatctctgatgg atgtaaggtaaccttctttaggtctgatacattctgcacaggatccaattttcggtgccctgtacgagt gcactcttatgcacgaggacgagatatgctacaacccactgcaaatttaaacccaaactttaaca 478 PotamipivirusB tttcaacgtcgtggctgacgttaaaaagccacaattccacttaccttttaccttttatgtttaatgtttgtta gttttgtgatctttaacaaatagatctaaataatttgttggtaaccaatctcggatgtttcggctgcattgt agtttatttatttcattttagttgtaggtggccactacgtcctggaatcatacatggtaacatgtacctcg gcggttatccactattacgctaatctaagaatatttaaatgaaaatgtaagtgttacggctgactttgg gcctgatagttaaatgctcgcactgacagatagtaccctcctttaggatcgattctgttacatgggatc cattttggtgccccactgattcaacctctttgttgaaaaagagttagcatactacaaattttccaaacaa aaaccctttttaatgactacaacttatgattttatgaattttactgctcttgaaaaagatattttgacattga tcgctgtactgtttcagacattcattgcatccatttttgttggctactcctcacaaactcaaaacttttcca cacgagaaaccttgtttattgaattttgcctttatattttggaacttgttgttggatttattgtttgcttaatta ttgacctcacacctgttttaaacactacaat 479 BeihaiConger gggacaaccccacagctggtacaaccattgtgggttggtctccaccctttttcaaccgtggcaactt Picornavirus cggttaaagttgcaaatcccccctctccctattccacctcccttactttcactccccatatatggtccca gattttattctacctctttatatttttatttagtacagtggtggtgaattactcccagcataaactttgctgg atcagtgttcatcaagcatactaattactaatgtactgagctatactattatctggcatctcacctggat aaccggtgtgaccatatttcctaggttgcctccctatgtattttgtagcacctgtgcatctgcacgttgg ggcgacaaattgtaggtttcctggcacgggtaagaattgtggaaagctagtatgcagttaatgcaa gggcgcgtttttcgctaccccgacactgctaaagtttttgggaggggtcccttaaacatttctagtatt gagtgatagctttgcggcaggtcaccacaaccttactataaataaacctgttgaatctcac 480 Porcine tacgcatgtattccacactcatttcccccctccacccttaaggtggttgtatccccataccttaccctcc Sapelovirus cttccacaatggacggacaaatggatttgacctcacggcaaacacatatggtatgatttcggataca JD2011 ccttaacggcagtagcgtggcgagctatggaaaaatcgcaattgtcgatagccatgttagtgacgc gcttcggcgtgctcctttggtgattcggcgactggttacaggagagtaggcagtgagctatgggca aacctctacagtattacttagagggaatgtgcaattgagacttgacgagcgtctcctcggagatgtg gcgcatgctcttggcattaccatagtgagcttccaggttgggaaacctggactgggcctatactacc tgatagggtcgcggctggccgcctgtaactagtatagtcagttgaaaccccccc 481 Porcine ttgaaatgggtgtggggtacatgcgtattacggtacgcatatattccacactcatttccccccctcca SapelovirusA2 cccttaaggtggttgtatccccataccttaccctcccttctaaaacagatggacaaatggatttgaact tatggcaagtgaatatggtatgactttggatacactttaacggcagtagcgtggcgagctatggaaa aatcgcaattgtcgatagccatgttagtgacgcgcttcggcgtgctcctttggtgattcggcgactgg ttacaggagagtaggcagtgagctatgggcaaacctctacagtattacttagagggaatgtgcaatt gagacttgacgagcgtctcttagagatgtggcgcatgctcttggcattaccatagtgagcttccagg ttgggaaacctggactgggcctatactacctgatagggtcgcggctggccgcctgtaactagtata gtcagttgaaacccccc 482 Simian ccaaggatctgttgcataggcgttgtatcccctaaccttttacctacccatcccaataggactggtatt Sapelovirus1 tcggttttgattgagtaatggatactgattctatacctgttacccattcaggggaaaaatggagtttcttt catggatctgacttgatatgaccaagagtcaacactttgcgtgttggccgtatggaatgctttaaggtt tattctttggattatgacttcagggttggccgcccaggataaaaggcaattgtggtaagtgatgttagt cattggtggttgaaacctgcctaagacgtcctaggtctacgctgtgcgggccgaagtaagcttagg aataacagggagtatgccattttctgctttcacccaacacgaccgtacacgaaagagctagaggca ctttggggcaaagggaaaagctttgcttagcccgaatgttcatttgagtccttgacgaatgcgtccc gtctgtcccgacggtgaggcgtatggcgcatgctcatggcattacccaatggtgtatctgtgaggg gggggctcctcacacttagtctagtgctacctgacagggccgcggctggtcgtttgtgtatggtata accagtagtaatcccccatggattgctttaacttcccctcctcccttaccaagacattctctaag 483 Simian ttttaacttgttatgacattcaaggaaaaaatgtctttttcattatgggactgacctgtttatgaacatgag Sapelovirus2 cagcggcactgctccacgggctatccgtgtaagaaatattgattattcttatggatcatgatttcagg gttggccgcccagtctaaaaggcaattgtggtaagctatgtaagtagttggctgttgaaaggagcc aagtacatcctaggtctacgctgtgcgggccgaagtaagacttggaacaactctgagtaggcagtt tttctctttagcccaacacgaccgcatactgaagagctagaggcactttggggcaaaggtaaaagc attgcttagaccgaatgttcaatgagaccttgacgagtgctgtcacagtgtcccctgatggcagtatg gcgcatgctcttggcattacccatatgtgtatctatagggggggggccccctatacttagtctagtgc tacctgacagggccgcggctggtcgtcggtgtgtggtataaccagtagtaatcccccatggattgc tttaactccccctcctccctcaacaaaactttctctaag 484 RabovirusC ccgggtataacccggagttttggggcaggtccaagccccacataggaacatacgatccacggatc gtgtgttcttttatgctttctaaccttaccctttgtaaccattacgctttacgccgcatggtgtttggcggc accatgacgtggacaagaggttacgccattacgatatgtaccctccccttttggggagagaccgac caattatggtacagtatccaactgtattgtggtcaagtacttctgtttccccggtgatgcgggataggc tgtacccacggccaaaacctgctgatccgttacccgactcacatctacgaggaggctagtaaaag gcatgaagttcaagagtatgatccaaccagatccccactggtaaactagtgatgagggttcccgttc cgaacatggcaacatgtgggttccctgcgttggcactaggccccttccgaggggtgctctgaagat ggattgttgatgaagaccaatttgtgcatgtgtttatcctccggccctctga 485 RabovirusA ccgaccccactggtcgaaggccacttggcaataagactggtggaacaaggtcgcctgtagttgatt NYC-B10 ggaaccttctttctaatgacttatgtcagcggtgctactcacaccgtaactctcctaccctatccccac gcttgtggaactaggaggggatgagtgattcaagtaagtactgtcagaatggtgaaaataatctgat tctgaaacgctatggatccatcgaaagatggggctacacgcctgcggaacaacacatggtaacat gtgccccaggggccgaaagccacggtgataggatcacccgtgtagtttgagatcatatcaatgttc atagtctagtaagatgatttgaaatctaactggtctgatggctaactgcttgtcttattgcggcctaagg atgtcctgcaggtacctttagagaaccttaagagactattgatctgagcaggagccaaggtggtcttt cccagccttggttaaaaagcgtctaagccgcggcaggggggggaggccccctttcctcccaaa ctataatatagattgt 486 ParabovirusC gatgtatccccatcccccagtgtgtatgccatactgcatagctcgcctatgccctatggattcacaac cctttcatataccctccctacccaaccccgtaaccacatgctttactccgcttggggttttgggcccc atgttgtgacgaaatggctacgcaatcaatgcggctaatggggcctgccgcttttaagtggcccca gttagaagtttatgcacacccgcccattaggaggccaccagccaggtggtcagagggcaagcac ttctgtttccccggtgaagtttgataagctgtgcccacggctgaagcagacagatccgttacccgcc tcactactacgagacggctagtagtgtgtaatatccgaatttcattgatccgggtgttccccccaccc agaaacgtgtgatgaggagcggcacccctcctatggcaacatagggcctctcctgcgctggcac acgggctctatgagcatgaaatcaggagaaagtcacacgaagaccttattgtgctagtgttgattcc tccgcccccctgaatgcggctaatcccaactccggagcgcccgctggcaaacccgccagaaga gcgtcgtaatgcgtaagtctggagcggaaccgactactttgggtgtggcgtgtttcctttatttccttt gtatttgtat 487 ParabovirusB aacccataatccattgtccatcaatgttttatgggggggaccctttctcccctccccctccaaatacct tttacccctctgtaaccaagagtgtgcaaaatctatttactagcccagaattgcggcttctggggagg tttattcctcatgcctaacaagatgttacgcaaactccgggctacggccctgggcttttgccctaaag atttagaagtttacactatcgtccaacaggaggacaacaaaccagttgttctaaggacaagcacttct gtttccccggtgagactggatagactgtacccacggttgaaactggttgatccgttacccgactcac tacttcgagaagattagtaggaaactgtgaaactgattccattgatccggatactttccccgtatcca gaaactactgatgagggttgacttcccgactacggcgacgtagtgtcatccctgcgctggcagtag gcctctttgaggatggaagatgtggatcggtaaccgaaggtcctattgagctagtgtttatacctccg gcctcctgaatgcggctaatcctaacccatgatctagtgctcacaaaccagtgagtagctagtcgta acgcgtaagtcgtgggcggaaccgactactttggagtgaccgtgtttcctattttacttttgtttg 488 ParabovirusA3 accgttacgcaccactcagttggtgtttggtggcaccaatgatggaacaaaaggctacaccacttg ggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactaggg ccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgtac ccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactgga aagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccacccc gaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgctcct actggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatccta acccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcggaacc gactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatctcga tctatagcgtttcttgagagatttccaaacatttattcaagtcgtacaattcttgtgtttaagcagtacagt gtaagg 489 Felipivirus127F gatgtcggatgacggctggccaccggggaaaaacggcaaatgtgcaccacctctgcaacccac gccgaccacgtttaaccatggcgttagtaggagtggaccactgcagtgggctctggtgtgcgaca gtcagtggtagagtagacagtcctgactgggcaatgggaccgcgttgcgtatccctaggtggcat cgagattcctctgctacccaccagcgtggactcctatggggggggccccataggctaggtctatac tgcctgatagggtcgcggctggtcgaccactgactgtataaccagttgtaactcact 490 BoosepivirusA ttgaaagacctcggcatatatcgttgtcacaacggtatatgtcgagatctttctccccaccccctcca attcccttttccccctcttgcaacttagaagtttgtacacacagggcaataggatacgtgatccagcc aggacacgtgagctcaagcacttctgtttccccgtccccttcacgtactacgggaatgttagtaattt gtgtgcactttagtaaggttgatccgggattaaccccaaatcccagaaactggtgatgagcgttacc acccccgccgggcgaccggaaggtttcgctgcgttggcaccagggcttcggcaccagaaaaag gtaaagcaaatgaaggcgctactgtgctacgagaagtttcctccaggcccctgaatgcggctaatc ctaaccagtgatccaccggtgcaaaaccatgtactaggtggtcgtaacgcgcaagtcgctggcgg aaccgactactttgggtgtcctgtgtttccatattttattttattcaattttatggtgacaagagtaaagag atacagatttgcagcc 491 BoosepivirusB ttttctcccctccccctccaactaccttttccccctcttgtaacgctagaagtttgtgcaaaccgcctgt agggtactgcaatccagcagtgcataggctaagcttttcttgttaccccaccccacattatactgagg aggattgtgaaattgtgttagtatgggttagtagcggtgacccgggtaaccccaacccagaaactc acggatgagatgaacaggaccccacatggtaacgtgtgtgttcgtctgccccgcaaggtgaggcc gtgagagctttgcacgcgaaaaccttgaaaacccaaaagtaccttgagctcttcgctattttgtgtttc ctccaggaccctgaatgcggctaaacctaacccgcgatccgcacgtagcaacccagctagagtgt ggtcgtaatgcgcaagttgcgggcggtaccgactactttggtgttcctgtgtttcctttattttattttga atttttatggtgacaacagctagaaaataagagtgaac 492 PhacovirusPf- gtgtgtcatttctcccctccccctcccaaaccttttccccctctaatcggattgattaacccggttaaag CHK1 atgattaatggtttgtgagttgatatgatggcccggcattgaatccgggaattcttaagtaatggaatt gcatccaatatgaaagtgagtgtggcaagctcacaagtagtacttggctctgcccattatttgagga ccaactcttcttgactacaatgtgtttaaagtaaactggaccacattgtgtatccagacaactccatttg ataatgtacgctggaaacgttttcagtgcatagggtcctaaagtggtgctgaaatattgcaagctcaa tgggatactgaacgctgaaaaccgccgctgttatcatatgggcccctagtgggtaaatgttggcttt aggcatatactgcttgggaatgcagtactggttgtagacagggtgatagcctaccggctggcgtag ttgagttggtatagccagttgattgccat 493 HRVC3QPM ttaaagctggatcatggttgttcccaccatgattacccacgcggtgcagtggtcttgtattacggtac atttccataccagttttatacaccccaccccgaaactcatagaagtttgtacacaatgaccaataggt ggtggccatccaggtcgctaatggtcaagcacttctgtttccccggcacccttgtatacgcttcaccc gaggcgaaaaatgaggttgtcgttatccgcaaagtgcctacgaaaagcctagtaacactttgaaaa cccatggttggtcgctcagctgtttacccaacagtagacctggcagatgaggctagacattcccca ccagcgatggtggtctagcctgcgtggctgcctgcacaccctgccgggtgtgaagccagaaagt ggacaaggtgtgaagagcctattgtgctcactttgagtcctccggcccctgaatgtggctaacccta accccgtagctgttgcatgtaacccaacatgtatgcagtcgtaatgggcaactatgggatgggacc aactactttgggtgtccgtgtttcctgttttactttttcattgcttatggtgacaattgtatctgatacacttg ttacc 494 HRVB27 ttaaaacagcggatgggtatcccaccatccgacccacagggtgtagtgctctggtattttgtaccttt gcacgcctgtttccccattgtacccctccttaaatttcctccccaagtaacgttagaagtttaaggaaa caaatgtacaataggaagcatcacatccagtggtgttatgtacaagcacttctgtttccccggagcg aggtataagtggtacccaccgccgaaagcctttaaccgttatccgccaatcaactacgtaatggcta gtattaccatgtttgtgacttggtgttcgatcaggtggttccccccactagtttggtcgatgaggctag gaactccccacgggtgaccgtgtcctagcctgcgtggcggccaacccagcttttgctgggacgcc tttttacagacatggtgtgaagacctgcatgtgcttgattgtgagtcctccggcccctgaatgcggct aaccttaaccccggagccttgcaacataatccaatgttgttgaggtcgtaatgagtaattctgggatg ggaccgactactttgggtgtccgtgtttccttttattctttatattgtcttatggtcacagcatatatagcat atatactgtgatc 495 HRVA73 ttaaaactgggtttgggttgttcccacccaaaccacccacgcggtgttgtacactgttattccggtaa ccttgtacgccagttttatatcccttcccccccttgtaacttagaagacatgcgaatcgaccaatagca ggcaatcaaccagattgtcaccggtcaagcacttctgtttccccggctctcgttgatatgctccaaca gggcaaaaacaattggagtcgttacccgcaagatgcctacgcaaaacctagtagcatcttcgaag atttttggttggtcgctcagttgctaccccagcaatagacctggcagatgaggctagaaatacccca ctggtgacagtgttctagcctgcgtggctgcctgcacacccacacgggtgtgaagccaaagattg gacaaggtgtgaagagtcacgtgtgctcatcttgagtcctccggcccctgaatgcggctaacctta accccgtagccattgctcgcaatccagcgagtatatggtcgtaatgagtaattacgggatgggacc gactactttgggtgtccgtgtttcactttttacttatcaatttgcttatggtgacaatatatatagatatatat tgacacc 496 EVL acatgggccagcccaccacacccactgggtgtagtagtctggttctatggaacctttctacgcctctt ttgcttccctcccccatttctccttcgattgctccacctgtgatctttgcaacttagaagaaataatgaac ccgcacaatagcgggcgctgagccacagcgtcaatgtgcaagcacttctgtttccccggaatggg cccataggctgtacccacggctgaaagggaccggcccgttacccgccttggtactgcgagaatgt tagtaactccctcgatagctttaggcgttacgctcagccctttgagcccgaagggtagttcgggtcg atgaggctcgtcattccccactggcgacagtgtgacttgcctgcgttggcggcccggggtggggg gcaacccccatccacgcctactgaaggacagggtgtgaaggcgctattgcgctactaaggagtcc tccggcccctgaatgcggctaacccgaaccccgagcccacggtggtaaacccgccacaagtgg gtcgtaatgagtaatttggggcagggaccgactactttgggtgtccgtgtttcctgtttttccatacgat ggctgcttatggtgacaaccataagcaattggattggccatccggtgttcatattgcgaat 497 EVK tcagcctgacgcaagtgcctccattggagtctctccaagccctccggggcttggagggcgccgac cccctgcctagggggagcccacgacacggctggagtccattggcacaccgcagccacgattca agccagaattgaaagcgggaagcacttctgtctccccggtgtggatcatacgctgtacccacggc gaaaagtgaagcatcgttacccgactcggtacttcgagaagcccagtacagttgtggatctctgca gggtatacgctcagcgtgacccctacgtagttccttgagatggctgagagaacaccccacgggcg accgtgtctctcggcgcgtggctcaaggccgggccttcagtggctcggtgccttgcagagtgaag cctccgaacagcctattgagctaccgtttagcctccgccctcttgaatgcggctaatcctaaccatg gagcgcccgcccacagtccagtgggtagagcgtcgtaacgcgcaagtccgtggcggaaccgac tactttagagtggcgtgtttccaatttatcctttataaagttgcttatggtgacaccacaagagatccac gatttcttgtttcttatcactgagacacaagtcatattcatcaatctttattgcggaattaacttggtgcgt ccaaacacatcagc 498 EVJ1631 caccctgagggcccacgtggcgtagtactctggtatcaaggtacctttgtacgcctattttatttccct tcccccacagtaacttagaagcttatctcatagttcaacagtagggtcactaaccaagtggctcagc gaacaagcacttctgtttccccggtcctagtacctgtgaagctgtacccacggcggaaggggaaa aagatcgttatccggccccctacttcggaaagcctagtaacaccattgaagcaatcgagtgttgcg ctcagcacagtaacccctgtgtagctttggttgatgagtctgggcactccccactggcgacagcgg cccaggctgcgttggcggccaaccgactcgggcaaccgggtcggacgctcgtttgtggacatgg tgtgaagagcctactgagctagagggtagtcctccggcccctgaatgcggataatcctaaccccg gagcacccacactcaatccagagtgcaggatgtcgtaacgcgtaagtctgggacggaaccgact actttgggtgtccgtgtttcctgttttacttactttggctgcttatggtgacaatctagtgttgttaccatat agctattggattggccatccggtgttttgaattgtgtgtttatactaattcttttacatatcacagacaacc aaat 499 EVJN125 cggtacctttgtacgcctattttacccccttccccttgtaacttagaagcaaagcaaaccagttcaata gtaagcaacacaacccagtgttgtgacgaacaagtacttctgtttccccgggagggtctgacggta agctgtacccacggctgaagtatgacctaccgttaaccggctacctacttcgagaagtctagtaata ccattgaagttttgttggcgttacgctcaacacactaccccgtgtgtagttttggctgatgagtcacgg cattccccacgggcgaccgtggccgtggctgcgttgcggccaaccaaggggcgcaagctccttg gacgtcacttaacagacatggtgtgaagaacctattgagctaggtagtagtcctccggcccctgaat gcggctaatcctaactccggagcacatcagtgcaacccagcatttggtgtgttgtaatacgcaagtc tggagcggaaccgactactttgggtgtccgtgtttcctgttttaccttatttggctgcttatggtgacaa tttgatattgttaccatatagctgttggattggccatccggatttttgaaagagacccaaaactttcttct ctacttcagattcaagtgcgaagttttccttttcatatattacttactaatttgaagtaccaaag 500 EVI ttagtactttctcacggggatagtggtatccctccctagtaatttagaagacttgaaaaaccgaccaat aggcacctcgcatccagcggggtaaaggtcaagcacttctgtttccccgggtcgagtagcgatag actgtgcccacggtcgaaggtgaaacaacccgttatccgactttgtacttcgggaagcctagtacc accaaagattatgcttggggtttcgctcagcacgaccctggtgtagatcaggccgatggatcaccg cattcctcacaggcgactgtggcggtggtcgcgtggcagcctgccgatggggcaacccatcgga cgccaagcatatgacagggtgtgaagagcctactgagctacaaagtattcctccggcccctgaat gcggctaatcccaaccacggagcatttgctaccaaaccaggtagtggaatgtcgtaacgggtaac tctgtggcggaaccgactactttgggtgtccgtgtttccttttaatttatcattctgtatatggtgacaact atagtgctatctcgatttgcattactattgttgagattaaaactttattacattgttgcattttaccctttgag tgagttttcacctgaacagattaatttactcatcctgtttatatattacaagcagaaatacttgcaaag 501 EVF1BEV261 gcaatgctgcaccagtgcactggtacgctagtaccttttcacggagtagatggtatcccttaccccg gaacctagaagattgcacacaaaccgaccaataggcgcaccgcatccagccgtgcagcggtca agcacttctgtctccccggtctgtaaagatcgttatccgcccgacccactacgaaaagcctagtaac tggccaagtgaacgcgaagttgcgctccgccacaaccccagtggtagctctggaagatggggct cgcaccacccccgtggtaacacggttgcctgcccgcgtgtgcttccgggttcggtctcgtgccgtt cacttcaacttcacgcaaccagccaagagcctattgtgctgggacggttttcctccggggccgtga atgctgctaatcccaacctccgagcgtgtgcgcacaatccagtgttgctacgtcgtaacgcgtaagt tggaggcggaacagactactttcggtaccccgtgtttcctctcattttatttaatattttatggtgacaat tgttgagatttgcgctcttgcaacgttgccattgaatattggcttatactatttggttgccttttacaaaac ctctgatatacccagttcttacattgatctgcttgtttttctcaatttgaagtatagactacaaatagcaaa 502 EVD94 cgtggcggccagtactctggtatcacggtacctttgtacgcctgttttatatccccttcccccgcaact tagaagaaaacaaatcaagttcactaggagggggtacaaaccagtaccaccacgaacaagcact tctgtttccccggtgatgtcgtatagactgtaaccacggttgaaaacgattgatccgttatccgctctt gtacttcgaaaagcccagtatcaccttggaatcttcgatgcgttgcgctcagcactcaaccccagag tgtagcttaggtcgatgagtctggacactcctcaccggcgacggtggtccaggctgcgttggcgg cctacctgtggtccaaagccacaggacgctagttgtgaacaaggtgtgaagagcctattgagctac aagagaatcctccggcccctgaatgcggctaatcctaaccacggagcaagggtacacaaaccag tgtatatcttgtcgtaacgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttccttttgt ttttatcatggctgcttatggtgacaatctaagattgttatcatatagctgttggattggccatccggtaa tttattgagatttgagcatttgcttgtttcttcaacaatttcacctattcattgcatttcagcagtcaaa 503 PV3 tacctttgtacgcctgttttatactccctcccccgcaacttagaagcatacaattcaagctcaatagga gggggtgcaagccagcgcctccgtgggcaagcactactgtttccccggtgaggccgcatagact gttcccacggttgaaagtggccgatccgttatccgctcatgtacttcgagaagcctagtatcgctctg gaatcttcgacgcgttgcgctcagcactcaaccccggagtgtagcttgggccgatgagtctggac agtccccactggcgacagtggtccaggctgcgctggcggcccacctgtggcccaaagccacgg gacgctagttgtgaacagggtgtgaagagcctattgagctacatgagagtcctccggcccctgaat gcggctaatcctaaccatggagcaggcagctgcaacccagcagccagcctgtcgtaacgcgcaa gtccgtggcggaaccgactactttgggtgtccgtgtttccttttattcttgaatggctgcttatggtgac aatcatagattgttatcataaagcgagttggattggccatccagtgtgaatcagattaattactcccttg tttgttggatccactcccgaaacgttttactccttaacttattgaaattgtttgaagacaggatttcagtgt caca 504 EVC102 ctttgtacgcctgttttacatcccctcccccacgtaactttagaagcaattcaacaagttcaatagagg gggtacaaaccagtatcaccacgaacaagcacttctgtttccccggtgattttacataagctgtgcc cacggctgaaagtgaatgatccgttacccgctcgagtacttcgaaaagcctagtatcgctttgggat cttcgacgcgttgcgctcagcactctaccccgagtgtagcttaggctgatgagtctgggcattcccc atcggcgacgatggcccaggctgcgttggcggcctacccatggctaacgccatgggacgctagt tgtgaacaaggtgtgaagagcctattgagctactcgagagtcctccggcccctgaatgcggctaat cccaaccacggatcaggtgcctccaacccaggaggtggcctgtcgtaacgcgcaagtctgtggc ggaaccgactactttgggtgtccgtgtttccttttatcttttaaatggctgcttatggtgacaatcataga ttgttatcataaagcgaattggattggccatccggtgaaatacaaacacattatttacttgtttgttggat ttactccgctcacacagcttactcctaagataatatttattgtattgctggtaaggagacactattata 505 EV30 aagcaaggcaaacctgaccaatagtaggtgtggcacaccagccgcattttggtcaagcacttctgt ttccccggaccgagtatcaataagctgctcacgcggctgaaggagaaaccgttcgttacccgacc agctacttcgagaaacctagtaacactatgaacgttgcggagtgtttcgttcagcacttcccccgtgt agatcaggtcgatgagtcaccgcattcctcacgggtgaccgtggcggtggctgcgttggcggcct gcctacgggttcgcccgtaggacgctctaataccgacatggtgtgaagagtccattgagctagctg gtagtcctccggcccctgaatgcggctaatcctaactgcggagcaggtgctcacagaccagtgag tagcctgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtttttccttttttcttctctta tattggctgcttatggtgacaattaaagaattgttaccatatagctattggattggccatccggtgacg agcagagccattgtttacctctttgttggatttgtacctttgaaccacaaagtcttgaataccattcatct cattttaaagttcaactcagctaaaagaaa 506 SA5 agtacttggtattccggtacctttgtacacctatttacaaaccctaccccttgtaaccttagaagcaatt atttaaccgctcactagggggtgtgctatccaagcacatcaagagcaagcacttctgtctccccgg gaggggctaatggtacgctgtgcccacggcggaaatgagccctaccgttaaccggcagtctactt cgggaagcccagtaactacattgaaactttgaggcgttacactcagcacataaccccaatgtgtagt tctggtcgatgagccttggcatcccccacaggcgactgtggccaaggctgcgttggcggccagc ctgcggaccaaaagtccgtaggacgcctaattgtggacatggtgtgaagagcctactgagctaga ctgtagtcctccggcccctgaatgcggctaatcctaaccctggagcatccgcgtgcaacccagtac gtagggtgtcgtaatgcgtaagtctgggatggaaccgactactttgggtgtccgtgtttcttgtttttca tactgggtcgcttatggttacaactaattgttgtaatcattggcagtgcgcgctgaccacgcgattatt gatatttccatttgttggatactccaatagtgtcaactcatatacacaacttttaccactgatcaagataa aa 507 EVA114 tgtgcgcctgttttgaaaccccctcccccaactcgaaacgtagaagtaatgtacactactgatcagta gcaggcgtggcgcaccagccatgtctcgatcaagcacttctgtttccccggactgagtatcaatag actgctcacgcggttgaaggtgaaaacgtccgttacccggctaactacttcgagaaacctagtagc accatagaaactgcagagtgtttcgctcagcacttcccccgtgtagatcaggtcgatgagtcactgc aatccccacgggtgaccgtggcagtggctgcgttggcggcctgcctatggggcaacccatagga cgctctaaggtggacatggtgtgaagagtctattgagctagttagtagtcctccggcccctgaatgc ggctaatcctaactgtggagcgcatactcccaaaccagggagcagtgcgtcgtaacgggcaactc cgcagcggaaccgactactttgggtgtccgtgtttccttttattcctatactggctgcttatggtgacaa ttgagagattgttaccatatagctattggattggccatccagtgtgtaatagagcaatcatttaccaatt tgttggatttactccattaacccacacgtctctcaacacactacatttcatcttactactgaacactaga aa 508 MobovirusA tattctcccacaaaccttcttgtaactctgttaagccttttacatccatgtaatttaattttctccacctaaa aggatttcccccatggtcctttttggctcgaacaaatgctacatagggtcttgttttctccccctggctc tcttgccagggttccataccccaattcctctatttccatgatttttcatcatggtttatttttactgtcttctta ttttctgaggtgaccaactcctaagccgactgggtcgcggaagcccggactcctcgcatcactagg gtgcgtagcgatgtaggcgaaaatattggttgctagatgcatacatatagtgaattgatactacacca aactctgttctttttgaaactagctattttctaagtaaggtaggctacgggtgaaaccttaccattgcag gtacgtgaaccgcaacggacatttggccgaagactggtgtacccacgtcagttataggacctcttc aacgttggtggacggcatgtcactgattagttaggctagtgaatttaagttcagggggtatcttttagc ttaagcgtgtattctagtaggacttgcagagcctccccacctaggaggatctctgtttatagccccttt tccttgttccgttagttttccacacttttacaatatttgatgatttgtt 509 BurpengaryVirus ctccccccccttccccttcccgagtaggagattggcatgtatgctctacatgcccgattctctcttgct cactctcttaaatcctggtggcggtctcggattaaacatttatgtcgtatctgggatcgtcttacttggt ggtaattcctctgttgcctagggacctccggactgccggattaaaggtctcaacagagggcaatgt acaaggaagtcattatacgctaattaagtatttgatgaatgactagtgtgacagggctgaggaactc cccccgggtaaccggtgcctcagcgtccgaaagacacgtggataggatccaccctgttataccca gcacgatgtaatagtcaaatacctctgatttgtgtaggatgtataaattgtgcattgtaaattttgggcg tagagatgctccgaaggtaccccgttttacgggatctgatcggaggctaattacccaatgcgcccta aataacttcatataatttctttttctttattcaaa 510 HunnivirusA1 taacgtttggcaagaaccctcacctgtcaattgggaccaccactttcagtgaccccatgcgaagtag tgagagagaataagctttcttacccttcatttgtgaacccttcagtcgaagccgcttggaataagata ggaggaaaagttcattctaaatggagtgaaacatgtacttcagaatttctagcacgcgctgggctttc ttgcgtgtgacggcactgtcttgccggagctctccacactgacaccccacgcttgtggaccttggtg gcagatgacaacactgcagctggaattgagtgtctggtacactctgtgtaacagtgaaaacaatgt gatcacttcggtgagctagtagcctgtggaccaacaactggtaacagttgcctcaggggccaaaa gccacggtgtttacagcaccctactggtttgattggagcaatccaagatgtcacagagttagtaattg ccaagcagtccgtactggtatcttgacataccgtgcagttttggatagtgaaggatgccctgacggt acccataggtaacaagtgacactatggatctaagcaggggctcactctacgctgctttacagctgg ctgtgagttaaaaaacgtctagctatccacaacctaggggactaggttttccttttatttagattacaatt at 511 HunnivirusA2 acagtttttgacaaggaccctcacctgtcaatcgggaccaccactttcagtgaccccgtgcgaagt gttgagagaaagtgagctttcttacccttcatttgtgaacccttcagtcgaagccgcttggaataagat ggaaggaaatgttcattctaaatggagtgaaacatatacttaatttccagtgtttagtggtctttccact agacaacggcactgtcttgccggaactctacacaccaacattccacgcttgtgggactcaaatgttg gatgacacagttgtagctggaactgagtgtttagtgcactctgtgtaacagtgaaaacaatgtgatca cttcggtgggctagtagcctgtggactaacaactggtaacagttgcctcaggggccaaaagccac ggtgttaacagcaccctactagtttgattggagcaatccatgatgttacagagttagtaactgccaaa cagattgtactggtatcttggcataccgtgcaacttaggatagtgaaggatgccctggcggtaccca taggtaacaagtgacactatggatctaaacaggggctcactctacgttgctttacaactagctgtga gttaaaaaacgtctaactatccacaacctaggggactaggttttcctttttatttttatacacaacta 512 IaIo ggtgtccgggtgtgtgggatagacccagatgtgcagtggatgcgagcatttgagtcagagtagga gcaagcccaggggcaaagggaccacattgtgtatcccgaatgaaggatcgagatttctctcctcat tacccggtgtcttgtcactgttgggggggcccaacagtcttagtcctatactgcctgatagggtcgc ggctggccggactcaagtgctatagtcagttgattttcactc 513 TauraSyndrome ctttaaaagtcgtgcgtggcttcaccacgcacgatcagtactatcagttaaccactcttgaatatgctc Virus aatgaccctattcaacactggtgtctcttagtacattattttagcacttaacgtgcatgagttttgcccat ttctttcaaaaaaatgagtattcgaggagacgtcccgctccccgtcttatttcaaccgtagactcgac atctattggtggacatttaattccagtcgccgtaagttgcttctgccccgcgctatattttcttatacttat ggttctataggtctggtttaaaacgtaaatagacggcccacaaactatagaacgcgtacccggaac gccaatcccggataagtccctggatatatagatgcaccgcaatataagcctgcagactgtctcatat act 514 ABPV cccgtcaaaataacaacttataacacgatgttacccgaagaaaccattttagtgtaacatttaagatta gaagtagttcatctaatagagataggcactattagaaggaggccttttctaaaggagccgttagtca gcccagacaagcgcagtactttagaagagagaagttccccgatagcgaccgaaaagacgcgtttt ccgtgctaactaatttaaatgtgggaacgaatattattattgaaattatgtgagccacgtagcaatcaa gtcatgtttttgtcactacgtttactcatctaatgtagataattttgtttaagtacctatttaggtgtcatccc accagagaagaaataatacgtaccggaacccagagtacaccccttattttaagccttactgggcttc tctgttagttagtaatctggcccacgttttgcgttgagtggggtcccaacagtaggaattcgacggac aagtagcaagcgagtcggtaccaattggtttagccttcgaaattactctgggcaggaagttactaaa cgagaactttctgcttaaatcccaacgcacaaacaaatagagtaaataaataattata 515 BRAV-2 tttgttttgcggctttgccgttgttcgggttttacctgttttcacacagcaaaacaggccttctagtttcgt gcttaaacgagatcatgctcgaactagaactacatagctggtcactggactcataccacaccttgtg gagctttatgggaaaggtggctagtgggctgtggaagtgactctgaccacatgcctctcaagtgtg ggaaatcacggatcggtgtagcgacgacaacaggccttgggacaccctctccagtaatggagac ccaaggggccaaaagccacgcctcgtgccctgttgttcacaaccccagtgcgacccgtgttagta cctatttgcgagaactgtgtctggacagctaaacacaaccctagtgggagactaaggatgcccag gaggtacccggaggtaacaagtgacactctggatctgacctggggagagagggcttgctttacag gcgcctctctttaaaaagcttctatgtctcatcaggcaccggaggccgggccttttccttttaaaatta cactta 516 BRBV-1 ccccccctacttaaagatgtacggttttgctgctttcacagagtaaagcagatagaggttctgaactg gcaaactttacctcgaaacacgcccgtttttctgctgtgtctcacagactgtcctgtcacacttgtggc ggcttgtgacactgtgaacatagtgagaccgaccaagacaacagtttcaagtgatgaacatcgaac gtctaaactggatccgtaactggacatgttagggcaaggacttcccccctggtaacaggagcctgg ctggccaaaagccccgctcattgagcctagcatgttgtcgaccctggactgttcagtttagttagtac atggaattcacttgtcacggttcttctgaactcggtctctagtatgacagcctaaggatgccctccag gtaccccggggtaacaagtgacacccgggatctgaggaggggactactttacgtagtttaaaaaa cgtctaagctgttatggtgaccagaggctggcacctttcacttttaaaattacactactgactacaatt gaagtgataacggttttacaggctttcaaactagttacacaagcactgttttcctgacacacacacttt 517 ERAV-1U188 aatattggcgcgcgcatttgcgcgcccccccccatttcagccccctgtcattgactggtcgaaggc gttcgcaataagactggtcgtcacttggctgttctatcgtttcaggctttagcgcgccctcgcgcggc gggccgtcaagcccgtgcgctgtatagcgccaggtaaccggacagcggcttgctggattttcccg gtgccattgctctggatggtgtcaccaagctgacaaatgcggactgaacctcacaaagcgacaca cctgtggtagcgctgcccaaaagggagcggaactcccccgccgcgaggcggtcctctctggcc aaaagcccagcgttaatagcgccttttgggatgcaggagccccacctgccaggtgtgaagtggag tgagtggatctccaatttggtctgttctgaactacaccatctactgctgtgaagaatgccctggaggc aagctggttacagccctgaccaggggccctgcccgtgactctcgatcggcgcagggtcaaaaatt gtctaagcagcagcaggaacgcgggagcgtttcttttccctttgtatcgac 518 GFTV atggggaagggtatgacgtgccccttccttcttcggagaactcgctctagtggtctttccacttctgg aaaagagtgagtgcacgtgatcaggaccgtcgaagacgacaaatacctggtgctctatctcatag acgtttcacagctgtagcgacccctcagtagcagcggaagccccctcctggtgacaggagcctct gcggccaaaagccacgtggataagatccactgctgagggcggtgcgaccctagcaccctgtgat gcatactagttgtagcgtgccggactattggtctgtcataagacacctgatagagagaccaagaat gtcctggaggtaccccgcgtgcgggatctgaccaggagaccattgcccaatgctttacaacgggt ctatggtttaaaaactgtcgcagtctctccaaaccaagtggtcttggttttcaattactttgaatatttca ct 519 SAFVV13C ttttcgacgtggttggaattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgt tttctgccctgctgggcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttc ccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatc cgtaaaaagggtacgtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaa caagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcac atcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgc ggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgat gatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcct gaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttgg tttaaaaattgtcacagcttctccaaaccaagtggtcttggttttccaattttgttgaatggcaat 520 SAVP-113 ggagatctaagtcaaccgactccgacgaaactaccatcatgcctccccgattatgtgatgctttctg ccctgctgggtggagcatcctcgggttgagaaaaccttcttcctttttccttggaccccggtcccccg gtctaagccgcttggaataagacagggttatcttcacctcttccttcttctacttcatagtgttctatact atgaaagggtatgtgtcgccccttccttctttggagaacacgcgcggcggtctttccgtctctcgaaa agcgcgtgtgcgacatgcagagaaccgtgaagaaagcagtttgcggactagctttagtgcccaca agaaaacagctgtagcgaccacacaaaggcagcggaccccccctcctggcaacaggagcctct gcggccaaaagccacgtggataagatccacctttgtgtgcggcacaaccccagtgccctggtttct tggtgacacttcagtgaaaacgcaaatggcgatctgaagcgcctctgtaggaaagccaagaatgt ccaggaggtaccccttccctcgggaagggatctgacctggagacacatcacatgtgctttacacct gtgcttgtgtttaaaaattgtcacagctttcccaaaccaagtggtcttggttttcactctttaaactgattt cact 521 VHEV aattccttcttcctttctccttggacctcggtcccccggtctaagccgctcggaatatgacagggttatt ttcacctcttctctcttctacttcatagtgttctatactatgaaagggtatgtgtcgccccttccttcttgga gaacgtgcgtggcggtctttccgtctctcgaaaaacgtgcgtgcgacatgcagagtaacgcaaag aaagcagttcttggtctagctctggtgcccacaagaaaacagctgtagcgaccacacaaaggcag cggaaaccccctcctggtaacaggagcctctgcggccaaaagccacgtggataagatccaccttt gtgtgcggtgcaaccccagcaccctggtttcttggtgacaccttagtgaaccctcgaatggcaatct caagcgcctctgtaggaaagccaagaatgtccaggaggtaccccttcctcatggagggatctgac ctggagacacatcacacgtgctatacacttgtgcttgtgtttaaaaattgtcacagctttcccaaacca agtggtcttggttttcccttaacttcgaaaagtcactatggcctgcaaacatggatacccagacgtgt gccct 522 TRVNGS910 atgcgacgtggttggagattaaaccgactccgacgaaagtgctatcatgcctccccgattatgtgat gttttctgccctgctgggcggagcattctcgggttgatacaccttgaatccttcatccttggacctcag gtcccccggtctaagccgcttggaatacgacagggttattttccaatcttctcctttctactttcatgag tcctattcatgaaaagggtctgtgctgccccttccttcttggagaatctgcgcggcggtctttccgtct ctcgaaaagcgcagatgcagcatgctggaaccggtgaagaaaacagttctttgtggaaacttaga gcagacatcgaaacagctgtagtgacctcacagtagcagcggaaccccctcctggtaacaggag cctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccct ggttcgatggttgttctctgtggaaccagagaatggtctttctcaagccctccagtagagaagccaa gaatgtcctgaaggtaccccgcatggggatctgatcaggagaccaatcgtcagtgctttacactg gcgctttggtttaaaaactgtcacagcttctccaaaccaagtggtcttggttttcacttttatcaaactgt ttc 523 EMCV2RD1338 aaatactggtcgaaaccgcttgggataagaccggggtttgttaatgtctcaatgttattctccaccca attgacgtcttttgtcaattggagggcagtgaaaccttgcccttgcttcttgcagaggattcccagtgg tctttccgctctcgacaagggaattcatgatccaccaaaagttgtgaagagagcaggtcccatgga agctttctgacgactgatgatgactgtagcgaccctttgcaggcagcggacccccccacctggtga caggtgcctctgcggccaaaagccacgtgtttaacagacacctgcaaaggcggcacaaccccag tgcctcatcaaaagtctgatgactgtggaaatagtcaaccggcttttcttaagcaaatttggtgtcgg ggctgaaggatgcccggaaggtaccacactggttgtgatctgatccggggccacagtacatgtgc tttacacatgtagctgcggttaaaaaacgtctaggccccccgaaccacggggacgtggttttccttt gaaaaccacgattacaat 524 EMCV1JZ1203 gtctgctcgatatcgcaggctgggtccgtgactacccactccccctttcaacgtgaaggctacgata gtgccaggggggtactgccgtaagtgccaccccaaaacaacaacaaaccccccctaacattact ggccgacgccgcttggaataaggccggtgtgcgtttgtctatatgttatttcccaccacattgccgtc ttttggcaatgtgtgggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttccc ctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttg aagacaaacaacgtctgtagcggccctttgcaggcagcggaaccccccacctggcgacaggtg cctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccac gttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctga aggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatg tgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaa cacgatgataat 525 EMCV1AnrB- atgtggtcgaagccacttggaataagaccggcgtgcgcttgtctatatgttacttccaccacattgcc 3741 gtcttttggcaatgtgagggcccggaacctggccctgtcttcttgacgaacattcctaggggactttc ccctctcgccaaaggaatgtaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttctt gaagacaaacagcgtctgtagcgaccctttgcaggcagcggaaccccccacctggtaacaggtg cctctgcggccgaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccac gttgtgcgttggatagttgtggaaagagtcaaatggctttccccaagcgtattcaacaaggggctga aggatgcccagaaggtaccccactggttgggatctgatctggggcctcggtgcaggtgctttacac ctgttgagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctagaaaa ccactatgacaat 526 CosavirusD1 cgtgctttacacggtttttgaaccccacaccggctgtttggcgcttgcaggacagtaggtatattttctt tcatttctcttttctagccgcgtaggttctatctacgcgggcggagtgatactcccgctccttcttggac aggcggcctccacgctctttgtggatcttaaggctgccaagtcactggtgtttgaagtgaagaatgg agagacactagggcgtttcatgtggctttgccagggattgtagcgatgctgtgtgtgtgtgcggattt cccctcgtggcgacacgagcctcacaggccaaaagccctgtccgaaaggacccacacagtggg gttgccccgacccctcccttcaaagctttgtgtaaacaaacttttgtttagactttcttaagcttctctca catcaggccccaaagatgtcctgaaggtaccctgtgtatctgaggatgagcaccaccaactaccc ggacttgtgggacgtgtcccacagacgcatgtggtattccagccccctccttttgaggagggggct tttgctcgctcagcacaggatctgatcaggagattcatctctggtgctttacaccagagcatggattta aaaattgcccaaggcctggcaaacaacctaggggactaggttttctctattttaaaagatgtcaat 527 CosavirusB1 cgtgctttacacggtttttgaaccccacaccggctgtttggcgcttgcaggacagcaggtttattttctt ttaactctctctttctagccacacacgatctatgtgtgtgggggagtgatactcccgttccttcttgga caggcggcctccacgccctttgtggatcttaaggctaccaagtcactggtgttggaaagtgaagag aaaggagttccttgggaactacatgtggcattgacagaggttgtagcgatgctgtgtgtgtgtgcgg attacccccgtggcgacacggaccccacaggccaaaagccctgtccgaaaggacccacacagt ggagcaaccccagctcccctcttcaatgttttgtgttagcaaccttggtattattttctctcaagcttcca atacaccgggccccaaagatgtcctgaaggtaccccgtgtatctgaggatgagcaccatcaacta cccggacttgttctttcgagaacagacgcatgtggtaacccagccccgatcctaaggggtcgggg cttttgctcactcagcacaggatctgatcaggagacctcccccccctgctttacagggggcggggg tttaaaaattgcccaaggcctggcaaataacctaggggactaggttttcctttttattttaaagttgtca at 528 CosavirusASH1 ccgtgctttacacggtttttgaaccccacaccggctgtttggcgcttgcaggacagcaggtttattttc ttatgctctttatttctagccaacagggttctatcctgttgggcggagtgatactcccgttccttcttgga cagattgcctccacgatctttgtggatctcaaggtgatcaagtcactggtaaatagagcgaaggttg aggaaacctgaggaatttccatgtggttttgccaggagttgtagcgatgctgtgtgtgtgtgcggatt tcccctcatggcaacatgagcctcacaggccaaaagccctgtccgaaaggacccacacagtgga gcaatcccagctccctcctacaaagctttgtgagaatgaactcacgtttattcttctttattctctgtttac atcaggccccaaagatgtcctgaaggtaccttgtgtatctgggcatgagcaccatcaactacccgg acttgcatttcggtgcagacacatgtggttacccagcccctctgctttggcagaggggcttttgctcg ctcagcacgagatctgatcaggagcccttcccagtgtgctttacacctggggggggttaaaaatt gcccaaggcctggcaaaataacctaggggactaggttttccttttattaacaatgtctgtcatt 529 MalagasivirusB ctttattttcttatgtaactcttctttttaagttttattttgcctacttgtgagcttatgcgggaccactgtctta gacaaccccacatttgtcatgagtaagtacacgcaaccattacgattactttttaaccgtctgacctttt gataacaactgaagttaggcgtgaaacatgcatttataccaaagtagccccgcatttccccactacg gtgggggggctaccctactggctttggaactgtagccattatgtgttgcctggctttcaggatctcac aacacaacagttctctcacaatggaatatgggtgagattgcagtgacatgaacaagtatctagtagt acatagactcaagcctagttgcctgcggaacaacatgtggtaacacatgccccagggtccaaaag acaagggttaacagccccttctaggtgtctgtgtgtgaagaatactttagtagtgttgttatgatctcac ctgttagtacagaatgagtatggcttggtgaaggatgtcctacaggtacccattatatggatctgagt aggagaccactagtggtggctttaccgccaggtgagtggtttaaaaagcgtctagccaagccaac agcactagggatagtgctttctatattttatattttcagtgtat 530 MosavirusA2 cccccccctcaaattgcaacgatatagctaatggcgagattgagatgctatatcacctcttctaagtt SZAL6 atagacctcatctgattgataaggacgtaatttggtcgaaaccgcttggaataagaccgatgcgcgt agtcatgatgatgatgtaagatctaggaacttatccaatctgcttatgtctatgtaagtagaggggca ggcctcattgccctaattctttctaccgagtatctgctagggtttctagcggcttgaatacttggattga gggatacaagatactactgatcgattgtcgattgggaaacttgtagatacttcaaagctaccagtag cgtggactcacagccagcggactacccctcatggtaacatgagcctctgggcccacaaggcacg tcgcaagacctgtgagacggcaaccccagcctagctttgttgaggaaacaagcgataacatgaca tgagagaccggaaggattcttgtattgtgagccgaaggatggcctctaggtacctcattttatgagat ctgaggaggtgctcttgagttggtgctttacactgacaacacagagttaaaaagcgtctaagctcac ccggaaattgggaaatttccgttatttccattttgtttgcaaagtcgttc 531 SVV ctgggccctcatgcccagtccttcctttccccttccggggggtaaaccggctgtgtttgctagaggc acagaggagcaacatccaacctgcttttgtggggaacggtgcggctccaattcctgcgtcgccaa aggtgttagcgcacccaaacggcgcatctaccaatgctattggtgtggtctgcgagttctagcctac tcgtttctcccctatccactcactcacgcacaaaaagtgtgctgtaattacaagatttagccctcgcac gagatgtgcgataaccgcaagattgactcaagcgcggaaagcgctgtaaccacatgctgttagtc ccttcatggctgcgagatggctatccacctcggatcactgaactggagctcgaccctccttagtaag ggaaccgagaggccttcttgcaacaagctccgacacagagtccacgtgattgctaccaccatgag tacatggttctcccctctcgacccaggacttctttttgaatatccacggctcgatccagagggtgggg catgatccccctagcatagcgagctacagcgggaactgtagctaggccttagcgtgccttggatac tgcctgatagggcgacggcctagtcgtgtcggttctataggtagcacatacaaat 532 PTVA actagtccttggacttttgttgtgtttaaacacagaaatttaattacctggccatgaattcattggattaa cccttctgaaagacttgctctggcgcgagctaaagcgcaattgtcaccaggtattgcaccagtggt ggcgacagggtacagaagagcaagtactcctgaccgggcaatgggactgcattgcatatcccta ggcacctattgagatttctctggggcccaccggcgtggagttcctgtatgggaatgcaggactgga cttgtgctgcctgacagggtcgcggctggccgtctgtactttgtatagtcagttgaaactcacc 533 PTVB cttccttttaattcgtaactgataagtgatagtccttggaagctaggtatttgttacgctagttttggatta tcttgtgcccaacatttgttttcgaacatatgttgtgtttaaacacagaaatctagtttctttggttatgagt ttaatggaatatccttttgaaagacttgccttggcgcgggctagagcgcaattgtcaccaggtattgc accaatggtggcgacagggtacagaagagcaagtactcctgactgggtaatgggactgcattgca tatccctaggcatctattgagatttctctggagcccaccagcatggagttcctgtatgggaatgcagg actggacttgtgctgcctgacagggtcgcggctggccgtctgtactttgtatagtcagttgaaactca tt 534 Tottorivirus cccctttacgtaactgcaacttaaagagtaccctactgcattggatgtgtggtaaacttttacgcacac atttgtagtagtgttagttatgttctacctaatgagtatgcatgcacccgtcgaaacacgcttgtgataa gataggtgagtccatgtgactaatctcattaagataaataagcaccctacaacgcacggcacgctc gtgtcttccgtgcggggccgggacaacagcggcctaaatcttctaggtgaccaccatgcttttggg actatggcaccactgtggacgtgagtacctggcagtaagtctgtgaaaagatggaaggtgtccca agctatggggcgtatgcatatagcctgcggaacaaacaacggcgacgttgtccccagggcccaa aaggcacgtggataagatccacctatatgtttaccccatagtgtaagtcactggaagtcctagtaatg gatgtctggagtaaggctcacggggtagggcgaaggatgcccagaaggtacccgtaggtaacct taagagactatggatctgatctggggaccggatggcgccatcaccatgacgtggaggccggttta aaaaacgtctaagcccgaccaacaacctaggggactaggttttccttttttattcatgtatgacgtt 535 Posavirus1 acatttccttgcgtgcgcacccgaaaatttattgaacttggcttgaatcataagtaatgcttcttatagc ggacactttgagaatataatgcttgtatggattaatgtatactgttttaaataacaaacctagacacgc agttgcgttgatggttgtatcaatcacatataagtgttgaactcgtgttaatcctcgatcgctatatgttt gccgcctacttccaataaaatagattacatgcgcgtcatgcctttgtgggttacctattggcctctgac aaaaacaagtcgtaagatttgtagcttcccggtgtaaaaagctgggcgcggtctggctctcgtagg gtggaaaggtccaccaatggctggttgagtgtaagctccggtgtcctggttgtcgcaattccaggc gtcgtaataacctatattgcatctgactctaactcttgtggctctactgtatctagttcttgttctactaact ctaataatactactggctctaatactgaaaaacttacatatgttaatatagataatatccttgatcctgat atccctcacgtcactgaagttcgccgaaaacgaatttcagatcatatcattgaatctcaaggatgtac ttgctctgaacctactataactcctcatgcgttttcattttctactcttggc 536 A105-675 ccacccacagcaagaatgccatcatctgtcctcacccccatttctcccctccttcccctgcaaccatt acgcttactcgcatgtgcattgagtggtgcacgtgttgaacaaacagctacactcacgtgggggcg ggttttcccgcccttcggcctctcgcgaggcccacccttccccttcctcccataactacagtgctttg gtaggtaagcatcctgatcccccgcggaagctgctcgcgtggcaactgtggggacccagacagg ttatcaaaggcacccggtctttccgcctccaggagtatccctgctagtgaattctagtggggctctgc ttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctct catgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagcaatggac gggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggt tggtgtgcccaacgcccgtactttggtgacaactcaagaccacccaggaatgccagggaggtacc ccgcctcacggcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccatttctttctt ctgttc 537 A110-675 acctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaaca gtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaataga ctgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaaca ccgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgc attccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatggg acgctctaatacagacatggtgcgaagagtctattgagctagttggtagtcctccggcccctgaatg cggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaac tctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtgac aattgagagatcgttaccatatagctattggattggccatccggtgactaatagagctattatatatcc ctttgttgggtttataccacttagcttgaaagaggttaaaacattacaattcattgttaagttgaatacag caaa 538 18-675 cccacagcaagaatgccatcatctgtcctcacccccaattttcccttttcttcccctgcaaccattacg cttactcgcatgtgcattgagtggtgcatgtgttgaacaaacagctacactcacatggggggggtt ttcccgccctacggcctctcgcgaggcccaccccttccctccccttataactacagtgctttggtag gtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatc aaaggcacccggtctttccgccttcaggagtatccctactagtgaattctagcggggctctgcttggt gccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgt gtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggc cagtgcgtgtagtcgtcttccggcttgtccggggcatgtttggtgaaccggtggggtaaggttggtg tgcccaacgcccgtactttggtgacacctcaagaccacccaggaatgccagggaggtaccccac ctcacggtgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttcttctgttc acg 539 A115-675 acctttgtgcgcctgttttataccccccccaacctcgaaacttagaagtaaagcaaacccgatcaata gcaggtgcggcgcaccagtcgcatcttgatcaagcacttctgtaaccccggaccgagtatcaata gactgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacccagta gcatcatgaaagttgcagagtgtttcgctcagcactacccccgtgtagatcaggccgatgagtcac cgcacttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatggggcaacccat aggacgctctaatacggacatggtgcgaagagtctattgagctagttagtagtcctccggcccctg aatgcggctaatcctaactgcggagcacatacccttaatccaaagggcagtgtgtcgtaacgggta actctgcagcggaaccgactactttgggtgtccgtgtttccttttaatttttactggctgcttatggtgac aattgaggaattgttgccatatagctattggattggccatccggtgactaacagagctattgtgttcca atttgttggatttaccccgctcacactcacagtcgtaagaacccttcattacgtgttatttctcaactcaa gaaa 540 A73-675 ttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatactccccccaccc cccttttgtaactaagtatgtgtgctcgtgatcttgactcccacggaacggaccgatccgttggtgaa caaacagctaggtccacatcctcccttcccctgggagggcccccgccctcccacatcctcccccc agcctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtcc ccccttcatcaagacaccaggtctttcctccttaaggctagccccggcgtgtgaattcacgttgggc aactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccct ggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccaacctggtga caggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaa ggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtac cccacctccggggggatctgagcctgggctaattgtctacgggtagtttcatttccaatccttttatgt cggagtc 541 Kobuvirus16317 ttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccatccc ctttctgcaacttaagtatgtgtgctcgtgatcttgactcccacggaatggatcgatccgctggagaa caaactgctagatccacatcctcccttcccctgggaggaccttggtcctcccacatcctccccccag cctgacgtaccacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtcccc ccttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaac tagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggc ccttcactatgtgcctggcaagcatacctgagaaggtgttccgctgtggctgccagcctggtaaca ggtgccccagtgtgcgtaaccttcttccgtcttcggacggtagtgattggttaagatttggtgtaaggt ccatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtacccc acccccggggggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgtcg gagtc 542 Aichivirus ttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacatccccatcccc Chshc7 tttctgtaacttaagtatgtgtgcttgtaatcttgactcccacggaatggatcgatccgctggagaaca aactgctagatccacatcctcccttcccctgggaggaccttggtcctcccacatcctccccccagcc tgacgtaccacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtcccccc ttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaacta gtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcc cttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacagg tgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttc atgtgccaacgccctgtgcgggatgaaatctctactgccctaggaatgccaggcaggtaccccac cctcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaatccttttatgtcgga gtc 543 Aichivirus actccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccatcccct Goiania tttttgcaacttaagtatgtgtgctcgtaatcttgactcccacggaatggatcgatccgctggagaaca aactgctagatccacatcctccctcccccctgggaggacctcggtcctcccacatcctccccccag cctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtcccc ccttcatcaagacaccaggtctttcctccttaaggctagtcccgatgtgtgaattcacatcgggcaac tagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggc ccttcactatgtgcctggcaagcatatctgagaaggcgttccgctgtggctgccagcctggtaaca ggtgccccagtgtgcgtaaccttcttccgtccccggacggtagtgattggttaagacttggcgtaag gttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtacc ccaccttcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttctaattctttcatgtc ggagtc 544 Aichivirus ttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccacccc ETHP4 ctttttgcaacttaagtatgtgtgctcgtgatcttgactcccacggaatggatcgatccgctggagaa caaactgctagatccacatcctcccttcccttgggaggacctcggtcctcccacatcctccccccag cctgacgtaccacaggctgtgtgaagcccccgcgaaagccgctcacgtggcaattgtgggtccc cccttcattaagacaccaggtctttcctccttaaggctagtcccgatgtgtgaattcacattgggcaac tagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggc ccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacag gtgccccagtgtgcgtaaccttcttccgtcttcggacggtagtgattggttaagatttggcgtaaggtt catgtgccaacgccctgtgcgggatgaaacctctactaccctaggaatgccaggcaggtacccca ccctcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttctatgtcgg agtc 545 Aichivirus tactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccaccccc DVI2169 ttttctgcaacttaagtatgtgtgctcgtaatcttgactcccacggaatggatcgatccgctggagaac aaactgctagatccacatcctcccttcccctgggaggaccccggtcctcccacatcctccccccag cctgacgtatcacaggctgtgtgaagtccccgcgaaagctgctcacgtggcaattgtgggtccccc cttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaact agtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggc ccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacag gtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggtt catgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtacccca ccttcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgtcgga gtc 546 Aichivirus gcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccacccccttttctgcaac DVI2321 ttaagtatgtgtgctcgtaatcttgactcccacggaatggatcgatccgctggagaacaaactgcta gatccacatcctcccttcccctgggaggaccccggtcctcccacatcctccccccagcctgacgta tcacaggctgtgtgaagtccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaa gacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaactagtggtgtc actgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactat gtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacaggtgcccca gtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgcc aacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccaccttcggg tgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgtcggagtc 547 Aichivirusrat08 tactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccactttcctgtcctctccc cttttctgtaactccaagtgtgtgctcgtaatcttgactcccgcggattgaccgctccgctggtgaaca aactgctaggtcatctcctccccacccttgggcgtccttccgggcgtccacaccctccccccagcc tgacgtgtcacaggctgtacaaagaccccgcgaaagctgctaacgtggcaattgtgggtcccccc tttgtaaaggaaccgagtctttctcccttaaggctagacccctgtgtgaattcacaggtggcaactag tggttccactgcatgctcccgacctcggccgcggagtgctgttccccaagtcgtaacactgaccttc acttatgtgcctggcaagcatatctgagaagatgttccgctgtggctgccaaacctggtaacaggtg ccccagtgtgcgtagtcttcttccgtcttcggacggtaggtgttaggtaaagatgcggcgtaaggtt caagtgccaacgccctggaagggatgacccttctactgccctaggaatgccgcgcaggtacccc aggttcgcctgggatctgagcgcgggctaattgtctacgggtagtttcatttccctcttcttccactgg catc 548 AichivirusRt386 actccattcagcttcttcggaacctgttcggaggaattaaacgggcacccactttcctgtcctctccc cctttctgcaactcaagtgtgtgctcgtaatcctgactcccacgggttgaccgccccgttggtgaac aaacagctaggtcattcccccctacccctgggcgccatttcagtgggttcatatcctccccccag cctgacgtgtcacaggctgtgcaaagtccccgcgaaagctgctcacgtggcaattgtgggtcccc cctttgtgaaggaaccgagtctttctcccttaaggctagacccctgtgtgaactcacaggtggcaac tagtggttccactgcatgctcccgacctcggccgcggagtgctgttccccaagtcgtgacactgac ctccacttatgtgcctggcaagcatatctgagaagatgttccgctgtggctgccaaacctggtaaca ggtgccccagtgcgtgtagtcttcttccgtctccggacggtaagtgtgtggtaaagatgcggcgtaa ggttcaagtgccaacgccctggaagggatgacccttctactgccctaggaatgccgcgcaggtac cccaggttcgcctgggatctgagcgcgggctaattgtctacgggtagtttcatttccctctcttttcact ggcatc 549 NorwayRat gtataagggttgggaaccttgtaccaagctacctctgccattcagtatttgggagtagaagtagatgt Pestivirus gtttacaaactcacacgtgtgggggggggatagactgtgccagcggtcgtgtaccagcacctac gcatacgtgtggactgcgaaccaggagagcacctaggtctgacaagctgtgagaacacagtagt cgtcagtgagtcagctggtaaggatcacccacctggatactcacgtggacgagggagtttcccag tcagaaacctacaccagaggaggggtcctctggagacatggatggtctgagtaacagactatcta ctggggtgtgctgcctgacagggtctcggctgatagcctggctagcagtataaaaatcagttgaatt ggcatatgagttgtgaacatctagtaaacaatgaaagacaaaaacaaaaaatgagcataataaaaa aattgtacaatccactactcaggtgtggctgcagactt 550 Porcine tttgaaaaggggggggggggcctcggccccctcaccctcttttccggtggccattcgcccgggc KobuvirusGS2 caccgttactccactccactccttcgggactggtttggaggaacacaacagggcttcccatccctgt ttaccctttattccatcatcctttccccaagtttaccctatccacaccccactgactgactcctttggattt tgacctcagaatgcctatttgacctcccactcgcctctcccttttcggattgccggtggtgcctggcg gaaaaagcacaagtgtgttgcaggctaccaaactcctacccgacaaaggtgcgtgtccgcgtgct gagtaatgggataggagatgcctacaacaggctcgcccatgagtagagcatggactgcggtgca tgtgacttcggtcaccacgggcatagcattgctcacccgtgaatcaagtcatcgagatttctctgacc tctgaagtgcactgtggttgcgtggctgggaatccacgcttgaccatgtactgcttgatagagtcgc ggctggccgactcatgggttaaagtcagttgacaagacac 551 Kobuvirus cctacccaagggttacatgggaccatattcctcctcccctgtaactttaagttttgtgcccgtattcttg SZAL6 actccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacactttcctccccccctct gggctgctccggcagtccactccctccccccagcgtaacatgccccgctggagtgatgcacctgg aagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaactggttgacccccac actcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatttgcagctggcaacta gtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaagcgtatgccttccttctg taagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtgccaacctggcgacag gtgacccagtgtgcgtagacttcttccggattcgtccggctcttctctaggaaacatgcgtgtaaggt tcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtgccgcaggtacccctac ttcggtagggatctgagcggtagctaattgtctacgggtagtttcatttccatcttctcttcaggtcgac atc 552 Kobuvirussheep gaccttctggtacttcttcgcctgggtcacaaaagcgaagaacctgcctctctaacgccagacgag TB3 cggcattaaacttgaacttctggcactctccactctcccttttccctgtccctttccccactgcgctctc aaggtcgcgcaatcctgggactagcccagttttaaaagttcctggcaccctttgcccctctaggccc ttaaggtaggaactgaccttgtgctgtgatctcggtgcgggagtgctaccacgtagtcatcgtaagc ctcgtttctggttctgccctggcaaggctacagagtaccgtgttccgctgtggatgccatccgggta accggacccccagtgtgtgtagcggtatgttcacggtccgccgtgttcaccagattcctgacctgg ctttgctagaaatggtgtgtgcccaatccctgtgaccagtatcaattacatcacctaggaatgctagg aaggtaccccagtcctgagctgggatctgatcctaggctaattgtctacggtgatgctccttttattttc ttacaactgctattgactgtctgattgctgattctgctcttgtgctcttctgctctggctcattctcaaggg ttctctttgtccaagatcctttggttctctccttgttccacttgccactgccaacgcttgtc 553 Pronghorn gtatacgcagttagttcatcctgtgtatacagattggagactctaaaaacaacgattcggaataggg antelope gcccgcggcgaagaccgaagacaggctaaccatgccgttagtagggctagcaccaaaacgcg pestivirus ggaactagacacttaggagagtggtctggctactctaagaggtgagtacaccttaaccgtcaagg gttctactcctcagttgaggactagagatgccctgtggacgggggcatgcccaagagttagcttag ccggggcgggggttgttccggtgaaagtagcaatattgaccacactgcctgatagggcggagca ggccccctaggtagtctagtataaaatgtctgctgtacatggcac 554 Porcinepestivirus tacgcggggtataacgacagtagttcaagtgtcgttatgcatcattggccataacaaattatctaattt isolate ggaatagggacctgcgacctgtacgaaggccgagcgtcggtagccattccgactagtaggacta Bungowannah gtacaaataggtcaactggttgagcaggtgagtgtgctgcagcggctaagcggtgagtacaccgt attcgtcaacaggtgctactggaaaggatcacccactagcgatgcctgtgtggacgaggacatgtc caagccaatgttatcagtagcgggggtcgttactgagaaagctgcccagaatgggtagttgcacat acagtctgataggatgccggcggatgccctgtattttgaccagtataaatattatccgttgtaaagcat 555 Porcinepestivirus gcagatatcggtggtggacctgggggttgggctcaccgtgccccttcatggggtagacctcactg 1 cttgatagagtgccggcggatgcctcaggtaagagtataaaatccgttgttcactaac 556 Pestivirusgiraffe- gtatacgagtttagctcaatcctcgtatacaatattgggcgtcaccaaatatagatttggcataggca 1 acaccccgatgcgaaggccgaaaagggctaaccatgcccttagtaggactagcaaaaaatcggg gactagcccaggtggtgagcttcctggatgaccgaagccctgagtacagggcagtcgtcaacagt tcaacacgcagaataggtttgcgtcttgatatgctgtgtggacgagggcatgcccacggtacatctt aacctatccgggggtcggataggcgaaagtccagtattggactgggagtacagcctgatagggtg ttgcagagacccatctgataggctagtataaaaaactctgctgtacatggcac 557 Classicalswine gtatacgaggttagttcattctcgtatgcatgattggacaaattaaaatttcaatttggatcagggcctc fevervirus cctccagcgacggccgaactgggctagccatgcccacagtaggactagcaaacggagggacta gccgtagtggcgagctccctgggtggtctaagtcctgagtacaggacagtcgtcagtagttcgac gtgagcagaagcccacctcgatatgctatgtggacgagggcatgcccaagacacaccttaaccct agcgggggtcgctagggtgaaatcacaccacgtgatgggagtacgacctgatagggtgctgcag aggcccactattaggctagtataaaaatctctgctgtacatggcac 558 Humanpegivirus tcagggttggtaggtcgtaaatcccggtcaccttggtagccactataggtgggtcttaagagaaggt isolateJD2B1I taagattcctcttgtgcctgcggcgagaccgcgcacggtccacaggtgttggccctaccggtggg aataagggcccgacgtcaggctcgtcgttaaaccgagcccgtcacccacctgggcaaacgacgc ccacgtacggtccacgtcgcccttca 559 Humanpegivirus cccggcactgggtgcaagccccagaaaccgacgcctatttaaacagacgttatgaaccggcgcc isolateGBV-C- gacccggcgaccggccaaaaggtggtggatgggtgatgccagggttggtaggtcgtaaatcccg ZJ gtcatcttggtagccactataggtgggtcttaagggttggtcaaggtccctctggcgcttgtggcga gaaagcgcacggtccacaggtgttggccctaccggtgtgaataagggcccgacgtcaggctcgt cgttaaaccgagcccattacccacctgggcaaacaacgcccacgtacggtccacgtcgccctaca atgtctctcttgaccaataggctttgccggcgagttgacaaggaccagtgggggctgggcgacgg gggtcgtataggaagaaaaatgccacccgccctcacccgaaggttcttgggctaccccggctgca ggccgccgcggagctggggtagcccaagaaccttcgggtgagggcgggtggcatttttcttccta taccgatc 560 Humanpegivirus tggtcaccttggtagccactataggtgggtcttaagagaaggttaagattcctcttgtgcctgcggcg isolateJD2B8C agaccgcgcacggtccacaggtgttggccctaccggtgtgaataagggcccgacgtcaggctcg tcgttaaaccgagcccatttcccgcctgggcaaacgacgcccacgtacggtccacgtcgccctttt aatgtctctcttgaccaataggttcatccggcgagttgacaaggaccagtgggggccgggggtca cagggatggaccctgggccctgcccttcccggcggggggggaaagcatggggccacccagct ccgcggcggcctgcagccggggtagcccaagaaccttcgggtgagggcgggtggcatttttctt cctataccgatc 561 HepatitisGB gccgggtggaaggcccggaaccgccccaccacctcaactaggtggtaagggtacgtctatcggt virusA ccggctggcccgaaaggcggtggatcctgtgtgttagggttcgtaggtggtaaatcccagcacag gtggtaatcgctatagggcaggcttatcccggtgaccgcttccctggatcctggagcgggtcgtgg cggcacggtccacaggagtggggcctccggtgtgaataagccctcgtctggagcatcagacgtt aaactgagacgtcccgaagagatcggaacgacgccccacgtatggcaacgccgcttaaaaccct tcggggacagctatgcgggttgacaatgccagtggggggccgggcccactattgttgtgggctcc gagttcctctagggatggccgaaaggcagccatggggccacccaggcggcgccgtgctacagg cggcaaggggaaaaatccttcgggtgaccccgggtggcattccctcccttagcagcatgagtgtg gtggtagctgcaacc 562 Simianpegivirus ggggaatctcaccccccgtccggttccggaagaatcggaaaccgacaccctgaccaatcattctt gatcatagagtggatgttagtgaaagccagacgaaagccggcggatgggtggtgacagggttgg taggtcgtaaatcccggccaccctggtacccggtataagttgggcggaagctgactgaagctccg tgctcttttctgtgcgttcttggtgcacggtccacaggtgacgcctataccggtgtgaataataggcc gactcgagcggagtcgttaaactgagaacctccatacggatggcaacttggcttgcgtacgggga cgccgctaaagtcacagtgggttaagtccggcgggttgacaaccccagcaaggcgagggggtc ctattgttggactctgccagttcccggtggaggtaggcatggggtggcccagctccgcggcgcgc tacagccggggtagcccaaaatccgaaaggtgaggggggccacatgtccgaaatttagtcaag c 563 PegivirusI agaatggtctaagtggttgccaccgtggtccgaaggggaggaggacctacgctgccagggttgg caggtcgtaaatcccgggtgtaggagatccctccttgttaggactgctggtagctggggggtcggt gaccccctgggcaaccgccaaacccggacgaccgggtggggctccatgttggcacggtccac aggtgtgaaccctaccggtgtgaataagggttggtggttgcggtccaccttaaacgtagtatgcatt gggcttggtaaaacaccgctcgtagtacggaacgccgcctttaaagacacagtaggcgtagccg gcgggttgacaatccatacggggggggggtgtggtcatggatctgtccacaccaccttcatgcg gccctctaagcaagccataccggggggaggcgcgcggcaccgcactgccgggcaaggggaa gaaccttcgggtgacccccccccaaccaccgtccgatcaatgctaatgttgcgtttaggcgtgaca ccggcaca 564 PegivirusK agaatggtgtgatccgtcgccgctccagcggaaagcgggcgggatctagtggttagggttgttcg cgtaaatcccacactagtggtacgctcgtataacgtgggagcagccggTggggtcgacccccc Acctggcggctgctgagcaccggacgaagcgcggggggtgaacgctaacccgcggcccggg ctgccaacgttaggcacgtcttggctggaagacgttaaacacagggccccccctcaaccctgatc cgaggccagagaccaaggtacgccgcccttttaaaggcgttactcgtccaataggatctctccgg cgggttgtcaaaccttgctggccctggtgatggttacgggagggggggggggggagtagaag ccccgcccggcatgggggtaccaagctcggcacgcccagcacgcgtggcgtaggggaaaaat ccttcgggtgacccctggtaccataaagtaattaacatgagcatgccgctagggtgtgctttttcttcc ttccttgggaaggcggtggcacc 565 Theiler'sdisease- tgataccgtgtcccggtacgacctcgcgcgtccccaagctcgccctgaggggggagcgtaaggg associatedvirus cgcgtagtggggtagccccccaaaccgagccaccctagtgagtgactttagaatggttagggaga ctaccgccttcgctgtttggggacctaatgatccgcgtgccagggttcttcgggtaaatcccggcgc ggtgttttgggttcagggcagtaggggcagacgggccagcagtcgctggttcctggtaccaccac cctatccggacgacctccctcacgaaaggtcgccacggtctgtggctcgacgacgcctataattca gtccgaggggcgcagccctcgttaaacttaggcaaggttcctcgccattgatttggccaggggttt aagtgaacgccgcccttttaatgtttaatagggttctttcccgggggttgacaaacacttccctggg ctcttcgttggcctcggttccttgatgcttcggcacccatgagcgcacaggggggggaccctgcga cagtccgccaagaggaaaatccttcgggtgacctcgtgcgcaacccaatcccttcttcttccacatg gcgtgtctgtggtgcatgctgtg 566 Rodentpegivirus ggacttcggtccccctgttactctgcgagccaccgcagagccagggttggtacgcccgaggtgtt agaccccggccgaaagctcctaaccatggggttagtaggacgtggtaaatgccactgaggggtt ggagagctggtagagcgagtaagtcggcgtaaggcccgagtacgggcctcccagcccgggtca gcctaaacctggctgtgatacccggtgcatggagggcgtgtcccaacgctcgatcgctgtagggt gggtccctgcagttgggtgtggctaccctgctcgtactgcttgatagagtcccggggacggacc agctctcgtcagtccgtggagttgcac 567 Humanpegivirus aactgttgttgtagcaatgcgcatattgctacttcggtacgcctaattggtaggcgcccggccgacc 2 ggccccgcaagggcctagtaggacgtgtgacaatgccatgagggatcatgacactggggtgag cggaggcagcaccgaagtcgggtgaactcgactcccagtgcgaccacctggcttggtcgttcatg gagggcatgcccacgggaacgctgatcgtgcaaagggatgggtccctgcactggtgccatgcg cggcaccactccgtacagcctgatagggtggcggcgggcccccccagtgtgacgtccgtggag cgcaac 568 GBvirus cccccggcactgggtgcaagccccagaaaccgacgcctatctaagtagacgcaatgactcggcg C/HepatitisG ccgactcggcgaccggccaaaaggtggtggatgggtgatgacagggttggtaggtcgtaaatcc virus cggtcaccttggtagccactataggtgggtcttaagagaaggttaagattcctcttgtgcctgcggc gagaccgcgcacggtccacaggtgttggccctaccggtgggaataagggcccgacgtcaggct cgtcgttaaaccgagcccgttacccacctgggcaaacgacgcccacgtacggtccacgtcgccct tcaatgtctctcttgaccaataggcgtagccggcgagttgacaaggaccagtgggggccggggg cttggagagggactccaagtcccgcccttcccggtgggccgggaaatgc 569 EquinePegivirus agaatggggagttaactcctggcactggcccgaagcatgaactgatcgcggtggcagggttcttc 1 gggtaaatcccggccgcgtgttgtgattgtgttagggcaggtgacagtcggcagggtcgaccccc tgcttcaggaccactgtcttcctggacgaccgttgctgaaaaagggccgccacggtctgtagctcg ccgacgcttctaattcaggccggaggaccacgctccgtaatcgagcccaagtactcaaaccccag cacccctgggtcacgccctacgccgcccttttaacgcttcggctaatagggtctatccgggggtt gacaaagggcgcagggttacctggtactacgagcttgggtgtccctgggagtaatcccagggtgc c 570 Culextheileri atataaatcccagtttggttaaacctatttcaaggcttaagttgtttattattttatcgccgctcgtgactat flavivirus aaagttgcctagcggagagagataaagaagaaggagttcaaggctcagggcagggcgcaagtt ccctggtccctaggccgctcgcaggaaggaggagtgaagaagaagaaagagaaggagaggac caccgccgaaagaaggcaggtgcctcacaagagggccaaccagcgtgttggaccagtggcca acgccggacggcgtggtggcctgctgggacgcctggggattggatggagtgccttcctacagga agacatcgttcaagccatc 571 Bussuquaravirus agtatttcttctgcgtgagaccattgcgacagttcgtaccggtgagttttgacttaacgcagtgagaa aagttttcgaggaaagacgagaagcgaattctctga 572 ZikaVirus agttgttgatctgtgtgagtcagactgcgacagttcgagtctgaagcgagagctaacaacagtatca acaggtttaatttggatttggaaacgagagtttctggtc 573 Yokosevirus agtaaattttgcgtgctagtcgctgagcgtcagaccgcaaagtgagtttttagtgatctaaagtgagg agttattcttactgtcatcaaacactacaaataaacacgttgaaattatttccggaagaacaactgtcc ggaatcaaagacg 574 Wesselsbron agtatattctgcgtgctaatcgttcgacgttagtccgtggagtgagcttctattagagtcgttaacacg virus tttgaataatttctactgaaaggagtagaagaaaggagattcattccca 575 Equine acctccgtgctatgcacggtgcgttgtcagcgttttgcgcttgcatgcgctacacgcgtcgtccaac hepacivirus gcggagggattcttccacattaccatgtgtcactccccctatggagggttccaccccgcccacacg gaaataggttaaccatacctatagtacgggtgagcgggtcctcctagggcccccccggcaggtcg agggagctgaaattcgtgaatccgtgagtacacggaaatcgcggcttgaacgtcatacgtgacctt cggagccgaaatttgggcgtgccccacgaaggaaggcgggggcggtgttgggccgccgcccc ctttatcccacggtctgataggatgcttgcgagggcacctgccggtctcgtagaccataggac 576 HepacivirusB accacaaacactccagtttgttacactccgctaggaatgctcctggagcaccccccctagcagggc gtgggggatttcccctgcccgtctgcagaagggtggagccaaccaccttagtatgtaggcggcgg gactcatgacgctcgcgtgatgacaagcgccaagcttgacttggatggccctgatgggcgttcatg ggttcggtggtggtggcgctttaggcagcctccacgcccaccacctcccagatagagcggcggc actgtagggaagaccggggaccggtcactaccaaggacgcagacctctttttgagtatcacgcct ccggaagtagttgggcaagcccacctatatgtgttgggatggttggggttagccatccataccgta ctgcctgatagggtccttgcgaggggatctgggagtctcgtagaccgtagcac 577 HepacivirusI cagggtttcgaccctggcccggatacctatcgccttacgccgaaaggtaacgagtaggagtcggg tccccaggcccttaccgccaccaagccaggtggggaggtatgggagccggggggtgcagctg gtagctccatgggggacgccccgtgagcggatgctgcatcgataccgggttagctctctgggaga gcggcacttgacaccacgaatccgggaaccggacaatcgccggcgtgggacgcgttgcctccg tggccgagcaatttggcatgcccgtggtgaagagtgatggtgggggggggccccccttccagta ccgtactgcctgatagggtcttgcctcaagcccagagagtcgaggctgaaaaccgccatc 578 HepacivirusJ gccgctcccgaaagggagtccggcgcgtcatcccactccgaggagtggggggcgtccccgtg tgccggggaaccatgaagcctaagggcatccacattttagaatgaacttgaagcttcgtttcgctgg ccggaaagtcctgggttcccatggccagggttccgcaggtgggtaaatcccggtggggttccatc caggatatacggcaggcgggcgtagtccggcggttcggacgacgtgtgggtcgcctacggtgg attgttcacaggatgggcactccggtgtgaataggccccgtcagggtgcgctgacgttaaactcag gccttgcctggtgttcggggaggattgcagggccacgccgcctctaagggccgtatggcacagta cttcttcggggggttgtcaaggccctccaacgcgacaccagtgcctcggcaggcatggggcca cccagctcggcgtcccgcacacagacggcgtaggggaaaatcagcaatgtgaccccgggtggc attttccttctctctacttccatgcatgatcaaccgcaatc 579 HepacivirusK gggaacaatggtccgtccgcggaacgactctagccatgagtctagtacgagtgcgtgccacccat tagcacaaaaaccactgactgagccacacccctcccggaatcctgagtacaggacattcgctcgg acgacgcatgagcctccatgccgagaaaattgggtatacccacgggtaaggggtggccacccag cgggaatctgggggctggtcactgactatggtacagcctgatagggtgctgccgcagcgtcagtg gtatgcggctgttcatggaac 580 Icavirus cgaagtttaagctaagcaccctcgggcgttcccggattatgtgatcacatcaatttgatggctggtca ccacgcaacgcctggagagatactcttacttttctcttaagatccccggtcatttgacgcttgtaggat gatagggttattttccactataaatactttcatactcttggatgttctatatccaagacgggaggaccta ccccgtaccccttagaggtgagatgccaagaacaggccctttctgttctctcgacaatggcatcata ggcaacaagcatcacaccaagattgctaagttttgttaagagttcttcaagctatagggtggctgtag cgaccttctgatgcctgcggatttccccacggagcgatccgtgccacaggggccaaaagccacg gctaacgcccatcaggagcggcacttaccccgtgccccacccttgaaacttgaatgttcacactgg cttctctcggctttctgaactgtctgcttgttggggccccgaaggatgccctggaggtaccccatttta tgggatctgaccaggggacacctcagctctctaagttgctggtgtttaaaaaacgtctaagggccc ccaccccttaggtggagggatccacctttcctttattttttaaaactcttttatggtcacaattgttt 581 Antarcticpenguin ctaggagactacgcagtgggataagatgactatgatgtcgtacgggcagaagccagtacagtcga virusA agtcgagaccgacgtcgaggatttgactctgcctgacctagtgccatc 582 Forestpouched tattggatccgcctccgggcaaaggttactttcttgtacctcggcttagccacagggtgaccccttgt giantrat acgtaggggccccgacgtagcactggtctgacaacaccttctcggcatttcaccttctgcccgctct arterivirus tccgggcggtggtgtcaagaagcagcagtgctcttctcttttcttcctgcagttcaccgagccctacg gggggtaggtg 583 AvisivirusPf- cattcccctttccccagccatgggttaaatggcccctcaccaggttcggtgctgtctaggcttccagt CHK1 aaagaagtcaaccgagcattgaacaaaacctcagtgggtatggtagttaaccccgtccactggac aactttgtcctcttaaaagtggatcaatccaccccaactccccccctagccacctgagccatggtgg atagcagtgacgaaactagggaccccaatacctctagtgccaagagaattcccccctcgcgagag gtgctcttgggcccgaaaggctagttggcagggtgaagtgaaggaagctgctagcgtggcaacc ttaagcgtagcccgaagctgaccttagaggttaaccctagtggaccactggatgaagctgtggag gtggtggataggaaagttggccacttgtgagtagatgcccagaaggcataaggctgatctggggc cagtgactataccgttccggtaaacctggtataaaaaccatgaaagcaagtgggtttaaaatttcttct aattccttcatttcagtagtgataactggcaga 584 Avian accaaacaaggactagataacccacgtgaccgttaactggaaaataagatgttgtaggggcgacc paramyxovirus tagttggaattcgaccccggctccgaaacctctaattgtggttattggcagtctagtctacttctaacg penguin 585 Newcastle accaaacagagaatctgtgaggtacgataaaaggcgaagaagcaatcgagatcgtacgggtaga diseasevirus aggtgtgaaccccgagcgcgaggccgaagctcgaacctgagggaaccttctaccgat 586 BatHp- ttaagcttcggcttgttgcataggaccggaaaggtactatctaccctaactcttgtagttagactctcta betacoronavirus aacgaactttaaaactggttgtgtccttcagtagtctgtatggccattggaggcacaccggtaattatc aaatactaagaagattcatagtacatccttgtctagcttttggttggcagtgagcctacggtttcgtcc gtgtcgctcacaattatccacacagtaggtttcgtccgctgtggttgagttgctagtccgttgctgtttc gtcagccatctacaactcgacacc 587 Basellaalba ggaatatggctaatcggcttattctaatcaaacgcaaaagacttatgacacagaccggacctgaac endornavirus gaggtgataaaacacctcgttcaggttcaaaacgtagaagattcattcctccgattagaaatacaact acgtctaagcacgatagagatggtatcaagatcggttttagaccacgagaaaatcggcaaatgaaa gtacaagttgggtggtttaaattaccaagaacagtaacattcaagaacaacggcaacccgtttgtta cctcatttcgtaaattgtttagaagtaacaaagataaattatttaatggtgggaagaacctaagtacag taccagccagaagtagtgaaatgacagaaatgtttatgttcatgtccacgctagagggccaattgtc aatccaagatcgagatccaaaaataatcaataagtctatatacatgatagaggta 588 Ballpython ccccttcacccataggcactaggagaacaggataacccctaacggggcatcctgcctgtgaccttt nidovirus cagattcgctagttagatatcttcacagactctgctaggcttctgacccagtccgttcccaaagtccgt taccgcccgagtagcgcttaggcgcgaaagggacggaagtacctccagtaagcgaaagctgaa gtaagggaaatacggcaagactaacttgttagtcttacagtgtggataacctggtagttatccccga caagagacctgactcgatgtgaaaacatccaactaggttggcttcaaactagctacaggcaggata tcccccaacggggcctccaaggaatcgagaaccaaccctcattccacgtctgtagtaagcaaaaa caggggcgatcttcaccgacacctctcaccacagagcacaccaacctctgtgaagccaatttcctc gtccaaggacaggttattgagggtcaactttcttccgaccagaagaagggatttcctaccaaaaga aaaaccaaatccaccaacaccacaaggtaaaacaacaacttgtgaagccaatacttagtcaaaga ctaactattgagggtcaactttctcttcaatagagaagggatttcctggtaaaacaaataacaacaac taacatcagcaact 589 Batsapelovirus gacaggtgttttggagggcggatgacgatatctggctggccaccagggaataacggcaaatgtct gatcatacggttcacaagtctaccggcgatagtggttcaacaccatgtgtagcagggattcttgcgt atgtgaaggcgacagtgc 590 BatPicornavirus taagcggaaagcattcttgtcccccggtcagtaacctataggctgttcccacggctgaaagggtga 3 acatccgttacccgcctcagtacttcgagaaacctagtacgcctgatgattccaaattggtatgatcc ggtcaaccccagaccagaaactgtggatgggggtcaccattcctagtatggcaacatacaggtgt ccccgcgtgtgtcacaggcccttacgggtgccatttcggatgagtctggccgaagagtctattgag ctactgttgatacctccggccccctgaatgcggctaatctcaaccccggagccactgggtggtgaa ccaaccacttggtggtcgtaatgagcaattctgggacggaaccgactactttggggtgtccgtgttt cttttgttcatattaaactgttttatggtcacaacacaacttggtacgatttgtgattattcactgctcactt gtcacagtaaatatacacaatcatc 591 BatPicornavirus gggttttacgaaacccgtatacaccagaccttttctcccctccccctccacctaccttttccccctcttt 2 ggaccgaaacaaggacacgtaagtggaaacgcgattttatatgtggttggccaccacggaataac ggcaattgtctacatgtgggaagtgcaacctccctagccgataacccctgaccgggtgtgtaggat aggaaaggtgcccactgtgggcgacaggttatggtagagtggatacctagccaggggcaatggg actgctttgcatatccctaatgaagtattgagatttctctgctcattacccggtgatggttgtgtggggg gggccccatacactagatccatactgcctgatagggtcgcggctggccgaccataacctgtatagt cagttgaattcagccaag 592 BatPicornavirus gaaacccgtatacaccggaccttttctcccctccctctccacttacctttttcccctcttcggcatgaaa 1 caaggattattcaagtggaaacgcgatttaatatgcggctggccaccgcggaataacggcaattgt gtatctgctggaagccaagcctgcctagccgatagcccttgaccgggtgtgtaggatagcccagg aaccagcaatacgcgacaggttatggtagagtagatacctagccaggggcaatgggactgcattg catatccctaatgaaccattgagatttctctggtcattacccggtgatggttactagaggggggcctc tagtactagatctatactgcctgatagggtcgcggctggccgaccatgacctgtatagtcagttgatt tgagcaat 593 BatIflavirus acgaatcggtatacgcttcggtacctattgttgcaagttcgttccctattttcgatttgcctgcccgaatt tgactcaaacaattgtgacatactatgtctctgtttgaaagcactacacgagtttgcgcccagcatgta tgttttcaagtcttttgtataagtctgcctctatagtggttttctttgaccttaaagccttgtcaaccatcct atatgctgcatcgagacttgatgtcaatctgcctctactacgcaaatgtctagtaattagttataaggtt ttactattttccctcatttccaattttagtttgtagtgtatgtgagtatcattcttactccgactgttaagaga aaccaatttatagtcgttaaatatgataaatggaatgaatgatggtgtcattttaaaaacactcttctcta taggcgtaagcattctcgctcttagagtcgtaaagaagaaatgccgtgtctatcagtatgttatgcga tttattttctgccacgcgcttctagtgcaatctagttgacatacagacattgcctaccactcgcgaggg tcgaccggtagtgtaaggagtaagtgatgatttccgcttattctgtaccctttgcctggtgaggacag atcctgactaattttaaatataaatgaacactagcttccaag 594 Batdicibavirus gtatagcaccggaatggtattttactactccaagtatacgtactaggagttaaaccctgtaatttacag gggatttagtgacttttatccgtaaaagtcgattggacgttaatcggtaacgaggccaagtaccgtg aaccaatttaaaaacgtattttctcatgtggtagaaccaacttggaaatagcatggcatataggttgttt aggg 595 Betacoronavirus gataaagtgtgaatcgcttccgtagcatcgcaccctcgatctcttgttagatctaatctaatctaaactt HKU24 tataaaaacactaggtccctgctagcctatgcctgagggtttaggcgttgcatactagtgtcttagga atttgactgataacacttccctgctaacggcgtgttgcactctcagtctaagcctcccacccatagga ggtatc 596 Betacoronavirus atttaagtgaatagcttggctatctcacttcccctcgttctcttgcagaactttgattttaacgaacttaaa England1 taaaagccctgttgtttagcgtattgttgcacttgtctggtgggattgtggcattaatttgcctgctcatc taggcagtggacatatgctcaacactgggtataattctaattgaatactatttttcagttagagcgtcgt gtctcttgtacgtctcggtcacaatacacggtttcgtccggtgcgtggcaattcggggcacatc 597 Boone tacgatcgctgtacattccactactgccaattagctcccccttcccgttgctcccctctataaggaga cardiovirus1 gccttctcttgcaaaggtgaagccttcacccccggtcgaagccgcttggaataagacagggttattt tctcctctcctcggcgcttgcctcttctaagctgaataggttctatctattcaggcggatggtctggtcc gttccttcttggacagagtgtgtatctgggttttccggatctcgaccacacactcaccagagctcagg agtgattaagtcaaggcccgatctgcggcgaaaaggaaatgaagtattttgcagctgtagcgacct ctcaaggccagcggatttccccacctggtgacaggtgcctctggggccaaaagccacgtgttaat agcacccttgagagcggtggtaccccaccaccctgcaaattatggatttgacttagtaactaaaaga ttgacttggcatacctcaacctgagcggcggctaaggatgccctgaaggtacccgtgttgaaatcg cttcggcgaccatggatctgatcaggggccctgcctggagtggttctatcccacacagcgtagggt taaaaaacgtctaaccgccccacaaagaccccggcagggatgccggtttcctttttaccaattcttg acact 598 Bredavirus atcacctagtacttacaagcgggtcaaaccgccctccggaacggtcataaccccctcccgaacgt gcgcttgacgtgactggtctttcagtctagctttctgagaaatactccggggttgtaacccaccatttt gacctttggtcagtttggtaacactccaaccaaacagcatctgacccacctccagcttgctgcaggc catttggacCaaacgggttcagatatcttgtggctaaacctctgacccacctccagtttactgcagg ccttttggactaaacgggtAcagactctttgtggttagtttttaactacccactgtttagccgccaacc tgatttttattgttacaaaattttgtgtttacacattattttcttacggttggcagtttgtttggttgtttgcaca gtttttgctgataccaatttttactgtgcttttggtgttttcggctaaggctgtttttcacatacttagtttgct tgaagtaaccttcacaacatctgttttgtttttggtttctaaggtaaagagtttcaggaaaaaacatagg cgcccatcttgtggtgtctagttttaattaatctggcaaacaagtatcaagtcatcgactccctttggag tgagacttacgagtaccaattcgcctattttggccatccatataaaa 599 Bovineviral gtatacgcccagttagttcaggtggacgtgtacgattgggtatcccaaattaataatttggtttaggga diarrheavirus3 ctaaatcccctggcgaaggccgaaacaggttaaccatacctttagtaggacgagcataatggggg actagtggtggcagtgagctccctggatcaccgaagccccgagtacggggtagtcgtcaatggtt cgacgcatcaaggaatgcctcgagatgccatgtggacgagggcgtgcccacggtgtatcttaacc caggcgggggccgcttgggtgaaatagggttgttatacaagcctttgggagtacagcctgatagg gtgttgcagagacctgctacaccactagtataaaaactctgctgtacatggcac 600 BovinerhinitisA ttttgcggctctgccgccgttcgggttttacctgttttcacagagcaaaacaggacctctagtttcgtg virus cttaaacgagatcatgctcgaactagaactataacgctggtcactggacccgtgccgcgccttgcg gatctttgcgggaatggtggctagtgggctgtggaagtgactctaaccacacgcccctcaagtgtg ggaaaacacgaactggtgtagcgacgacgataggccttgggacaccctctccagtgatggagac ccaaggggccaaaagccacgccttgtgccctgtcgttcacaaccccagtgcagttcgtgccagta cctgcttttgggaagtgtgctttggacagctgaaaacagtcctagtgggagactaaggatgcccag gaggtacccggaggtaacaagtgacactctggatctgacttggggagagcgggtctgctttacag acgccactctttaaaaaacttctatgtctcgtcaggcaccggaggccgggccttttcctttaaaacaa tacacttt 601 Bovine ttggatctgagcaggggccccctttgggttgctttacaactcaactgggggttaaaaaacgtctaac picornavirus ccgacacgccagagggatctggtttccttttatttcttttactcaccactggatgcagattgacgataa isolateTCH6 acgttgttgtttgtgactattgacttgatctgcttctacgggtttactttcactgttatacttcttgctttgttt ggtgttcactgtactttgtctccttctacatttcaca 602 Bovinenidovirus caccaatagattagtcaagctgtctataggcataaactaacccccaaccccattaccccggggcca TCH5 ggtgggccgccgccttcgggcaaacccgtgcgctggtataatcaaggttcacagccagattcact gccggttagctagtggggcggtagcctggcaaaacccgaagaggttggaaagggaacttcagg gtagtttatcctaggctagcgtagctacagttcggtcaagataaccgtcctggtgctagggctagta gagacagtggtaacttggacaagggtccagggccactttagggaataccctacggaaggctagg tccgtaaggaagacccccgcagttgtccgcggttgagcagagctcctgcgtagacaaaaggcaa aaagtggattacattcgcctgcaggaaaaggcaaacgtcgttggagtcggagctaaagtactgga cgattgataccacgcctgctgcggtagataaa 603 Bovine ccatcgacactccaggctcacggattaagttaggttccgccgaagcgggctaaccaggcccctag hepacivirus taggaggcgcctatcccgtgagccctttccccacggattgagtggagctggagctgggaaggac cgagtacggtccaatcgagaagaaccctgatgaacattccaggcctcttcggtagatttggatatat ccaccagtgaaggcggggtcgtgggtacaggccccctagtccacacagcctgatagggtcctgc cgcaggatccgtgggtgcggctgtacatgtacc 604 Botrytiscinerea gccccgccgaccttcctatttatttctaaataggaaggtcccgactagtcggataattcggtttaacg mitovirus4RdRp aattatggttagatctattaaagttaaaatagattgaatttctttctcatcctccttattcctatactttggga gtaaatgacaaatgtctatcctcaaaccgaaatggcttaagtgatgaatttgaaagaaaggtaggttt taagaatataaggcatcaatatattatacccttgaatgttaagtgaccacggcgtgacgattagggct atcttaggatagacagccatctaacgcgacagcagtggaaatcagcttagcatctcaagatcatgta taatatatacataaccttacaattataaaaccaaaccaaaacacactataattttatataaattatagaa gtatcggacctggacggtacctactattaactgatagtagccaaatgcaggaagctc 605 Botrytiscinerea ggaacttttcagttccagaagtggctttattaagccttcaaagtttacactttgaacctgcgattaattcc mitovirus2RdRp ccatagtgactcttgttactgtgaattaggaatagttgtagttcaacttctaatgaggtgaacaatataa taactcatcttattaaccctatacgtagacaattgtccaaagagacagttggaattctgccaatctgga atgtttggtacgcgtagaagataataagagaccctctattcccctgcctcatgactaagtcatggccc ggggtgtaatagagatacttttatatattatacaatc 606 Canine cgctctttatacaaatctgtcaaccctttgtataactctaagccgaacaattatagctaggctttttattat picodicistrovirus ataacattaattaggcattagcgttgtcgccaatctcttggtaatcctaaggatacctttcctgttgacta strain209 agatgaagcgccttcggttaccgatgcccggtgtccacgaagccatcgtggtcggccgcgtcccc cacctctcccaacttggactccatgttttcagtaggtgtaatgattagtattattgattctgctcgttcaat gtgtttatcttcacgatctgggacccaacacatgcttcactcatgtttaaatgttggttccctcattttga agacacccaaaccatagagtgcgagaatgaggatttctacttccattctggtaacagaaatgaattc ctgcgtgtgtctcgtaaatggaatctttaagaacttcagataaatcgaacaatacactaatacaagttg ttttctaccaacatgttcaatgcggctaatctgaccgtggagctgtgaagcgctcaaacccgagtgtt gtatacagtcgtaatgcgtaagtccatgaggaaccgactactgttacctctgttggtgtgtttctccttt CCtctcttttattattatttgttattgcaaatactacaactttgatcaac 607 Caninedistemper accagacaaagttggctaaggatagttaaattattgaatattttattaaaaacttagggtcaatgatcct virus accttaaagaacaaggctagggttcagacctaccaat 608 Caninekobuvirus tttaagtgttgtgcccaatctcttgactcctgctggaaccaccgaccagtagtgtccaaaatgccagg tggaaaatcctcccttcccctctgggcttcatgcccggcatcctccccccagcctgacgtgccaca ggctgtgcaaagaccccgcgaaagctgccaaaagtggcaattgtgggtcccccctttgtcaaggc gtcgagtctttctcccttaaggctagtcctgtcagtgaactctgtcgggcaactagtgacgccactgc atgcctccgacctcggccgcggagtgctgccccccaagtcatgcccctgaccacaagttgtgctg tctggcaaacattgtctgtgagaatgttccgctgtggctgccaagcctggtaacaggctgccccagt gtgcgtaattctcatccagacttcggtctggcaacttgctgttaagacatggcgtaaggggcgtgtg ccaacgccctggaacgagtgtccactctaataccccgaggaatgctacgcaggtacccctggctc gccagggatctgagcgtaggctaattgtctaagggtattttcatttcccaccctcttcttcttgttcata 609 Camel cttaagtgtcttatctatctatagatagaaaagtcgctttttagactttgtgtctactcttctcaactaaac alphacoronavirus gaaatttttgctacggccggcatctctgatgctggagtcgtggcgtaattgaaatttcatttgggttgc aacagtttggaaataagtgctgtgcgtcctagtctaagggttctgtgttctgtcacgggattccattct acaaacgccttactcgaggttctgtctcgtgtttgtgtggaagcaaagttctgtctttgtggaaaccag taactgttccta 610 Cripavirus gcaaaatcggtagtacgttaaacgtacgaccaccgatgagactgaaatgacactagttgagattatt tcaatatcctagtgttataaagtcaatatttgttggttgatcgtttcgtcaatcgatggcgctgacagcc ggaaagacggcaataataaaaaccaagatttagtttttaagttttgattgaattgcaaaagctatcttg aatagacaatcaaaatattaagtaaagcaaaagcttcttaaagaagacaatatttaattagttagtaac caaacctcatcgtgcccctaagggttaaccggttacgtaaaagcgtagaggtattaaggtcactgc ggagacctaaaatccgcaattttatgttttgtaatgttttagttatagacttagatgtaactataagagttt ataaatacttgtttcaagatttatagacaagatctgatcctatggattttagataaccttcatgttagtgg atagtgtgtgtacctatctaaacgcataaggctcttatttcatatttaaagtaggactatgtattacggc gcatctaacggtaacgttagtcaagaccggagaatctcggaatgaattttagtaattcccaaatttata 611 Human acctttgtgcgcctgttttatatccccaccccgagtaaacgttagaagttacgcaaccccgatcaata coxsackievirus gtaggtgtagcactccagctgcatcgagatcaagcacttctgtctccccggaccgagtatcaatag A2 actgctaacgcggttgaaggagaaaacgttcgttacccggccaattacttcgagaagcccagtagt gccgtgaaagttgcggagtgtttcgctcagcacttcccccgtgtagatcaggctgatgagtcaccg cgatccccacaggtgactgtggcggtggctgcgttggcggcctgcctatggggcaacccatagg acgctctaatacagacatggtgcgaagagcctattgagctaattggtagtcctccggcccctgaatg cggctaatcctaactgcggagcacatgccctcaaaccagggggtggtgtgtcgtaacgggtaact ctgcagcggaaccgactactttgggtgtccgtgtttctttttattcttataatggctgcttatggtgacaa ttaaagaattgttaccatatagctattggattggccatccggtgactaacaaatcgctcatataccagt ttgttggttttgttcccttatcacatacagctcataacaccctcttatatttactacaattgaatagcaaga a 612 Coronavirus agaaacaagtagtgttttaaaaaccttcaaattagtgcctgtaacatctttgcaatgaaagtagcgctc AcCoV-JC34 actagcctctatgcaaagaatgttaaaagaaatacgaagcatttaaagaatacaatctatctaggata ggtacaattctcctccccctcttgacttcggtcaactcaactcaactaaacgaaatcccccttgcatg gttccgacccgtgtaaggttgtgtatttcgtgcagtcgttgcccttactagtgtaagcgtaacggcatc taggtttgcacgtcttggaggaaacggtgtgtacgtttctagtgtttacgccgtatcggttccggccc gataggtattgcattagacgtcctgggtggttctgcctgcccttgtgtgattcggctgttccgtcagttt ggtcacctcacacgtccttaagac 613 Chicken gggtatggtggttaaccccgtccactgggcatctttgccctctcagaagtggatcaatccaccccaa picornavirus3 ctccccccctggttacctgagccacagtggactccggtgacgaagctagggaccccaatacctca agtgccaagagagtccccccctcgcgagaggtgctcttgggcccaaaaggctagttggcagagt gaagtgaaggaagctgctaacgtggtgaccttaagcgtaattcgaagctgacctttgaggttaacc ctagtggaccactggaggaatctgtggaggtggtggttaggaaagttggccacttgtgagtagatg cccagaaggcataaggctgatctggggccagtgactataccgttccggtaaacctggtataaaaa ccatgaaagcaagtgggtgaaattttctctttttatccttcattcagcagttgatattggcaaa 614 Chicken gtggccgacttgcagaacttcctaccgaaccaccacctcacccccataactccttccctctacacct picornavirus1 tccgctatggtggttaccactgctttattgccttgactgagaatggccaccccctcgacacctgcccc ctactgccccaccgcgcaaccttttgcttgtccactcggttggagaggcatgggggccccgttcat atccccagtccagttggtgaccttccccctccccgtccggtagatggtccagagggctttgccgac gccctctatgatgcttgcttgtctttcctccgtcagcgcgagcatgcacttgtcgagcccacggaaa cacagcctagcttttgcttctctcaccctgcgtaccctgggcgccttccgctcgagattcgctttgttc gacaccctggcgtccccccaccgctacgtgattttactcgtggcatacaccgccctggcgttcagt acattccactgcctaatttgggtggcctccctcaatctcccgcaccccccattgcgcacgtcatcac cgccgccgctaacgcgatccggcgcggttctcactggcactgtcccctcgtccgccgggtttcca ctcatggttggcttttcacttattctggttactggtgttccatcctacttcatgatggtcgccatgaccat gac 615 Chickenorivirus aaaccctcacgagtgcttgtggtaggtcccaggccaatattcttcgtaaggcttggttccaattttcca 1 ccactcgtgtttgggttctggcctatggtacccagaggggcggtttgggggaattaactccccctcc cctgtggtcctataccaccccacacctctgtgggctttctttactatcttcttgttttccgacttttaaaca ctaggcaggcgcgcctagtcatacaccgcccggctggtctttccagctcttgtgggcggtgcgcg ctggtccatcgtgcccagcgacatagcaccttgtggacacctccgaacgccctcccctgtatggg gtggtgcccaggggtttcagtgtggtgacacactccctggggcccgaaaggctagtgtgcaacag gtgaggtacagccagctgcccccgtggctggagggaccaagcttgtgaagcacacctcaccttct tggggggggctagtaagtggtgaaagcatagtgtccgtgtcgctggccaacactttgggtcaagt ccagccactcagtgagtagatgcccaggaggtacccctagtggatctgacttggggcctgttactt aatgcaggttaaaaactatgaaagctgagtagtgtagcccggctggtggcttctcttccttattcattc tatttt 616 Chicken ggttaacttgtttaaccaaggcttccgtgcagggcttgcacgttaggagttcatgttgattcatgctcc gallivirus1 aatgcccaaaactttgtgtgtttgtttatgtctttcccaaagtttcccccaaggtttcggtactcaaacct taattcctagtcccatcttttgggccttagtatctaggaaatgtacccgtgccttgacgaacgtaagaa agctgtcttttattgaacggttctaatgaactaagtataactggctcgcgccacctggtgtgtgccgtt ggaattcccccatggtaacatggtccaacgggcccgaaaggctagtgggcaatcggtcctccaa ggaaggggttcccaccccgacctgaacaggatttgatgaagctcacctcccaggctcctaacccc aaggaagtttacttatagtaattagaatttagtatgtaattgctggcaatcttgctagtagtcaggaacg ttatgaccaaatgagtagacccccagaaggtaccccattatatgggatctgatctgggcctcatact gtgtgtctccccacatatgaggttaaaaccatgaaagtttggtccaaaatattcttttccttttatcttttct ttagtggtgacgccattatatcagcagtttgctg 617 Chicken ggtgcatcatcactgaacaccctcgggcagagatgcaagggtggaagtcactcctgccccctgg calicivirus caacatgcaggtgcccgatcccaagcttagttctgacttcctctcctgggtggtgcaacactccaag gttgatgaacaaacctggagggacctctgggcaacctggtctctcgaggatctccggcgcatctcc acagactacctcatgctcccggaacctgagaagaacactgatgcctatgatggctggctgatggtc ggcgagatgttgacctttgccggtctcattggctgggagg 618 Carppicornavirus agctacaggaaagagagagataatcacagcacataaatacaactacagaagagagcctttccctg 1 agcactatttacagcaaaccacgctgggaaaagtggtagcatgacccacttacgggttacttagtat aggattttaatatcttcgattctttattttcttttcttaactaagttcgcccggactttatccgtgttttatttta gtttaagcaaaattgagtaactaagtttttaacctgccaaatggtgagaagtaactctgtgaaaatacc atttgtgcatgaaattgtcttgaaaactcttaggcttttggggggtcccactgctgtttggaggactatt gacagactctattgtagttgagtagtgactaatgataacgatttgcgtattacgcaatgggctgtacc cgttagatttagtatgccggggggaggggtcccactggattgcactatgtaacctgacagggcgtc tgccgacgcactacaatgaggataagatcggctgtttttattt 619 Falcon cgcttggaataagttgagaggaattatgcatgctagttgtgtttgtttacaactaattgttctaatccaa picornavirus gtgaagctcttcgcttggggcggcacgacacttgccgtaattcttctaccgtccctccacaccttgtg gatgaagggccggatgtgtggcctctggctaacccctctctctggggtgatgctactggatgtttta ctcctagaccaaatcacatgaactcctcttgatccacttcggtggggctatgagcctgcggattaata gctggcgacagctaccccaggggccaaaagccacggtgttagcagcaccctcatagtctgatgc ccaagggctgatgttgggagctagtagtgtgtgtctggcctatgtttaggacttctggccaagcgca gaggagtggggctgaaggatgcccagaaggtacccgtaggtaaccttaagagactatggatctg atctggggccccctcacgtggctttaccacgtgttgggggttaaaaaacgtctaggccccaccagc ccacgggagtgggctttcccttaaaaagcccaacaatatttatggtgacaattcactgtttcttctttgc aatttttgtattcttggactccttattttttgtttgcttgattttagtggacattcagattcaaatacaaa 620 EquinerhinitisB cgacaggcacaggtcgctccgagttctagtagtgtgggaacttgttactactgatgaaacgaggta virus1 gtgacactcagtacctgcgaacgaggtcggggccctcccttcttccttcacccaactttcacttttcgt tccactttagcaggggtcttctttctatccccctggcggcattggaactagccgtcgcgtcttaacgc gcagccctgaaggccccacaccttgtggatcttgccgtgggtatgtttctggcatgtgtttctcaagc ctgcaaccgaagccgaacagccacatgaacagtttgagcgtggtagcgctgtgtgagttggcggt ggatccccctcgtggtaacacgagcccccgtggccaaaagcccagtgtttacagcacctctcaca tccaggacgaccccatcctggcgctcactcttagtagtatggcttagtacgcattaggtggtaagcc gagctctccctcggccttgttctgaatgcacacatgtctaggggctaaggatgtcctacaggtaccc gcacgtaaccttcagagagtgcggatctgagtaggagaccgtggtgcactgctttacagatgcag cccggtttaaaaagcgtctatgcccctacagggtagcggtgggccgcgccctttccttttaaaacta cttgttct 621 EquinerhinitisA aagggttactgctcgtaatgagagcacatgacattttgccaagatttcctggcaattgtcacgggag virus agaggagcccgttctcgggcacttttctctcaaacaatgttggcgcgcctcggcgcgcccccccttt ttcagccccctgtcattgactggtcgaaggcgctcgcaataagactggtcgttgcttggcttttctatt gtttcaggctttagcgcgcccttgcgcggcgggccgtcaagcccgtgtgctgtacagcaccaggt aaccggacagcggcttgctggattttcccggtgccattgctctggatggtgtcaccaagctggcag atgcggagtgaaccttacgaagcgacacacctgtggtagcgctgcccagaagggagcggagct cccccgccgcgaggcggtcctctctggccaaaagcccagcgttaatagcgccttctgggatgca ggaaccccacctgccaggtgtgaagtggactaagtggatctccaatttggcctgttctgaactacac catctactgctgtgaagaatgtcctgaaggcaagctggttacagccctgatcaggagccccgctcg tgactctcgatcgacgcggggtcaaaaactgtctaagcagcagcagaaacgcgggagcgtttcttt ttcctcatttgtttc 622 Equinearteritis gctcgaagtgtgtatggtgccatatacggctcaccaccatatacactgcaagaattactattcttgtg virus ggcccctctcggtaaatcctagagggctttcctctcgttattgcgagattcgtcgttagataacggca agttccctttcttactatcctattttcatcttgtggcttgacgggtcactgccatcgtcgtcgatctctatc aactacccttgcgact 623 Enterovirussp. actctggtatcacggtacctttgcacgcctattttataccccttccccatcgtaacttagaagcaacaa isolateCPML acaaactgcccaatagcagcacaacacccagttgtgttaggggcaagcacttctgtttccccggaa gggtctgacggtatgctgtacccacggcagaagtatgacctaccgttaaccggccatgtacttcga gaagcctagtaccattatgaaggttgattgatgttacgctccccagcaaccccagctggtagttctg gtcgatgagtctcggcattccccacgggcgaccgtggccgaggctgcgttggcggccagcctac accatacggtgtaggacgtcaagatactgacatggtgtgaagagcctattgagctacgtggtagtc ctccggcccctgaatgcggctaatcctaactccggagcatccgccagtaagcccactggaagggt gtcgtaatgcgaaagtctggagcggaaccgactactttgggtgtccgtgtttcctgttttacttattgtt tggctgcttatggtgacaacttatagttatcatcataagctacttggtcttgccaaccggagaattattt ggttatttgttggtttcataaacctacagtcgtattttcctgtcttattaattgttctcaaaattaacaaca 624 Enterovirus taccgctgcaccagtgagctggtacgctagtaccttcgcacggagtagatggcatcccccacccc AN12 gtaacttagaagcaaagtacacatctggccaatagtggcgctgcatccagccgcgcaacggtcaa gcacttctgtttccccggtccgcaagggtcgttatccgcccagtccactacggaaagcctactaacc attgaagctatcgagaggttgcgctcggccacgaccccggtggtagctctgagtgatggggctcg caaacacccccgtggtaacacggatgcttgcccgcgcgtgcactcgggttcagcctattggttgtt cacctcaacatagtgtaaatggccaagagcctactgtgctggattggttttcctccggagccgtgaa tgctgctaatcccaacctccgagcgtgtgcgcacaatccagtgttgctacgtcgtaacgcgtaagtt ggaggcggaacagactactttcggtactccgtgtttcttttgttttattttgaattttatggtgacaattgc tgagatttgcgaattagcgactctaccgctgaacattgccctgtactacctaatcgcatttcacaaaa cctcagagataccaagctcttacattgatctgcttgttttcctgaatctcaaatataaattggaacaagc aaa 625 Dolphin accagacaaagctggctaggggtagaataacagataatgataaattatcatacttaggattaatgat morbillivirus cctatcaattggcacaggatttggataaaggttcacagtc 626 Diankevirus tgttttcaaccataatactactactacaagtataaaaccccgtccgtctgtcggagacgctaaactctg accaccaatctagccacatcagttgcttaaagaacctcttgagacactctcccacttaacatcttttag gaatcttcgatgctacaacaacttggctagtgaacaataaatccgtacaattcacagttgtaagagg ccataggtccagactttgaaaggtttgtttctattgttacaaatacttagattaacagaggctatttaata gtgctcatcacgttaacagagtaaccttgtgcaatagtatgagcttgttgtaaaacgtcttgatacgac acc 627 Guereza cactcaatactacactccgcatttggggagaagcgctggcgttcgcggaaccgcgttaaccatac hepacivirus gcgtagtacgagtgcgacagaccccggtgctactggtggtagcgagacacgagccgaagtctgt ggggggaactccacttagagggcatgcccgggcgtaggcttctgagttgggatgggccccaact tggcccctgagtgggggggtgttacgacctgatagggtgcgggctggcgcctaccactttccagt cgtacatgagtc 628 Grapevine gcccggggggtgcagtcctgtgaaagggtctgcaccatactatatatgtatatgattacatcccaaa associated aggcgacttcgttcaggttttaaatctgacgtaggtccagtaaataagcatgtcaaaacatgtaagttt narnavirus-1 atcctgtaatctactctcataagatgagataagatgatattgcagttcccatgtaaataaatccattatg aattcattcatataaggtagaagtggtaactatggttgaaacattaatataaaacggtcattttgcatga acgtcattaaggaactggcataccaatgtctatttagtgactatgatatttagagtatcccttatattaat taacaattattccttttagcatatcatccgacaacaaattttaaaagaagaaatattactcattaaaa 629 Goattorovirus gtacttacaagcgggttaaaccgccctccggaacggttacaaccccctcccgaacgtgcgcttga cgtgactggttctcagtctggctttctgagaaatactccagggttgtatcccaccatcttgacctctgg tcattttggtaacaccataaccaaacaaactctacacacctaacccacctccagcttgctgcaggcc ttttggactaaacgggtttaggtgttttgtgaccaactcgtctacccacctccagttttactgcaggcct ttttggactaaacggCtttagacttttgtggttagtttttaactacccactgtttagccgccaacctgatt ttcattgttgtaaaattttgtgtttatacactattttcatacggttggcagtttgtttggttgtttgcacagttt ttgttgataccaatttttactgtgcttttagtgtattctgctaaggctgtattttacatacttagtttggttga agcaatttttacaacatttatattgtttttgatttctaaggtaaagagtcttaggaaacaccatagacgcc attcttgtggtgtctagttccaactaatctggcaaacaagtaccaagtcattgactcactttggagtga gacttacgagtaccaatttgcctatttcggacatccatataaag 630 Foot-and-mouth acaagcttgacaccgcctgtcccggcgttaaagggaagtaaccacaagcttacaaccgcctaccc diseasevirusO cggtgttaatgggatgtaaccacaagatacaccttcacccggaagtaaaacggcaaattcacaca isolate gttttgcccgtttttcatgagaaacgggacgtctgcgcacgaaacgcgccgtcgcttgaggaggac ttgtacaaacacgatctaaacaggtttccccaactgacatacaccgtgcaatttgaaactccgcctg gtctttccaggtctagaggggtaacactttgtactgtgcttgactccacgctcggtccactggcgagt gttagtaacagcactggtgcttcgtagcggagcatggtggccgtgggaactcctccttggtaacaa ggacccacggggccgaaagccacgtcctgacggacccaccatgtgtgcaaccccagcacggc aacttttctgtgaaactcactctaaggtgacactgatactggtattcaagtactggtgacaggctaag gatgcccttcaggtaccccgaggtaacacgcgacactcgggatctgagaaggggactggggctt ctgtaaaagcgcccagtttaaaaagcttctatgcctggataggtgaccggaggccggcgcctttcc attataactactgacttt 631 Felineinfectious acttttaaagtaaagtgagtgtagcgtggctataactcttcttttactttaactagccttgtgctagatttg peritonitisvirus tcttcggacaccaactcgaactaaacgaaatatttgtctctctatgaaaccatagaagacaagcgttg attatttcaccagtttggcaatcactcctaggaacggggttgagagaacggcgcaccagggttccg tccctgtttggtaagtcgtctagtattagctgcggcggttccgcccgtcgtagttgggtagaccgggt tccgtcctgtgatctccctcgccggccgccaggaga 632 Farmingtonvirus acgacgcataagcagagaaacataagagactatgttcatagtcaccctgtattcattattgacttttat gacctattattagacccttcacgggtaaatccttctccttgcagttctcgccaagtacctccaaagtca gaacg 633 Avianinfectious acttaagatagatattaatatatatctattacactagccttgcgctagatttttaacttaacaaaacggac bronchitisvirus ttaaatacctacagctggtcctcataggtgttccattgcagtgcactttagtgccctggatggcacctg gccacctgtcaggtttttgttattaaaatcttattgttgctggtatcactgcttgttttgccgtgtctcacttt atacatctgttgcttgggctacctagtgtccagcgtcctacgggcgtcgtggctggttcgagtgcga ggaacctctggttcatctagcggtaggcgggtgtgtggaagtagcacttcagacgtaccggttctgt tgtgtgaaatacggggtcacctccccccacatacctctaagggcttttgagcctagcgttgggctac gttctcgcataaggtcggctatacgacgtttgtagggggtagtgccaaacaacccctgaggtgaca ggttctggtggtgtttagtgagcagacatacaatagacagtgacaac 634 Human ttaaaactgggtgtgggttgttcccacccacaccacccaatgggtgttgtactctgttattccggtaac rhinovirus1 tttgtacgccagtttttccctcccctccccatccttttacgtaacttagaagttttaaatacaagaccaat agtaggcaactctccaggttgtctaaggtcaagcacttctgtttccccggttgatgttgatatgctcca acagggcaaaaacaacagataccgttatccgcaaagtgcctacacagagcttagtaggattctga aagatctttggttggtcgttcagctgcatacccagcagtagaccttgcagatgaggctggacattcc ccactggtaacagtggtccagcctgcgtggctgcctgcgcacctctcatgaggtgtgaagccaaa gatcggacagggtgtgaagagccgcgtgtgctcactttgagtcctccggcccctgaatgcggcta accttaaacctgcagccatggctcataagccaatgagtttatggtcgtaacgagtaattgcgggatg ggaccgactactttgggtgtccgtgtttcactttttcctttattaattgcttatggtgacaatatatatattg atatatattggcatc 635 EV22 ccttataacccgacttgctgagcttctataggaaaaaaccctttcccagccttggggggctggtcaa taaaaacccccatagtaaccaacacctaagacaatttgatcaaccctatgcctggtccccactattc gaaggcaacttgcaataagaagagtggaacaaggatgcttaaagcatagtgtaaatgatcttttcta acctgtattatgtacagggtggcagatggcgtgccataaatctattagtgggataccacgcttgtgg accttatgcccacacagccatcctctagtaagtttgtaaaatgtctggtgagatgtgggaacttattgg aaacaacaatttgcttaatagcatcctagtgccagcggaacaacatctggtaacagatgcctctggg gccaaaagccaaggtttgacagacccattaggattggtttcaaaacctgaattgttgtggaagatatt cagtacctatcaatctggtagtggtgcaaacactagttgtaaggcccacgaaggatgcccagaag gtacccgcaggtaacaagagacactgtggatctgatctggggccaactacctctatcaggtgagtt agttaaaaaacgtctagtgggccaaacccaggggggatccctggtttccttttattgttaatattgaca tt 636 HumanTMEV- tccgacgtggttggaattaacatcttttccgacgaaagtgctattatgcctccccgattgtgtgatgctt likecardiovirus tctgccctgctgggcggagcgtcctcgggttgagaaaccttgaatcttttcctttggagccttggctc ccccggtctaagccgcttggaatatgacagggttattttccaaactctttatttctactttcatgggttct atccatgaaaagggtatgtgttgccccttccttctttggagaatctgcgcggcggtctttccgtctctc aacaggcgtggatgcaacatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtg gacatcgaaacagctgtagcgacctcacagtagcagcggattcccctcttggcgacaagagcctc tgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttc gatggccattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgt cctgaaggtaccccgcgcgcgggatctgatcaggagaccaattggcagtgctttacgctgccactt tggtttaaaaactgtcacagcttctccaaaccaagtggtcttggttttccaattttgttgactgacaat 637 Human acttaagtaccttatctatctacagatagaaaagttgctttttagactttgtgtctacttttctcaactaaac coronavirus229E gaaatttttgctatggccggcatctttgatgctggagtcgtagtgtaattgaaatttcatttgggttgca acagtttggaagcaagtgctgtgtgtcctagtctaagggtttcgtgttccgtcacgagattccattcta caaacgccttactcgaggttccgtctcgtgtttgtgtggaagcaaagttctgtctttgtggaaaccagt aactgttccta 638 Hubeizhaovirus- gtgcaggatggcctttcccatcttaagtggtttgtaggatttcgtgggtccataccccccgatttcttg likevirus1 gtacgtattccatgcacggagaatacgaccaaaactcttatttcaaaaaatattattttttactcttgtgg gctgagtgcgacccaccagttccagcttagcaacctggaagttgttggagatttatggaaccaaatt acacatgcgtggagtgccgccactccgtatctgttcactcattacgcgattaagttctgcgacgaga cgagcgaa 639 Hubeitombus- ggaccatccaggcaggtgtaggctagtaccctcacctgacctgtcgcgatgtttggctttgtgagg likevirus9 cttgtgggaggatcccttggcccatgcattgctgctgtcatcgtgaaaaatgttgtatgctcgcacct ggcgtggaggaaacggcatttgacggatgctaaggaggatttgaaggtcctcaactcccatgcctt attacaagtcccctttccttcaagcttcgagcccacgtctgtgacgagcagaggtgaggttggagtc aagaggagtcctattcaagctgttcgcagcaagaaccataaaactttcattgctcaatgggcaaga gcggctaaggctcggttctcgtttgcacgacagtgtgagcggagccctgtaaatgtcgcggccctt catcggtggtttgcatcggagtttaagaaaattggcatgaacttgctccaggtttcgtacgtgatcgat gaggttgttgaacttagtttggaaccaacgtatgagcgtatggtgtccgaaactaagaagcaattcc gtcatcgggcacgtatggaatactacaacgagaaaaaatgccttgaaaagatccactaggaacgc 640 Hubeitombus- ttcgggtttacccgcgtaagcggccacactgactggttgtcggtgttgaatttgtataccagatgagg likevirus32 agacgttaccaccgtctcggcagtgctacgtctgggaaaggactgtgatagtggacagtcctacc gtctttagatacattgcgttgtgtatagcccgggagggattaactaatagcaacgcaatgcacacgg cggttcggatttgcttgactgatggaaagacatctaagttctaattgaacc 641 Hubeisobemo- gagatgtttgcgtgggccgttgcgctgcgggcggcccacctccctacggggaccgtgagacacc likevirus3 gctggggaaggcccccacccccggccaaggggatcctgccgagaggcaggagaaagaggcc cagccctctggggcgcctttaggggTgcctgggagggaagtacccgagccgggcggccggtc gggtgcggctgtgcagttcgaggctaaccgtaaggaaggcctgagctgcctcggcttgtcggaa aggaagacgaaggcacttatcaaggctttcaggaagcaggagcgcaattacagcgcgcgccgt gcggcctggattaaccgcatttggccg 642 Hubeipicorna- agcaacttctttctgaaaactagctagagttcgacgatctctctggctaatgacaaataaccaatcaa likevirus2 aaagtcaaatgttcatgtatatatatatttagtagtgacctttatttagaaaaactttagatgatttatcgtc aagttgccctttgtgaagcgatcagctttttatatcgtttcatttttgtatacgtcttaattgacgttgtaag tacgttttgcatacctcacattgaggatagtatcgtttcctgactaagaagttaaactagtctaccaata gcaaccatataggatatagctttgtttttaacaaggtttaatctgatcttatgctcttgcttacggtgtttta tgtatagaaaaGtttttataaaaactacataattgtcaaaagaaaaagcgttacgtactgacgcattta tgttcacagtgtgacacaaaccttctatattttgattgtaaaataggctttgcctgaccatttatcaaata caaactgtttcaaacgcctctccgagccatattggccgacgcgaatcgacataacagggtgagttt acagctgcagttgcagccgaggatccacttatttgagagagaattactcttaacgaaattttgaagtc acttctacacagttaagtctgagtaggcgttatccaaacgtaaa 643 HepacivirusP acatgggggggggctgacagtgagtacactgtgccaagcaggtgctacgctatgcctaggtgct gctgtaggccaaggacatgtcccagtcatcccaggtgagggggggggttcccctcaccgctgcc actgcctgatagggtcctgccggagggtctcggtgtccggctgtac 644 Harrier gatgtgtgacggtgtaattactttccggatcccactttcctattataactctttcatcccaaggttaggg picornavirus1 aaagaacctggctcggtaccaccagaccctccgccacgctagtggactctccggagataacggt accccctttgtagtcacctgtgctggtgaagaaccacctagtattgcttgggtgcgtgccgcctagct tccatttcttctggagcactgtgcaatgaggtttccccacttggtaacaagtgcctcaggtcccgcaa ggatactgtggggtggtgtgaccgcagggagctgtctccacggctcctctaatgttacgccgctat ccacaggccagtgcgtgtcatcgatcccggatgacagagctagtattgcgaacccccaagtaaga aaagtggctagtaacctgatagctggtgaagagggtgggtcagttgagtagatgccctagaggta cccgaaaggatctgactagggacccgtgactatacattaggtaaaccgggtataaaaaccatgaa aaactgaccacttttcttttaacctcttctttctttttatgtgtgttaagtgttttgagtttggactgtaccttg cccgcctttcttggattttctctatcgctcttcttacacctactgttatcaaggcactctttagagata 645 Kunsagivirus1 tttcaaatcggactccggtagttataccggagcccggtttggacgcttgggccgcgttaacagccc cccacccctttcccactgactgatttctcggattggactcatttgcattgctaactctgattctggatttc cccgtttatgtcgtcgcggtcggaagtgcacgtacttcgacgttgatctgatggcgtttgtttccagg ggggaggtggcggcagaaacgcccccgccgtaaacttcgggggccacgcctgtcaagccact ccctggggccgagcgcctgaggtgatacagagagataagcacactgggcgctgacaacgcccg ggacctcagtgagaagagcagtagggccgtgtttatgggactccattggatatcccccgcttgtcg gaactcacggctactccgggttgggaagcccgcgactggtactgtactgggtgatagcctggtgc cttccctctcactgttgtatgaaggctgaaaaccccct 646 Kagoshima-2-24- tatagtttgcctgtttctcgcaccgttaccgctcgttcgggaatgtgaaactggcacccctcctctccc KoV ctaccaccctttctccttcgcccccattcataatttacaacgccgcacacagcggggccgccaag ggctagcctggcggttataaaaggaacctgggtctttccctcttcaagccaaaaggtaggttccctg tgtccctgaatgctcggtgaggaatgctgcaccgtaacgctttgtgaagtgtttgcaagttctggccc ggcaagcctacagagtgctgtgatccgctgcggacgccatcctggtaacaggacccccagtgtg cgcaacagtatgttcagacttcggtttgttcacttgctttcatggaccattgcgcgaaagtgcgtgcg ccatatccctgtacttcaggtgtgcttctctggaccctaggaatgctgcgaaggtaccccgtttcggc gggatctgatcgcaggctaattgtctatgggttcagtttcctttttctttactccacaattgactgcttaa ctgactctggatcttgtgcttccactgctctttctgctctcaaaacggcttcacttaccaactctcacctt tcgaccaacaccatttacacactaacttttttcgactcttctgactcctggcttggtgaagac 647 Kashmirbee tacgtacaattttgacgcttcgttcatgcaacaatgaactcacatgtggcgctcggtagtaaccagag virus gggcgtcattcccccgtatggagtggagaatataagctaccgactcgagctgtagaataattcagc aacttataacgaacacgaattttagtcgacgaaaccattttagcctttaattctatgatttgattataatg ataaacagctcatgtaactgtctaaactacataaatacaactggattacgaacctttaagtaactatctt agatgaagtctagtagtctcccaatatttccgtgaaaagaatgagggacgagatagctctatttaaa gacgtgaggctttaaattctgataaatacattacctgagaattcctcctttaggagttagaatttgaaaa gattagtacctatacttaatagaatttaaatatgttaataatgctgaagacaagtatttcgattattaacct ctatatttctatataaagtatctgtgagtctcagtggacatcacagtaaggtcgcagttaacttgtaatc ttttcattcctgtgtcggagcagtggtaatggagccggacgatttcgccaaaac 648 Jingmenpicorna- tgtgtttttgtcaagataattgttctgtgattaacagtgattgtggttcgtgtaatgcgacgcagtcaaat likevirus gctagttttgatgaagtgtatgagagagtggaaaacttatctcataagaagattgaagagtgtgtaga tcaggctattgatcgagcttctaagcttcgtgattacaagcttaatgttcacaatggctcccgacggg aatcatctgatcctctctttatttcgccccattcgttgttatcgcttggggtatctaagtttgttgcgtttga gcagcatcacagtttcgcttcagttgagtctctgaagttgcttgctctgtct 649 Mumpsvirus accaaggggaaaatgaagatgggatgttggtagaacaaatagtgtaagaaacagtaagcccgga agtggtgttttgcgatttcgaggccgggctcgatcctcacctttcattgtcgataggggacattttgac actacctggaaa 650 MouseMosavirus gaagttgatcatgaacttggttattggtggaacgcacatgaactcccaacaatgatcttgaagacac agcgtggtaacaattaccatgcccttgtggctgcccaagacattgatggctattgggtgatttatgat gac 651 Miniopterus gttgtcgacccgttgcttggtttaagcatgaggtggattcccccgattatgtctacccgttactatggc schreibersii gggcggtcgtttcagggtgtttctactgaggactgcaccaagtctttatcttttattctcagatctccgg picornavirus1 ctgtttgacgcttgtaggacagcaggactattttctcttaatctttttctacccactagggtcctatccta gtggagggagggtgccaccccttctctctttagagagtgcgcctggcggtctttccgtctctggaaa aaggagcacatggcatgctacaattggcacaagaaaacaagctttgcggattctttctttgtactaga ggaagctgtagcgaccctgtatggcgagcggactcccctctcggcgacgagagcctctcgggcc aaaagccaagtgttaatagcacccatacaggggcagtaccccactgccctttctcaacatacaat gactgatgaaccactgttggtttttctgacacctttgtagtaggattccaaggaatgtcctgaaggtac cctgttagcttacgcgcaggatctgatcaggagtctctttacagtgctgtacactgtgcaagggatta aaaattgtttgaggaatccccgagatagtggtctatctcttcctattttgttttacagacacg 652 Lindavirus gtatagcagcagtagctcaaggctgctatacgattggacataccaaattccaattggtgttagggac cacctaggtgaaggccgacgacaggtagccattcctgttagtaggacgaaccgttatggtggact ggttgctcaggtgagcaggctgcaatgcgtaagtggtgagtacaccacagccgtcaaaggtgcc actggtaaggatcacccactggcgatgccttgtggacgggggcgtgcccaacgcaatgttagcg gtggcgggggctgccatcgtgaaagctaggtcttgatggaccttgttgcctgtacagtctgatagg atgccggcggatgccctgtgacagccagtataaagaatatccgttgtgattgcac 653 Lesavirus2 tctttctttattttcttatgtaactcttctttttaagttttattttgcctacttgtgagcttatgcgggaccactg tcttagacaaccccacatttgtcatgagtaagtacacgcaaccattacgattactttttaaccgtctga ccttttgataacaactgaagttaggcgtgaaacatgcatttataccaaagtagccccgcatttcccca ctacggtgggggggctaccctactggctttggaactgtagccattatgtgttgcctggctttcaggat ctcacaacacaacagttctctcacaatggaatatgggtgagattgcagtgacatgaacaagtatcta gtagtacatagactcaagcctagttgcctgcggaacaacatgtggtaacacatgccccagggtcca aaagacaagggttaacagccccttctaggtgtctgtgtgtgaagaatactttagtagtgttgttatgat ctcacctgttagtacagaatgagtatggcttggtgaaggatgtcctacaggtacccattatatggatc tgagtaggagaccactagtggtggctttaccgccaggtgagtggtttaaaaagcgtctagccaagc caacagcactagggatagtgctttctatattttatattttcagtgtatatggtgacaa 654 Lesavirus1 gtaactaataagcaagttttactgcctgcaaactgcttcaatgggaccaccgcttcggcgaccccttt gttgagtttgtatgtttttaagtaatctttgcaaccatacgattattttagccgcctctctataatgatcttgt tatagtgggacgtgaaacattggtttttctcacacacgtccggtcacccgggcgtgtgttcttccgta agtcctatccacataccatcgtgggtaggccagcatgtttgcacaggctgtgacagtgtgggggg ctttccacctctcaacaacacactgaattgcaatgcactcacggaggaaatgacaatttggttatagt tttgaactgtgctagtaattttttcacattaagccatgttgcctgcggaatcacatgtggtaacacatg cctcagggcccaaaaggcacgggttagcagccccttcatggtgtgttagaagtgaaaacacatag tatgagctataatatctttgttgtcttctctgtagtgtaccccgccaaatgtaaggatgcccagcaggt acccattttatggatctgagctggggattgatagtgtatctataaatgcactgatcaatttaaaaagcg tctaagtttggcacaaacactggggacagtgtttttcctttattcttttatttgatta 655 Phopivirusstrain gggagtaaacctcaccaccgtttgccgtggtttacggctacctatttttggatgtaaatattaattcctg NewEngland caggttcaggtctcttgaattatgtccacgctagtggcactctcttacccataagtgacgccttagcg gaacctttctacacttgatgtggttaggggttacattatttccctgggccttctttggccctttttcccctg cactatcattctttcttccgggctctcagcatgccaatgttccgaccggtgcgcccgccggggttaa ctccatggttagcatggagctgtaggccctaaaagtgctgacactggaactggactattgaagcat acactgttaactgaaacatgtaactccaatcgatcttctacaaggggtaggctacgggtgaaacccc ttaggttaatactcatattgagagatacttctgataggttaaggttgctggataatggtgagtttaacga caaaaaccattcaacagctgtgggccaacctcatcaggtagatgcttttggagccaagtgcgtagg ggtgtgtgtggaaatgcttcagtggaaggtgccctcccgaaaggtcgtaggggtaatcaggggca gttaggtttccacaattacaatttgaa 656 Pestivirusstrain gtatacgagattagctcatactcgtgtacaaattggacgtagcaaatttaaaaattcggatagggtcc Aydin ccatccagcgacggccgaacggggttaaccatacctctagtaggactagcagacggatggacta gccacagtggtgagctccctgggaggtctaagttctgagtacagaacagtcgtcagtagttcaacg ctggtaaaccccagccttgagatgctacgtggacgagggcatgcccaagacacaccttaacctgg acgggggtcgtccaggtgaaagtacccatctttgggtgctgggagtacagcctgatagggcgctg cagaggcccactgacaggctagtataaaaatctctgctgtacatggaac 657 Quail tttgcatcagttcgcccctcccctcaccatacctttttcccctctttaggactgatacttggttatgatga picornavirus gcagaggatttcgcaagttatgcttcttgataaaaagtaattcacgaatcatgggattatagcctgga QPV1 agtgaacactcatgtggcaagtgggttagtagctctccatgttcccatgtgcagtggactgacaaca gtgagttcggggttgtgtagtaaagggaaagtattacttacccgcacctgctatacgtggtgtacgta ggatacgagttagtagtgcttagcaactttaaactggtgctgaaatattgcaaggtcactgaagttgt gaacgcgaacgctccgccactgccatgtatagcgtgcaatgcataaatggtgcactacatgatacg agggaatgggaaaccctccatggccgaatgcagggtgacagcctgccggcggatgcctgttgtt agtataatccgttgtttgccac 658 Porcine acactcatttcccccctccacccttaaggtggttgtatcccctacaccctaccctcccttccacatagg sapelovirus1 acgaataaacggacttgagattaaggcaagtacataaggtatggtttttggatacacttaaatggca gtagcgtggcgagctatggaaaaatcgcaattgtcgatagccatgttagcgacgcgcttcggcgtg ctcctttggtgattcggcgactggttacaggagagtagacagtgagctatgggcaaacccctacag tattacttaggggaatgtgcaattgagacttgacgagcgtctctttgagatgtggcgcatgctcttgg cattaccatagtgagcttccaggttgggaaacctggactgggtctatactgcctgatagggtcgcg gctggccgcctgtaactagtatagtcagttgaaaacccccc 659 Porcine atgacgtataggtgttggctctatgccttggcatttgtattgtcaggagctgtgaccattggcacagc reproductiveand ccaaaacttgctgcacagaaacacccttctgtgatagcctccttcaggggagcttagggtttgtccct respiratory agcaccttgcttccggagttgcactgctttacggtctctccacccctttaacc syndromevirus2 660 Porcine Gaaccttagaagtttacacaaacaaagaccaataggagtccaacacccagttggattgcggtcaa enterovirus9 gcacttctgtttccccggacctagtagtgataggctgtacccacggccgaagatgaacccgtccgtt atccggctacctacttcgggaagcctagtaacattctgaagtctctgaggcgtttcgctcagcacga ccccggtgtagatcgggctgatgggtctccgcataccccacgggcgaccgtggcggaggccgc gttggcggcccgcctatggcgaaagccataggacgcctcttagatgacagggtgtgaagagcct actgagctgggtagtagtcctccggcccctgaatgcggctaatcctaaccacggagcgtccacca gcaatccagctggcagggcgtcgtaacgggcaactctgtggcggaaccgactactttgggtgtcc gtgtttccttttgatcctattttggctgcttatggtgacaacgataagttgttatcataaagctcttgggtt ggccacctggaaaaagttatcagtgtttgatattgttcggctctcacgcctaccaataagacaagcc ctatatttacttgttgcattttactcgtcagaagaaatcacagagtatcatttggatttgttactcacatta aggacaag 661 Pigeon tttatttagctgttaagttttatttgtgccgagAccccatagtaggatcttggtgttccacattaagctct picornavirusB Cccgaccacacatccaaacgataggcggtgtaagggctccctggctaagtgtttactcattgctag ggaagtgttgcgacccgttaccagtaggaatacaggaggtcttagttgcctaaccagataaagtgg tgctgaaatattgcaagctcaatgtctggcgaacgacggactaccgttgaactattgttaacgcccg cgtgtgtaggcaacacacgggttagtaggtcacttacattgacatccgtgccgggaaagcggatct gagctatcgattgcctgatagggtgccggcgggcgcggtacgtgtggtatagtccgctgtcttggg gtatggcgtctactcggttttgttgtcgttttggaatgtcccatgtcggagatgtcgttaccggtgtttg ctttacttgtgcacgaataagaaaacagagtttggagttttggaagttaatggataacatcaagtttcc attgcgttcctcgtgcattttaggccagaaaatcgaccacaaaatcgaga 662 Picornavirus aaacgggaggatcggctttggcttatcctcttaatagtctacaaaactgggctgactggtggggga HK21 gctactagatccgggagaggactagttaccccgcgtaacttccctttttgtcactccctccacctacc ccttccctgtcccttagactcttaagtaggtgtgcaggcctggccccccggaaatgggcaaatcgg acgtttgcgtgtagggtcgttctgaaaggtggggcccactccaccgtagtaggatcttcctgtttcac gtttaaacctctccgggcaggtatcgctagacactaggctgtataggatggcacgcacttaggtttc gcaccttcctagtgcgccaagatcccgcttttgagctcaagtacatggttctttgtaagatgtctaacc agaggaggtggtgctgaaatattgcaagccactctggcgaacgtccactgcattgcctggaacag gctctctagcgccctccactggtagcgcggtggtgggttagtaggatacctatatggacaggggat gcgggaatacccctcactagctagtgactggttgatcgactggcggcggatccagtgatacttgca taatccgcagacttgggag 663 Picornavirales agccatgaactagtgtgcgattcccacagtgttggtcgaaagccccgcactgtctactcgcatattt Tottori-HG1 gactccagtccttccgcagccagcggttaggttctggttaaagtgcattatgtgcacggcgccacg cagaactgccagaaatggtaagctgcgcccaacgccaacggtttgtgtatcccgttagtcacacgt ttacagctgttcgttaacagtagggttttgtcgacccggaccccttaatgcgcgcgaattcaaccgc gcctagtcggcaatggtatcatttaatcccatgcactacgggagaaatttgagaccaaagaattcct gagggccactgcttgctctaagtgcaatgcctcgggagacttctgtcaggagcctagcggctttca accgcgacagctaactcctgcgggatgtttggtgtccatacttactggcgtcctcacaacgctaagt ggatgttgtccacaggtaggcaaacaccgagccccacattcaggagacctgtatgaacgatcctat cagcattagagttggaattgggtgtgctaacgtccgcataagtgcaccccgtggtaacgctgggaa actatccagcgcaacgtactgtcctcaatgtctagggaaggaccgccctaagcgtacaaccgggc catgtgtcgagc 664 Rodent ttcaaagaggggtccgggattttcctggtccccctctttgggcacccttggctcgggggtgtgaata hepatovirus ccgtgctcgcgtttgccgtgcgttaacggcttcatttatgtttgtttgtctgttttattatgttGgtttgtct gtttgttatgttggtattgttcgtgtttaatgttatgaccacattacactccagccaatgaagaacagatg gtgcggttattgctggcggaattcctaacgtcctggatccgttggtacgcatcacaaaacaatttgca gagagagtggtgaaacggcttgggaatccctgagtacagggaaatcacactgatagctcatcttg gctgttttcagtcatggaccttatgcagtgtaatttgggtgtaccccccatagcttaggaggaatgttc tgtcttggcactagagtgggacgctgatgcctccgtgtctaggatggtctaagggacagaatgggg tgcctctgatgccatactacctgatagggtgctctcacggcctctgcatcttagtgagaagttcaattt t 665 Rinderpestvirus accaaacaaagttgggtaaggatcggtctatcaatgattatgatttagcacacttaggattcaagatc ctatcgactggagcaggcttaaggtaaaggttctttaaa 666 RabovirusA ctacggatatttgcatgacccgctttctatcgccccaacaatcccctttgtaaccacaagctttactca ggctagcagcccgactagctgtttggaagaaaaggctagggcacacaccaacaacaccgaccc cactggtcgaaggccgcttggcaataagactggtggaacagggtcgcctgtagttgtttggaacat tctttctaatgactttgtcagcggtgctactcacaccgtaactcttctaccctatccccacgcttgtgga actaggaggggatgagtgattcaagtaagtactgtcagaatggtgaaaatgatctgattctgaaacg ctatggatccatcgaaagatggggctacacgcctgcggaacaacacatggtaacatgtgccccag gggccgaaagccacggtgataggatcacccgtgtagtttgagatcatatcaatgttcatagtctagt aagatgatttgaaatctaactgagctgatggctaactgcttgtcttattgcggcctaaggatgtcctgc aggtacctttagataaccttaagagactattgatctgagcaggagccaaagtggtctttcccagcttt ggttaaaaaacgtctaagccgcggcaggggggggaggccccctttcctcccaaaacttaatatt gattgt 667 Shingleback ctgtgagtaccgacaggctcgaagtctattatgaggcgtcgaaacagaaaacctgtaacaactccg nidovirus1 gtttcatctatcactgccgtcaagaggcagaagaggacgaccacgtgtcaccagatcacttgtatct gtttcagtcaggaagtcaacttttcgacgaagttcgaccattcatcgacccgctgaaaagcgtagaa gtcgatgaagatgagctccaaagagccatcgccgatttcgacaaccaaagtgactgtttccactcct tcgagctcgtgaatttcgagctgaaacaacaaatcaacgagaacgagtggtacggttattataatta cgacaaccaaaactgcaaagttcagttgccagtcacatgtcgaatcgaggacgtaacctgggatc aggtttacgtg 668 Senecavalley tttgaaatggggggctgggccctgatgcccagtccttcctttccccttccggggggttaaccggctg virus tgtttgctagaggcacagaggggcaacatccaacctgcttttgcggggaacggtgcggctccgatt cctgcgtcgccaaaggtgttagcgcacccaaacggcgcacctaccaatgttattggtgtggtctgc gagttctagcctactcgtttctcccccgaccattcactcacccacgaaaagtgtgttgtaaccataag atttaacccccgcacgggatgtgcgataaccgtaagactggctcaagcgcggaaagcgctgtaac cacatgctgttagtccctttatggctgcaagatggctacccacctcggatcactgaactggagctcg accctccttagtaagggaaccgagaggccttcgtgcaacaagctccgacacagagtccacgtga ctgctaccaccatgagtacatggttctcccctctcgacccaggacttctttttgaatatccacggctcg atccagagggtggggcatgacccctagcatagcgagctacagcgggaactgtagctaggcctta gcgtgccttggatactgcctgatagggcgacggcctagtcgtgtcggttctataggtagcacatac aaat 669 Sclerotinia ttgaattaatcttttacgtttacgcgcataaaatcaggacacatctcttgtatactttagtatatcaatgat sclerotiorum gtttttgttttatgcgattaatcgtaagagaacttctttccatccgcctgtatgggcgggataataagttc dsRNA accgccttggtcgaggcgcaaacttgtatgtgcaaaggtgagctatatgctcgaaatagtcgtaact mycovirus-L aacacacagccactacctgtagagctctattgatccggaatcctttagtgggaatgcagagctcaca ccggacctgcgggtatcttcggcgttagggacttctgtttcagccttgaatcatttacctttataccttct ctgaggcgcctgggccgggcgcgatattaagtacaagtcaaggacatcgcgggtagtggtctaat cagccgctagtcctgctggagagttccaacttagttgggtgtggtgcatactagctggatagagtag gtatgtattgctaacgtatgccggaggctatccgtcctcggtagaacgtgccgaggagtagtctctg cagacccccgaacgcgtggggtctttacttaaatgtaggcggagggagcgctcgtaggtggaac gactgcctcccagtcgaatgcaagattttgcacgcggaccagtctgcccggcaattcccgggtg 670 Yakenterovirus ctccggcacagccgcaccagtgcactggtacgctagtaccttttcacggggtagtcggtatccccc cccgtaacttagaagcatgtaacaaaccgaccaataggtgcgcggcagccagctgcgttgcggtc aagcacttctgtctccccggtccgcaaggatcgttacccgcccactccactacgaggagcctagta actggccaagtgattgcggagttgcgttcagccacaaccccagtggtagctctggaagatggggc tcgcacatcccccgtggtaacacggttgcttgcccgcgtgtgcttccgggttcagtctccgactgttc acttcaacatcacgcaaccagccaagagccgattgtgctggagtggtcttcctccggggccgtga atgctgctaatcctaacctccgagcgtgtgcgcacaatccagtgttgctacgtcgtaacgcgtaagt tggaggcggaacagactactttcggcaccccgtgtttcctttattttattcttattttatggtgacaattg cagagatttgtgatattgcgactttaccgttaaacatagcactgcattacctggttgcattccacaaaa cttcagagattcctagttcctacattgacctacttgtttatttgaatcttaaatacaaacttgagcaagtg aa 671 Wobblypossum cggctgtgagtgcttagcatatgctagagtactacagccgggtgttggagtcatatgcactggttgc diseasevirus ctgtataatagtcgggatctgtctgacctacattatctttgggagttgcttatcacgacaattctcgaag tgtctgtcgacagcttacccccgattcgacaaggccccttgtccaccgcagacctatcgattttcaac gagaacactatcagaggtttaaatttaaaactcaccaaca 672 Avian gctttttcaatcccttgtgtcgatgttcccgtatgtcatctggttcatgtaacggtgcaacttctatttttg orthoreovirus gtaacgttcactgtcaggcagcgcaaaactcggcgggtggtgatcttcaagcgacttcctctcttgtt segmentS1 gcttattggccctacttggctgcgggcggtggtctgctcgttgttattattataattgttggcgctgtttg ttgttgcaaggctaaggttaaagcggacgcagcccgaaacgttttctaccgagagctgttcgcactt aattcgggtaaaagtgatgcaggacctccgatttaccaggtttagtgtacgacgatttgagttttcac ctttcgtcttagaggagtgcactactccatctttcacgactataactaataccgatccggctctctactt taacattgagtttccgtcaagtcatcgtctctcccccttcattccagaactgttgtctcagccttgtacc gttcacgtttcattgattcggagattcgctctctgtgcaaccttatctagtatttgtgaatacgactgtgc gctactgccatccatcaacgctattacgacgatccctacaccaggtgcgtcatcatctctgattgttc attggg 673 Caprine ggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatacttcccttcact Kobuvirusd10 ccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttccca accccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtg tgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaa cgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggtt ccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaa gtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccc cagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaa ctctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttg attgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 674 Caprine ccccctcaccctcttttccggtggccacgcccgggccaccgatacttcccttcactccttcgggact Kobuvirusd20 gttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccg ccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccagg tcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtgtcct ggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgt gtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtg cccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacag ctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgct gacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttga ctgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 675 Caprine ctcttttccggtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggagg Kobuvirusd30 aacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggt ggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgt acggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactat gacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatct tccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctac aaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgac cggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactg actatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgc ttttcttctcacaatccttgctcagttcaaagtc 676 Caprine gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac Kobuvirusd40 agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg ggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgc atgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtc ctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttg attctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggct aattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccact tgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctca caatccttgctcagttcaaagtc 677 Caprine ccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacagggctcccc Kobuvirusd50 tgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgg gtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcc cacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctg cggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttgg cgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcacca ccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacg ggtggtgtttcctttttttttcacacaactctactgctgacaactcactgactatccacttgctctcttgt gcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgct cagttcaaagtc 678 Picornaviralessp. tttgctcagcgtaacttctccgggttacgtggagaccaaaaggctacggagactcgggctacggc isolateRtMruf- cctggagcacctaggtgctcctaaagacgttagaagttgtacaaactcgcccaatagggcccccc PicoV aaccaggggggtagcgggcaagcacttctgtttccccggtatgatctcataggctgtacccacgg ctgaaagagagattatcgttacccgcctcactacttcgagaagcccagtaatggttcatgaagttgat ctcgttgacccggtgtttcccccacaccagaaacctgtgatgggggtggtcatcccggtcatggcg acatgacggacctccccgcgccggcacagggcctcttcggaggacgagtgacatggattcaacc gtgaagagcctattgagctagtgttgattcctccgcccccgtgaatgcggctaatcccaactccgga gcaggcgggcccaaaccagggtctggcctgtcgtaacgcgaaagtctggagcggaaccgacta ctttcgggaaggcgtgtttccttttgttccttttatcaagttttatggtgacaactcctggtagacgttttat tgcgtttattgagagatttccaacaattgaacagactagaaccacttgttttatcaaaccctcacagaa taagataaca 679 Apodemus ttactcagcgtaactactccgggttacgtgatgaagaagaggctacggagattctcgggctacggc agrarius cctggagccactccggctcctaaagatttagaagtttgagcacacccgcccactagggcccccca picornavirus tccaggggggcaacgggcaagcacttctgtttccccggtatgatctgataggctgtaaccacggct strainLongquan- gaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatgatgggtgaaattgaatc Aa118 cgttgatccggtgtctcccccacaccagaaactcatgatgagggttgccatcccggctacggcga cgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatgatatggatcttgtcgtg aagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggctaatcctaaccccggagc aggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctgggacggaaccgactactttc gggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctgggtaaacgttctattgc gtttattgagagattcccaacaattgaacaaacgagaactacctgttttattaaatttacacagagaag aattaca 680 Niviventer ccctttcataaccccccccttttaacccaacccttcgtaaccgtacgcttcactcgcctttgggtatag confucianus cggcccaatgtgctgaagaaaaggatacgctataaggggccaacgggtggtggcccttaagacc picornavirus acccaacctagaagcttgtacactcgggcaatagtgaggcccacatccagtgggtcaagcccaaa gcattcttgttccccggtatgatctcataagctgtacccacggctgaaagagtgattatcgttatccca ctcagtacttcggagagcctagtacaccacttggaaatggaagtctgtgatccggggttgaccctg aaccccagaaactcatgatgaggctaaccttcccgaacacggcgacgtgtggttagcctgcgctg gcatgaggcctctttgtaggcagactgaaatggaagggtgacgaagagccgactgagctactgttt tattcctccggccccctgaatgcggctaatcctaactcctggtccagtacttgtaacccaacaggtg gctggtcgtaatgcgtaagccgggagcggaaccgactactttggggcgtccgtgtttctcaatatta ttcatttctagcttatggtgacaatttatgattgcagagattgtgctgtatttgtgtctgagagaagaagt aacaat 681 Batpicornavirus tttcaaaaggccctgggcatacggcgttattcgtaacgtcgtatgtccagggcggtagcatcaggc isolateBtRs- caaggcctgatgctaccacgtgtggactaaaccacacactcttcttgtgacacgttgtgtcacctatc PicoV cctttcttggtaacttagaagcttgtacacttacgcacgtaggtgccccacatccagtggggtttgtg caaagcaatcttgttccccggtaaaccctgataggctgtaaccacggccgaaacaaggtttgtcgtt acccgactcactactacacaaagcctagtaaagttcaatgaaagtgcgcagcgtgatccggtcaaa acccccttgaccagaaacacatgatgagggtcaccaacccccactggcgacagtgtggtgtccct gcgttggcatgtggcctcgtagaggcgttgcaatctggatttgctccgaagagccccgtgtgctagt gtttatacctccggccccttgaatgcggctaatcctaacccccgagcatgtacacacaagccagtgt gtagcatgtcgtaatgagcaatttggggatggaaccgactactttagggtgtccgtgtttctcattatt ctttgtttgatgtttt 682 Rhinolophus ttttttttctcaggggtagcatccagccaaggcctgatgctaccaacgtgtgactaaaccacactct picornavirus ctttttgtgatacattgtgtcacctatccctttcttggtaacttagaagcttgtacacccacgcacgtag strainGuizhou- gtaccccacatccagtggggtttgtgcaaagcattcttgttccccggtaaaccctgataggctgtaa Rr100 ccacggctgaaacaaggtttgtcgttacccgactcactactacgcaaagcctagtaaagttcaatga aagtgcgcagcgtgatccggtcaaaacccccttgaccagaaacacatgatgagggtcaccaacc cccactggcgacagtgtggtgtccctgcgttggcatgtggcctcatagaggcgttgcaatctggatt tgctccgaagagccccgtgtgctagtgtttatacctccggccccttgaatgcggctaatcctaaccc ccgagcatgtacacacacgccagtgtgcagcatgtcgtaatgagcaatttggggatggaaccgac tactttagggtgtccgtgtttctcattattctttgtttgatgttttatggtgacaaca 683 Rhinolophus cggaacgttgtatgctcagggcgtaggcaccacccacgggtggtgcctacacgtgtggactaaac picornavirus cacacactcttttcagcacttagtgctgctatctctttttgtaacttagaagtttgtacacaatgcgttag strainHenan- ggccacacatccagtgtggtatcgcaaagcacttctgtttccccggtgctagtaggagggtggctg Rf265 ctccacggccacttgccgaacccatcgttacccgactcattacttcgcaaagcctagtaacccagtt gaagcaagcccggcgtgttccggtcaggaaaaaccccccctggccagaaacatgtgatgagggt gggctatccccactggtgacagtgagccctccctgcgttggcacatggcccgatctgggcgtggtt cttgtggatgctgccgaagagccccgtgagctagtgtttataccgccggcctcgtgaatgcggcta accctaaccccggagcagaggctactgaagccacagtagtcgctgtcgtaacgagtaattctggg atgggaccgactactttcgagtgtccgtgtttcctttattcttttattgttgtttatggtgacaaac 684 Human cccctaggatccactggatgtcagtacactggtatcgtggtacctttgtacgcctgttttataccccctt enterovirusC105 ccccgcaactttagaagcatcaaaagcaccgctcaatagtcaccacacccccagtgtggtttcgag caagcacttctgttttcccggttgcgtcccatatgctgtgcaaacggcaaaaagggacaatatcgtta cccgcttgtatactacgggaaacctagtaccaccattgattgtgttgagagttgcgctcatcacctttc cccggtgtagctcaggccgatgaggctcagaatcccccacaggtgactgtgtctgagcctgcgtt ggcggcctgccctcgccttatggcgtgggacgcttgatacatgacatggtgcgaagagtctactgt gctatgcaagagtcctccggcccctgaatgtggctaatcctaaccactgatcccacgcacgcaaac cagtgtgtagtgggtcgtaacgcgcaagtcggtggcggaaccaactactttgggtgaccgtgtttc ctttattacttattgaatgtttatggtgacaattgtttgattcagttgttgccattctctacattcatttaccca gcatcaaaccaattgaactgttaca 685 Human agtctggacatccctcaccggcgacggtggtccaggctgcgctggcggcctacctgtggtccaaa poliovirus1 gccacgggacgctacatgtgaacaaggtgtgaagagcctattgagctacaaaagagtcctccgg strain cccctgaatgcggctaatcccaaccacggatcaagggtgcacaaaccagtgtacaccttgtcgta NIE1116623 acgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttcctttttaattttgatggctgctt atggtgacaatcatagattgttatcataaagctaattggattggccatccggtgagagtgaaatatatt gtttacctccctgttgggtttactctaactaacttctccatttataaacttgtcatcacagttttaataatta gaagtgcagtttaca 686 Human tttaaaacagcctgggggttgttcccacccccagggcccactgggcgttagtactctggtatcgcg enterovirus109 gtaccttagtatgcctgttttatgtctcctttcccccgcaactttagaagtaatcaagttatggctcaaca gtcgccacacccccagtgtggttccgagcaagcacttctgttccccggttgcgtcttatatgctgtgt gaacggcagaaagggacaatatcgttatccgctcaactactacgggaagcctagtaccaccatgg attgacctgaaagttgcgttcagcgcacccccagcgcagctcaggccgatgaggctccgaatacc ccacgggcgaccgtgtcggagcctgcgttggcggcctgcccacgttgcaaaacgtgggacgctc atttcatgacatggtgcgaagagcctactgtgctagttgagagtcctccggcccctgaatgtggata atcctaaccactgaacctacgggcgcaaaccagcgtctggtaggccgtaacgcgcaagtcggtg gcggaaccaactactttgggtgtccgtgtttccttttatctttttgaatgtttatggtgacaattgttgtgta cagttgttaccatagtttgcattcagaaataaacctaacactttccaattatttgttaca 687 Human ttgtgcgcctgttttatattccccccccgcaacttagaagcacgaaaccaagttcaatagaaggggg poliovirus2 tacaaaccagtaccaccacgaacaagcacttctgtttccccggtgacattgcatagactgctcacgc strain ggttgaaagtgatcgatccgttacccgcttgtgtacttcgaaaagcctagtattgccttggaatcttcg NIE0811460 acgcgttgcgctcagcacccgaccccggggtgtagcttaggctgatgagtctggacattcctcacc ggtgacggtggtccaggctgcgttggcggcctacctatggctaatgccataggacgctagatgtg aacaaggtgtgaagagcctattgagctacataagagtcctccggcccctgaatgcggctaatccta accacggagcaggcggtcgcaaaccagtgactagcttgtcgtaacgcgcaagtctgtggcggaa ccgactactttgggtgtccgtgtttcctgttatttttattatggctgcttatggtgacaatcagagattgtt atcataaagcgaattggattggccatccggtgagtgttgtgtcaggtatacaactgtttgttggaacc actgtgttagtttaacctctctttcaaccaattagtcaaaaacaatacgaagatagaacaacaatacta ca 688 Bovine ttttctcccctccccctccaactaccttttccccctcttgtaacgctagaagtttgtgcaaaccgcctgt picornavirus agggtactgcaatccagcagtgcataggctaagcttttcttgttaccccaccccacattatactgagg aggattgtgaaattgtgttagtatgggttagtagcggtgacccgggtaaccccaacccagaaactc acggatgagatgaacaggaccccacatggtaacgtgtgtgttcgtctgccccgcaaggtgaggcc gtgagagctttgcacgcgaaaaccttgaaaacccaaaagtaccttgagctcttcgctattttgtgtttc ctccaggaccctgaatgcggctaaacctaacccgcgatccgcacgtagcaacccagctagagtgt ggtcgtaatgcgcaagttgcgggcggtaccgactactttggtgttcctgtgtttcctttattttattttga atttttatggtgacaacagctagaaaataagagtgaac 689 Human acccttgtacgcctgttttatactcccctccccgtaacttagaagaaacaaaataagttcaataggag poliovirus1 ggggtacaaaccagtaccaccacgaacaagcacttctgtctccccggtgacattgcatagactgtc strain cccacggttgaaagcaattgatccgttacccgctcttgtacttcgagaagcctagtaccatcttggaa EQG1419328 tcatcgatgcgttgcgctccacactcagtcccagagtgtagcttaggctgatgagtctggacattcct caccggcgacggtggtccaggctgcgttggcggcctacctgtggcccaaagccacaggacgct agatgtgaacaaggtgtgaagagcctattgagctataagagagtcctccggcccctgaatgcggc taatcccaaccacggatcaagggtgcacgaaccagtgtataccttgtcgtaacgcgcaagtccgtg gcggaaccgactactttgggtgaccgtgtttccttttattatttcaatggctgcttatggtgacaatcatt gattgttatcataaagcgaattggactggccatccggtgaaagtgaaacatattgtttgcctcctcgtt gggtctacttcaaccaatctttacttacaatcttaccactacagttttgctggttagaagtgtgtttcacg 690 Human ttgtgcgcctgttttatactcccctcccgcaacttagaagcacgaaaccaagttcaatagaaggggg poliovirus2 tacaaaccagtaccactacgaacaagcacttctgtttccccggtgacattgcatagactgctcacgc isolateIS_061 ggttgaaagtgatcgatccgttacccgcttgtgtacttcgaaaagcctagtatcgccttggaatcttc gacgcgttgcgctcagcacccgaccccggggtgtagcttaggccgatgagtctggacattcctca ccggtgacggtggtccaggctgcgttggcggcctacctatggctaacgccataggacgttagatg tgaacaaggtgtgaagagcctattgagctacataagagtcctccggcccctgaatgcggctaatcc taaccacggagcaggcggtcgcgaaccagtgactggcttgtcgtaacgcgcaagtctgtggcgg aaccgactactttgggtgtccgtgtttcctgttatttttatcatggctgcttatggtgacaatcagagatt gttatcataaagcgaattggattggccatccggtgagtgttgtgtcaggtatacaactgtttgttggaa ccactgtgttagctttgcttctcatttaaccaattaatcaaaaacaatacgaggataaaacaacaatac taca 691 Coxsackievirus cctttgtgcgcctgttttatgcccccttcccccaattgaaacttagaagttacacacaccgatcaacag B5 cgggcgtggcataccagccgcgtcttgatcaagcactcctgtttccccggaccgagtatcaataga ctgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacctagtagca tcatgaaagttgcgaagcgtttcgctcagcacatccccagtgtagatcaggtcgatgagtcaccgc attccccacgggcgaccgtggcggtggctgcgttggcggcctgcctacggggcaacccgtagg acgcttcaatacagacatggtgcgaagagtcgattgagctagttagtagtcctccggcccctgaatc cggctaatcctaactgcggagcacataccctcaacccagggggcattgtgtcgtaacgggtaact ctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttataatggctgcttatggtgaca attgaaagattgttaccatatagctattggattggccatccggtgtctaacagagctattatatacctct ttgttggatttgtaccacttgatctaaaggaagtcaagacactacaattcatcatacaattgaacacag caaa 692 Coxsackievirus tttgtgcgcctgttttacaacccttccccaacttgtaacgtagaagtaatacacactactgatcaatag A10 caggcatggcgcgccagtcatgtctcgatcaagcacttctgttcccccggactgagtatcaataga ctgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacctagtagca ccatagaagctgcagagtgtttcgctcagcacttcccccgtgtagatcaggctgatgagtcactgca atccccacgggtgaccgtggcagtggctgcgttggcggcctgcctatggggcaacccataggac gctctaatgtggacatggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcg gctaatcctaactgcggagcacatgccttcaacccagaaggtagtgtgtcgtaacgggcaactctg cagcggaaccgactactttgggtgtccgtgtttctttttattcctatattggctgcttatggtgacaatca cggaattgttgccatatagctattggattggccatccggtgtctaatagagctattgtgtacctatttgtt ggatttactccgctatcacataaatctctgaacactttgtgctttatattgaacttaaacacccgaaa

[1245] In some embodiments, an IRES of the invention is an IRES having a sequence as listed in Table 17 (SEQ ID NOs: 1-72 and 348-389). In some embodiments, an IRES is a Salivirus IRES. In some embodiments, an IRES is a Salivirus SZ1 IRES. In some embodiments, an IRES is a AP1.0 (SEQ ID NO:348). In some embodiments, an IRES is a CK1.0 (SEQ ID NO:349). In some embodiments, an IRES is a PV1.0 (SEQ ID NO:350). In some embodiments, an IRES is a SV1.0 (SEQ ID NO:351).

TABLE-US-00022 TABLE18 Anabaenapermutationsite5intronfragmentsequences. SEQ Permutation IDNO site Sequence 73 L2-1 GAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACC TAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAG TAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCG GAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGAC GAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGT AGCGAAAGCTGCAAGAGAATGAAAATCCGT 74 L2-2 AAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCT AAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGT AGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGG AAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACG AGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTA GCGAAAGCTGCAAGAGAATGAAAATCCGT 75 L2-3 AGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTA AATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTA GTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGA AGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGA GGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAG CGAAAGCTGCAAGAGAATGAAAATCCGT 76 L5-1 GTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATT AGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGC AGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAA AGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAG CTGCAAGAGAATGAAAATCCGT 77 L5-2 TTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTA GTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCA GAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAA GAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGC TGCAAGAGAATGAAAATCCGT 78 L5-3 TATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAG TAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAG AGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAG AGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCT GCAAGAGAATGAAAATCCGT 79 L5-4 ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTG CAAGAGAATGAAAATCCGT 80 L5-5 TAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTA AGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAG ACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAG AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGT 81 L6-1 ACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCG ACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAG TCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGA GAATGAAAATCCGT 82 L6-2 CAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGA CGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTC CAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGA ATGAAAATCCGT 83 L6-3 AATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGAC GGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCC AATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAA TGAAAATCCGT 84 L6-4 ATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACG GGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCA ATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAAT GAAAATCCGT 85 L6-5 TAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGG GAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAA TTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATG AAAATCCGT 86 L6-6 AGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGG AGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAAT TCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGT 87 L6-7 GATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGA GCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATT CTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGT 88 L6-8 ATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAG CTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTC TCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAA AATCCGT 89 L6-9 TGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGC TACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCT CAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAA ATCCGT 90 L8-1 CAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATA GGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 91 L8-2 AAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAG GCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 92 L8-3 AGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGG CAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 93 L8-4 GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 94 L8-5 ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCA GTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 95 L9a-1 AATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 96 L9a-2 ATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 97 L9a-3 TAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 98 L9a-4 AGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 99 L9a-5 GGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 100 L9-1 GAAAGCTGCAAGAGAATGAAAATCCGT 101 L9-2 AAAGCTGCAAGAGAATGAAAATCCGT 102 L9-3 AAGCTGCAAGAGAATGAAAATCCGT 103 L9-4 AGCTGCAAGAGAATGAAAATCCGT 104 L9-5 GCTGCAAGAGAATGAAAATCCGT 105 L9-6 CTGCAAGAGAATGAAAATCCGT 106 L9-7 AAGAGAATGAAAATCCGT 107 L9-8 AGAGAATGAAAATCCGT 108 L9-9 GAGAATGAAAATCCGT 109 L9a-6 GCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 110 L9a-7 AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 111 L9a-8 GTAGCGAAAGCTGCAAGAGAATGAAAATCCGT

[1246] In some embodiments, a 5 intron fragment is a fragment having a sequence listed in Table 18. Typically, a construct containing a 5 intron fragment listed in Table 18 will contain a corresponding 3 intron fragment as listed in Table 19 (e.g., both representing fragments with the L.sup.9a-8 permutation site).

TABLE-US-00023 TABLE19 Anabaenapermutationsite3intronfragmentsequences. SEQ Permutation IDNO site Sequence 112 L2-1 ACGGACTTAAATAATTGAGCCTTAAA 113 L2-2 ACGGACTTAAATAATTGAGCCTTAAAG 114 L2-3 ACGGACTTAAATAATTGAGCCTTAAAGA 115 L5-1 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTA 116 L5-2 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAG 117 L5-3 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGT 118 L5-4 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT 119 L5-5 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTA 120 L6-1 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT A 121 L6-2 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AA 122 L6-3 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AAC 123 L6-4 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACA 124 L6-5 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAA 125 L6-6 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAAT 126 L6-7 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATA 127 L6-8 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAG 128 L6-9 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGA 129 L8-1 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGT 130 L8-2 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTC 131 L8-3 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCA 132 L8-4 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAA 133 L8-5 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAG 134 L9a-1 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCC 135 L9a-2 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCA 136 L9a-3 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAA 137 L9a-4 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAAT 138 L9a-5 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATA 139 L9-1 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGC 140 L9-2 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCG 141 L9-3 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGA 142 L9-4 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAA 143 L9-5 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAA 144 L9-6 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAG 145 L9-7 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC 146 L9-8 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCA 147 L9-9 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCA A 148 L9a-6 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAG 149 L9a-7 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGC 150 L9a-8 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCA

[1247] S06831 In some embodiments, a 3 intron fragment is a fragment having a sequence listed in Table 19. In some embodiments, a construct containing a 3 intron fragment listed in Table 19 will contain a corresponding 5 intron fragment as listed in Table 18 (e.g., both representing fragments with the L9a-8 permutation site).

TABLE-US-00024 TABLE20 Non-anabaenapermutationsite5intronfragmentsequences. SEQ IDNO Intron Sequence 151 Azop1 tgcgccgatgaaggtgtagagactagacggcacccacctaaggcaaacgctatggtgaaggcatagtcca gggagtggcgaaagtcacacaaaccggaatccgt 152 Azop2 ccgggcgtatggcaacgccgagccaagcttcggcgcctgcgccgatgaaggtgtagagactagacggc acccacctaaggcaaacgctatggtgaaggcatagtccagggagtggcgaaagtcacacaaaccggaat ccgt 153 Azop3 acggcacccacctaaggcaaacgctatggtgaaggcatagtccagggagtggcgaaagtcacacaaacc ggaatccgt 154 Azop4 acgctatggtgaaggcatagtccagggagtggcgaaagtcacacaaaccggaatccgt 155 S795pl attaaagttatagaattatcagagaatgatatagtccaagccttatggtaacatgagggcacttgaccctggta g 156 Twortp1 aagatgtaggcaatcctgagctaagctcttagtaataagagaaagtgcaacgactattccgataggaagtag ggtcaagtgactcgaaatggggattacccttctagggtagtgatatagtctgaacatatatggaaacatatag aaggataggagtaacgaacctattcgtaacataattgaacttttagttat 157 Twortp2 taataagagaaagtgcaacgactattccgataggaagtagggtcaagtgactcgaaatggggattacccttc tagggtagtgatatagtctgaacatatatggaaacatatagaaggataggagtaacgaacctattcgtaacat aattgaacttttagttat 158 Twortp3 taggaagtagggtcaagtgactcgaaatggggattacccttctagggtagtgatatagtctgaacatatatgg aaacatatagaaggataggagtaacgaacctattcgtaacataattgaacttttagttat 159 Twortp4 ctagggtagtgatatagtctgaacatatatggaaacatatagaaggataggagtaacgaacctattcgtaaca taattgaacttttagttat 160 LSUp1 agttaataaagatgatgaaatagtctgaaccattttgagaaaagtggaaataaaagaaaatcttttatgataac ataaattgaacaggctaa 161 Phip1 caaagactgatgatatagtccgacactcctagtaataggagaatacagaaaggatgaaatcc 162 Nostoc agtcgagggtaaagggagagtccaattctcaaagcctattggcagtagcgaaagctgcgggagaatgaaa atccgt 163 Nostoc agccgagggtaaagggagagtccaattctcaaagccaataggcagtagcgaaagctgcgggagaatgaa aatccgt 164 Nodularia agccgagggtaaagggagagtccaattctcaaagccgaaggttattaaaacctggcagcagtgaaagctg cgggagaatgaaaatccgt 165 Pleurocapsa agctgagggtaaagagagagtccaattctcaaagccagcagatggcagtagcgaaagctgcgggagaat gaaaatccgt 166 Planktothrix agccgagggtaaagagagagtccaattctcaaagccaattggtagtagcgaaagctacgggagaatgaaa atccgt

[1248] In some embodiments, a 5 intron fragment is a fragment having a sequence listed in Table 20. A construct containing a 5 intron fragment listed in Table 20 will contain a corresponding 3 intron fragment in Table 21 (e.g., both representing fragments with the Azop1 intron).

TABLE-US-00025 TABLE21 Non-anabaenapermutationsite3'intronfragmentsequences. SEQ IDNO Intron Sequence 167 Azop1 gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaac ctaagcgcccgcccgggcgtatggcaacgccgagccaagcttcggcgcc 168 Azop2 gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaac ctaagcgcccgc 169 Azop3 gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaac ctaagcgcccgcccgggcgtatggcaacgccgagccaagcttcggcgcctgcgccgatgaaggtgta gagactag 170 Azop4 gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaac ctaagcgcccgcccgggcgtatggcaacgccgagccaagcttcggcgcctgcgccgatgaaggtgta gagactagacggcacccacctaaggcaa 171 S795pl aggattagatactacactaagtgtcccccagactggtgacagtctggtgtgcatccagctatatcggtgaa accccattggggtaataccgagggaagctatattatatatatattaataaatagccccgtagagactatgta ggtaaggagatagaagatgataaaatcaaaatcatc 172 Twortp1 actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacac ctaaacaaact 173 Twortp2 actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacac ctaaacaaactaagatgtaggcaatcctgagctaagctcttag 174 Twortp3 actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacac ctaaacaaactaagatgtaggcaatcctgagctaagctcttagtaataagagaaagtgcaacgactattcc ga 175 Twortp4 actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacac ctaaacaaactaagatgtaggcaatcctgagctaagctcttagtaataagagaaagtgcaacgactattcc gataggaagtagggtcaagtgactcgaaatggggattaccctt 176 LSUp1 cgctagggatttataactgtgagtcctccaatattataaaatgttggtaatatattgggtaaatttcaaagaca acttttctccacgtcaggatatagtgtatttgaagcgaaacttattttagcagtgaaaaagcaaataaggac gttcaacgactaaaaggtgagtattgctaacaataatccttttttttaatgcccaacatctttattaact 177 Phip1 gtgggtgcataaactatttcattgtgcacattaaatctggtgaactcggtgaaaccctaatggggcaatacc gagccaagccatagggaggatatatgagaggcaagaagttaattcttgaggccactgagactggctgta tcatccctacgtcacacaaacttaatgccgatggttatttcagaaagaaaaccaatggcgtcttagagatgt atcacagaacggtgtggaaggagcataacggagacatacctgatggcttcgagatagaccataagtgtc gcaatagggcttgctgtaatatagagcatttacagatgcttgagggtacagcccacactgttaagaccaat cgtgaacgctacgcagacagaaaggaaacagctagggaatactggctggagactggatgtaccggcc tagcactcggtgagaagtttggtgtgtcgttctcttctgcttgtaagtggattagagaatggaaggcgtaga gactatccgaaaggagtagggccgagggtgagactccctcgtaacccgaagcgccagacagtcaact 178 Nostoc acggacttaagtaattgagccttaaagaagaaattctttaagtggcagctctcaaactcagggaaacctaa atctgttcacagacaaggcaatcctgagccaagccgaaagagtcatgagtgctgagtagtgagtaaaat aaaagctcacaactcagaggttgtaactctaagctagtcggaaggtgcagagactcgacgggagctac cctaacgtaa 179 Nostoc acggacttaaactgaattgagccttagagaagaaattctttaagtgtcagctctcaaactcagggaaacct aaatctgttgacagacaaggcaatcctgagccaagccgagaactctaagttattcggaaggtgcagaga ctcgacgggagctaccctaacgtca 180 Nodularia acggacttagaaaactgagccttgatcgagaaatctttcaagtggaagctctcaaattcagggaaacctaa atctgtttacagatatggcaatcctgagccaagccgaaacaagtcctgagtgttaaagctcataactcatc ggaaggtgcagagactcgacgggagctaccctaacgtta 181 Pleurocapsa acggacttaaaaaaattgagccttggcagagaaatctgtcatgcgaacgctctcaaattcagggaaacct aagtctggcaacagatatggcaatcctgagccaagccttaatcaaggaaaaaaacatttttaccttttacctt gaaaggaaggtgcagagactcaacgggagctaccctaacaggtca 182 Planktothrix acggacttaaagataaattgagccttgaggcgagaaatctctcaagtgtaagctgtcaaattcagggaaa cctaaatctgtaaattcagacaaggcaatcctgagccaagcctaggggtattagaaatgagggagtttcc ccaatctaagatcaatacctaggaaggtgcagagactcgacgggagctaccctaacgtta

[1249] In some embodiments, a 3 intron fragment is a fragment having a sequence listed in Table 21. A construct containing a 3 intron fragment listed in Table 21 will contain the corresponding 5 intron fragment as listed in Table 20 (e.g., both representing fragments with the Azop1 intron).

TABLE-US-00026 TABLE22 SpacerandAnabaena5intronfragmentsequences. SEQ IDNO Spacer Sequence 183 T25L10 agtatataagaaacaaaccacTAGATGACTTACAACTAATCGGAAGGTGC AGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAA AGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAG CTGCAAGAGAATGAAAATCCGTggctcgcagc 184 T25L20 ctgaaattatacttatactcaaacaaaccacTAGATGACTTACAACTAATCGGAA GGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAG GGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGC GAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 185 T25L30 ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA (I80-10) TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA [Control] GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 186 T25L40 catcaacaatatgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTAC AACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAA CGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCA ATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggct cgcagc 187 T25L50 catcaacaatatgaaactatacttatactcagtatatgaagcattatcgcaaacaaaccacTAGATG ACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTA CCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCA AAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAAT CCGTggctcgcagc 188 T50L10 tagcgtcagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGC AGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAA AGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAG CTGCAAGAGAATGAAAATCCGTggctcgcagc 189 T50L20 atactcatactagcgtcagcaaacaaacaaaTAGATGACTTACAACTAATCGGA AGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGA GGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAG CGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 190 T50L30 gtgtgaagctatactcatactagcgtcagcaaacaaacaaaTAGATGACTTACAACTA ATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCA AGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGG CAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 191 T50L40 cctcacctgagtgtgaagctatactcatactagcgtcagcaaacaaacaaaTAGATGACTTA CAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTA ACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCC AATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgg ctcgcagc 192 T50L50 ccgaatgatgcctcacctgagtgtgaagctatactcatactagcgtcagcaaacaaacaaaTAGAT GACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCT ACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTC AAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAA TCCGTggctcgcagc 193 T75L10 cggtgcgagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTG CAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTA AAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAA GCTGCAAGAGAATGAAAATCCGTggctcgcagc 194 T75L20 cgctccgacccagtgcgagcaaacaaacaaaTAGATGACTTACAACTAATCGG AAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACG AGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTA GCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 195 T25L30 ctgaaattatactAatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 1MM TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 196 T25L30 ctgaaaAtatactAatactcaCtatatgacaaacaaaccacTAGATGACTTACAACTA 3MM ATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCA AGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGG CAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 197 T25L30 ctgaTaAtataGtAatactcaCtatatgacaaacaaaccacTAGATGACTTACAACT 5MM AATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTC AAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAG GCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 198 T25L30 ctgaTaAtaAaGtAatacAcaCtataAgacaaacaaaccacTAGATGACTTACAA 8MM CTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACG TCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAAT AGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgc agc 199 T25L30 ctgaaattatacttatactctctaagttacaaacaaaccacTAGATGACTTACAACTAAT OffTarget10 CGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAG ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCA GTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 200 T25L30 ctgaaattatgtgtgttacAtctaagttacaaacaaaccacTAGATGACTTACAACTAA OffTarget20 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 201 T25L30 gttgatcggtgtgtgttacAtctaagttacaaacaaaccacTAGATGACTTACAACTAA OffTarget30 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 202 T25L30I25- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 10 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattaaacag 203 T25L30I25- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 20 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattcacaatataaa ttacg 204 T25L30I50- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 10 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggatcatagc 205 T25L30I50- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 20 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggatcgcagcataa tatccg 206 T25L30I80- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 20 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagcgcg cctaccg 207 T25L30I80- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 20x2 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagcgcg cctaccgaaagccggcgtcgacgttagcgc 208 T25L30I50- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 20x2 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggatcgcagcataa tatccgaaacgaggatacaagtgacatgc 209 T25L30I25- ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 20x2 TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattcacaatctaaa ttacgaaacgataaatgataactctaac 210 T0L0 aaacaaaccacTAGATGACTTACAACTAATCGGAAGGTGCAGAGAC TCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAA GAGAATGAAAATCCGTggctcgcagc 211 T100L5 cgggcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAG AGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAG AGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCT GCAAGAGAATGAAAATCCGTggctcgcagc 212 T75L30 cgctccgacgagcttccggccagtgcgagcaaacaaacaaaTAGATGACTTACAACT AATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTC AAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAG GCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 213 T0L0a aaacaaaccacGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCC GTggctcgcagc 214 T25L10a agtatataagaaacaaaccacGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 215 T25L20a ctgaaattatacttatactcaaacaaaccacGGCAGTAGCGAAAGCTGCAAGAG AATGAAAATCCGTggctcgcagc 216 T25L30a ctgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC (I80-10) AAGAGAATGAAAATCCGTggctcgcagc [Control] 217 T50L10a tagcgtcagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 218 T50L20a atactcatactagcgtcagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAG AATGAAAATCCGTggctcgcagc 219 T50L30a gtgtgaagctatactcatactagcgtcagcaaacaaacaaaGGCAGTAGCGAAAGCTG CAAGAGAATGAAAATCCGTggctcgcagc 220 T75L10a cggtgcgagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 221 T75L20a cgctccgacccagtgcgagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGA GAATGAAAATCCGTggctcgcagc 222 T75L30a cgctccgacgagcttccggccagtgcgagcaaacaaacaaaGGCAGTAGCGAAAGCT GCAAGAGAATGAAAATCCGTggctcgcagc 223 T0L0b aaacaaaccacAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAA GCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCC GTggctcgcagc 224 T25L10b agtatataagaaacaaaccacAAGACGAGGGTAAAGAGAGAGTCCAATTC TCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAA AATCCGTggctcgcagc 225 T25L20b ctgaaattatacttatactcaaacaaaccacAAGACGAGGGTAAAGAGAGAGTC CAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGA ATGAAAATCCGTggctcgcagc 226 T25L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG (I80-10) AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC [Control] AAGAGAATGAAAATCCGTggctcgcagc 227 T50L10b tagcgtcagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATT CTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 228 T50L20b atactcatactagcgtcagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGT CCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAG AATGAAAATCCGTggctcgcagc 229 T50L30b gtgtgaagctatactcatactagcgtcagcaaacaaacaaaAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTG CAAGAGAATGAAAATCCGTggctcgcagc 230 T75L10b cggtgcgagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATT CTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 231 T75L20b cgctccgacccagtgcgagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAG TCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGA GAATGAAAATCCGTggctcgcagc 232 T75L30b cgctccgacgagcttccggccagtgcgagcaaacaaacaaaAAGACGAGGGTAAAG AGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCT GCAAGAGAATGAAAATCCGTggctcgcagc 233 T25L30I0-0 ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGC AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 234 T25L30aI0- ctgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC 0 AAGAGAATGAAAATCCGT 235 T25L30a ctgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I25-10 AAGAGAATGAAAATCCGTgattaaacag 236 T25L30a ctgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I25-20 AAGAGAATGAAAATCCGTgattcacaatataaattacg 237 T25L30a ctgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I50-10 AAGAGAATGAAAATCCGTggatcatagc 238 T25L30a ctgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I50-20 AAGAGAATGAAAATCCGTggatcgcagcataatatccg 239 T25L30a ctgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I80-20 AAGAGAATGAAAATCCGTggctcgcagcgcgcctaccg 240 T25L30bI0- ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG 0 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGT 241 T25L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I25-10 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGTgattaaacag 242 T25L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I25-20 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGTgattcacaatataaattacg 243 T25L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I50-10 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGTggatcatagc 244 T25L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I50-20 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGTggatcgcagcataatatccg 245 T25L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I80-20 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGTggctcgcagcgcgcctaccg

[1250] In some embodiments, a spacer and 5 intron fragment are spacers and fragments having sequences as listed in Table 22.

TABLE-US-00027 TABLE23 SpacerandAnabaena3intronfragmentsequences. SEQ IDNO Spacer Sequence 246 T25L10 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaacttatatact 247 T25L20 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagagtataagtataatttcag 248 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC (I80-10) TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT [Control] ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 249 T25L40 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcatattgttgatg 250 T25L50 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagcgataatgcttcatatactgagtataagtatagtttcatattg ttgatg 251 T50L10 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctgacgcta 252 T50L20 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctgacgctagtatgagtat 253 T50L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctgacgctagtatgagtatagcttcacac 254 T50L40 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctgacgctagtatgagtatagcttcacactcaggtgagg 255 T50L50 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctgacgctagtatgagtatagcttcacactcaggtgaggc atcattcgg 256 T75L10 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctcgcaccg 257 T75L20 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctcgcactgggtcggagcg 258 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 1MM TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 259 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 3MM TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 260 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 5MM TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 261 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 8MM TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 262 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC OffTarget10 TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtaacttagagagtataagtataatttcag 263 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC OffTarget20 TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtaacttagaTgtaacacacataatttcag 264 T25L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC OffTarget30 TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtaacttagaTgtaacacacaccgatcaac 265 T25L30I25- ctgtttaatcACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCT 10 TTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 266 T25L30I25- cgtaatttatattgtgaatcACGGACTTAAATAATTGAGCCTTAAAGAAGA 20 AATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATC TAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAA TTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 267 T25L30I50- gctatgatccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 10 TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 268 T25L30I50- cggatattatgctgcgatccACGGACTTAAATAATTGAGCCTTAAAGAAG 20 AAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAA TCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGT AATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 269 T25L30I80- cggtaggcgcgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAA 20 GAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAA ATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 270 T25L30I80- gcgctaacgtcgacgccggcaaacggtaggcgcgctgcgagccACGGACTTAAATAA 20x2 TTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAAC TCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAG CCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcat atactgagtataagtataatttcag 271 T25L30I50- gcatgtcacttgtatcctogaaacggatattatgctgcgatccACGGACTTAAATAATTG 20x2 AGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTC AGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCC AAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatac tgagtataagtataatttcag 272 T25L30I25- gttagagttatcatttatcgaaacgtaatttagattgtgaatcACGGACTTAAATAATTGA 20x2 GCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCA GGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCA AGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactg agtataagtataatttcag 273 T0L0 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaa 274 T100L5 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagcccg 275 T75L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagctcgcactggccggaagctcgtcggagcg 276 T0L0a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAcacaaacacaa 277 T25L10a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAcacaaacacaacttatatact 278 T25L20a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagagtataagtataatttc ag 279 T25L30a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC (I80-10) TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT [Control] ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataa gtataatttcag 280 T50L10a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctgacgcta 281 T50L20a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctgacgctagtatga gtat 282 T50L30a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctgacgctagtatga gtatagcttcacac 283 T75L10a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctogcaccg 284 T75L20a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctcgcactgggtcgg agcg 285 T75L30a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctcgcactggccgga agctcgtcggagcg 286 T0L0b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCcacaaacacaa 287 T25L10b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCcacaaacacaacttatatact 288 T25L20b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCcacaaacacaagagtataagtataatt tcag 289 T25L30b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC (I80-10) TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT [Control] ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCcacaaacacaagtcatatactgagtat aagtataatttcag 290 T50L10b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctgacgcta 291 T50L20b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctgacgctagtatg agtat 292 T50L30b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctgacgctagtatg agtatagcttcacac 293 T75L10b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctogcaccg 294 T75L20b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctcgcactgggtcg gagcg 295 T75L30b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctcgcactggccg gaagctcgtcggagcg 296 T25L30I0-0 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAAcacaaacacaagtcatatactgagtataagtataatttcag 297 T25L30aI0- ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG 0 TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTC GACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGA GTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataagtataat ttcag 298 T25L30a ctgtttaatcACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCT I25-10 TTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataa gtataatttcag 299 T25L30a cgtaatttatattgtgaatcACGGACTTAAATAATTGAGCCTTAAAGAAGA I25-20 AATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATC TAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAA TTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGT GCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGT AAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatac tgagtataagtataatttcag 300 T25L30a gctatgatccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC I50-10 TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGA GAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataa gtataatttcag 301 T25L30a cggatattatgctgcgatccACGGACTTAAATAATTGAGCCTTAAAGAAG I50-20 AAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAA TCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGT AATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAG GTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGG GTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcat atactgagtataagtataatttcag 302 T25L30a cggtaggcgcgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAA I80-20 GAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAA ATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAA GGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAG GGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtc atatactgagtataagtataatttcag 303 T25L30bI0- ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG 0 TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGA CAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTC GACGGGAGCTACCCTAACGTCcacaaacacaagtcatatactgagtataagtataat ttcag 304 T25L30b ctgtttaatcACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCT I25-10 TTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCcacaaacacaagtcatatactgagtat aagtataatttcag 305 T25L30b cgtaatttatattgtgaatcACGGACTTAAATAATTGAGCCTTAAAGAAGA I25-20 AATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATC TAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAA TTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGT GCAGAGACTCGACGGGAGCTACCCTAACGTCcacaaacacaagtcatat actgagtataagtataatttcag 306 T25L30b gctatgatccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC I50-10 TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGA GACTCGACGGGAGCTACCCTAACGTCcacaaacacaagtcatatactgagtat aagtataatttcag 307 T25L30b cggatattatgctgcgatccACGGACTTAAATAATTGAGCCTTAAAGAAG I50-20 AAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAA TCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGT AATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAG GTGCAGAGACTCGACGGGAGCTACCCTAACGTCcacaaacacaagtc atatactgagtataagtataatttcag 308 T25L30b cggtaggcgcgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAA I80-20 GAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAA ATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAG TAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAA GGTGCAGAGACTCGACGGGAGCTACCCTAACGTCcacaaacacaagt catatactgagtataagtataatttcag

[1251] In some embodiments, a spacer and 3 intron fragment is a spacer and intron fragment having sequences as listed in Table 23.

TABLE-US-00028 TABLE24 CARsequences SEQ IDNO CAR Sequence 309 FMC63-4- ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC 1BB ATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGAC CACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCAT CAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG GTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTA CCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTC TGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAA CCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGG CAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGA AATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGG CGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTG GCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTG TACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTG GATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGT GATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAA GTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGT GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCAT CTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGC CATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCT GCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACA ACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAG CACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTT TCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAG CCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCAAGAGA GGCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGC GACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCC GCTTTCCAGAAGAGGAGGAGGGCGGGTGCGAACTGtaa 310 FMC63-CD28 ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC ATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGAC CACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCAT CAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG GTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTA CCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTC TGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAA CCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGG CAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGA AATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGG CGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTG GCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTG TACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTG GATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGT GATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAA GTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGT GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCAT CTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGC CATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCT GCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACA ACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAG CACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTT TCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAG CCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGC AAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACC CCTAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTAC GCTCCTCCTAGAGACTTCGCCGCCTACCGGTCCtaa 311 FMC63- ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC CD28-zeta ATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGAC CACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCAT CAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG GTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTA CCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTC TGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAA CCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGG CAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGA AATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGG CGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTG GCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTG TACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTG GATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGT GATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAA GTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGT GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCAT CTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGC CATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCT GCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACA ACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAG CACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTT TCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAG CCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGC AAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACC CCTAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTAC GCTCCTCCTAGAGACTTCGCCGCCTACCGGTCCAGAGTGAAG TTCAGCAGATCCGCCGATGCTCCCGCCTATCAGCAGGGCCAA AACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGA GTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAA TGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTG TATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAG CGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGAC ACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATA CCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 312 FMC63-zeta ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC ATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGAC CACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCAT CAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG GTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTA CCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTC TGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAA CCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGG CAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGA AATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGG CGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTG GCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTG TACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTG GATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGT GATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAA GTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGT GTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCAT CTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGC CATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCT GCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACA ACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAG CACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTT TCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAG CCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCAGAGTG AAGTTCAGCAGATCCGCCGATGCTCCCGCCTATCAGCAGGGC CAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGA AGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTG AAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGC CTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTAC AGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGG ACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGG ATACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 313 CircKymriah- ATGGCTCTCCCGGTCACAGCCCTTCTCCTGCCCCTGGCACTCT Q388 TGCTGCATGCGGCACGACCCGACATCCAGATGACCCAGACCA CAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCA GCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGT ATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACC ACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTG GCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACC TGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCA ACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAA TCACCGGTGGAGGTGGTTCTGGCGGAGGGGGATCTGGTGGAG GCGGTTCAGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGG TGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCG GAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGC CTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCA GCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGA CCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCG CCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATT GGGGCCAGGGCACCAGCGTGACCGTTTCTTCTACCACAACGC CCGCCCCGCGACCGCCTACTCCCGCTCCCACAATTGCATCACA ACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCAGCTGCCGGT GGCGCGGTTCACACGCGGGGGCTCGATTTCGCCTGTGATATA TATATATGGGCCCCATTGGCTGGAACATGCGGAGTATTGCTTC TGAGCCTGGTGATTACCCTCTACTGTAAGAGAGGCCGGAAGA AACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCA GACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGA AGAGGAGGAGGGCGGGTGCGAACTGAGAGTGAAGTTCAGCA GATCCGCCGATGCTCCCGCCTATCAGCAGGGCCAAAACCAGC TGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGAC GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGG CAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGA GCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCG GAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGA CTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGAC GCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 314 CircKymriah- ATGGCTCTCCCGGTCACAGCCCTTCTCCTGCCCCTGGCACTCT K388 TGCTGCATGCGGCACGACCCGACATCCAGATGACCCAGACCA CAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCA GCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGT ATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACC ACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTG GCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACC TGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCA ACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAA TCACCGGTGGAGGTGGTTCTGGCGGAGGGGGATCTGGTGGAG GCGGTTCAGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGG TGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCG GAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGC CTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCA GCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGA CCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCG CCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATT GGGGCCAGGGCACCAGCGTGACCGTTTCTTCTACCACAACGC CCGCCCCGCGACCGCCTACTCCCGCTCCCACAATTGCATCACA ACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCAGCTGCCGGT GGCGCGGTTCACACGCGGGGGCTCGATTTCGCCTGTGATATA TATATATGGGCCCCATTGGCTGGAACATGCGGAGTATTGCTTC TGAGCCTGGTGATTACCCTCTACTGTAAGAGAGGCCGGAAGA AACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCA GACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGA AGAGGAGGAGGGCGGGTGCGAACTGAGAGTGAAGTTCAGCA GATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAAAACCAGC TGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGAC GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGG CAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGA GCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCG GAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGA CTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGAC GCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 315 CircM971- ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC CD22 ATCCTGCCTTTCTGCTGATCCCCCAGGTTCAACTCCAGCAGTC TGGTCCCGGCCTCGTTAAACCAAGCCAGACTTTGTCTCTTACC TGTGCTATCAGTGGCGATAGCGTGTCTAGTAATTCAGCCGCAT GGAACTGGATCCGACAATCACCGAGTAGGGGACTTGAATGGC TGGGTAGAACCTATTACCGGTCCAAATGGTACAATGACTATG CAGTGTCTGTAAAAAGCAGGATCACGATCAACCCTGATACGT CTAAAAACCAGTTTTCTCTGCAACTTAATAGTGTGACCCCTGA AGACACCGCTGTGTATTACTGTGCACGGGAGGTTACCGGTGA TCTTGAAGATGCTTTTGATATATGGGGCCAAGGTACGATGGT CACGGTGTCTAGTgggggaggcggcagcGACATACAGATGACGCAG AGCCCATCCAGTCTCTCCGCGTCTGTTGGTGACAGAGTGACTA TTACATGTAGGGCGTCTCAGACCATTTGGTCTTACCTCAATTG GTATCAACAGCGACCAGGCAAAGCACCGAACTTGCTCATTTA CGCTGCCAGCTCACTCCAAAGTGGTGTGCCGTCCAGATTTAGT GGTAGGGGCAGTGGCACTGATTTCACTCTGACTATTTCAAGTC TTCAAGCTGAGGATTTTGCCACATACTACTGCCAGCAAAGTT ACTCAATACCTCAGACTTTTGGACAGGGGACAAAATTGGAGA TTAAAtccggaACCACAACGCCCGCCCCGCGACCGCCTACTCCC GCTCCCACAATTGCATCACAACCCCTGTCTTTGAGACCCGAA GCTTGTCGACCAGCTGCCGGTGGCGCGGTTCACACGCGGGGG CTCGATTTCGCCTGTGATATATATATATGGGCCCCATTGGCTG GAACATGCGGAGTATTGCTTCTGAGCCTGGTGATTACCCTCTA CTGTAAGAGAGGCCGGAAGAAACTTCTTTATATATTCAAGCA GCCCTTTATGCGACCCGTTCAGACTACCCAAGAGGAAGATGG ATGCAGTTGCCGCTTTCCAGAAGAGGAGGAGGGCGGGTGCGA ACTGAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTA TAAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGG GGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGC AGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCC TCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGG CCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGA AGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCAC CGCCACCAAGGATACCTATGACGCCCTGCACATGCAGGCCCT GCCTCCAAGAtaa 316 CircCD19_22 ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC Bispecific29 ATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGAC CACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCAT CAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG GTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTA CCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTC TGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAA CCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGG CAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGA AATCACCggcggcggaggatccCAGGTTCAACTCCAGCAGTCTGGTC CCGGCCTCGTTAAACCAAGCCAGACTTTGTCTCTTACCTGTGC TATCAGTGGCGATAGCGTGTCTAGTAATTCAGCCGCATGGAA CTGGATCCGACAATCACCGAGTAGGGGACTTGAATGGCTGGG TAGAACCTATTACCGGTCCAAATGGTACAATGACTATGCAGT GTCTGTAAAAAGCAGGATCACGATCAACCCTGATACGTCTAA AAACCAGTTTTCTCTGCAACTTAATAGTGTGACCCCTGAAGAC ACCGCTGTGTATTACTGTGCACGGGAGGTTACCGGTGATCTTG AAGATGCTTTTGATATATGGGGCCAAGGTACGATGGTCACGG TGTCTAGTGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTG GCGAGGGATCTACCAAGGGCGACATACAGATGACGCAGAGC CCATCCAGTCTCTCCGCGTCTGTTGGTGACAGAGTGACTATTA CATGTAGGGCGTCTCAGACCATTTGGTCTTACCTCAATTGGTA TCAACAGCGACCAGGCAAAGCACCGAACTTGCTCATTTACGC TGCCAGCTCACTCCAAAGTGGTGTGCCGTCCAGATTTAGTGGT AGGGGCAGTGGCACTGATTTCACTCTGACTATTTCAAGTCTTC AAGCTGAGGATTTTGCCACATACTACTGCCAGCAAAGTTACT CAATACCTCAGACTTTTGGACAGGGGACAAAATTGGAGATTA AAgggggaggcggcagcGAAGTGAAACTGCAAGAGTCTGGCCCTGG ACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTC AGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGA CAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGG GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTG AAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTAT TGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGAT TATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTtccggaACC ACAACGCCCGCCCCGCGACCGCCTACTCCCGCTCCCACAATT GCATCACAACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCA GCTGCCGGTGGCGCGGTTCACACGCGGGGGCTCGATTTCGCC TGTGATATATATATATGGGCCCCATTGGCTGGAACATGCGGA GTATTGCTTCTGAGCCTGGTGATTACCCTCTACTGTAAGAGAG GCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGCG ACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCCG CTTTCCAGAAGAGGAGGAGGGGGGTGCGAACTGAGAGTGA AGTTCAGCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCC AAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAA GAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGA AATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCC TGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACA GCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGA CACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGAT ACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 317 CircCD19_22 ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC Bispecific30 ATCCTGCCTTTCTGCTGATCCCCCAGGTTCAACTCCAGCAGTC TGGTCCCGGCCTCGTTAAACCAAGCCAGACTTTGTCTCTTACC TGTGCTATCAGTGGCGATAGCGTGTCTAGTAATTCAGCCGCAT GGAACTGGATCCGACAATCACCGAGTAGGGGACTTGAATGGC TGGGTAGAACCTATTACCGGTCCAAATGGTACAATGACTATG CAGTGTCTGTAAAAAGCAGGATCACGATCAACCCTGATACGT CTAAAAACCAGTTTTCTCTGCAACTTAATAGTGTGACCCCTGA AGACACCGCTGTGTATTACTGTGCACGGGAGGTTACCGGTGA TCTTGAAGATGCTTTTGATATATGGGGCCAAGGTACGATGGT CACGGTGTCTAGTgggggaggcggcagcGACATACAGATGACGCAG AGCCCATCCAGTCTCTCCGCGTCTGTTGGTGACAGAGTGACTA TTACATGTAGGGCGTCTCAGACCATTTGGTCTTACCTCAATTG GTATCAACAGCGACCAGGCAAAGCACCGAACTTGCTCATTTA CGCTGCCAGCTCACTCCAAAGTGGTGTGCCGTCCAGATTTAGT GGTAGGGGCAGTGGCACTGATTTCACTCTGACTATTTCAAGTC TTCAAGCTGAGGATTTTGCCACATACTACTGCCAGCAAAGTT ACTCAATACCTCAGACTTTTGGACAGGGGACAAAATTGGAGA TTAAAGGGGGAGGCGGATCCGGCGGTGGTGGCTCCGGCGGTG GTGGTTCTGGAGGCGGCGGAAGCGGTGGGGGTGGTAGCGAC ATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTG GGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATC AGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACC GTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGC GTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTAC AGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACC TACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCG GAGGCACCAAGCTGGAAATCACCGGCTCTACAAGCGGCAGC GGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGCGAAGT GAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCA GTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCT GATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGC CTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTAC TACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGAC AACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAG ACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTAC TACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACC AGCGTGACCGTTTCTTCTtccggaACCACAACGCCCGCCCCGCGA CCGCCTACTCCCGCTCCCACAATTGCATCACAACCCCTGTCTT TGAGACCCGAAGCTTGTCGACCAGCTGCCGGTGGCGCGGTTC ACACGCGGGGGCTCGATTTCGCCTGTGATATATATATATGGG CCCCATTGGCTGGAACATGCGGAGTATTGCTTCTGAGCCTGGT GATTACCCTCTACTGTAAGAGAGGCCGGAAGAAACTTCTTTA TATATTCAAGCAGCCCTTTATGCGACCCGTTCAGACTACCCAA GAGGAAGATGGATGCAGTTGCCGCTTTCCAGAAGAGGAGGA GGGCGGGTGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCG ATGCTCCCGCCTATAAGCAGGGCCAAAACCAGCTGTACAACG AGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGAC AAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAG ACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAA AGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGG GCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAG GGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCAC ATGCAGGCCCTGCCTCCAAGAtaa 318 FMC63CD28 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCR anti-CD19 ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG humanCAR TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQV FLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR 319 FMC634- MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR 1BBanti- ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG CD19human TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG CAR GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVS WIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR 320 Anti-BCMA MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSLGERATINCR humanCAR ASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGS GSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTS GSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFT DYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLD TSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVS SAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR 321 1D3?mouse MGVPTQLLGLLLLWITDAICDIQMTQSPASLSTSLGETVTIQCQA CD19CAR; SEDIYSGLAWYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGT Caprine QYSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLELKGGGGSG Kobuvirus GGGSGGGGSEVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYM IRES HFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNT AYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSSIEFMYP PPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYG LLVTVALCVIWTNSRRNRGGQSDYMNMTPRRPGLTRKPYQPYA PARDFAAYRPRAKFSRSAETAANLQDPNQLFNELNLGRREEFDV LEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIG TKGERRRGKGHDGLFQGLSTATKDTFDALHMQTLAPR 322 WT?(wild MGVPTQLLGLLLLWITDAICDIQMTQSPASLSTSLGETVTIQCQA typezeta) SEDIYSGLAWYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGT mouseCAR QYSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLELKGGGGSG GGGSGGGGSEVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYM HFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNT AYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSSIEFMYP PPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYG LLVTVALCVIWTNSRRNRGGQSDYMNMTPRRPGLTRKPYQPYA PARDFAAYRPRAKFSRSAETAANLQDPNQLYNELNLGRREEYD VLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEI GTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR

[1252] In some embodiments, a CAR is encoded by a nucleotide sequence as listed in Table 24.

TABLE-US-00029 TABLE25 CARdomainsequences. SEQ IDNO Protein Sequence 318 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 319 CD3? RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPE intracellular MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD domain GLYQGLSTATKDTYDALHMQALPPR 320 CD28 QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQAPGQ intracellular GLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLR signaling SDDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGS domain GGGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRYYLSWYQQKP GKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CLQTYTTPDFGPGTKVEIK 321 FMC63VH EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLE WLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTA IYYCAKHYYYGGSYAMDYWGQGTSVTVSS 322 FMC63VL DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEIT

[1253] In some embodiments, a CAR domain encoded by an inventive polynucleotide has a sequence as listed in Table 25.

TABLE-US-00030 TABLE26 PD-1orPD-L1sequences. SEQ IDNO Description Sequence 323 Pembrolizumabheavy QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV chain RQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSST TTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK 324 Pembrolizumablight EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWY chain QQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISS LEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 325 Nivolumabheavy QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVR chain QAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSK NTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK 326 Nivolumablight EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP chain GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPE DFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 327 Atezolizumabheavy EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQ chain APGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNT AYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 328 Atezolizumablight DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQK chain PGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 329 Avelumabheavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQ chain APGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 330 Avelumablightchain QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQ HPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGL QAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTV TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPV KAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 331 Durvalumabheavy EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVR chain QAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAK NSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 332 Durvalumablight EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQK chain PGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

[1254] In some embodiments, a cleavage site separating expression sequences encoded by an inventive polynucleotide has a sequence listed in Table 26.

TABLE-US-00031 TABLE27 Cytokinesequences. SEQ IDNO Cytokine Sequence 333 IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINV IVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 334 IL-12A RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSE EIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASR KTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQN MLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIR AVTIDRVMSYLNAS 335 IL-12B IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSS EVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGI WSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSV KSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPA AEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTD KTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS 336 IL-7 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHI CDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLN CTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLL QEIKTCWNKILMGTKEH 337 IL-10 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKA HVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQE KGIYKAMSEFDIFINYIEAYMTMKIRN 338 IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE EKNIKEFLQSFVHIVQMFINTS 339 IL-18 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTI FIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKD TKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKK EDELGDRSIMFTVQNED 340 IL-27beta RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATY RLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVH PWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPF PEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQV AAQDLTDYGELSDWSLPATATMSLGK 341 IFNgamma QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKI MQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKR DDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQ MLFRG 342 TGFbetal ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFC LGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS

[1255] In some embodiments, a cytokine encoded by an inventive polynucleotide has a sequence as listed in Table 27.

TABLE-US-00032 TABLE28 Transcriptionfactorsequences. SEQ Transcription IDNO factor Sequence 343 FOXP3 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGT FQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGP LPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLT PPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPR KDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADH LLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKM ALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAV RRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILE APEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFV RVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP 344 FOXP3 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGT FQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGP LPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLT PPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPR KDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADH LLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKM ALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAV RRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILE APEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFV RVESEKGAVWTVDELEFRKKR 345 FOXP3 GGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQAL LQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATG VFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLS AVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEK GRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKA SSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLW GSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQ RTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESE KGAVWTVDELEFRKKR 346 STAT5B MAVWIQAQQLQGEALHQMQALYGQHFPIEVRHYLSQWIESQA WDSVDLDNPQENIKATQLLEGLVQELQKKAEHQVGEDGFLLKI KLGHYATQLQNTYDRCPMELVRCIRHILYNEQRLVREANNGSSP AGSLADAMSQKHLQINQTFEELRLVTQDTENELKKLQQTQEYFII QYQESLRIQAQFGPLAQLSPQERLSRETALQQKQVSLEAWLQRE AQTLQQYRVELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLA GNGGPPEGSLDVLQSWCEKLAEIIWQNRQQIRRAEHLCQQLPIPG PVEEMLAEVNATITDIISALVTSTFIIEKQPPQVLKTQTKFAATVR LLVGGKLNVHMNPPQVKATIISEQQAKSLLKNENTRNDYSGEIL NNCCVMEYHQATGTLSAHFRNMSLKRIKRSDRRGAESVTEEKF TILFESQFSVGGNELVFQVKTLSLPVVVIVHGSQDNNATATVLW DNAFAEPGRVPFAVPDKVLWPQLCEALNMKFKAEVQSNRGLTK ENLVFLAQKLFNNSSSHLEDYSGLSVSWSQFNRENLPGRNYTFW QWFDGVMEVLKKHLKPHWNDGAILGFVNKQQAHDLLINKPDG TFLLRFSDSEIGGITIAWKFDSQERMFWNLMPFTTRDFSIRSLADR LGDLNYLIYVFPDRPKDEVYSKYYTPVPCESATAKAVDGYVKPQ IKQVVPEFVNASADAGGGSATYMDQAPSPAVCPQAHYNMYPQ NPDSVLDTDGDFDLEDTMDVARRVEELLGRPMDSQWIPHAQS 347 HELIOS METEAIDGYITCDNELSPEREHSNMAIDLTSSTPNGQHASPSHMT STNSVKLEMQSDEECDRKPLSREDEIRGHDEGSSLEEPLIESSEVA DNRKVQELQGEGGIRLPNGKLKCDVCGMVCIGPNVLMVHKRSH TGERPFHCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCSYACRR RDALTGHLRTHSVGKPHKCNYCGRSYKQRSSLEEHKERCHNYL QNVSMEAAGQVMSHHVPPMEDCKEQEPIMDNNISLVPFERPAVI EKLTGNMGKRKSSTPQKFVGEKLMRFSYPDIHFDMNLTYEKEA ELMQSHMMDQAINNAITYLGAEALHPLMQHPPSTIAEVAPVISS AYSQVYHPNRIERPISRETADSHENNMDGPISLIRPKSRPQEREAS PSNSCLDSTDSESSHDDHQSYQGHPALNPKRKQSPAYMKEDVK ALDTTKAPKGSLKDIYKVFNGEGEQIRAFKCEHCRVLFLDHVMY TIHMGCHGYRDPLECNICGYRSQDRYEFSSHIVRGEHTFH

[1256] In some embodiments, a transcription factor encoded by an inventive polynucleotide has a sequence as listed in Table 28.

TABLE-US-00033 TABLE29 AdditionalAccessorySequences SEQ IDNO IRES Sequence 390 CK3UTRScr ccctgcagccgtcaccgtaagtttgaagttaccgcatatcagcctctgcttcccagcgcgtccaatt cctgttcttattgtttcccctccaggcgttacgcgtgacgacgaactgtgtcgcagctaccacattatt ccggagccttcattctcgcggctctgatcgt 391 CK3UTRS2M ggagaccgcggccacgccgagtaggatcgagggtacagtctcc 392 CK3UTR gacaccaggatcactcttgctctgacccgccctgtgtagaatagactcatgcttccctaagacctgg atttcttcccaggcactttcacccgcctgccctgctccttcagtggactgcacccagggaggcggtc tctgactgtcctttactttctattctggattgc 393 CK5UTR1 AAACCCCCCTAAGCCGCCGCCGCCGCCACC 394 CK5UTR2 CCCCCCCAACCCGTCACG 395 CK5UTR3 GTCACG 396 SZ13UTRScr tctgcgcactcgtaatcagtactaacccccctttgtcggacactatgcgataatcgatccgcctttttc accgccttcggaattttatttacctcaactgatcctggagtctctcttggttttcacggaggcctccgcc ca 397 SZ1S2M ggagaccgcggccacgccgagtaggatcgagggtacagtctcc 398 SZ13UTR ccccttgaaacccccgccccaggttcagtctctcttcatccctctgtcctgcatggtgatacaaagac cctttgtggaccctaagccatgtagttgctgctccctccttccagttgtgaatattggtttctgttaatca ca 399 SZ15UTR1 AAACCCCCCTAAGCCGCCGCCGCCGCCACC 400 SZ15UTR2 CCCCCCCAACCCGTCACG 401 SZ15UTR3 GTCACG 402 UTR1 gTcacG 403 UTR2 AATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGC CACC 404 UTR3 cgaactagtattcttctggtccccacagactcagagagaacccgccacc 405 UTR4 Agccacc 406 STOP1 tgatAGctAaCtaG 407 STOP2 tagtAGctAaCtaG 408 STOP3 tGatGActGaGtGA 409 STOP4 tagtagctagGtag 410 STOP5 taa 411 STOP6 taatagCtaaCtag 412 STOP7 taaCtagCtaaCtag 718 CCL1 MQIITTALVCLLLAGMWPEDVDSKSMQVPFSRCCFSFAEQEIP LRAILCYRNTSSICSNEGLIFKLKRGKEACALDTVGWVQRHRK MLRHCPSKRK 719 CCL2 MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKIS VQRLAS 720 CCL3 MQVSTAALAVLLCTMALCNQFSASLAADTPTACCFSYTSRQI PQNFIADYFETSSQCSKPGVIFLTKRSRQVCADPSEEWVQKYV SDLELSA 721 CCL4 MKLCVTVLSLLMLVAAFCSPALSAPMGSDPPTACCFSYTARK LPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQE YVYDLELN 722 CCL5 MKVSAAALAVILIATALCAPASASPYSSDTTPCCFAYIARPLPR AHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYIN SLEMS 723 CCL6 MRNSKTAISFFILVAVLGSQAGLIQEMEKEDRRYNPPIIHQGFQ DTSSDCCFSYATQIPCKRFIYYFPTSGGCIKPGIIFISRRGTQVCA DPSDRRVQRCLSTLKQGPRSGNKVIA 724 CCL7 MKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKI PKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDF MKHLDKKTQTPKL 725 CCL8 MKVSAALLCLLLMAATFSPQGLAQPDSVSIPITCCFNVINRKIP IQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSM KHLDQIFQNLKP 726 CCL9/CCL10 MKPFHTALSFLILTTALGIWAQITHATETKEVQSSLKAQQGLEI EMFHMGFQDSSDCCLSYNSRIQCSRFIGYFPTSGGCTRPGIIFIS KRGFQVCANPSDRRVQRCIERLEQNSQPRTYKQ 727 CCL11 MKVSAALLWLLLIAAAFSPQGLAGPASVPTTCCFNLANRKIPL QRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSM KYLDQKSPTPKP 728 CCL12 MKISTLLCLLLIATTISPQVLAGPDAVSTPVTCCYNVVKQKIHV RKLKSYRRITSSQCPREAVIFRTILDKEICADPKEKWVKNSINH LDKTSQTFILEPSCLG 729 CCL13 MKVSAVLLCLLLMTAAFNPQGLAQPDALNVPSTCCFTFSSKKI SLQRLKSYVITTSRCPQKAVIFRTKLGKEICADPKEKWVQNY MKHLGRKAHTLKT 730 CCL14 MKISVAAIPFFLLITIALGTKTESSSRGPYHPSECCFTYTTYKIPR QRIMDYYETNSQCSKPGIVFITKRGHSVCTNPSDKWVQDYIKD MKEN 731 CCL15 MKVSVAALSCLMLVAVLGSQAQFINDAETELMMSKLPLENP VVLNSFHFAADCCTSYISQSIPCSLMKSYFETSSECSKPGVIFLT KKGRQVCAKPSGPGVQDCMKKLKPYSI 732 CCL16 MKVSEAALSLLVLILIITSASRSQPKVPEWVNTPSTCCLKYYEK VLPRRLVVGYRKALNCHLPAIIFVTKRNREVCTNPNDDWVQE YIKDPNLPLLPTRNLSTVKIITAKNGQPQLLNSQ 733 CCL17 MAPLKMLALVTLLLGASLQHIHAARGTNVGRECCLEYFKGAI PLRKLKTWYQTSEDCSRDAIVFVTVQGRAICSDPNNKRVKNA VKYLQSLERS 734 CCL18 MKGLAAALLVLVCTMALCSCAQVGTNKELCCLVYTSWQIPQ KFIVDYSETSPQCPKPGVILLTKRGRQICADPNKKWVQKYISD LKLNA 735 CCL19 MALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIV RNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRL QRTSAKMKRRSS 736 CCL20 MCCTKSLLLAALMSVLLLHLCGESEAASNFDCCLGYTDRILH PKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYI VRLLSKKVKNM 737 CCL21 MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAK VVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQL MQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTER SQTPKGP 738 CCL22 MDRLQTALLVVLVLLAVALQATEAGPYGANMEDSVCCRDY VRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRDKEICADPRVP WVKMILNKLSQ 739 CCL23 MKVSVAALSCLMLVTALGSQARVTKDAETEFMMSKLPLENP VLLDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFL TKKGRRFCANPSDKQVQVCVRMLKLDTRIKTRKN 740 CCL24 MAGLMTIVTSLLFLGVCAHHIIPTGSVVIPSPCCMFFVSKRIPE NRVVSYQLSSRSTCLKAGVIFTTKKGQQFCGDPKQEWVQRY MKNLDAKQKKASPRARAVAVKGPVQRYPGNQTTC 741 CCL25 MNLWLLACLVAGFLGAWAPAVHTQGVFEDCCLAYHYPIGW AVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRKVCGNPKSRE VQRAMKLLDARNKVFAKLHHNTQTFQAGPHAVKKLSSGNSK LSSSKFSNPISSSKRNVSLLISANSGL 742 CCL26 MMGLSLASAVLLASLLSLHLGTATRGSDISKTCCFQYSHKPLP WTWVRSYEFTSNSCSQRAVIFTTKRGKKVCTHPRKKWVQKYI SLLKTPKQL 743 CCL27 MKGPPTFCSLLLLSLLLSPDPTAAFLLPPSTACCTQLYRKPLSD KLLRKVIQVELQEADGDCHLQAFVLHLAQRSICIHPQNPSLSQ WFEHQERKLHGTLPKLNFGMLRKMG 744 CCL28 MQQRGLAIVALAVCAALHASEAILPIASSCCTEVSHHISRRLLE RVNMCRIQRADGDCDLAAVILHVKRRRICVSPHNHTVKQWM KVQAAKKNGKGNVCHRKKHHGKRNSNRAHQGKHETYGHK TPY 745 CXCL1 MARAALSAAPSNPRLLRVALLLLLLVAAGRRAAGASVATELR CQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKAC LNPASPIVKKIIEKMLNSDKSN 746 CXCL2 MARATLSAAPSNPRLLRVALLLLLLVAASRRAAGAPLATELR CQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKA CLNPASPMVKKIIEKMLKNGKSN 747 CXCL3 MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAGASVVTELR CQCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKAC LNPASPMVQKIIEKILNKGSTN 748 CXCL4 MAYYEESIIFDTDNSSEESGDFDVDFQGPCERAPNYDFHKIFLP TVFGIIFILGIFGNGLVVIVMGFQNKCKTSMTDKYRLHLSVAD LMFVLTLPFWAVDAASSWYFGGFLCKAVHMIYTVNLYSSVLI LAFISLDRYLAVVRATATSSQATRKLLAGKVIYVCVWLPAAIL TVPDLVFASSFEVELDMGGSRMICQRIYPLESNHIWVAAFRFQ HILVGFVLPGLVILTCYCIIISKLSQGSKGQALKRKALKTTVILI LCFFCCWLPYCAGIFVDTLMNLEVIPHSCALEQGVMKWISITE VLAYFHCCLNPILYAFLGVKFKKSARNALTFSSRSSHKMLTKK RGAISSASTESESSSVLYS 749 CXCL5 MSLLSSRAARVPGPSSSLCALLVLLLLLTQPGPIASAGPAAAVL RELRCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKN GKEICLDPEAPFLKKVIQKILDGGNKEN 750 CXCL6 MSLPSSRAARVPGPSGSLCALLALLLLLTPPGPLASAGPVSAV LTELRCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKN GKQVCLDPEAPFLKKVIQKILDSGNKKN 751 CXCL7 MSLRLDTTPSCNSARPLHALQVLLLLSLLLTALASSTKGQTKR NLAKGKEESLDSDLYAELRCMCIKTTSGIHPKNIQSLEVIGKGT HCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDESAD 752 CXCL8 MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKP FHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQR VVEKFLKRAENS 753 CXCL9 MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQ SLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKK WEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT 754 CXCL10 MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLE KLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVS KERSKRSP 755 CXCL11 MSVKGMAIALAVILCATVVQGFPMFKRGRCLCIGPGVKAVK VADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSKQARLI IKKVERKNF 756 CXCL12 MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARA NVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLE KALNKRFKM 757 CXCL13 MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIP RRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRM MEVLRKRSSSTLPVPVFKRKIP 758 CXCL14 MSLLPRRAPPVSMRLLAAALLLLLLALYTARVDGSKCKCSRK GPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHCL HPKLQSTKRFIKWYNAWNEKRRVYEE 759 CXCL15 MAAQGWSMLLLAVLNLGIFVRPCDTQELRCLCIQEHSEFIPLK LIKNIMVIFETIYCNRKEVIAVPKNGSMICLDPDAPWVKATVG PITNRFLPEDLKQKEFPPAMKLLYSVEHEKPLYLSFGRPENKRI FPFPIRETSRHFADLAHNSDRNFLRDSSEVSLTGSDA 760 CXCL16 MGRDLRPGSRVLLLLLLLLLVYLTQPGNGNEGSVTGSCYCGK RISSDSPPSVQFMNRLRKHLRAYHRCLYYTRFQLLSWSVCGG NKDPWVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQA SEGASSDIHTPAQMLLSTLQSTQRPTLPVGSLSSDKELTRPNET TIHTAGHSLAAGPEAGENQKQPEKNAGPTARTSATVPVLCLL AIIFILTAALSYVLCKRRRGQSPQSSPDLPVHYIPVAP 761 CXCL17 MKVLISSLLLLLPLMLMSMVSSSLNPGVARGHRDRGQASRRW LQEGGQECECKDWFLRAPRRKFMTVSGLPKKQCPCDHFKGN VKKTRHQRHHRKPNKHSRACQQFLKQCQLRSFALPL 762 XCL1 MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSR IKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRK SNTRNNMIQTKPTGTQQSTNTAVTLTG 763 XCL2 MRLLILALLGICSLTAYIVEGVGSEVSHRRTCVSLTTQRLPVSR IKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRK SNTRNNMIQTKPTGTQQSTNTAVTLTG 764 CX3CL1 MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKI PVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDA MQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVV LEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPP TPKAQDGGPVGTELFRVPPVSTAATWQSSAPHQPGPSLWAEA KTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRP ENSLEREEMGPVPAHTDAFQDWGPGSMAHVSVVPVSSEGTPS REPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRR QAVGLLAFLGLLFCLGVAMFTYQSLQGCPRKMAGEMAEGLR YIPRSCGSNSYVLVPV 765 Tbet HLLWSKFNQHQTEMIITKQGRRMFPFLSFTVAGLEPTSHYRMF VDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSP NTGAHWMRQEVSFGKLKLTNNKGASNNVTQMIVLQSLHKY QPRLHIVEVNEGEPETVCNASNTHIFTFQETQFIAVTAYQNAEI TQLKIDNNPFAKGFRENFESMYASVDTSVPSPPGPNCQLLGGD PYSPLLSNQYPVP 766 GATA3 MEVTADQPRWVSHHHPAVLNGQHPDTHHPGLSHSYMDAAQ YPLPEEVDVLFNIDGQGNHVPPYYGNSVRATVQRYPPTHHGS QVCRPPLLHGSLPWLDGGKALGSHHTASPWNLSPFSKTSIHH GSPGPLSVYPPASSSSLSGGHASPHLFTFPPTPPKDVSPDPSLST PGSAGSARQDEKECLKYQVPLPDSMKLESSHSRGSMTALGGA SSSTHHPITTYPPYVPEYSSGLFPPSSLLGGSPTGFGCKSRPKAR SSTGRECVNCGATSTPLWRRDGTGHYLCNACGLYHKMNGQN RPLIKPKRRLSAARRAGTSCANCQTTTTTLWRRNANGDPVCN ACGLYYKLHNINRPLTMKKEGIQTRNRKMSSKSKKCKKVHD SLEDFPKNSSFNPAALSRHMSSLSHISPFSHSSHMLTTPTPMHP PSSLSFGPHHPSSMVTAMG 767 RORgt MDRAPQRQHRASRELLAAKKTHTSQIEVIPCKICGDKSSGIHY GVITCEGCKGFFRRSQRCNAAYSCTRQQNCPIDRTSRNRCQHC RLQKCLALGMSRDAVKFGRMSKKQRDSLHAEVQKQLQQRQ QQQQEPVVKTPPAGAQGADTLTYTLGLPDGQLPLGSSPDLPE ASACPPGLLKASGSGPSYSNNLAKAGLNGASCHLEYSPERGK AEGRESFYSTGSQLTPDRCGLRFEEHRHPGLGELGQGPDSYGS PSFRSTPEAPYASLTEIEHLVQSVCKSYRETCQLRLEDLLRQRS NIFSREEVTGYQRKSMWEMWERCAHHLTEAIQYVVEFAKRL SGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTVFFE GKYGGMELFRALGCSELISSIFDFSHSLSALHFSEDEIALYTAL VLINAHRPGLQEKRKVEQLQYNLELAFHHHLCKTHRQSILAK LPPKGKLRSLCSQHVERLQIFQHLHPIVVQAAFPPLYKELFSTE TESPVGLSK 768 Cd25 MEPCLLMWGILTFITVSGYTTDLCDDDPPNLKHATFKALTYK TGTVLNCDCERGFRRISSYMHCTGNSSHASWENKCQCKSISPE NRKGKVTTKPEEQKGENPTEMQSQTPPMDEVDLVGHCREPPP WEHENSKRIYHFVVGQTLHYQCMQGFTALHRGPAKSICKTIF GKTRWTQPPLKCISESQFPDDEELQASTDAPAGRDTSSPFITTS TPDFHKHTEVATTMESFIFTTEYQIAVASCVLLLISIVLVSGLT WQRRRRKSRTI 769 Skp1 MPSIKLQSSDGEIFEVDVEIAKQSVTIKTMLEDLGMDDEGDDD PVPLPNVNAAILKKVIQWCTHHKDDPPPPEDDENKEKRTDDIP VWDQEFLKVDQGTLFELILAANYLDIKGLLDVTCKTVANMIK GKTPEEIRKTFNIKNDFTEEEEAQVRKENQWCEEK 770 Spy MRKLTALFVASTLALGAANLAHAADTTTAAPADAKPMMHH KGKFGPHQDMMFKDLNLTDAQKQQIREIMKGQRDQMKRPPL EERRAMHDIIASDTFDKAKAEAQIAKMEEQRKANMLAHMET QNKIYNILTPEQKKQFNANFEKRLTERPAAKGKMPATAE 771 Fkpa MKSLFKVTLLATTMAVALHAPITFAAEAAKPATAADSKAAFK NDDQKSAYALGASLGRYMENSLKEQEKLGIKLDKDQLIAGV QDAFADKSKLSDQEIEQTLQAFEARVKSSAQAKMEKDAADN EAKGKEYREKFAKEKGVKTSSTGLVYQVVEAGKGEAPKDSD TVVVNYKGTLIDGKEFDNSYTRGEPLSFRLDGVIPGWTEGLK NIKKGGKIKLVIPPELAYGKAGVPGIPPNSTLVFDVELLDVKPA PKADAKPEADAKAADSAKK 772 Sura MKNWKTLLLGIAMIANTSFAAPQVVDKVAAVVNNGVVLESD VDGLMQSVKLNAAQARQQLPDDATLRHQIMERLIMDQIILQM GQKMGVKISDEQLDQAIANIAKQNNMTLDQMRSRLAYDGLN YNTYRNQIRKEMIISEVRNNEVRRRITILPQEVESLAQQVGNQ NDASTELNLSHILIPLPENPTSDQVNEAESQARAIVDQARNGA DFGKLAIAHSADQQALNGGQMGWGRIQELPGIFAQALSTAKK GDIVGPIRSGVGFHILKVNDLRGESKNISVTEVHARHILLKPSPI MTDEQARVKLEQIAADIKSGKTTFAAAAKEFSQDPGSANQGG DLGWATPDIFDPAFRDALTRLNKGQMSAPVHSSFGWHLIELL DTRNVDKTDAAQKDRAYRMLMNRKFSEEAASWMQEQRASA YVKILSN 773 Hsp60 MLRVNSKSSIKTFVRHLSHKELKFGVEGRAALLKGVNTLADA VSVTLGPKGRNVLIEQQFGAPKITKDGVTVAKAITLEDKFEDL GAKLLQEVASKTNESAGDGTTSATVLGRSIFTESVKNVAAGC NPMDLRRGSQAAVEAVIEFLQKNKKEITTSEEIAQVATISANG DKHIGDLLANAMEKVGKEGVITVKEGKTLEDELEVTEGMKF DRGFISPYFITNTKTGKVEFENPLILLSEKKISSIQDILPSLELSN QTRRPLLIIAEDVDGEALAACILNKLRGQVQVCAVKAPGFGD NRKNTLGDIAILSGGTVFTEELDIKPENATIEQLGSAGAVTITK EDTVLLNGEGSKDNLEARCEQIRSVIADVHTTEYEKEKLQERL AKLSGGVAVIKVGGASEVEVGEKKDRYEDALNATRAAVEEG ILPGGGTALIKATKILDEVKEKAVNFDQKLGVDTIRAAITKPA KRIIENAGEEGAVIVGKIYDEPEFNKGYDSQKGEFTDMIAAGII DPFKVVKNGLVDASGVASLLATTECAIVDAPQPKGSPAAPPA PGMGGMPGMF 774 Hsp70 MAPAVGIDLGTTYSCVGIFRDDRIEIIANDQGNRTTPSFVAFTD TERLIGDAAKNQVAMNPANTVFDAKRLIGRKFADPEVQADM KHFPFKITDKGGKPVIQVEFKGETKEFTPEEISSMVLTKMRETA EAYLGGTVNNAVVTVPAYFNDSQRQATKDAGLIAGLNVLRII NEPTAAAIAYGLDKKADGERNVLIFDLGGGTFDVSLLTIEEGIF EVKSTAGDTHLGGEDFDNRLVNHFVSEFKRKFKKISPAERAR ALRRSPTACERAKRTLSSAAQTSIEIDSLYEGIDFYTSITRARFE ELCQDLFRSTMEPVERVLRDAKIDKSSVHEIVLVGGSTRIPRIQ KLVSDFFNGKEPNKSINPDEAVAYGAAVQAAILSGDTSSKSTN EILLLDVAPLSLGIETAGGVMTPLIKRNTTIPTKKSETFSTFSDN QPGVLIQVFEGERARTKDNNLLGKFELTGIPRARGVPQIEVTF DVDANGIMNVSALEKGTRKTNKIVITNDKGRLSKEEIERMLA EAEKYKAEDEAEASRIRPKNGLESYAYSLRNSLRHSKVDEKL EAGDKEKLKSEIDKTVQWLDENQTATKEEYESQQKELEAVA NPIMMKFYAGGEGAPGGFPGAGGPGGFPGGPGAGHASGGGD DGPTVEEVDLKFPMLPLPWQLSVRKMHRPFFLFLLFLIFLIFLIL FLFYFFLPVRFNESCFS 775 GroEL MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVV LDKSFGAPTITKDGVSVAREIELEDKFENMGAQMVKEVASKA NDAAGDGTTTATVLAQAIITEGLKAVAAGMNPMDLKRGIDK AVTAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEA MDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGYLSPYFIN KPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIA EDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQDI ATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGV GEEAAIQGRVAQIRQQIEEATSDYDREKLQERVAKLAGGVAV IKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVA LIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQIVLNCGE EPSVVANTVKGGDGNYGYNAATEEYGNMIDMGILDPTKVTR SALQYAASVAGLMITTECMVTDLPKNDAADLGAAGGMGGM GGMGGMM 776 GroES MNIRPLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLA VGNGRILENGEVKPLDVKVGDIVIFNDGYGVKSEKIDNEEVLI MSESDILAIVEA 777 Hsp90-alpha MPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIF LRELISNSSDALDKIRYESLTDPSKLDSGKELHINLIPNKQDRTL TIVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIG QFGVGFYSAYLVAEKVTVITKHNDDEQYAWESSAGGSFTVRT DTGEPMGRGTKVILHLKEDQTEYLEE RRIKEIVKKHSQFIGYPITLFVEKERDKEVSDDEAEEKEDKEEE KEKEEKESEDKPEIEDVGSDEEEEKKDGDKKKKKKIKEKYIDQ EELNKTKPIWTRNPDDITNEEYGEFYKSLTNDWEDHLAVKHF SVEGQLEFRALLFVPRRAPFDLFENRKKKNNIKLYVRRVFIMD NCEELIPEYLNFIRGVVDSEDLPLNISR EMLQQSKILKVIRKNLVKKCLELFTELAEDKENYKKFYEQFSK NIKLGIHEDSQNRKKLSELLRYYTSASGDEMVSLKDYCTRMK ENQKHIYYITGETKDQVANSAFVERLRKHGLEVIYMIEPIDEY CVQQLKEFEGKTLVSVTKEGLELPEDEEEKKKQEEKKTKFEN LCKIMKDILEKKVEKVVVSNRLVTSPCCIV TSTYGWTANMERIMKAQALRDNSTMGYMAAKKHLEINPDHS IIETLRQKAEADKNDKSVKDLVILLYETALLSSGFSLEDPQTHA NRIYRMIKLGLGIDEDDPTADDTSAAVTEEMPPLEGDDDTSR MEEVD 778 HtpG MKGQETRGFQSEVKQLLHLMIHSLYSNKEIFLRELISNASDAA DKLRFRALSNPDLYEGDGELRVRVSFDKDKRTLTISDNGVGM TRDEVIDHLGTIAKSGTKSFLESLGSDQAKDSQLIGQFGVGFYS AFIVADKVTVRTRAAGEKPENGVFWESAGEGEYTVADITKED RGTEITLHLREGEDEFLDDWRVRSIISKY SDHIALPVEIEKREEKDGETVISWEKINKAQALWTRNKSEITDE EYKEFYKHIAHDFNDPLTWSHNRVEGKQEYTSLLYIPSQAPW DMWNRDHKHGLKLYVQRVFIMDDAEQFMPNYLRFVRGLIDS SDLPLNVSREILQDSTVTRNLRN ALTKRVLQMLEKLAKDDAEKYQTFWQQFGLVLKEGPAEDFA NQEAIAKLLRFASTHTDSSAQTVSLEDYVSRMKEGQEKIYYIT ADSYAAAKSSPHLELLRKKGIEVLLLSDRIDEWMMNYLTEFD GKPFQSVSKVDESLEKLADEVDESAKEAEKALTPFIDRVKALL GERVKDVRLTHRLTDTPAIVSTDADEMSTQM AKLFAAAGQKVPEVKYIFELNPDHVLVKRAADTEDEAKFSE WVELLLDQALLAERGTLEDPNLFIRRMNQLLVS 779 Hsp40 MGKDYYQTLGLARGASDEEIKRAYRRQALRYHPDKNKEPGA EEKFKEIAEAYDVLSDPRKREIFDRYGEEGLKGSGPSGGSGGG ANGTSFSYTFHGDPHAMFAEFFGGRNPFDTFFGQRNGEEGMD IDDPFSGFPMGMGGFTNVNFGRSRSAQEPARKKQDPPVTHDL RVSLEEIYSGCTKKMKISHKRLNPDGKSIRNEDKILTIEVKKG WKEGTKITFPKEGDQTSNNIPADIVFVLKDKPHNIFKRDGSDVI YPARISLREALCGCTVNVPTLDGRTIPVVFKDVIRPGMRRKVP GEGLPLPKTPEKRGDLIIEFEVIFPERIPQTSRTVLEQVLPI 780 ClpP MWPGILVGGARVASCRYPALGPRLAAHFPAQRPPQRTLQNGL ALQRCLHATATRALPLIPIVVEQTGRGERAYDIYSRLLRERIVC VMGPIDDSVASLVIAQLLFLQSESNKKPIHMYINSPGGVVTAG LAIYDTMQYILNPICTWCVGQAASMGSLLLAAGTPGMRHSLP NSRIMIHQPSGGARGQATDIAIQAEEIMKLKKQLYNIYAKHTK QSLQVIESAMERDRYMSPMEAQEFGILDKVLVHPPQDGEDEP TLVQKEPVEAAPAAEPVPAST 781 ClpX MPSCGACTCGAAAVRLITSSLASAQRGISGGRIHMSVLGRLGT FETQILQRAPLRSFTETPAYFASKDGISKDGSGDGNKKSASEGS SKKSGSGNSGKGG NQLRCPKCGDLCTHVETFVSSTRFVKCEKCHHFFVVLSEADS KKSIIKEPESAAEAVKLAFQQKPPPPPKKIYNYLDKYVVGQSF AKKVLSVAVYNHYKRIYNNIPANLRQQAEVEKQTSLTPRELEI RRREDEYRFTKLLQIAGISPHG NALGASMQQQVNQQIPQEKRGGEVLDSSHDDIKLEKSNILLL GPTGSGKTLLAQTLAKCLDVPFAICDCTTLTQAGYVGEDIESV IAKLLQDANYNVEKAQQGIVFLDEVDKIGSVPGIHQLRDVGG EGVQQGLLKLLEGTIVNVPEKNSRKLRGETVQVDTTNILFVAS GAFNGLDRIISRRKNEKYLGFGTPSNLGKG RRAAAAADLANRSGESNTHQDIEEKDRLLRHVEARDLIEFGM IPEFVGRLPVVVPLHSLDEKTLVQILTEPRNAVIPQYQALFSMD KCELNVTEDALKAIARLALERKTGARGLRSIMEKLLLEPMFEV PNSDIVCVEVDKEVVEGKKEPGYIRAPTKESSEEEYDSGVEEE GWPRQADAANS 782 CDKN1C MSDASLRSTSTMERLVARGTFPVLVRTSACRSLFGPVDHEELS RELQARLAELNAEDQNRWDYDFQQDMPLRGPGRLQWTEVD SDSVPAFYRETVQVGRCRLLLAPRPVAVAVAVSPPLEPAAESL DGLEEAPEQLPSVPVPAPASTPPPVPVLAPAPAPAPAPVAAPV AAPVAVAVLAPAPAPAPAPAPAPAPVAAPAPAPAPAPAPAPA PAPAPDAAPQESAEQGANQGQRGQEPLADQLHSGISGRPAAG TAAASANGAAIKKLSGPLISDFFAKRKRSAPEKSSGDVPAPCPS PSAAPGVGSVEQTPRKRLR 783 BAXinhibitor MNIFDRKINFDALLKFSHITPSTQQHLKKVYASFALCMFVAAA GAYVHMVTHFIQAGLLSALGSLILMIWLMATPHSHETEQKRL GLLAGFAFLTGVGLGPALEFCIAVNPSILPTAFMGTAMIFTCFT LSALYARRRSYLFLGGILMSALSLLLLSSLGNVFFGSIWLFQAN LYVGLVVMCGFVLFDTQLIIEKAEHGD 784 codA MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPIT ENSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIE RWAERKALLTHDDVKQRAWQTLKWQIANGIQHVRTHVDVS DATLTALKAMLEVKQEVAPWIDLQIVAFPQEGIQDYIWHCIDL FLDFITVFRKLMMILAMNEKDKKKEKKLSYPNGEALLEEALR LGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCD EIDDEQSRFVETVAALAHHEGMGARVTASHTTAMHSYNGAY TSRLFRLLKMSGINFVANPLVNIHLQGRFDTYPKRRGITRVKE MLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQL MGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAEN GFDALRRQVPVRYSVRGGKVIASTQPAQTTVYLEQPEAIDYK R

[1257] In some embodiments, a circular RNA or a precursor RNA (e.g., linear precursor RNA) disclosed herein comprises a sequence as listed in Table 29.

[1258] In some embodiments, a polynucleotide or a protein encoded by a polynucleotide contains a sequence with at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% similarity to one or more sequences disclosed herein. In some embodiments, a polynucleotide or a protein encoded by a polynucleotide contains a sequence that is identical to one or more sequences disclosed herein.

[1259] Preferred embodiments are described herein. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

EXAMPLES

[1260] Wesselhoeft et al., (2019) RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In vivo. Molecular Cell. 74(3), 508-520 and Wesselhoeft et al., (2018) Engineering circular RNA for Potent and Stable Translation in Eukaryotic Cells. Nature Communications. 9, 2629 are incorporated by reference in their entirety.

[1261] The invention is further described in detail by reference to the following examples but are not intended to be limited to the following examples. These examples encompass any and all variations of the illustrations with the intention of providing those of ordinary skill in the art with complete disclosure and description of how to make and use the subject invention and are not intended to limit the scope of what is regarded as the invention.

Example 1

Example 1A: External Homology Regions Allow for Circularization of Long Precursor RNA Using the Permuted Intron Exon (PIE) Circularization Strategy

[1262] A 1,100 nt sequence containing a full-length encephalomyocarditis virus (EMCV) IRES, a Gaussia luciferase (GLuc) expression sequence, and two short exon fragments of the permuted intron-exon (PIE) construct were inserted between the 3 and 5 introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage. Precursor RNA was synthesized by run-off transcription. Circularization was attempted by heating the precursor RNA in the presence of magnesium ions and GTP, but splicing products were not obtained.

[1263] Perfectly complementary 9 nucleotide and 19 nucleotide long homology regions were designed and added at the 5 and 3 ends of the precursor RNA. Addition of these homology arms increased splicing efficiency from 0 to 16% for 9 nucleotide homology regions and to 48% for 19 nucleotide homology regions as assessed by disappearance of the precursor RNA band.

[1264] The splicing product was treated with RNase R. Sequencing across the putative splice junction of RNase R-treated splicing reactions revealed ligated exons, and digestion of the RNase R-treated splicing reaction with oligonucleotide-targeted RNase H produced a single band in contrast to two bands yielded by RNase H-digested linear precursor. This shows that circular RNA is a major product of the splicing reactions of precursor RNA containing the 9 or 19 nucleotide long external homology regions.

Example 1B: Spacers that Conserve Secondary Structures of IRES and PIE Splice Sites Increase Circularization Efficiency

[1265] A series of spacers was designed and inserted between the 3 PIE splice site and the IRES. These spacers were designed to either conserve or disrupt secondary structures within intron sequences in the IRES, 3 PIE splice site, and/or 5 splice site. The addition of spacer sequences designed to conserve secondary structures resulted in 87% splicing efficiency, while the addition of a disruptive spacer sequences resulted in no detectable splicing.

Example 2

Example 2A: Internal Homology Regions in Addition to External Homology Regions Creates a Splicing Bubble and Allows for Translation of Several Expression Sequences

[1266] Spacers were designed to be unstructured, non-homologous to the intron and IRES sequences, and to contain spacer-spacer homology regions. These were inserted between the 5 exon and IRES and between the 3 exon and expression sequence in constructs containing external homology regions, EMCV IRES, and expression sequences for Gaussia luciferase (total length: 1289 nt), Firefly luciferase (2384 nt), eGFP (1451 nt), human erythropoietin (1313 nt), and Cas9 endonuclease (4934 nt). Circularization of all 5 constructs was achieved. Circularization of constructs utilizing T4 phage and Anabaena introns were roughly equal. Circularization efficiency was higher for shorter sequences. To measure translation, each construct was transfected into HEK293 cells. Gaussia and Firefly luciferase transfected cells produced a robust response as measured by luminescence, human erythropoietin was detectable in the media of cells transfected with erythropoietin circRNA, and EGFP fluorescence was observed from cells transfected with EGFP circRNA. Co-transfection of Cas9 circRNA with sgRNA directed against GFP into cells constitutively expressing GFP resulted in ablated fluorescence in up to 97% of cells in comparison to an sgRNA-only control.

Example 2B: Use of CVB3 IRES Increases Protein Production

[1267] Constructs with internal and external homology regions and differing IRES containing either Gaussia luciferase or Firefly luciferase expression sequences were made. Protein production was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. The Coxsackievirus B3 (CVB3) IRES construct produced the most protein in both cases.

Example 2C: Use of polyA or polyAC Spacers Increases Protein Production

[1268] Thirty nucleotide long polyA or polyAC spacers were added between the IRES and splice junction in a construct with each IRES that produced protein in example 2B. Gaussia luciferase activity was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. Both spacers improved expression in every construct over control constructs without spacers.

Example 3

HEK293 or HeLa Cells Transfected with Circular RNA Produce More Protein than Those Transfected with Comparable Unmodified or Modified Linear RNA

[1269] HPLC-purified Gaussia luciferase-coding circRNA (CVB3-GLuc-pAC) was compared with a canonical unmodified 5 methylguanosine-capped and 3 polyA-tailed linear GLuc mRNA, and a commercially available nucleoside-modified (pseudouridine, 5-methylcytosine) linear GLuc mRNA (from Trilink). Luminescence was measured 24 h post-transfection, revealing that circRNA produced 811.2% more protein than the unmodified linear mRNA in HEK293 cells and 54.5% more protein than the modified mRNA. Similar results were obtained in HeLa cells and a comparison of optimized circRNA coding for human erythropoietin with linear mRNA modified with 5-methoxyuridine.

[1270] Luminescence data was collected over 6 days. In HEK293 cells, circRNA transfection resulted in a protein production half-life of 80 hours, in comparison with the 43 hours of unmodified linear mRNA and 45 hours of modified linear mRNA. In HeLa cells, circRNA transfection resulted in a protein production half-life of 116 hours, in comparison with the 44 hours of unmodified linear mRNA and 49 hours of modified linear mRNA. CircRNA produced substantially more protein than both the unmodified and modified linear mRNAs over its lifetime in both cell types.

Example 4

Example 4A: Purification of circRNA by RNase Digestion, HPLC Purification, and Phosphatase Treatment Decreases Immunogenicity. Completely Purified Circular RNA is Significantly Less Immunogenic than Unpurified or Partially Purified Circular RNA. Protein Expression Stability and Cell Viability are Dependent on Cell Type and Circular RNA Purity

[1271] Human embryonic kidney 293 (HEK293) and human lung carcinoma A549 cells were transfected with: [1272] products of an unpurified GLuc circular RNA splicing reaction, [1273] products of RNase R digestion of the splicing reaction, [1274] products of RNase R digestion and HPLC purification of the splicing reaction, or [1275] products of RNase digestion, HPLC purification, and phosphatase treatment of the splicing reaction.

[1276] RNase R digestion of splicing reactions was insufficient to prevent cytokine release in A549 cells in comparison to untransfected controls.

[1277] The addition of HPLC purification was also insufficient to prevent cytokine release, although there was a significant reduction in interleukin-6 (IL-6) and a significant increase in interferon-?1 (IFN-?1) compared to the unpurified splicing reaction.

[1278] The addition of a phosphatase treatment after HPLC purification and before RNase R digestion dramatically reduced the expression of all upregulated cytokines assessed in A549 cells. Secreted monocyte chemoattractant protein 1 (MCP1), IL-6, IFN-?1, tumor necrosis factor ? (TNF?), and IFN? inducible protein-10 (IP-10) fell to undetectable or untransfected baseline levels.

[1279] There was no substantial cytokine release in HEK293 cells. A549 cells had increased GLuc expression stability and cell viability when transfected with higher purity circular RNA. Completely purified circular RNA had a stability phenotype similar to that of transfected 293 cells.

Example 4B: Circular RNA does not Cause Significant Immunogenicity and is not a RIG-I Ligand

[1280] A549 cells were transfected with the products of a splicing reaction:

[1281] A549 cells were transfected with: [1282] unpurified circular RNA, [1283] high molecular weight (linear and circular concatenations) RNA, [1284] circular (nicked) RNA, [1285] an early fraction of purified circular RNA (more overlap with nicked RNA peak), [1286] a late fraction of purified circular RNA (less overlap with nicked RNA peak), [1287] introns excised during circularization, or [1288] vehicle (i.e. untransfected control).

[1289] Precursor RNA was separately synthesized and purified in the form of the splice site deletion mutant (DS) due to difficulties in obtaining suitably pure linear precursor RNA from the splicing reaction. Cytokine release and cell viability was measured in each case.

[1290] Robust IL-6, RANTES, and IP-10 release was observed in response to most of the species present within the splicing reaction, as well as precursor RNA. Early circRNA fractions elicited cytokine responses comparable to other non-circRNA fractions, indicating that even relatively small quantities of linear RNA contaminants are able to induce a substantial cellular immune response in A549 cells. Late circRNA fractions elicited no cytokine response in excess of that from untransfected controls. A549 cell viability 36 hours post-transfection was significantly greater for late circRNA fractions compared with all of the other fractions.

[1291] RIG-I and IFN-?1 transcript induction upon transfection of A549 cells with late circRNA HPLC fractions, precursor RNA or unpurified splicing reactions were analyzed. Induction of both RIG-I and IFN-?1 transcripts were weaker for late circRNA fractions than precursor RNA and unpurified splicing reactions. RNase R treatment of splicing reactions alone was not sufficient to ablate this effect. Addition of very small quantities of the RIG-I ligand 3p-hpRNA to circular RNA induced substantial RIG-I transcription. In HeLa cells, transfection of RNase R-digested splicing reactions induced RIG-I and IFN-?1, but purified circRNA did not. Overall, HeLa cells were less sensitive to contaminating RNA species than A549 cells.

[1292] A time course experiment monitoring RIG-I, IFN-?1, IL-6, and RANTES transcript induction within the first 8 hours after transfection of A549 cells with splicing reactions or fully purified circRNA did not reveal a transient response to circRNA. Purified circRNA similarly failed to induce pro-inflammatory transcripts in RAW264.7 murine macrophages.

[1293] A549 cells were transfected with purified circRNA containing an EMCV IRES and EGFP expression sequence. This failed to produce substantial induction of pro-inflammatory transcripts. These data demonstrate that non-circular components of the splicing reaction are responsible for the immunogenicity observed in previous studies and that circRNA is not a natural ligand for RIG-I.

Example 5

Circular RNA Avoids Detection by TLRs

[1294] TLR 3, 7, and 8 reporter cell lines were transfected with multiple linear or circular RNA constructs and secreted embryonic alkaline phosphatase (SEAP) was measured.

[1295] Linearized RNA was constructed by deleting the intron and homology arm sequences. The linear RNA constructs were then treated with phosphatase (in the case of capped RNAs, after capping) and purified by HPLC.

[1296] None of the attempted transfections produced a response in TLR7 reporter cells. TLR3 and TLR8 reporter cells were activated by capped linearized RNA, polyadenylated linearized RNA, the nicked circRNA HPLC fraction, and the early circRNA fraction. The late circRNA fraction and m1?-mRNA did not provoke TLR-mediated response in any cell line.

[1297] In a second experiment, circRNA was linearized using two methods: treatment of circRNA with heat in the presence of magnesium ions and DNA oligonucleotide-guided RNase H digestion. Both methods yielded a majority of full-length linear RNA with small amounts of intact circRNA. TLR3, 7, and 8 reporter cells were transfected with circular RNA, circular RNA degraded by heat, or circular RNA degraded by RNase H, and SEAP secretion was measured 36 hours after transfection. TLR8 reporter cells secreted SEAP in response to both forms of degraded circular RNA, but did not produce a greater response to circular RNA transfection than mock transfection. No activation was observed in TLR3 and TLR7 reporter cells for degraded or intact conditions, despite the activation of TLR3 by in vitro transcribed linearized RNA.

Example 6

Unmodified Circular RNA Produces Increased Sustained In Vivo Protein Expression than Linear RNA

[1298] Mice were injected and HEK293 cells were transfected with unmodified and m1?-modified human erythropoietin (hEpo) linear mRNAs and circRNAs. Equimolar transfection of m1?-mRNA and unmodified circRNA resulted in robust protein expression in HEK293 cells. hEpo linear mRNA and circRNA displayed similar relative protein expression patterns and cell viabilities in comparison to GLuc linear mRNA and circRNA upon equal weight transfection of HEK293 and A549 cells.

[1299] In mice, hEpo was detected in serum after the injection of hEpo circRNA or linear mRNA into visceral adipose. hEpo detected after the injection of unmodified circRNA decayed more slowly than that from unmodified or m1?-mRNA and was still present 42 hours post-injection. Serum hEpo rapidly declined upon the injection of unpurified circRNA splicing reactions or unmodified linear mRNA. Injection of unpurified splicing reactions produced a cytokine response detectable in serum that was not observed for the other RNAs, including purified circRNA.

Example 7

Circular RNA can be Effectively Delivered In Vivo or In Vitro Via Lipid Nanoparticles

[1300] Purified circular RNA was formulated into lipid nanoparticles (LNPs) with the ionizable lipidoid cKK-E12 (Dong et al., 2014; Kauffman et al., 2015). The particles formed uniform multilamellar structures with an average size, polydispersity index, and encapsulation efficiency similar to that of particles containing commercially available control linear mRNA modified with 5moU.

[1301] Purified hEpo circRNA displayed greater expression than 5moU-mRNA when encapsulated in LNPs and added to HEK293 cells. Expression stability from LNP-RNA in HEK293 cells was similar to that of RNA delivered by transfection reagent, with the exception of a slight delay in decay for both 5moU-mRNA and circRNA. Both unmodified circRNA and 5moU-mRNA failed to activate RIG-I/IFN-?1 in vitro.

[1302] In mice, LNP-RNA was delivered by local injection into visceral adipose tissue or intravenous delivery to the liver. Serum hEpo expression from circRNA was lower but comparable with that from 5moU-mRNA 6 hours after delivery in both cases. Serum hEpo detected after adipose injection of unmodified LNP-circRNA decayed more slowly than that from LNP-5moU-mRNA, with a delay in expression decay present in serum that was similar to that noted in vitro, but serum hEpo after intravenous injection of LNP-circRNA or LNP-5moU-mRNA decayed at approximately the same rate. There was no increase in serum cytokines or local RIG-I, TNF, or IL-6 transcript induction in any of these cases.

Example 8

Example 8A: Expression and Functional Stability by IRES in HEK293, HepG2, and 1C1C7 Cells

[1303] Constructs including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and varying IRES were circularized. 100 ng of each circularization reaction was separately transfected into 20,000 HEK293 cells, HepG2 cells, and 1C1C7 cells using Lipofectamine MessengerMax. Luminescence in each supernatant was assessed after 24 hours as a measure of protein expression. In HEK293 cells, constructs including Crohivirus B, Salivirus FHB, Aichi Virus, Salivirus HG-J1, and Enterovirus J IRES produced the most luminescence at 24 hours (FIG. 1A). In HepG2 cells, constructs including Aichi Virus, Salivirus FHB, EMCV-Cf, and CVA3 IRES produced high luminescence at 24 hours (FIG. 1B). In 1C1C7 cells, constructs including Salivirus FHB, Aichi Virus, Salivirus NG-J1, and Salivirus A SZ-1 IRES produced high luminescence at 24 hours (FIG. 1C).

[1304] A trend of larger IRES producing greater luminescence at 24 hours was observed. Shorter total sequence length tends to increase circularization efficiency, so selecting a high expression and relatively short IRES may result in an improved construct. In HEK293 cells, a construct using the Crohivirus B IRES produced the highest luminescence, especially in comparison to other IRES of similar length (FIG. 2A). Expression from IRES constructs in HepG2 and 1C1C7 cells plotted against IRES size are in FIGS. 2B and 2C.

[1305] Functional stability of select IRES constructs in HepG2 and 1C1C7 cells were measured over 3 days. Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after transfection of 20,000 cells with 100 ng of each circularization reaction, followed by complete media replacement. Salivirus A GUT and Salivirus FHB exhibited the highest functional stability in HepG2 cells, and Salivirus N-J1 and Salivirus FHB produced the most stable expression in 1C1C7 cells (FIGS. 3A and 3B).

Example 8B: Screening of Additional IRES

[1306] Functional stability of additional IRES constructs in HEK293 cells were measured. Briefily, 5 untranslated regions (UTRs) of interest were identified from GenBank. Selected UTRs UTRs were truncated to 675 nt from the 5 end and inserted into a circular RNA backbone construct encoding Gaussia Luciferase (Gluc) and in front of the Gluc coding region. The circular RNAs were transfected into HEK293 cells. After 24 hours, the supernatants were collected and the luminescence from secreted Gluc protein was measured using commercially available reagents. The results are depicted in FIGS. 1D and 1E and Table 30, suggesting that many natural IRES sequences enhance the protein expression in a circular RNA context.

TABLE-US-00034 TABLE 30 SEQ ID NO IRES Expression 413 RhPV 1.10E+05 414 Halastavi arva (1x mut) 9.46E+04 415 Oscivirus 4.55E+07 416 Cadicivirus B 2.10E+05 417 PSIV (2x mut for Xbal) 9.70E+04 418 PSIV IGR 1.01E+05 419 PV Mahoney 1.09E+05 420 REV A 9.44E+04 421 Tropivirus A 9.52E+04 422 Symapivirus A 1.27E+05 423 Sakobuvirus A FFUP1 (1x mut) 8.82E+06 424 Rosavirus C NFSM6F 6.84E+05 425 Rosavirus 2 GA7403 5.05E+06 426 Rhimavirus A 8.42E+05 427 Rafivirus LPXYC222841 2.22E+05 428 Rafivirus WHWGGF74766 4.53E+06 429 Poecivirus BCCH-449 3.43E+05 430 Megirivirus A LY 1.80E+06 431 Megirivirus E 1.10E+07 432 Megirivirus C 1.24E+05 433 Ludopivirus 1.05E+05 434 Livupivirus 2.10E+05 435 Aichivirus A FSS693 6.25E+07 436 Aichivirus KVGH 1.72E+07 437 Aichivirus DV 7.79E+07 438 Murine Kobuvirus 1 1.60E+07 439 Porcine Kobuvirus K-30 N/A 440 Porcine Kobuvirus XX 1.32E+07 441 Caprine Kobuvirus 12Q108 2.87E+08 442 Rabbit Kobuvirus 3.73E+07 443 Aalivirus 2.65E+05 444 Grusopivirus A 1.09E+05 445 Grusopivirus B 2.12E+05 446 Yancheng osbecks grenadier anchovy 1.57E+06 picornavirus 447 Turkey Gallivirus M176 4.37E+05 448 Falcovirus A1 1.48E+05 449 Tremovirus B 1.31E+05 450 Didelphis aurita HAV 1.38E+05 451 Hepatovirus G1 1.41E+05 452 Hepatovirus D 1.47E+06 453 Hepatovirus H2 1.08E+05 454 Hepatovirus I 8.79E+05 455 Hepatovirus C 5.08E+05 456 Fipivirus A 2.69E+05 457 Fipivirus C 1.09E+05 458 Fipivirus E 1.10E+05 459 Aquamavirus 4.51E+06 460 Avisivirus A 1.91E+05 461 Avisivirus B 8.68E+04 462 Crohivirus A 9.96E+04 463 Kunsagivirus B 8.01E+04 464 Limnipivirus A 8.30E+04 465 Limnipivirus C 1.35E+05 466 Orivirus 6.09E+05 467 HAV FH1 1.24E+05 468 HAV HM175 4.96E+05 469 Parechovirus F 6.56E+05 470 Parechovirus D 3.10E+05 471 Parechovirus C 1.24E+06 472 Ljungan Virus 87-012 2.00E+06 473 Parechovirus A2 1.80E+07 474 Parechovirus A3 3.58E+06 475 Parechovirus A8 1.61E+07 476 Parechovirus A17 1.20E+06 477 Potamipivirus A 8.43E+05 478 Potamipivirus B 7.20E+05 479 Beihai Conger Picornavirus 1.15E+06 480 Porcine Sapelovirus JD2011 N/A 481 Porcine Sapelovirus A2 4.34E+06 482 Simian Sapelovirus 1 6.55E+07 483 Simian Sapelovirus 2 4.24E+07 484 Rabovirus C 2.49E+06 485 Rabovirus A NYC-B10 1.24E+06 486 Parabovirus C 1.83E+07 487 Parabovirus B 7.85E+06 488 Parabovirus A3 2.44E+08 489 Felipivirus 127F 8.92E+06 490 Boosepivirus A 7.07E+07 491 Boosepivirus B 1.17E+08 492 Phacovirus Pf-CHK1 5.87E+06 493 HRVC3 QPM 1.64E+07 494 HRVB27 2.04E+08 495 HRVA73 1.08E+08 496 EV L 6.49E+07 497 EV K 7.52E+07 498 EV J 1631 9.88E+07 499 EV JN125 2.90E+07 500 EV I 1.31E+08 501 EV F1 BEV 261 1.12E+07 502 EV D94 9.25E+07 503 PV3 1.25E+08 504 EV C102 8.85E+07 505 EV 30 5.48E+06 506 SA5 1.61E+08 507 EV A114 1.50E+08 508 Mobovirus A 3.44E+06 509 Burpengary Virus 1.09E+07 510 Hunnivirus A1 1.61E+06 511 Hunnivirus A2 6.38E+06 512 Ia Io 1.35E+06 513 Taura Syndrome Virus 8.30E+05 514 ABPV 6.48E+05 515 BRAV-2 3.98E+06 516 BRBV-1 3.34E+06 517 ERAV-1 U188 N/A 518 GFTV 1.23E+06 519 SAFV V13C 9.32E+07 520 SAV P-113 4.37E+07 521 VHEV 1.74E+08 522 TRV NGS910 3.84E+07 523 EMCV2 RD1338 1.97E+06 524 EMCV1 JZ1203 N/A 525 EMCV1 AnrB-3741 2.55E+06 526 Cosavirus D1 2.11E+06 527 Cosavirus B1 1.91E+06 528 Cosavirus A SH1 2.16E+06 529 Malagasivirus B 5.05E+06 530 Mosavirus A2 SZAL6 8.27E+06 531 SVV 1.06E+06 532 PTV A 7.29E+05 533 PTV B 6.02E+06 534 Tottorivirus 2.76E+07 535 Posavirus 1 1.55E+06 536 A105-675 2.18E+07 537 A110-675 1.24E+08 538 18-675 6.04E+07 539 A115-675 5.93E+07 540 A73-675 1.30E+08 541 Kobuvirus 16317 2.03E+07 542 Aichivirus Chshc7 1.87E+07 543 Aichivirus Goiania 1.66E+07 544 Aichivirus ETHP4 1.78E+07 545 Aichivirus DVI2169 2.98E+06 546 Aichivirus DVI2321 6.63E+07 547 Aichivirus rat08 3.51E+07 548 Aichivirus Rt386 5.71E+07 549 Norway Rat Pestivirus N/A 550 Porcine Kobuvirus GS2 44200000 551 Kobuvirus SZAL6 98850000 552 Kobuvirus sheep TB3 N/A 553 Pronghorn antelope pestivirus 1.35E+06 554 Porcine pestivirus isolate Bungowannah 1.10E+07 555 Porcine pestivirus 1 9.46E+04 556 Pestivirus giraffe-1 4.72E+05 557 Classical swine fever virus 3.16E+05 558 Human pegivirus isolate JD2B1I 6.85E+05 559 Human pegivirus isolate GBV-C-ZJ N/A 560 Human pegivirus isolate JD2B8C 5.36E+05 561 Hepatitis GB virus A N/A 562 Simian pegivirus 8.56E+04 563 Pegivirus I 8.02E+04 564 Pegivirus K 8.07E+04 565 Theiler's disease-associated virus 7.84E+04 566 Rodent pegivirus 1.79E+05 567 Human pegivirus 2 3.14E+05 568 GB virus C/Hepatitis G virus 1.36E+05 569 Equine Pegivirus 1 8.80E+04 570 Culex theileri flavivirus 8.52E+04 571 Bussuquara virus 8.20E+04 572 Zika Virus 8.61E+04 573 Yokose virus 8.55E+04 574 Wesselsbron virus N/A 575 Equine hepacivirus 8.40E+04 576 Hepacivirus B 8.84E+04 577 Hepacivirus I 7.50E+04 578 Hepacivirus J 7.65E+04 579 Hepacivirus K 8.91E+04 580 Icavirus 4.41E+06 581 Antarctic penguin virus A 8.42E+04 582 Forest pouched giant rat arterivirus N/A 583 Avisivirus Pf-CHK1 1.19E+05 584 Avian paramyxovirus penguin 9.91E+04 585 Newcastle disease virus 8.86E+04 586 Bat Hp-betacoronavirus 8.47E+04 587 Basella alba endornavirus 7.65E+04 588 Ball python nidovirus 8.25E+04 589 Bat sapelovirus 8.05E+04 590 Bat Picornavirus 3 N/A 591 Bat Picornavirus 2 7.99E+07 592 Bat Picornavirus 1 1.85E+07 593 Bat Iflavirus 9.76E+04 594 Bat dicibavirus 7.43E+04 595 Betacoronavirus HKU24 8.96E+04 596 Betacoronavirus England 1 8.74E+04 597 Boone cardiovirus 1 2.62E+06 598 Breda virus 1.16E+05 599 Bovine viral diarrhea virus 3 2.70E+06 600 Bovine rhinitis A virus 3.62E+06 601 Bovine picornavirus isolate TCH6 1.21E+05 602 Bovine nidovirus TCH5 1.17E+05 603 Bovine hepacivirus 1.89E+05 604 Botrytis cinerea mitovirus 4 RdRp 9.68E+04 605 Botrytis cinerea mitovirus 2 RdRp 8.73E+04 606 Canine picodicistrovirus strain 209 2.79E+06 607 Canine distemper virus 3.02E+05 608 Canine kobuvirus 1.48E+08 609 Camel alphacoronavirus 2.48E+05 610 Cripavirus 1.95E+05 611 Human coxsackievirus A2 7.75E+07 612 Coronavirus AcCoV-JC34 1.82E+05 613 Chicken picornavirus 3 9.13E+04 614 Chicken picornavirus 1 1.21E+05 615 Chicken orivirus 1 3.16E+05 616 Chicken gallivirus 1 1.51E+07 617 Chicken calicivirus 1.28E+05 618 Carp picornavirus 1 1.13E+05 619 Falcon picornavirus 3.08E+06 620 Equine rhinitis B virus 1 1.01E+05 621 Equine rhinitis A virus 3.73E+05 622 Equine arteritis virus 1.89E+05 623 Enterovirus sp. isolate CPML 6.83E+07 624 Enterovirus AN12 3.87E+06 625 Dolphin morbillivirus 1.22E+05 626 Dianke virus 1.35E+05 627 Guereza hepacivirus 1.38E+05 628 Grapevine associated narnavirus-1 1.30E+05 629 Goat torovirus 1.19E+05 630 Foot-and-mouth disease virus O isolate 1.12E+05 631 Feline infectious peritonitis virus 1.35E+05 632 Farmington virus 1.22E+05 633 Avian infectious bronchitis virus 2.84E+05 634 Human rhinovirus 1 7.40E+07 635 EV22 1.95E+07 636 Human TMEV-like cardiovirus 4.48E+07 637 Human coronavirus 229E N/A 638 Hubei zhaovirus-like virus 1 1.03E+05 639 Hubei tombus-like virus 9 9.28E+04 640 Hubei tombus-like virus 32 9.23E+04 641 Hubei sobemo-like virus 3 1.17E+05 642 Hubei picorna-like virus 2 1.95E+05 643 Hepacivirus P 6.04E+05 644 Harrier picornavirus 1 1.47E+05 645 Kunsagivirus 1 4.15E+05 646 Kagoshima-2-24-KoV 9.30E+07 647 Kashmir bee virus 1.65E+05 648 Jingmen picorna-like virus 9.32E+04 649 Mumps virus 1.47E+05 650 Mouse Mosavirus 9.00E+04 651 Miniopterus schreibersii picornavirus 1 6.05E+06 652 Linda virus 7.37E+05 653 Lesavirus 2 3.67E+07 654 Lesavirus 1 6.37E+06 655 Phopivirus strain NewEngland 1.06E+05 656 Pestivirus strain Aydin 3.11E+06 657 Quail picornavirus QPV1 6.55E+07 658 Porcine sapelovirus 1 N/A 659 Porcine reproductive and respiratory syndrome 1.29E+05 virus 2 660 Porcine enterovirus 9 3.20E+07 661 Pigeon picornavirus B 1.24E+05 662 Picornavirus HK21 4.09E+05 663 Picornavirales Tottori-HG1 9.54E+04 664 Rodent hepatovirus 1.39E+05 665 Rinderpest virus 4.26E+05 666 Rabovirus A 2.88E+06 667 Shingleback nidovirus 1 2.62E+05 668 Seneca valley virus 1.46E+07 669 Sclerotinia sclerotiorum dsRNA mycovirus-L 1.69E+05 670 Yak enterovirus 6.19E+06 671 Wobbly possum disease virus 2.60E+05 672 Avian orthoreovirus segment S1 4.37E+05 673 Caprine Kobuvirus d10 2.20E+08 674 Caprine Kobuvirus d20 2.00E+08 675 Caprine Kobuvirus d30 1.87E+08 676 Caprine Kobuvirus d40 2.15E+08 677 Caprine Kobuvirus d50 9.65E+07 678 Picornavirales sp. isolate RtMruf-Pico V 2.26E+08 679 Apodemus agrarius picornavirus strain 1.90E+08 Longquan-Aa118 680 Niviventer confucianus picornavirus 6.10E+07 681 Bat picornavirus isolate BtRs-Pico V 1.13E+06 682 Rhinolophus picornavirus strain Guizhou-Rr100 N/A 683 Rhinolophus picornavirus strain Henan-Rf265 3.85E+05 684 Human enterovirus C105 5.49E+05 685 Human poliovirus 1 strain NIE1116623 3.94E+05 686 Human enterovirus 109 4.92E+05 687 Human poliovirus 2 strain NIE0811460 2.59E+07 688 Bovine picornavirus 3.82E+06 689 Human poliovirus 1 strain EQG1419328 2.44E+05 690 Human poliovirus 2 isolate IS_061 5.84E+06 691 Coxsackievirus B5 N/A 692 Coxsackievirus A10 N/A

Example 9

Expression and Functional Stability by IRES in Jurkat Cells

[1307] 2 sets of constructs including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized. 60,000 Jurkat cells were electroporated with 1 ?g of each circularization reaction. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation. A CVB3 IRES construct was included in both sets for comparison between sets and to previously defined IRES efficacy. CVB1 and Salivirus A SZ1 IRES constructs produced the most expression at 24 h. Data can be found in FIGS. 4A and 4B.

[1308] Functional stability of the IRES constructs in each round of electroporated Jurkat cells was measured over 3 days. Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 ?g of each circularization reaction, followed by complete media replacement (FIGS. 5A and 5B).

[1309] Salivirus A SZ1 and Salivirus A BN2 IRES constructs had high functional stability compared to other constructs.

Example 10

Expression, Functional Stability, and Cytokine Release of Circular and Linear RNA in Jurkat Cells

[1310] A construct including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and a Salivirus FHB IRES was circularized. mRNA including a Gaussia luciferase expression sequence and a ?150 nt polyA tail, and modified to replace 100% of uridine with 5-methoxy uridine (5moU) is commercially available and was purchased from Trilink. 5moU nucleotide modifications have been shown to improve mRNA stability and expression (Bioconjug Chem. 2016 Mar. 16; 27(3):849-53). Expression of modified mRNA, circularization reactions (unpure), and circRNA purified by size exclusion HPLC (pure) in Jurkat cells were measured and compared (FIG. 6A). Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 60,000 cells with 1 ?g of each RNA species.

[1311] Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 ug of each RNA species, followed by complete media replacement. A comparison of functional stability data of modified mRNA and circRNA in Jurkat cells over 3 days is in FIG. 6B.

[1312] IFN? (FIG. 7A), IL-6 (FIG. 7B), IL-2 (FIG. 7C), RIG-I (FIG. 7D), IFN-?1 (FIG. 7E), and TNF? (FIG. 7F) transcript induction was measured 18 hours after electroporation of 60,000 Jurkat cells with 1 ?g of each RNA species described above and 3p-hpRNA (5 triphosphate hairpin RNA, which is a known RIG-I agonist).

Example 11

Expression of Circular and Linear RNA in Monocytes and Macrophages

[1313] A construct including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and a Salivirus FHB IRES was circularized. mRNA including a Gaussia luciferase expression sequence and a ?150 nt polyA tail, and modified to replace 100% of uridine with 5-methoxy uridine (5moU) was purchased from Trilink. Expression of circular and modified mRNA was measured in human primary monocytes (FIG. 8A) and human primary macrophages (FIG. 8B). Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 60,000 cells with 1 ?g of each RNA species. Luminescence was also measured 4 days after electroporation of human primary macrophages with media changes every 24 hours (FIG. 8C). The results can be found in FIG. 8. The difference in luminescence was statistically significant in each case (p<0.05).

Example 12

Expression and Functional Stability by IRES in Primary T Cells

[1314] Constructs including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 primary human CD3+ T cells were electroporated with 1 ?g of each circRNA. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation (FIG. 9A). Aichi Virus and CVB3 IRES constructs had the most expression at 24 hours.

[1315] Luminescence was also measured every 24 hours after electroporation for 3 days in order to compare functional stability of each construct (FIG. 9B). The construct with a Salivirus A SZ1 IRES was the most stable.

Example 13

Expression and Functional Stability of Circular and Linear RNA in Primary T Cells and PBMCs

[1316] Constructs including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and a Salivirus A SZ1 IRES or Salivirus FHB IRES were circularized. mRNA including a Gaussia luciferase expression sequence and a ?150 nt polyA tail, and modified to replace 100% of uridine with 5-methoxy uridine (5moU) and was purchased from Trilink. Expression of Salivirus A SZ1 IRES HPLC purified circular and modified mRNA was measured in human primary CD3+ T cells. Expression of Salivirus FHB HPLC purified circular, unpurified circular and modified mRNA was measured in human PBMCs. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 150,000 cells with 1 ?g of each RNA species. Data for primary human T cells is shown in FIGS. 10A and 10B, and data for PBMCs is shown in FIG. 10C. The difference in expression between the purified circular RNA and unpurified circular RNA or linear RNA was significant in each case (p<0.05).

[1317] Luminescence from secreted Gaussia luciferase in primary T cell supernatant was measured every 24 hours after electroporation over 3 days in order to compare construct functional stability. Data is shown in FIG. 10B. The difference in relative luminescence from the day 1 measurement between purified circular RNA and linear RNA was significant at both day 2 and day 3 for primary T cells.

Example 14

Circularization Efficiency by Permutation Site in Anabaena Intron

[1318] RNA constructs including a CVB3 IRES, a Gaussia luciferase expression sequence, Anabaena intron/exon regions, spacers, internal homology regions, and homology arms were produced. Circularization efficiency of constructs using the traditional Anabaena intron permutation site and 5 consecutive permutations sites in P9 was measured by HPLC. HPLC chromatograms for the 5 consecutive permutation sites in P9 are shown in FIG. 11A.

[1319] Circularization efficiency was measured at a variety of permutation sites. Circularization efficiency is defined as the area under the HPLC chromatogram curve for each of: circRNA/(circRNA+precursor RNA). Ranked quantification of circularization efficiency at each permutation site is in FIG. 11B. 3 permutation sites (indicated in FIG. 11B) were selected for further investigation.

[1320] Circular RNA in this example was circularized by in vitro transcription (IVT) then purified via spin column. Circularization efficiency for all constructs would likely be higher if the additional step of incubation with Mg2+ and guanosine nucleotide were included; however, removing this step allowed for comparison between, and optimization of, circular RNA constructs. This level of optimization is especially useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors.

Example 15

Circularization Efficiency of Alternative Introns

[1321] Precursor RNA containing a permuted group 1 intron of variable species origin or permutation site and several constant elements including: a CVB3 IRES, a Gaussia luciferase expression sequence, spacers, internal homology regions, and homology arms were created. Circularization data can be found in FIG. 12. FIG. 12A shows chromatograms resolving precursor, CircRNA and introns. FIG. 12B provides ranked quantification of circularization efficiency, based on the chromatograms shown in FIG. 12A, as a function of intron construct.

[1322] Circular RNA in this example was circularized by in vitro transcription (IVT) then spin column purification. Circularization efficiency for all constructs would likely be higher if the additional step of incubation with Mg2+ and guanosine nucleotide were included; however, removing this step allows for comparison between, and optimization of, circular RNA constructs. This level of optimization is especially useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors.

Example 16

Circularization Efficiency by Homology Arm Presence or Length

[1323] RNA constructs including a CVB3 IRES, a Gaussia luciferase expression sequence, Anabaena intron/exon regions, spacers, and internal homology regions were produced. Constructs representing 3 Anabaena intron permutation sites were tested with 30 nt, 25% GC homology arms or without homology arms (NA). These constructs were allowed to circularize without an Mg.sup.2+ incubation step. Circularization efficiency was measured and compared. Data can be found in FIGS. 13A and 13B. Circularization efficiency was higher for each construct lacking homology arms. FIG. 13A provides ranked quantification of circularization efficiency; FIG. 13B provides chromatograms resolving precursor, circRNA and introns.

[1324] For each of the 3 permutation sites, constructs were created with 10 nt, 20 nt, and 30 nt arm lengths and 25%, 50%, and 75% GC content. Splicing efficiency of these constructs was measured and compared to constructs without homology arms (FIG. 14). Splicing efficiency is defined as the proportion of free introns relative to the total RNA in the splicing reaction.

[1325] FIG. 15 A (left) shows HPLC chromatograms indicating the contribution of strong homology arms to improved splicing efficiency. Top left: 75% GC content, 10 nt homology arms. Center left: 75% GC content, 20 nt homology arms. Bottom left: 75% GC content, 30 nt homology arms.

[1326] FIG. 15 A (right) shows HPLC chromatograms showing increased splicing efficiency paired with increased nicking, appearing as a shoulder on the circRNA peak. Top right: 75% GC content, 10 nt homology arms. Center right: 75% GC content, 20 nt homology arms. Bottom right: 75% GC content, 30 nt homology arms.

[1327] FIG. 15 B (left) shows select combinations of permutation sites and homology arms hypothesized to demonstrate improved circularization efficiency.

[1328] FIG. 15 B (right) shows select combinations of permutation sites and homology arms hypothesized to demonstrate improved circularization efficiency, treated with E. coli polyA polymerase.

[1329] Circular RNA in this example was circularized by in vitro transcription (IVT) then spin-column purified. Circularization efficiency for all constructs would likely be higher if an additional Mg2+ incubation step with guanosine nucleotide were included; however, removing this step allowed for comparison between, and optimization of, circular RNA constructs. This level of optimization is especially useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors.

Example 17

Circular RNA Encoding Chimeric Antigen Receptors

[1330] Constructs including Anabaena intron/exon regions, a Kymriah chimeric antigen receptors (CAR) expression sequence, and a CVB3 IRES were circularized. 100,000 human primary CD3+ T cells were electroporated with 500 ng of circRNA and co-cultured for 24 hours with Raji cells stably expressing GFP and firefly luciferase. Effector to target ratio (E:T ratio) 0.75:1. 100,000 human primary CD3+ T cells were mock electroporated and co-cultured as a control (FIG. 16).

[1331] Sets of 100,000 human primary CD3+ T cells were mock electroporated or electroporated with 1 ?g of circRNA then co-cultured for 48 hours with Raji cells stably expressing GFP and firefly luciferase E:T ratio 10:1 (FIG. 17).

[1332] Quantification of specific lysis of Raji target cells was determined by detection of firefly luminescence (FIG. 18). 100,000 human primary CD3+ T cells either mock electroporated or electroporated with circRNA encoding different CAR sequences were co-cultured for 48 hours with Raji cells stably expressing GFP and firefly luciferase. % Specific lysis defined as 1-[CAR condition luminescence]/[mock condition luminescence]. E:T ratio 10:1.

Example 18

Expression and Functional Stability of Circular and Linear RNA in Jurkat Cells and Resting Human T Cells

[1333] Constructs including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 Jurkat cells were electroporated with 1 ?g of circular RNA or 5moU-mRNA. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation (FIG. 19A left). 150,000 resting primary human CD3+ T cells (10 days post-stimulation) were electroporated with 1 ?g of circular RNA or 5moU-mRNA. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation (FIG. 19A right).

[1334] Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after electroporation, followed by complete media replacement. Functional stability data shown in FIG. 19B. Circular RNA had more functional stability than linear RNA in each case, with a more pronounced difference in Jurkat cells.

Example 19

IFN-?1, RIG-I, IL-2, IL-6, IFN?, and TNF? Transcript Induction of Cells Electroporated with Linear RNA or Varying Circular RNA Constructs

[1335] Constructs including Anabaena intron/exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 CD3+ human T cells were electroporated with 1 ?g of circular RNA, 5moU-mRNA, or immunostimulatory positive control poly inosine:cytosine. IFN-?1 (FIG. 20A), RIG-I (FIG. 20B), IL-2 (FIG. 20C), IL-6 (FIG. 20D), IFN? (FIG. 20E), and TNF? (FIG. 20F) transcript induction was measured 18 hours after electroporation.

Example 20

Specific Lysis of Target Cells and IFN? Transcript Induction by CAR Expressing Cells Electroporated with Different Amounts of Circular or Linear RNA; Specific Lysis of Target and Non-Target Cells by CAR Expressing Cells at Different E:T Ratios

[1336] Constructs including Anabaena intron/exon regions, an anti-CD19 CAR expression sequence, and a CVB3 IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 human primary CD3+ T cells either mock electroporated or electroporated with different quantities of circRNA encoding an anti-CD19 CAR sequence were co-cultured for 12 hours with Raji cells stably expressing GFP and firefly luciferase at an E:T ratio of 2:1. Specific lysis of Raji target cells was determined by detection of firefly luminescence (FIG. 21A). % Specific lysis was defined as 1-[CAR condition luminescence]/[mock condition luminescence]. IFN? transcript induction was measured 24 hours after electroporation (FIG. 21B).

[1337] 150,000 human primary CD3+ T cells were either mock electroporated or electroporated with 500 ng circRNA or m1?-mRNA encoding an anti-CD19 CAR sequence, then co-cultured for 24 hours with Raji cells stably expressing firefly luciferase at different E:T ratios. % Specific lysis of Raji target cells was determined by detection of firefly luminescence (FIG. 22A). % Specific lysis was defined as 1-[CAR condition luminescence]/[mock condition luminescence].

[1338] CAR expressing T cells were also co-cultured for 24 hours with Raji or K562 cells stably expressing firefly luciferase at different E:T ratios. Specific lysis of Raji target cells or K562 non-target cells was determined by detection of firefly luminescence (FIG. 22B). % Specific lysis is defined as 1-[CAR condition luminescence]/[mock condition luminescence].

Example 21

Specific Lysis of Target Cells by T Cells Electroporated with Circular RNA or Linear RNA Encoding a CAR

[1339] Constructs including Anabaena intron/exon regions, an anti-CD19 CAR expression sequence, and a CVB3 IRES were circularized and reaction products were purified by size exclusion HPLC. Human primary CD3+ T cells were electroporated with 500 ng of circular RNA or an equimolar quantity of m1?-mRNA, each encoding a CD19-targeted CAR. Raji cells were added to CAR-T cell cultures over 7 days at an E:T ratio of 10:1. % Specific lysis was measured for both constructs at 1, 3, 5, and 7 days (FIG. 23).

Example 22

Specific Lysis of Raji Cells by T Cells Expressing an Anti-CD19 CAR or an Anti-BCMA CAR

[1340] Constructs including Anabaena intron/exon regions, anti-CD19 or anti-BCMA CAR expression sequence, and a CVB3 IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 primary human CD3+ T cells were electroporated with 500 ng of circRNA, then were co-cultured with Raji cells at an E:T ratio of 2:1. % Specific lysis was measured 12 hours after electroporation (FIG. 24).

Example 23

Example 23A: Synthesis of Compounds

[1341] Synthesis of representative ionizable lipids of the invention are described in PCT applications PCT/US2016/052352, PCT/US2016/068300, PCT/US2010/061058, PCT/US2018/058555, PCT/US2018/053569, PCT/US2017/028981, PCT/US2019/025246, PCT/US2018/035419, PCT/US2019/015913, and US applications with publication numbers 20190314524, 20190321489, and 20190314284, the contents of each of which are incorporated herein by reference in their entireties.

Example 23B: Synthesis of Compounds

[1342] Synthesis of representative ionizable lipids of the invention are described in US patent publication number US20170210697A1, the contents of which is incorporated herein by reference in its entirety.

Example 24

Protein Expression by Organ

[1343] Circular or linear RNA encoding FLuc was generated and loaded into transfer vehicles with the following formulation: 50% ionizable lipid 15 in Table 10b, 10% DSPC, 1.5% PEG-DMG, 38.5% cholesterol. CD-1 mice were dosed at 0.2 mg/kg and luminescence was measured at 6 hours (live IVIS) and 24 hours (live IVIS and ex vivo IVIS). Total Flux (photons/second over a region of interest) of the liver, spleen, kidney, lung, and heart was measured (FIGS. 25 and 26).

Example 25

Distribution of Expression in the Spleen

[1344] Circular or linear RNA encoding GFP is generated and loaded into transfer vehicles with the following formulation: 50% ionizable lipid 15 in Table 10b, 10% DSPC, 1.5% PEG-DMG, 38.5% cholesterol. The formulation is administered to CD-1 mice. Flow cytometry is run on spleen cells to determine the distribution of expression across cell types.

Example 26

Production of Nanoparticle Compositions

[1345] In order to investigate safe and efficacious nanoparticle compositions for use in the delivery of circular RNA to cells, a range of formulations are prepared and tested. Specifically, the particular elements and ratios thereof in the lipid component of nanoparticle compositions are optimized.

[1346] Nanoparticles can be made in a 1 fluid stream or with mixing processes such as microfluidics and T-junction mixing of two fluid streams, one of which contains the circular RNA and the other has the lipid components.

[1347] Lipid compositions are prepared by combining an ionizable lipid, optionally a helper lipid (such as DOPE, DSPC, or oleic acid obtainable from Avanti Polar Lipids, Alabaster, AL), a PEG lipid (such as 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, AL), and a structural lipid such as cholesterol at concentrations of about, e.g., 40 or 50 mM in a solvent, e.g., ethanol. Solutions should be refrigerated for storage at, for example, ?20? C. Lipids are combined to yield desired molar ratios (see, for example, Tables 31a and 31b below) and diluted with water and ethanol to a final lipid concentration of e.g., between about 5.5 mM and about 25 mM.

TABLE-US-00035 TABLE 31a Formulation number Description 1 Aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE, Chol and DMG- PEG2K (40:30:25:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1? PBS (pH 7.4), concentrated and stored at 2-8? C. 2 Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE, cholesterol and DMG-PEG2K (18:56:20:6) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1? PBS (pH 7.4), concentrated and stored at 2-8? C. Final concentration = 1.35 mg/mL EPO circRNA (encapsulated). Zave = 75.9 nm (Dv(50) = 57.3 nm; Dv(90) = 92.1 nm). 3 Aliquots of 50 mg/mL ethanolic solutions of HGT4003, DOPE, cholesterol and DMG-PEG2K (50:25:20:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1? PBS (pH 7.4), concentrated and stored at 2-8? C. 4 Aliquots of 50 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K (70:25:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1? PBS (pH 7.4), concentrated and stored at 2-8? C. 5 Aliquots of 50 mg/mL ethanolic solutions of HGT5000, DOPE, cholesterol and DMG-PEG2K (40:20:35:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1? PBS (pH 7.4), concentrated and stored at 2-8? C. Final concentration = 1.82 mg/mL EPO mRNA (encapsulated). Zave = 105.6 nm (Dv(50) = 53.7 nm; Dv(90) = 157 nm). 6 Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE, cholesterol and DMG-PEG2K (40:20:35:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1? PBS (pH 7.4), concentrated and stored at 2-8? C.

[1348] In some embodiments, transfer vehicle has a formulation as described in Table 31a.

TABLE-US-00036 TABLE 31b Composition (mol %) Compone 40:20:38.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 45:15:38.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 50:10:38.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 55:5:38.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 60:5:33.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 45:20:33.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 50:20:28.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 55:20:23.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 60:20:18.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 40:15:43.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 50:15:33.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 55:15:28.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 60:15:23.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 40:10:48.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 45:10:43.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 55:10:33.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 60:10:28.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 40:5:53.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 45:5:48.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 50:5:43.5:1.5 Compound:Phospholipid:Phytosterol*:PEG-DMG 40:20:40:0 Compound:Phospholipid:Phytosterol*:PEG-DMG 45:20:35:0 Compound:Phospholipid:Phytosterol*:PEG-DMG 50:20:30:0 Compound:Phospholipid:Phytosterol*:PEG-DMG 55:20:25:0 Compound:Phospholipid:Phytosterol*:PEG-DMG 60:20:20:0 Compound:Phospholipid:Phytosterol*:PEG-DMG 40:15:45:0 Compound:Phospholipid:Phytosterol*:PEG-DMG

[1349] In some embodiments, transfer vehicle has a formulation as described in Table 31b.

[1350] For nanoparticle compositions including circRNA, solutions of the circRNA at concentrations of 0.1 mg/ml in deionized water are diluted in a buffer, e.g., 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution. Alternatively, solutions of the circRNA at concentrations of 0.15 mg/ml in deionized water are diluted in a buffer, e.g., 6.25 mM sodium acetate buffer at a pH between 3 and 4.5 to form a stock solution.

[1351] Nanoparticle compositions including a circular RNA and a lipid component are prepared by combining the lipid solution with a solution including the circular RNA at lipid component to circRNA wt:wt ratios between about 5:1 and about 50:1. The lipid solution is rapidly injected using, e.g., a NanoAssemblr microfluidic based system at flow rates between about 10 ml/min and about 18 ml/min or between about 5 ml/min and about 18 ml/min into the circRNA solution, to produce a suspension with a water to ethanol ratio between about 1:1 and about 4:1.

[1352] Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange. Formulations are dialyzed twice against phosphate buffered saline (PBS), pH 7.4, at volumes 200 times that of the primary product using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, IL) with a molecular weight cutoff of 10 kDa or 20 kDa. The formulations are then dialyzed overnight at 4? C. The resulting nanoparticle suspension is filtered through 0.2 ?m sterile filters (Sarstedt, N?mbrecht, Germany) into glass vials and sealed with crimp closures. Nanoparticle composition solutions of 0.01 mg/ml to 0.15 mg/ml are generally obtained.

[1353] The method described above induces nano-precipitation and particle formation.

[1354] Alternative processes including, but not limited to, T-junction and direct injection, may be used to achieve the same nano-precipitation. B. Characterization of nanoparticle compositions

[1355] A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the nanoparticle compositions in 1?PBS in determining particle size and 15 mM PBS in determining zeta potential.

[1356] Ultraviolet-visible spectroscopy can be used to determine the concentration of circRNA in nanoparticle compositions. 100 ?L of the diluted formulation in 1?PBS is added to 900 ?L of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of circRNA in the nanoparticle composition can be calculated based on the extinction coefficient of the circRNA used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.

[1357] A QUANT-IT? RIBOGREEN? RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluate the encapsulation of circRNA by the nanoparticle composition. The samples are diluted to a concentration of approximately 5 ?g/mL or 1 ?g/mL in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 ?L of the diluted samples are transferred to a polystyrene 96 well plate and either 50 ?L of TE buffer or 50 ?L of a 2-4% Triton X-100 solution is added to the wells. The plate is incubated at a temperature of 37? C. for 15 minutes. The RIBOGREEN? reagent is diluted 1:100 or 1:200 in TE buffer, and 100 ?L of this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilabel Counter; Perkin Elmer, Waltham, MA) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free circRNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100). C.

In Vivo Formulation Studies:

[1358] In order to monitor how effectively various nanoparticle compositions deliver circRNA to targeted cells, different nanoparticle compositions including circRNA are prepared and administered to rodent populations. Mice are intravenously, intramuscularly, intraarterially, or intratumorally administered a single dose including a nanoparticle composition with a lipid nanoparticle formulation. In some instances, mice may be made to inhale doses. Dose sizes may range from 0.001 mg/kg to 10 mg/kg, where 10 mg/kg describes a dose including 10 mg of a circRNA in a nanoparticle composition for each 1 kg of body mass of the mouse. A control composition including PBS may also be employed.

[1359] Upon administration of nanoparticle compositions to mice, dose delivery profiles, dose responses, and toxicity of particular formulations and doses thereof can be measured by enzyme-linked immunosorbent assays (ELISA), bioluminescent imaging, or other methods. Time courses of protein expression can also be evaluated. Samples collected from the rodents for evaluation may include blood and tissue (for example, muscle tissue from the site of an intramuscular injection and internal tissue); sample collection may involve sacrifice of the animals.

[1360] Higher levels of protein expression induced by administration of a composition including a circRNA will be indicative of higher circRNA translation and/or nanoparticle composition circRNA delivery efficiencies. As the non-RNA components are not thought to affect translational machineries themselves, a higher level of protein expression is likely indicative of a higher efficiency of delivery of the circRNA by a given nanoparticle composition relative to other nanoparticle compositions or the absence thereof.

Example 27

Characterization of Nanoparticle Compositions

[1361] A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the transfer vehicle compositions in 1?PBS in determining particle size and 15 mM PBS in determining zeta potential.

[1362] Ultraviolet-visible spectroscopy can be used to determine the concentration of a therapeutic and/or prophylactic (e.g., RNA) in transfer vehicle compositions. 100 ?L of the diluted formulation in 1?PBS is added to 900 ?L of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of therapeutic and/or prophylactic in the transfer vehicle composition can be calculated based on the extinction coefficient of the therapeutic and/or prophylactic used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.

[1363] For transfer vehicle compositions including RNA, a QUANT-IT? RIBOGREEN? RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluate the encapsulation of RNA by the transfer vehicle composition. The samples are diluted to a concentration of approximately 5 ?g/mL or 1 ?g/mL in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 ?L of the diluted samples are transferred to a polystyrene 96 well plate and either 50 ?L of TE buffer or 50 ?L of a 2-4% Triton X-100 solution is added to the wells. The plate is incubated at a temperature of 37? C. for 15 minutes. The RIBOGREEN? reagent is diluted 1:100 or 1:200 in TE buffer, and 100 ?L of this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, MA) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence values of the reagent blank are

[1364] subtracted from that of each of the samples and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

Example 28

T Cell Targeting

[1365] To target transfer vehicles to T-cells, T cell antigen binders, e.g., anti-CD8 antibodies, are coupled to the surface of the transfer vehicle. Anti-T cell antigen antibodies are mildly reduced with an excess of DTT in the presence of EDTA in PBS to expose free hinge region thiols. To remove DTT, antibodies are passed through a desalting column. The heterobifunctional cross-linker SM(PEG)24 is used to anchor antibodies to the surface of circRNA-loaded transfer vehicles (Amine groups are present in the head groups of PEG lipids, free thiol groups on antibodies were created by DTT, SM(PEG)24 cross-links between amines and thiol groups). Transfer vehicles are first incubated with an excess of SM(PEG)24 and centrifuged to remove unreacted cross-linker. Activated transfer vehicles are then incubated with an excess of reduced anti-T cell antigen antibody. Unbound antibody is removed using a centrifugal filtration device.

Example 29

RNA Containing Transfer Vehicle Using RV88

[1366] In this example RNA containing transfer vehicles are synthesized using the 2-D vortex microfluidic chip with the cationic lipid RV88 for delivery of circRNA.

##STR01513##

TABLE-US-00037 TABLE 32a Materials and Instrument Vendor Cat # 1M Tris-HCl, pH 8.0, Sterile Teknova T1080 5M Sodium Chloride solution Teknova S0250 QB Citrate buffer, pH 6.0 Teknova Q2446 (100 nM) Nuclease-free water Ambion AM9937 Triton X-100 Sigma-Aldrich T8787-100ML RV88 GVK bio DSPC Lipoid 556500 Cholesterol Sigma C3045-5G PEG2K Avanti Polar Lipids 880150 Ethanol Acros Organic 615090010 5 mL Borosilicate glass vials Thermo Scientific ST5-20 PD MiniTrap G-25 GE Healthcare VWR Cat. Desalting Columns #95055-984 Quant-IT RiboGreen Molecular R11490 RNA Assay kit Probes/Life Technologies Black 96-well microplates Greiner 655900

[1367] RV88, DSPC, and cholesterol all being prepared in ethanol at a concentration of 10 mg/ml in borosilica vials. The lipid 14.0-PEG2K PE is prepared at a concentration of 4 mg/ml also in a borosilica glass vial. Dissolution of lipids at stock concentrations is attained by sonication of the lipids in ethanol for 2 min. The solutions are then heated on an orbital tilting shaker set at 170 rpm at 37? C. for 10 min. Vials are then equilibrated at 26? C. for a minimum of 45 min. The lipids are then mixed by adding volumes of stock lipid as shown in Table 32b. The solution is then adjusted with ethanol such that the final lipid concentration was 7.92 mg/ml.

TABLE-US-00038 TABLE 32b Stock Ethanol Composition MW % nmoles mg (mg/ml) ul (ul) RV88 794.2 40% 7200 5.72 10 571.8 155.3 DSPC 790.15 10% 1800 1.42 10 142.2 Cholesterol 386.67 48% 8640 3.34 10 334.1 PEG2K 2693.3 2% 360 0.97 4 242.4

[1368] RNA is prepared as a stock solution with 75 mM Citrate buffer at pH 6.0 and a concentration of RNA at 1.250 mg/ml. The concentration of the RNA is then adjusted to 0.1037 mg/ml with 75 mM citrate buffer at pH 6.0, equilibrated to 26? C. The solution is then incubated at 26? C. for a minimum of 25 min.

[1369] The microfluidic chamber is cleaned with ethanol and neMYSIS syringe pumps are prepared by loading a syringe with the RNA solution and another syringe with the ethanolic lipid. Both syringes are loaded and under the control of neMESYS software. The solutions are then applied to the mixing chip at an aqueous to organic phase ratio of 2 and a total flow rate of 22 ml/min (14.67 ml/min for RNA and 7.33 ml/min for the lipid solution. Both pumps are started synchronously. The mixer solution that flowed from the microfluidic chip is collected in 4?1 ml fractions with the first fraction being discarded as waste. The remaining solution containing the RNA-liposomes is exchanged by using G-25 mini desalting columns to 10 mM Tris-HCl, 1 mM EDTA, at pH 7.5. Following buffer exchange, the materials are characterized for size, and RNA entrapment through DLS analysis and Ribogreen assays, respectively.

Example 30

RNA Containing Transfer Vehicle Using RV94

[1370] In this example, RNA containing liposome are synthesized using the 2-D vortex microfluidic chip with the cationic lipid RV94 for delivery of circRNA.

##STR01514##

TABLE-US-00039 TABLE 33 Materials and Instrument Vendor Cat # 1M Tris-HCl, pH 8.0, Sterile Teknova T1080 5M Sodium Chloride solution Teknova S0250 QB Citrate buffer, pH 6.0 Teknova Q2446 (100 mM) Nuclease-free water Ambion AM9937 Triton X-100 Sigma-Aldrich T8787-100ML RV94 GVKbio DSPC Lipoid 556500 Cholesterol Sigma C3045-5G PEG2K Avanti Polar Lipids 880150 Ethanol Acros Organic 615090010 5 mL Borosilicate vials Thermo Scientific ST5-20 PD MiniTrap G-25 GE Healthcare VWR Cat. Desalting Columns #95055-984 Quant-IT RiboGreen Molecular R11490 RNA Assay kit Probes/Life Technologies Black 96-well microplates Greiner 655900

[1371] The lipids were prepared as in Example 29 using the material amounts named in Table 34 to a final lipid concentration of 7.92 mg/ml.

TABLE-US-00040 TABLE 34 Stock Ethanol Composition MW % nmoles mg (mg/ml) ul (ul) RV94 806.22 40% 2880 2.33 10 232.6 155.3 DSPC 790.15 10% 720 0.57 10 56.9 Cholesterol 386.67 48% 3456 1.34 10 133.6 PEG2K 2693.3 2% 144 0.39 4 97.0

[1372] The aqueous solution of circRNA is prepared as a stock solution with 75 mM Citrate buffer at pH 6.0 the circRNA at 1.250 mg/ml. The concentration of the RNA is then adjusted to 0.1037 mg/ml with 75 mM citrate buffer at pH 6.0, equilibrated to 26? C. The solution is then incubated at 26? C. for a minimum of 25 min.

[1373] The microfluidic chamber is cleaned with ethanol and neMYSIS syringe pumps are prepared by loading a syringe with the RNA solution and another syringe with the ethanolic lipid. Both syringes are loaded and under the control of neMESYS software. The solutions are then applied to the mixing chip at an aqueous to organic phase ratio of 2 and a total flow rate of 22 ml/min (14.67 ml/min for RNA and 7.33 ml/min for the lipid solution. Both pumps are started synchronously. The mixer solution that flowed from the microfluidic chip is collected in 4?1 ml fractions with the first fraction being discarded as waste. The remaining solution containing the circRNA-transfer vehicles is exchanged by using G-25 mini desalting columns to 10 mM Tris-HCl, 1 mM EDTA, at pH 7.5, as described above. Following buffer exchange, the materials are characterized for size, and RNA entrapment through DLS analysis and Ribogreen assays, respectively. The biophysical analysis of the liposomes is shown in Table 35.

TABLE-US-00041 TABLE 35 RNA RNA Ratio encapsulation encapsulation Sample N:P TFR (aqueous/ amount yield size Name Ratio ml/min org phase) (?g/ml) % d .Math. nm PDI SAM- 8 22 2 31.46 86.9 113.1 0.12 RV94

Example 31

General Protocol for in Line Mixing

[1374] Individual and separate stock solutions are preparedone containing lipid and the other circRNA. Lipid stock containing a desired lipid or lipid mixture, DSPC, cholesterol and PEG lipid is prepared by solubilized in 90% ethanol. The remaining 10% is low pH citrate buffer. The concentration of the lipid stock is 4 mg/mL. The pH of this citrate buffer can range between pH 3 and pH 5, depending on the type of lipid employed. The circRNA is also solubilized in citrate buffer at a concentration of 4 mg/mL. 5 mL of each stock solution is prepared.

[1375] Stock solutions are completely clear and lipids are ensured to be completely solubilized before combining with circRNA. Stock solutions may be heated to completely solubilize the lipids. The circRNAs used in the process may be unmodified or modified oligonucleotides and may be conjugated with lipophilic moieties such as cholesterol.

[1376] The individual stocks are combined by pumping each solution to a T-junction. A dual-head Watson-Marlow pump was used to simultaneously control the start and stop of the two streams. A 1.6 mm polypropylene tubing is further downsized to 0.8 mm tubing in order to increase the linear flow rate. The polypropylene line (ID=0.8 mm) are attached to either side of a T-junction. The polypropylene T has a linear edge of 1.6 mm for a resultant volume of 4.1 mm.sup.3. Each of the large ends (1.6 mm) of polypropylene line is placed into test tubes containing either solubilized lipid stock or solubilized circRNA. After the T-junction, a single tubing is placed where the combined stream exited. The tubing is then extended into a container with 2? volume of PBS, which is rapidly stirred. The flow rate for the pump is at a setting of 300 rpm or 110 mL/min. Ethanol is removed and exchanged for PBS by dialysis. The lipid formulations are then concentrated using centrifugation or diafiltration to an appropriate working concentration.

[1377] C57BL/6 mice (Charles River Labs, MA) receive either saline or formulated circRNA via tail vein injection. At various time points after administration, serum samples are collected by retroorbital bleed. Serum levels of Factor VII protein are determined in samples using a chromogenic assay (Biophen FVTI, Aniara Corporation, OH). To determine liver RNA levels of Factor VII, animals are sacrificed and livers are harvested and snap frozen in liquid nitrogen. Tissue lysates are prepared from the frozen tissues and liver RNA levels of Factor VII are quantified using a branched DNA assay (QuantiGene Assay, Panomics, CA).

[1378] FVII activity is evaluated in FVTI siRNA-treated animals at 48 hours after intravenous (bolus) injection in C57BL/6 mice. FVII is measured using a commercially available kit for determining protein levels in serum or tissue, following the manufacturer's instructions at a microplate scale. FVII reduction is determined against untreated control mice, and the results are expressed as % Residual FVII. Two dose levels (0.05 and 0.005 mg/kg FVII siRNA) are used in the screen of each novel liposome composition.

Example 32

circRNA Formulation Using Preformed Vesicles

[1379] Cationic lipid containing transfer vehicles are made using the preformed vesicle method. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol at a molar ratio of 40/10/40/10, respectively. The lipid mixture is added to an aqueous buffer (50 mM citrate, pH 4) with mixing to a final ethanol and lipid concentration of 30% (vol/vol) and 6.1 mg/mL respectively and allowed to equilibrate at room temperature for 2 min before extrusion. The hydrated lipids are extruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22? C. using a Lipex Extruder (Northern Lipids, Vancouver, BC) until a vesicle diameter of 70-90 nm, as determined by Nicomp analysis, is obtained. For cationic lipid mixtures which do not form small vesicles, hydrating the lipid mixture with a lower pH buffer (50 mM citrate, pH 3) to protonate the phosphate group on the DSPC headgroup helps form stable 70-90 nm vesicles.

[1380] The FVII circRNA (solubilised in a 50 mM citrate, pH 4 aqueous solution containing 30% ethanol) is added to the vesicles, pre-equilibrated to 35? C., at a rate of ?5 mL/min with mixing. After a final target circRNA/lipid ratio of 0.06 (wt wt) is achieved, the mixture is incubated for a further 30 min at 35? C. to allow vesicle re-organization and encapsulation of the FVII RNA. The ethanol is then removed and the external buffer replaced with PBS (155 mM NaCl, 3 mM Na2HP04, 1 mM KH2P04, pH 7.5) by either dialysis or tangential flow diafiltration. The final encapsulated circRNA-to-lipid ratio is determined after removal of unencapsulated RNA using size-exclusion spin columns or ion exchange spin columns.

Example 33

Expression of Trispecific Antigen Binding Proteins from Engineered Circular RNA

[1381] Circular RNAs are designed to include: (1) a 3 post splicing group I intron fragment; (2) an Internal Ribosome Entry Site (IRES); (3) a trispecific antigen-binding protein coding region; and (4) a 3 homology region. The trispecific antigen-binding protein regions are constructed to produce an exemplary trispecific antigen-binding protein that will bind to a target antigen, e.g., GPC3.

Generation of a scFv CD3 Binding Domain

[1382] The human CD3epsilon chain canonical sequence is Uniprot Accession No. P07766. The human CD3gamma chain canonical sequence is Uniprot Accession No. P09693. The human CD3delta chain canonical sequence is Uniprot Accession No. P043234. Antibodies against CD3epsilon, CD3gamma or CD3delta are generated via known technologies such as affinity maturation. Where murine anti-CD3 antibodies are used as a starting material, humanization of murine anti-CD3 antibodies is desired for the clinical setting, where the mouse-specific residues may induce a human-anti-mouse antigen (HAMA) response in subjects who receive treatment of a trispecific antigen-binding protein described herein. Humanization is accomplished by grafting CDR regions from murine anti-CD3 antibody onto appropriate human germline acceptor frameworks, optionally including other modifications to CDR and/or framework regions.

[1383] Human or humanized anti-CD3 antibodies are therefore used to generate scFv sequences for CD3 binding domains of a trispecific antigen-binding protein. DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. The order in which the VL and VH domains appear in the scFv is varied (i.e. VL-VH, or VH-VL orientation), and three copies of the G4S or G.sub.4S subunit (G.sub.4S).sub.3 connect the variable domains to create the scFv domain. Anti-CD3 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD3-expressing cells.

Generation of a scFv Glypican-3 (GPC3) Binding Domain

[1384] Glypican-3 (GPC3) is one of the cell surface proteins present on Hepatocellular Carcinoma but not on healthy normal liver tissue. It is frequently observed to be elevated in hepatocellular carcinoma and is associated with poor prognosis for HCC patients. It is known to activate Wnt signalling. GPC3 antibodies have been generated including MDX-1414, HN3, GC33, and YP7.

[1385] A scFv binding to GPC-3 or another target antigen is generated similarly to the above method for generation of a scFv binding domain to CD3.

Expression of Trispecific Antigen-Binding Proteins In Vitro

[1386] A CHO cell expression system (Flp-In?, Life Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968; 60(4):1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies.

[1387] For adaption to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.

[1388] Recombinant CHO cell lines stably expressing secreted trispecific antigen-binding proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0.1?10.sup.6 viable cells/mL. Cell pools stably expressing trispecific antigen-binding proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.

[1389] Trispecific antigen-binding proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant. Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.

[1390] Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.

Purification of Trispecific Antigen-Binding Proteins

[1391] Trispecific antigen-binding proteins are purified from CHO cell culture supernatants in a two-step procedure. The constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step. Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL Purity and homogeneity (typically >90%) of final samples are assessed by SDS PAGE under reducing and non-reducing conditions, followed by immunoblotting using an anti-(half-life extension domain) or anti idiotype antibody as well as by analytical SEC, respectively. Purified proteins are stored at aliquots at ?80? C. until use.

Example 34

Expression of Engineered Circular RNA with a Half-Life Extension Domain has Improved Pharmacokinetic Parameters than without a Half-Life Extension Domain

[1392] The trispecific antigen-binding protein encoded on a circRNA molecule of example 23 is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection intramuscularly. Another cynomolgus monkey group receives a comparable protein encoded on a circRNA molecule in size with binding domains to CD3 and GPC-3, but lacking a half-life extension domain. A third and fourth group receive a protein encoded on a circRNA molecule with CD3 and half-life extension domain binding domains and a protein with GPC-3 and half-life extension domains, respectively. Both proteins encoded by circRNA are comparable in size to the trispecific antigen-binding protein. Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD3 and/or GPC-3.

[1393] Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae.sup.?at+Be.sup.?Pt, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and a and ? are the apparent first-order rate constants for the distribution and elimination phases, respectively. The a-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or ?-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(a?k21)/(a?p), B=D/V(p?k21)/(a?p), and a and ? (for ?>?) are roots of the quadratic equation: r.sup.2+(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose.

[1394] Data analysis: Graphs of concentration versus time profiles are made using KaleidaGraph (KaleidaGraph? V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) are not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin? Professional V. 3.1 WinNonlin? Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif). Pharmacokinetic parameters are computed as described in Ritschel W A and Kearns G L, 1999, EST: Handbook Of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D C.

[1395] It is expected that the trispecific antigen-binding protein encoded on a circRNA molecule of Example 23 has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a half-life extension domain.

Example 35

Cytotoxicity of the Trispecific Antigen-Binding Protein

[1396] The trispecific antigen-binding protein encoded on a circRNA molecule of Example 23 is evaluated in vitro on its mediation of T cell dependent cytotoxicity to GPC-3+ target cells.

[1397] Fluorescence labeled GPC3 target cells are incubated with isolated PBMC of random donors or T-cells as effector cells in the presence of the trispecific antigen-binding protein of Example 23. After incubation for 4 h at 37? C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorimeter. Target cells incubated without the trispecific antigen-binding protein of Example 23 and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.

[1398] Based on the measured remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1?(number of living targets(sample)/number of living targets(spontaneous))]?100%. Sigmoidal dose response curves and EC50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism software.

Example 36

Synthesis of Ionizable Lipids

38.1 Synthesis of ((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate)(Lipid 27, Table 10a) and ((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate))(Lipid 26, Table 10a)

[1399] In a 100 mL round bottom flask connected with condenser, 3-(1H-imidazol-1-yl)propan-1-amine (100 mg, 0.799 mmol) or 3-(2-methyl-1H-imidazol-1-yl)propan-1-amine (0.799 mmol), 6-bromohexyl 2-hexyldecanoate (737.2 mg, 1.757 mmol), potassium carbonate (485 mg, 3.515 mmol) and potassium iodide (13 mg, 0.08 mmol) were mixed in acetonitrile (30 mL), and the reaction mixture was heated to 80? C. for 48 h. The mixture was cooled to room temperature and was filtered through a pad of Celite. The filtrate was diluted with ethyl acetate. After washing with water, brine and dried over anhydrous sodium sulfate. The solvent was evaporated and the crude residue was purified by flash chromatography (SiO.sub.2:CH.sub.2Cl.sub.2=100% to 10% of methanol in CH.sub.2Cl.sub.2) and colorless oil product was obtained (92 mg, 15%). Molecular formula of ((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate)) is C.sub.50H.sub.95N.sub.3O.sub.4 and molecular weight (M.sub.w) is 801.7.

[1400] Reaction scheme for synthesis of ((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate)) (Lipid 26, Table 10a).

##STR01515##

[1401] Characterization of Lipid 26 was performed by LC-MS. FIG. 27A-C shows characterization of Lipid 26. FIG. 27A shows the proton NMR observed for Lipid 26. FIG. 27B is a representative LC/MS trace for Lipid 26 with total ion and UV chromatograms shown.

38.2 Synthesis of Lipid 22-S14

38.2.1 Synthesis of 2-(tetradecylthio)ethan-1-ol

[1402] To a mixture of 2-sulfanylethanol (5.40 g, 69.11 mmol, 4.82 mL, 0.871 eq) in acetonitrile (200 mL) was added 1-Bromotetradecane (22 g, 79.34 mmol, 23.66 mL, 1 eq) and potassium carbonate (17.55 g, 126.95 mmol, 1.6 eq) at 25? C. The reaction mixture was warmed to 40? C. and stirred for 12 hr. TLC (ethyl acetate/petroleum ether=25/1, R.sub.f=0.3, stained by I.sub.2) showed the starting material was consumed completely and a new main spot was generated. The reaction mixture was filtered and the filter cake was washed with acetonitrile (50 mL) and then the filtrate was concentrated under vacuum to get a residue which was purified by column on silica gel (ethyl acetate/petroleum ether=1/100 to 1/25) to afford 2-(tetradecylthio)ethan-1-ol (14 g, yield 64.28%) as a white solid.

[1403] .sup.1H NMR (ET36387-45-P1A, 400 MHz, CHLOROFORM-d) ? 0.87-0.91 (m, 3H) 1.27 (s, 20H) 1.35-1.43 (m, 2H) 1.53-1.64 (m, 2H) 2.16 (br s, 1H) 2.49-2.56 (m, 2H) 2.74 (t, J=5.93 Hz, 2H) 3.72 (br d, J=4.89 Hz, 2H). FIG. 28 shows corresponding Nuclear Magnetic Resonance (NMR) spectrum.

38.2.2 Synthesis of 2-(tetradecylthio)ethyl acrylate

[1404] To a solution of 2-(tetradecylthio)ethan-1-ol (14 g, 51.00 mmol, 1 eq) in dichloromethane (240 mL) was added triethylamine (7.74 g, 76.50 mmol, 10.65 mL, 1.5 eq) and prop-2-enoyl chloride (5.54 g, 61.20 mmol, 4.99 mL, 1.2 eq) dropwise at 0? C. under nitrogen. The reaction mixture was warmed to 25? C. and stirred for 12 hr. TLC (ethyl acetate/petroleum ether=25/1, Rf=0.5, stained by I.sub.2) showed the starting material was consumed completely and a new main spot was generated. The reaction solution was concentrated under vacuum to get crude which was purified by column on silica gel (ethyl acetate/petroleum ether=1/100 to 1/25) to afford 2-(tetradecylthio)ethyl acrylate (12 g, yield 71.61%) as a colorless oil.

[1405] .sup.1H NMR (ET36387-49-P1A, 400 MHz, CHLOROFORM-d) ? 0.85-0.93 (m, 3H) 1.26 (s, 19H) 1.35-1.43 (m, 2H) 1.53-1.65 (m, 2H) 2.53-2.62 (m, 2H) 2.79 (t, J=7.03 Hz, 2H) 4.32 (t, J=7.03 Hz, 2H) 5.86 (dd, J=10.39, 1.47 Hz, 1H) 6.09-6.19 (m, 1H) 6.43 (dd, J=17.30, 1.41 Hz, 1H). FIG. 29 shows corresponding Nuclear Magnetic Resonance (NMR) spectrum.

38.2.3 Synthesis of bis(2-(tetradecylthio)ethyl) 3,3-((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)dipropionate (Lipid 22-S14)

[1406] A flask was charged with 3-(2-methyl-1H-imidazol-1-yl)propan-1-amine (300 mg, 2.16 mmol) and 2-(tetradecylthio)ethyl acrylate (1.70 g, 5.17 mmol). The neat reaction mixture was heated to 80? C. and stirred for 48 hr. TLC (ethyl acetate, R.sub.f=0.3, stained by I.sub.2, one drop ammonium hydroxide added) showed the starting material was consumed completely and a new main spot was formed. The reaction mixture was diluted with dichloromethane (4 mL) and purified by column on silica gel (petroleum ether/ethyl acetate=3/1 to 0/1, 0.1% ammonium hydroxide added) to get bis(2-(tetradecylthio)ethyl) 3,3-((3-(2-methyl-TH-imidazol-1-yl)propyl)azanediyl)dipropionate (501 mg, yield 29.1%) as colorless oil.

[1407] .sup.1H NMR (ET36387-51-PTA, 400 MHz, CHLOROFORM-d) ? 0.87 (t, J=6.73 Hz, 6H) 1.25 (s, 40H) 1.33-1.40 (m, 4H) 1.52-1.61 (m, 4H) 1.81-1.90 (m, 2H) 2.36 (s, 3H) 2.39-2.46 (m, 6H) 2.53 (t, J=7.39 Hz, 4H) 2.70-2.78 (m, 8H) 3.84 (t, J=7.17 Hz, 2H) 4.21 (t, J=6.95 Hz, 4H) 6.85 (s, 1H) 6.89 (s, 1H). FIG. 30 shows corresponding Nuclear Magnetic Resonance (NMR) spectrum.

38.3 Synthesis of bis(2-(tetradecylthio)ethyl) 3,3-((3-(1H-imidazol-1-yl)propyl)azanediyl)dipropionate (Lipid 93-S14)

[1408] A flask was charged with 3-(1H-imidazol-1-yl)propan-1-amine (300 mg, 2.40 mmol, 1 eq) and 2-(tetradecylthio)ethyl acrylate (1.89 g, 5.75 mmol, 2.4 eq). The neat reaction mixture was heated to 80? C. and stirred for 48 hr. TLC (ethyl acetate, R.sub.f=0.3, stained by 12, one drop ammonium hydroxide added) showed the starting material was consumed completely and a new main spot was formed. The reaction mixture was diluted with dichloromethane (4 mL) and purified by column on silica gel (petroleum ether/ethyl acetate=1/20-0/100, 0.1% ammonium hydroxide added) to get bis(2-(tetradecylthio)ethyl) 3,3-((3-(1H-imidazol-1-yl)propyl)azanediyl)dipropionate (512 mg, yield 27.22%) as colorless oil.

[1409] .sup.1H NMR (ET36387-54-P1A, 400 MHz, CHLOROFORM-d) ? 0.89 (t, J=6.84 Hz, 6H) 1.26 (s, 40H) 1.34-1.41 (m, 4H) 1.58 (br t, J=7.50 Hz, 4H) 1.92 (t, J=6.62 Hz, 2H) 2.36-2.46 (m, 6H) 2.55 (t, J=7.50 Hz, 4H) 2.75 (q, J=6.84 Hz, 8H) 3.97 (t, J=6.95 Hz, 2H) 4.23 (t, J=6.95 Hz, 4H) 6.95 (s, 1H) 7.06 (s, 1H) 7.51 (s, 1H). FIG. 31 shows corresponding Nuclear Magnetic Resonance (NMR) spectrum.

38.4 Synthesis of heptadecan-9-yl 8-((3-(2-methyl-1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 54, Table 10a) 38.4.1 Synthesis of nonyl 8-bromooctanoate (3)

[1410] ##STR01516##

[1411] To a mixture of 8-bromooctanoic acid (2) (18.6 g, 83.18 mmol) and nonan-1-ol (1) (10 g, 69.32 mmol) in CH.sub.2Cl.sub.2 (500 mL) was added DMAP (1.7 g, 13.86 mmol), DIPEA (48 mL, 277.3 mmol) and EDC (16 g, 83.18 mmol). The reaction was stirred at room temperature overnight. After concentration of the reaction mixture, the crude residue was dissolved in ethyl acetate (500 mL), washed with 1N HCl, sat. NaHCO.sub.3, water and Brine. The organic layer was dried over anhydrous Na.sub.2SO.sub.4. The solvent was evaporated and the crude residue was purified by flash chromatography (SiO.sub.2:Hexane=100% to 30% of EtOAc in Hexane) and colorless oil product 3 was obtained (9 g, 37%).

38.4.2 Synthesis of heptadecan-9-yl 8-bromooctanoate (5)

[1412] ##STR01517##

[1413] To a mixture of 8-bromooctanoic acid (2) (10 g, 44.82 mmol) and heptadecan-9-ol (4) (9.6 g, 37.35 mmol) in CH.sub.2Cl.sub.2 (300 mL) was added DMAP (900 mg, 7.48 mmol), DIPEA (26 mL, 149.7 mmol) and EDC (10.7 g, 56.03 mmol). The reaction was stirred at room temperature overnight. After concentration of the reaction mixture, the crude residue was dissolved in ethyl acetate (300 mL), washed with 1N HCl, sat. NaHCO.sub.3, water and Brine. The organic layer was dried over anhydrous Na.sub.2SO.sub.4. The solvent was evaporated and the crude residue was purified by flash chromatography (SiO.sub.2:Hexane=100% to 30% of EtOAc in Hexane) and colorless oil product 5 was obtained (5 g, 29%).

38.4.3 Synthesis of heptadecan-9-yl 8-((3-(2-methyl-1H-imidazol-1-yl)propyl)amino)octanoate (7)

[1414] ##STR01518##

[1415] In a 100 mL round bottom flask connected with condenser, heptadecan-9-yl 8-bromooctanoate (5) (860 mg, 1.868 mmol) and 3-(2-methyl-1H-imidazol-1-yl)propan-1-amine (6) (1.3 g, 9.339 mmol) were mixed in ethanol (10 mL). The reaction mixture was heated to reflux overnight. MS (APCI) showed the expected product. The mixture was cooled to room temperature and concentrated. The crude residue was purified by flash chromatography (SiO.sub.2:CH.sub.2Cl.sub.2=100% to 10% of methanol+1% NH.sub.4OH in CH.sub.2Cl.sub.2) and colorless oil product 7 was obtained (665 mg, 69%).

38.4.4 Synthesis of heptadecan-9-yl 8-((3-(2-methyl-1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 54, Table 10a)

[1416] ##STR01519##

[1417] In a 100 mL round bottom flask connected with condenser, heptadecan-9-yl 8-((3-(2-methyl-1H-imidazol-1-yl)propyl)amino)octanoate (7) (665 mg, 1.279 mmol) and nonyl 8-bromooctanoate (3) (536 mg, 1.535 mmol) were mixed in ethanol (10 mL), then DIPEA (0.55 mL, 3.198 mmol) was added. The reaction mixture was heated to reflux overnight. Both MS (APCI) and TLC (10% MeOH+1% NH.sub.4OH in CH.sub.2Cl.sub.2) showed the product and some unreacted starting material. The mixture was cooled to room temperature and concentrated. The crude residue was purified by flash chromatography (SiO.sub.2:CH.sub.2Cl.sub.2=100% to 10% of methanol+1% NH.sub.4OH in CH.sub.2Cl.sub.2) and colorless oil was obtained (170 mg, 17%).

38.5 Synthesis of heptadecan-9-yl 8-((3-(1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 53, Table 10a)

[1418] ##STR01520##

[1419] Lipid 53 from Table 10a is synthesized according to the scheme above. Reaction conditions are identical to Lipid 54 with the exception of 3-(1H-imidazol-1-yl)propan-1-amine as the imidazole amine.

Example 37

Lipid Nanoparticle Formulation with Circular RNA

[1420] Lipid Nanoparticles (LNPs) were formed using a Precision Nanosystems Ignite instrument with a NextGen mixing chamber. Ethanol phase contained ionizable Lipid 26 from Table 10a, DSPC, Cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio was combined with an aqueous phase containing circular RNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous to ethanol mixing ratio was used. The formulated LNP then were dialyzed in 1 L of water and exchanged 2 times over 18 hours. Dialyzed LNPs were filtered using 0.2 ?m filter. Prior to in vivo dosing, LNPs were diluted in PBS. LNP sizes were determined by dynamic light scattering. A cuvette with 1 mL of 20 ?g/mL LNPs in PBS (pH 7.4) was measured for Z-average using the Malvern Panalytical Zetasizer Pro. The Z-average and polydispersity index were recorded.

39.1 Formulation of Lipids 26 and 27 from Table 10a

[1421] Lipid Nanoparticles (LNPs) were formed using a Precision Nanosystems Ignite instrument with a NextGen mixing chamber. Ethanol phase contained ionizable Lipid 26 or Lipid 27 from Table 10a, DOPE, Cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio was combined with an aqueous phase containing circular RNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous to ethanol mixing ratio was used. The formulated LNPs were then dialyzed in 1 L of water and exchanged 2 times over 18 hours. Dialyzed LNPs were filtered using 0.2 ?m filter. Prior to in vivo dosing, LNPs were diluted in PBS. LNP sizes were determined by dynamic light scattering. A cuvette with 1 mL of 20 ?g/mL LNPs in PBS (pH 7.4) was measured for Z-average using the Malvern Panalytical Zetasizer Pro. The Z-average and polydispersity index were recorded.

39.2 Formulation of Lipids 53 and 54 from Table 10a

[1422] Lipid Nanoparticles (LNPs) were formed using a Precision Nanosystems Ignite instrument with a NextGen mixing chamber. Ethanol phase contained ionizable Lipid 53 or 54 of Table 10a, DOPE, Cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a molar ratio of 50:10:38.5:1.5 was combined with an aqueous phase containing circular RNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous to ethanol mixing ratio was used. The formulated LNPs were then dialyzed in 1 L of 1?PBS and exchanged 2 times over 18 hours. Dialyzed LNPs were filtered using 0.2 ?m filter. Prior to in vivo dosing, LNPs were diluted in PBS. LNP sizes were determined by dynamic light scattering. A cuvette with 1 mL of 20 ?g/mL LNPs in PBS (pH 7.4) was measured for Z-average using the Malvern Panalytical Zetasizer Pro. The Z-average and polydispersity index were recorded.

[1423] LNP zeta potential was measured using the Malvern Panalytical Zetasizer Pro. A mixture containing 200 ?L of the particle solution in water and 800 ?L of distilled RNAse-free water with a final particle concentration of 400 ?g/mL was loaded into a zetasizer capillary cell for analysis.

[1424] RNA encapsulation was determined using a Ribogreen assay. Nanoparticle solutions were diluted in tris-ethylenediaminetetraacetic acid (TE) buffer at a theoretical oRNA concentration of 2 ?g/mL. Standard oRNA solutions diluted in TE buffer were made ranging from 2 ?g/mL to 0.125 ?g/mL. The particles and standards were added to all wells and a second incubation was performed (37? C. at 350 rpm for 3 minutes). Fluorescence was measured using a SPECTRAmax? GEMINI XS microplate spectrofluorometer. The concentration of circular RNA in each particle solution was calculated using the standard curve. The encapsulation efficiency was calculated from the ratio of oRNA detected between lysed and unlysed particles.

TABLE-US-00042 TABLE 36a Characterization of LNPs Encapsulation Zeta Data Ionizable Lipid Size (nm) PDI Efficiency (%) Potential (mV) 22-S14 88 0.09 96 3.968 93-S14 119 0.02 96 ?6.071 Lipid 26, Table 10a 86 0.08 92 ?15.24

TABLE-US-00043 TABLE 36b Characterization of LNPs RNA Ionizable Lipid Z-Average(nm) PDI Entrapment(%) 22-S14 64 0.05 97 93-S14 74 0.04 95 Lipid 26, Table 10a 84 0.04 96

Example 38

In Vivo Analysis

[1425] Female CD-1 or female c57BL/6J mice ranging from 22-25 g were dosed at 0.5 mg/kg RNA intravenously. Six hours after injection, mice were injected intraperitoneally with 200 ?L of D-luciferin at 15 mg/mL concentration. 5 minutes after injection, mice were anesthetized using isoflurane, and placed inside the IVIS Spectrum In Vivo Imaging System (Perkin Elmer) with dorsal side up. Whole body total IVIS flux of Lipids 22-514, 93-514, Lipid 26 (Table 10a) is presented in FIG. 32A. Post 10 minutes injection, mice were scanned for luminescence. Mice were euthanized and organs were extracted within 25 minutes of luciferin injection to scan for luminescence in liver, spleen, kidneys, lungs, and heart. Images (FIGS. 33A-B, 34A-B, 35A-B) were analyzed using Living Images (Perkin Elmer) software. Regions of interest were drawn to obtain flux and average radiance and analyzed for biodistribution of protein expression (FIG. 32A-B).

[1426] FIG. 32A illustrates the increased whole-body total flux observed from luciferase oRNA with Lipid 26 (Table 10a) LNPs compared to LNPs made with lipids 22-S14 and 93-S14. FIG. 32B shows the ex vivo IVIS analysis of tissues further highlighting the increased overall expression with Lipid 26 (Table 10a) while maintaining the desired spleen to liver ratios observed with lipids 22-S14 and 93-S14 despite the significant structural changes designed to improve expression. These data highlight the improvements afforded by Lipid 26 (Table 10a) compared to previously reported lipids.

[1427] Similar analysis as described above was also performed with oRNA encapsulated in LNPs formed with Lipid 15 from Table 10b or Lipid 53 or 54 from Table 10a. FIGS. 36A-C show the ex vivo IVIS analysis of tissues, respectively highlighting the overall expression with Lipid 15, 53, and 54 while maintaining the desired spleen to liver ratios despite the significant structural changes designed to improve expression. FIG. 36D shows the results for PBS control. These data demonstrates the improvements afforded by Lipids 15, 53, and 54 from Table 10a compared to previously reported lipids such as 93-S14 and 22-S14.

Example 39

Delivery of Luciferase

[1428] Human peripheral blood mononuclear cells (PBMCs) (Stemcell Technologies) were transfected with lipid nanoparticles (LNP) encapsulating firefly luciferase (fluc) circular RNA and examined for luciferase expression. PBMCs from two different donors were incubated in vitro with five different LNP compositions, containing circular RNA encoding for firefly luciferase (200 ng), at 37? C. in RPMI, 2% human serum, IL-2 (10 ng/mL), and 50 uM BME. PBMCs incubated without LNP were used as a negative control. After 24 hours, the cells were lysed and analyzed for firefly luciferase expression based on bioluminescence (Promega BrightGlo).

[1429] Representative data are presented in FIGS. 37A and 37B, showing that that the tested LNPs are capable of delivering circular RNA into primary human immune cells resulting in protein expression.

Example 40

In Vitro Delivery of Green Fluorescent Protein (GFP) or Chimeric Antigen Receptor (CAR)

[1430] Human PBMCs (Stemcell Technologies) were transfected with LNP encapsulating GFP and examined by flow cytometry. PBMCs from five different donors (PBMC A-E) were incubated in vitro with one LNP composition, containing circular RNA encoding either GFP or CD19-CAR (200 ng), at 37? C. in RPMI, 2% human serum, IL-2 (10 ng/mL), and 50 uM BME. PBMCs incubated without LNP were used as a negative control. After 24, 48, or 72 hours post-LNP incubation, cells were analyzed for CD3, CD19, CD56, CD14, CD1 Ib, CD45, fixable live dead, and payload (GFP or CD19-CAR).

[1431] Representative data are presented in FIGS. 38A and 38B, showing that the tested LNP is capable of delivering circular RNA into primary human immune cells resulting in protein expression.

Example 41

Multiple IRES Variants can Mediate Expression of Murine CD19 CAR In Vitro

[1432] Multiple circular RNA constructs, encoding anti-murine CD19 CAR, contains unique IRES sequences and were lipotransfected into 1C1C7 cell lines. Prior to lipotransfection, 1C1C7 cells are expanded for several days in complete RPMI Once the cells expanded to appropriate numbers, 1C1C7 cells were lipotransfected (Invitrogen RNAiMAX) with four different circular RNA constructs. After 24 hours, 1C1C7 cells were incubated with His-tagged recombinant murine CD19 (Sino Biological) protein, then stained with a secondary anti-His antibody. Afterwards, the cells were analyzed via flow cytometry.

[1433] Representative data are presented in FIGS. 39, showing that IRES sourced from the indicated virus (apodemus agrarius picornavirus, caprine kobuvirus, parabovirus, and salivirus) are capable of driving expression of an anti-mouse CD19 CAR in murine T cells.

Example 42

Murine CD19 CAR Mediates Tumor Cell Killing In Vitro

[1434] Circular RNA encoding anti-mouse CD19 CAR were electroporated into murine T cells to evaluate CAR-mediated cytotoxicity. For electroporation, T cells were electroporated with circular RNA encoding anti-mouse CD19 CAR using ThermoFisher's Neon Transfection System then rested overnight. For the cytotoxicity assay, electroporated T cells were co-cultured with Fluc+ target and non-target cells at 1:1 ratio in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME and incubated overnight at 37? C. Cytotoxicity was measured using a luciferase assay system 24 hours post-co-culture (Promega Brightglo Luciferase System) to detect lysis of Fluc+ target and non-target cells. Values shown are calculated relative to the untransfected mock signal.

[1435] Representative data are presented in FIG. 40, showing that an anti-mouse CD19 CAR expressed from circular RNA is functional in murine T cells in vitro.

Example 43

Functional Depletion of B Cells with a Lipid Encapsulated Circular RNA Encoding Murine CD19 CAR

[1436] C57BL/6J mice were injected with LNP formed with Lipid 15 in Table 10b, encapsulating circular RNA encoding anti-murine CD19 CAR. As a control, Lipid 15 in Table 10b encapsulating circular RNA encoding firefly luciferase (fLuc) were injected in different group of mice. Female C57BL.6J, ranging from 20-25 g, were injected intravenously with 5 doses of 0.5 mg/kg of LNP, every other day. Between injections, blood draws were analyzed via flow cytometry for fixable live/dead, CD45, TCRvb, B220, CD11b, and anti-murine CAR. Two days after the last injection, spleens were harvested and processed for flow cytometry analysis. Splenocytes were stained with fixable live/dead, CD45, TCRvb, B220, CD11b, NK1.1, F4/80, CD11c, and anti-murine CAR. Data from mice injected with anti-murine CD19 CAR LNP were normalized to mice that received fLuc LNP.

[1437] Representative data are presented in FIGS. 41A, 41B, and 41C, showing that an anti-mouse CD19 CAR expressed from circular oRNA delivered in vivo with LNPs is functional in murine T cells in vivo.

Example 44

CD19 CAR Expressed from Circular RNA has Higher Yield and Greate Cytotoxic Effect Compared to that Expressed from mRNA

[1438] Circular RNA encoding encoding anti-CD19 chimeric antigen antigen receptor, which includes, from N-terminus to C-terminus, a FMC63-derived scFv, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 intracellular domain, were electroporated into human peripheral T cells to evaluate surface expression and CAR-mediated cytotoxicity. For comparison, circular RNA-electroporated T cells were compared to mRNA-electroporated T cells in this experiment. For electroporation, CD3+ T cells were isolated from human PBMCs using commercially available T cell isolation kits (Miltenyi Biotec) from donor human PBMCs. After isolation, T cells were stimulated with anti-CD3/anti-CD28 (Stemcell Technologies) and expanded over 5 days at 37? C. in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME. Five days post stimulation, T cells were electroporated with circular RNA encoding anti-human CD19 CAR using ThermoFisher's Neon Transfection System and then rested overnight. For the cytotoxicity assay, electroporated T cells were co-cultured with Fluc+ target and non-target cells at 1:1 ratio in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME and incubated overnight at 37? C. Cytotoxicity was measured using a luciferase assay system 24 hours post-co-culture (Promega Brightglo Luciferase System) to detect lysis of Fluc+ target and non-target cells. Furthermore, an aliquot of electroporated T cells were taken and stained for live dead fixable staining, CD3, CD45, and chimeric antigen receptors (FMC63) at the day of analysis.

[1439] Representative data are presented in FIGS. 42 and 43. FIGS. 42A and 42B show that an anti-human CD19 CAR expressed from circular RNA is expressed at higher levels and longer than an anti-human CD19 CAR expressed from linear mRNA. FIGS. 43A and 43B show that an anti-human CD19 CAR expressed from circular RNA is exerts a greater cytotoxic effect relativea to anti-human CD19 CAR expressed from linear mRNA.

Example 45

Functional Expression of Two CARs from a Single Circular RNA

[1440] Circular RNA encoding chimeric antigen receptors were electroporated into human peripheral T cells to evaluate surface expression and CAR-mediated cytotoxicity. The purpose of this study is to evaluate if circular RNA encoding for two CARs can be stochastically expressed with a 2A (P2A) or an IRES sequence. For electroporation, CD3+ T cells were commercially purchased (Cellero) and stimulated with anti-CD3/anti-CD28 (Stemcell Technologies) and expanded over 5 days at 37? C. in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME. Four days post stimulation, T cells were electroporated with circular RNA encoding anti-human CD19 CAR, anti-human CD19 CAR-2A-anti-human BCMA CAR, and anti-human CD19 CAR-IRES-anti-human BCMA CAR using ThermoFisher's Neon Transfection System then rested overnight. For the cytotoxicity assay, electroporated T cells were co-cultured with Fluc+K562 cells expressing human CD19 or BCMA antigens at 1:1 ratio in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME and incubated overnight at 37? C. Cytotoxicity was measured using a luciferase assay system 24 hours post-co-culture (Promega BrightGlo Luciferase System) to detect lysis of Fluc+ target cells.

[1441] Representative data are presented in FIG. 44, showing that two CARs can be functionally expressed from the same circular RNA construct and exert cytotoxic effector function.

Example 46

In Vivo Circular RNA Transfection Using Cre Reporter Mice

[1442] Circular RNAs encoding Cre recombinase (Cre) are encapsulated into lipid nanoparticles as previously described. Female, 6-8 week old B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9) mice were dosed with lipid nanoparticles at 0.5 mg/kg RNA intravenously. Fluorescent tdTomato protein was transcribed and translated in Ai9 mice upon Cre recombination, meaning circular RNAs have been delivered to and translated in tdTomato+ cells. After 48 hr, mice were euthanized and the spleens were harvested, processed into a single cell suspension, and stained with various fluorophore-conjugated antibodies for immunophenotyping via flow cytometry.

[1443] FIG. 45A shows representative FACS plots with frequencies of tdTomato expression in various spleen immune cell (CD45+, live) subsets, including total myeloid (CDT1b+), B cells (CD19+), and T cells (TCR-B+) following treatment with LNPs formed with Lipid 27 or 26 from Table 10a or Lipid 15 from Table 10b. Ai9 mice injected with PBS represented background tdTomato fluorescence. FIG. 45B quantifies the proportion of myeloid cells, B cells, and T cells expressing tdTomato (mean+std. dev., n=3), which is equivalent to the proportion of each cell population which has been successfully transfected with Cre circular RNA. LNPs made with Lipids 27 and 26 from Table 10a exhibit significantly higher myeloid and T cell transfection compared with Lipid 93-S14, highlighting the improvements conferred by lipid structural modifications.

[1444] FIG. 45C illustrates the proportion of additional splenic immune cell populations expressing tdTomato with Lipids 27 and 26 from Table 10a (mean+std. dev., n=3), which also include NK cells (NKp46+, TCR-B-), classical monocytes (CD11b+, Ly-6G-, Ly-6C_hi), nonclassical monocytes (CD11b+, Ly-6G-, Ly-6C_lo), neutrophils (CD11b+, Ly-6G+), and dendritic cells (CD 11c+, MHC-II+). These experiments demonstrate that LNPs made with Lipids 27 and 26 from Table 10a and Lipid 15 from Table 10b are effective at delivering circular RNAs to many splenic immune cell subsets in mice and lead to successful protein expression from the circular RNA in those cells.

Example 47

Example 47A: Built-In polyA Sequences and Affinity-Purification to Produce Immue-Silent Circular RNA

[1445] PolyA sequences (20-30 nt) were inserted into the 5 and 3 ends of the RNA construct (precursor RNA with built-in polyA sequences in the introns). Precursor RNA and introns can alternatively be polyadenylated post-transcriptionally using, e.g., E. coli. polyA polymerase or yeast polyA polymerase, which requires the use of an additional enzyme.

[1446] Circular RNA in this example was circularized by in vitro transcription (IVT) and affinity-purified by washing over a commercially available oligo-dT resin to selectively remove polyA-tagged sequences (including free introns and precursor RNA) from the splicing reaction. The IVT was performed with a commercial IVT kit (New England Biolabs) or a customerized IVT mix (Orna Therapeutics), containing guanosine monophosphate (GMP) and guanosine triphosphate (GTP) at different ratios (GMP:GTP=8, 12.5, or 13.75). In some embodiments, GMP at a high GMP:GTP ratio may be preferentially included as the first nucleotide, yielding a majority of monophosphate-capped precursor RNAs. As a comparison, the circular RNA product was alternatively purified by the treatment with Xrn1, Rnase R, and Dnase I (enzyme purification).

[1447] Immunogenicity of the circular RNAs prepared using the affinity purification or enzyme purification process were then assessed. Briefly, the prepared circular RNAs were transfected into A549 cells. After 24 hours, the cells were lysed and interferon beta-1 induction relative to mock samples was measured by qPCR. 3p-hpRNA, a triphosphorylated RNA, was used as a positive control.

[1448] FIGS. 46B and 46C show that the negative selection affinity purification removes non-circular products from splicing reactions when polyA sequences are included on elements that are removed during splicing and present in unspliced precursor molecules. FIG. 46D shows circular RNAs prepared with tested IVT conditions and purification methods are all immunoquiescent. These results suggest the negative selection affinity purification is equivalent or superior to enzyme purification for circular RNA purification and that customized circular RNA synthesis conditions (IVT conditions) may reduce the reliance on GMP excess to achieve maximal immunoquiescence.

Example 47B: Dedicated Binding Site and Affinity-Purification for Circular RNA Production

[1449] Instead of polyA tags, one can include specifically design sequences (DBS, dedicated binding site).

[1450] Instead of a polyA tag, a dedicated binding site (DBS), such as a specifically designed complementary oligonucleotide that can bind to a resin, may be used to selectively deplete precursor RNA and free introns. In this example, DBS sequences (30 nt) were inserted into the 5 and 3 ends of the precursor RNA. RNA was transcribed and the transcribed product was washed over a custom complementary oligonucleotide linked to a resin.

[1451] FIGS. 47B and 47C demonstrates that including the designed DBS sequence in elements that are removed during splicing enables the removal of unspliced precursor RNA and free intron components in a splicing reaction, via negative affinity purification.

Example 47C: Production of a Circular RNA Encoding Dystrophin

[1452] A 12 kb12,000 nt circular RNA encoding dystrophin was produced by in vitro transcription of RNA precursors followed by enzyme purification using a mixture of Xrn1, DNase 1, and RNase R to degrade remaining linear components. FIG. 48 shows that the circular RNA encoding dystrophin was successfully produced.

Example 48

5 Spacer Between 3 Intron Fragment and the IRES Improves Circular RNA Expression

[1453] Expression level of purified circRNAs with different 5 spacers between the 3 intron fragment and the IRES in Jurkat cells were compared. Briefly, luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 60,000 cells with 250 ng of each RNA.

[1454] Additionally, stability of purified circRNAs with different 5 spacers between the 3 intron fragment and the IRES in Jurkat cells were compared. Briefly, luminescence from secreted Gaussia luciferase in supernatant was measured over 2 days after electroporation of 60,000 cells with 250 ng of each RNA and normalized to day 1 expression.

[1455] The results are shown in FIGS. 49A and 49B, indicating that adding a spacer can enhance IRES function and the importance of sequence identity and length of the added spacer. A potential explanation is that the spacer is added right before the IRES and likely functions by allowing the IRES to fold in isolation from other structured elements such as the intron fragments.

Example 49

[1456] This example describes deletion scanning from 5 or 3 end of the caprine kobuvirus IRES. IRES borders are generally poorly characterized and require empirical analysis, and this example can be used for locating the core functional sequences required for driving translation. Briefly, circular RNA constructs were generated with truncated IRES elements operably linked to a Gaussia luciferase coding sequence. The truncated IRES elements had nucleotide sequences of the indicated lengths removed from the 5 or 3 end. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 and 48 hours after electroporation of primary human T cells with RNA. Stability of expression was calculated as the ratio of the expression level at the 48-hour time point relative to that at the 24-hour time point.

[1457] As shown in FIG. 50, deletion of more than 40 nucleotides from the 5 end of the IRES reduced expression and disrupted IRES function. Stability of expression was relatively unaffected by the truncation of the IRES element but expression level was substantially reduced by deletion of 141 nucleotides from the 3 end of the IRES, whereas deletion of 57 or 122 nucleotides from the 3 end had a positive impact on the expression level.

[1458] It was also observed that deletion of the 6-nucleotide pre-start sequence reduced the expression level of the luciferase reporter. Replacement of the 6-nucleotide sequence with a classical kozak sequence (GCCACC) did not have a significant impact but at least maintained expression.

Example 50

[1459] This example describes modifications (e.g., truncations) of selected IRES sequences, including Caprine Kobuvirus (CKV) IRES, Parabovirus IRES, Apodemus Picornavirus (AP) IRES, Kobuvirus SZAL6 IRES, Crohivirus B (CrVB) IRES, CVB3 IRES, and SAFV IRES. The sequences of the IRES elements are provided in SEQ ID NOs: 348-389. Briefly, circular RNA constructs were generated with truncated IRES elements operably linked to a Gaussia luciferase coding sequence. HepG2 cells were transfected with the circular RNAs. Luminescence in the supernatant was assessed 24 and 48 hours after transfection. Stability of expression was calculated as the ratio of the expression level at the 48-hour time point relative to that at the 24-hour time point.

[1460] As shown in FIG. 51, truncations had variable effects depending on the identity of the IRES, which may depend on the initiation mechanism and protein factors used for translation, which often differs between IRESs. 5 and 3 deletions can be effectively combined, for example, in the context of CKV IRES. Addition of a canonical Kozak sequence in some cases significantly improved expression (as in SAFV, Full vs Full+K) or diminished expression (as in CKV, 5d40/3d122 vs 5d40/3d122+K).

Example 51

[1461] This example describes modifications of CK-739, AP-748, and PV-743 IRES sequences, including mutations altative translation initiation sites. Briefly, circular RNA constructs were generated with modified IRES elements operably linked to a Gaussia luciferase coding sequence. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 and 48 hours after transfection of 1C1C7 cells with RNA.

[1462] CUG was the most commonly found alternative start site but many others were also characterized. These triplets can be present in the IRES scanning tract prior to the start codon and can affect translation of correct polypeptides. Four alternative start site mutations were created, with the IRES sequnces provided in SEQ ID NOs: 378-380. As shown in FIG. 52, mutations of alternative translation initiation sites in the CK-739 IRES affected translation of correct polypeptides, positively in some instances and negatively in other instances. Mutation of all the alternative translation initiation sites reduced the level of translation.

[1463] Alternative Kozak sequences, 6 nucleotides before start codon, can also affect expression levels. The 6-nucleotide sequence upstream of the start codon were gTcacG, aaagtc, gTcacG, gtcatg, gcaaac, and acaacc, respectively, in CK-739 IRES and Sample Nos. 1-5 in the 6 nt Pre-Start group. As shown in FIG. 52, substitution of certain 6-nucleotide sequences prior to the start codon affected translation.

[1464] It was also observed that 5 and 3 terminal deletions in AP-748 and PV-743 IRES sequences reduced expression. However, in the CK-739 IRES, which had a long scanning tract, translation was relatively unaffected by deletions in the scanning tract.

Example 52

[1465] This example describes modifications of selected IRES sequences by inserting 5 and/or 3 untranslated regions (UTRs) and creating IRES hybrids. Briefly, circular RNA constructs were generated with modified IRES elements operably linked to a Gaussia luciferase coding sequence. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 and 48 hours after transfection of HepG2 cells with RNA.

[1466] IRES sequences with UTRs inserted are provided in SEQ ID NOs: 390-401. As shown in FIG. 53, insertion of 5 UTR right after the 3 end of the IRES and before the start codon slightly increased the translation from Caprine Kobuvirus (CK) IRES but in some instances abrogated translation from Salivirus SZ1 IRES. Insertion of 3 UTR right after the stop cassette had no impact on both IRES sequences.

[1467] Hybrid CK IRES sequences are provided in SEQ ID NOs: 390-401. CK IRES was used as a base, and specific regions of the CK IRES were replaced with similar-looking structures from other IRES sequences, for example, SZ1 and AV (Aichivirus). As shown in FIG. 53, certain hybrid synthetic IRES sequences were functional, indicating that hybrid IRES can be constructed using parts from distinct IRES sequences that show similar predicted structures while deleting these structures completely abrogates IRES function.

Example 53

[1468] This example describes modifications of circular RNAs by introducing stop codon or cassette variants. Briefly, circular RNA constructs were generated with IRES elements operably linked to a Gaussia luciferase coding sequence followed by variable stop codon cassettes, which included a stop codon in each frame and two stop codons in the reading frame of the Gaussia luciferase coding sequence. 1C1C7 cells were transfected with the circular RNAs. Luminescence in supernatant was assessed 24 and 48 hours after transfection.

[1469] The sequences of the stop codon cassettes are set forth in SEQ ID NOs: 406-412. As shown in FIG. 54, certain stop codon cassettes improved expression levels, although they had little impact on expression stability. In particular, a stop cassette with two frame 1 (the reading frame of the Gaussia luciferase coding sequence) stop codons, the first being TAA, followed by a frame 2 stop codon and a frame 3 stop codon, is effective for promoting functional translation.

Example 54

[1470] This example describes modifications of circular RNAs by inserting 5 UTR variants. Briefly, circular RNA constructs were generated with IRES elements with 5 UTR variants inserted between the 3 end of the IRES and the start codon, the IRES being operably linked to a Gaussia luciferase coding sequence. 1C1C7 cells were transfected with the circular RNAs. Luminescence in supernatant was assessed 24 and 48 hours after transfection.

[1471] The sequences of the 5 UTR variants are set forth in SEQ ID NOs: 402-405. As shown in FIG. 55, a CK IRES with a canonical Kozak sequence (UTR4) was more effective when a 36-nucleotide unstructured/low GC spacer sequence was added (UTR2), suggesting that the GC-rich Kozak sequences may interfere with core IRES folding. Using a higher-GC/structured spacer with a kozak sequence did not show the same benefit (UTR3), possibly due to interference with IRES folding by the spacer itself. Mutating the kozak sequence to gTcacG (UTR1) enhanced translation to the same level as the Kozak+spacer alternative without the need for a spacer.

Example 55

[1472] This example describes the impact of miRNA target sites in circular RNAs on expression levels. Briefly, circular RNA constructs were generated with IRES elements operably linked to a human erythropoietin (hEPO) coding sequence, where 2 tandem miR-122 target sites were inserted into the construct. miR-122-expressing Huh7 cells were transfected with the circular RNAs. hEPO expression in supernatant was assessed 24 and 48 hours after transfection by sandwich ELISA.

[1473] As shown in FIG. 56, the hEPO expression level was obrogated where the miR-122 target sites were inserted into the circular RNA. This result demonstrates that expression from circular RNA can be regulated by miRNA. As such, cell type- or tissue-specific expression can be achieved by incorporating target sites of the miRNAs expressed in the cell types in which expression of the recombinant protein is undesirable.

Example 56

Example 56A: Expression of Two Proteins from One circRNA Containing a 2A Site and Functional RNA Stability

[1474] Constructs including Anabaena intron/exon regions, varying IRES, a first expression sequence encoding Gaussia luciferase, a 2A self-cleaving peptide, and a second expression sequence encoding GFP or EGFP are circularized. 20,000-40,000 293 cells are transfected with 40 ng of circRNA. Luminescence from secreted Gaussia luciferase in supernatant is measured every 24 hours after transfection with complete media replacements at each time point. Fluorescence from expressed GFP or EGFP is measured 24 hours after transfection.

Example 56B: Expression of Two Proteins from One circRNA Containing 2 IRES Sequences and Functional RNA Stability

[1475] Constructs including Anabaena intron/exon regions, a first IRES, a first expression sequence encoding Gaussia luciferase, optionally a spacer, a second IRES, and a second expression sequence encoding GFP or EGFP, with the first and/or second IRES varying by construct are circularized. 293 cells are electroporated with 1 ?g of each circularization reaction. Luminescence/fluorescence from secreted Gaussia luciferase and GFP or EGFP in supernatant is measured 24 hours after electroporation. Functional stability of the IRES constructs in each round of electroporated 293 cells is measured over 3 days. Luminescence/fluorescence from secreted Gaussia luciferase and GFP or EGFP in supernatant is measured every 24 hours after electroporation of cells with 1 ?g of each circularization reaction, followed by complete media replacement.

Example 57

Expression and Functional Stability of Circular and Linear RNA Containing 2 Expression Sequences Separated by a Cleavage Site

[1476] Constructs including Anabaena intron/exon regions, a first expression sequence encoding Gaussia luciferase, a 2A self-cleaving peptide, an expression sequence encoding GFP or EGFP is circularized. mRNA encoding a first expression sequence encoding Gaussia luciferase, a 2A self-cleaving peptide, a second expression sequence encoding EGFP, a ?150 nt polyA tail, and modified to replace 100% of uridine with 1-methyl pseudouridine is produced. Expression of circular and modified mRNA is measured in 293 cells. Luminescence/fluorescence from secreted Gaussia luciferase and GFP or EGFP in supernatant is measured 24 hours after electroporation of cells with 1 ?g of each RNA species. Luminescence/fluorescence from secreted Gaussia luciferase and GFP or EGFP in supernatant is measured every 24 hours after electroporation over 3 days in order to compare construct functional stability.

Example 58

Example 58A: Comparison of Expression and Functional Stability of Dual Expression with Single Expression Constructs

[1477] A construct including an Anabaena intron/exon region, an IRES, a Gaussia luciferase expression sequence, a 2A self-cleaving peptide, and an GFP or EGFP expression sequence is circularized. Constructs containing a single expression sequence (encoding Gaussia luciferase or GFP or EGFP) are also circularized. Linear constructs containing a single expression sequence (encoding Gaussia luciferase or GFP or EGFP) are also generated. 293 cells are electroporated with 1 ?g of each circularization reaction or an equivalent amount of linear RNA. Fluorescence from secreted Gaussia luciferase and GFP or EGFP positivity in supernatant is measured 24 hours after electroporation.

[1478] Functional stability of the constructs in each round of electroporated 293 cells is measured over 3 days. Luminescence from secreted Gaussia luciferase in supernatant is measured every 24 hours after electroporation of cells with 1 ?g of each circularization reaction or equivalent mRNA, followed by complete media replacement.

Example 58B: Comparison of Expression and Functional Stability of Dual Expression with Single Expression Constructs

[1479] A construct including an Anabaena intron/exon region, a first IRES, a Gaussia luciferase expression sequence, a second IRES, and an GFP or EGFP expression sequence is circularized. Constructs containing a single expression sequence (encoding Gaussia luciferase or GFP or EGFP) are also circularized. Linear constructs containing a single expression sequence (encoding Gaussia luciferase or GFP or EGFP) are also generated. 293 cells are electroporated with 1 ?g of each circularization reaction or an equivalent amount of linear RNA. Fluorescence from secreted Gaussia luciferase and GFP or EGFP positivity in supernatant is measured 24 hours after electroporation.

[1480] Functional stability of the constructs in each round of electroporated 293 cells is measured over 3 days. Luminescence from secreted Gaussia luciferase in supernatant is measured every 24 hours after electroporation of cells with 1 ?g of each circularization reaction or equivalent mRNA, followed by complete media replacement.

Example 59

Example 59A: Functional Interaction Between TCR Payloads Expressed Using an IRES and a Cleavage Site

[1481] Constructs including Anabaena intron/exon regions, an IRES, a TCR alpha chain expression sequence, a 2A self-cleaving peptide, and a TCR beta chain expression sequence are circularized.

[1482] Human primary CD3+ T cells are electroporated with the circRNA and co-cultured for 24 hours with cells stably expressing the TCR target, GFP and firefly luciferase. Human primary CD3+ T cells are mock electroporated and co-cultured as a control. Quantification of specific lysis of target cells is determined by detection of firefly luminescence. % Specific lysis defined as 1?[TCR condition luminescence]/[mock condition luminescence].

Example 59B: Functional Interaction Between TCR Payloads Expressed Using 2 IRES

[1483] Constructs including Anabaena intron/exon regions, a first IRES, a TCR alpha chain expression sequence, optionally a spacer, a second IRES, and a TCR beta chain expression sequence are circularized.

[1484] Human primary CD3+ T cells are electroporated with the circRNA and co-cultured for 24 hours with cells stably expressing the TCR target, GFP and firefly luciferase. Human primary CD3+ T cells are mock electroporated and co-cultured as a control. Quantification of specific lysis of target cells is determined by detection of firefly luminescence. % Specific lysis defined as 1?[TCR condition luminescence]/[mock condition luminescence].

Example 60

Example 60A: CAR Payloads Expressed Using an IRES and a Cleavage Site

[1485] Constructs including Anabaena intron/exon regions, an IRES, an anti-CD19 CAR expression sequence, a 2A self-cleaving peptide, and an anti-C20 CAR expression sequence are circularized.

[1486] Human primary CD3+ T cells are electroporated with the circRNA and co-cultured for 24 hours with cells stably expressing the both CAR targets, GFP and firefly luciferase. Human primary CD3+ T cells are mock electroporated and co-cultured as a control. Quantification of specific lysis of target cells is determined by detection of firefly luminescence. % Specific lysis defined as 1?[TCR condition luminescence]/[mock condition luminescence].

Example 60B: TCR Payloads Expressed Using 2 IRES

[1487] Constructs including Anabaena intron/exon regions, a first IRES, an anti-CD19 CAR expression sequence, optionally a spacer, a second IRES, and an anti-CD20 CAR expression sequence are circularized.

[1488] Human primary CD3+ T cells are electroporated with the circRNA and co-cultured for 24 hours with cells stably expressing both CAR targets, GFP and firefly luciferase. Human primary CD3+ T cells are mock electroporated and co-cultured as a control. Quantification of specific lysis of target cells is determined by detection of firefly luminescence. % Specific lysis defined as 1?[TCR condition luminescence]/[mock condition luminescence].

Example 61

LNP and Circular RNA Construct Containing Anti-CD19 CAR Reduces B Cells in the Blood and Spleen In Vivo

[1489] Circular RNA constructs encoding an anti-CD19 CAR expression were encapsulated within lipid nanoparticles as described above. For comparison, circular RNAs encoding luciferase expression were encapsulated within separate lipid nanoparticle.

[1490] C57BL/6 mice at 6 to 8 weeks old were injected with either LNP solution every other day for a total of 4 LNP injections within each mouse. 24 hours after the last LNP injection, the mice's spleen and blood were harvested, stained, and analyzed via flow cytometry. As shown in FIG. 57A and FIG. 57B, mice containing LNP-circular RNA constructs encoding an anti-CD19 CAR led to a statistically significant reduction in CD19+ B cells in the peripheral blood and spleen compared to mice treated with LNP-circular RNA encoding a luciferase.

Example 62

IRES Sequences Contained within Circular RNA Encoding CARs Improves CAR Expressions and Cytotoxicity of T-Cells

[1491] Activated murine T-cells were electroporated with 200 ng of circular RNA constructs containing a unique IRES and a murine anti-CD19 1D3? CAR expression sequence. The IRES contained in these constructs were derived either in whole or in part from a Caprine Kobuvirus, Apodemus Picornavirus, Parabovirus, or Salivirus. A Caprine Kobuvirus derived IRES was additionally codon optimized. As a control, a circular RNA containing a wild-type zeta mouse CAR with no IRES was used for comparison. The T-cells were stained for the CD-19 CAR 24 hours post electroporation to evaluate for surface expression and then co-cultured with A20 Fluc target cells. The assay was then evaluated for cytotoxic killing of the Fluc+A20 cells 24 hours after co-culture of the T-cells with the target cells.

[1492] As seen in FIGS. 58A, 58B, 58C, and 59, the unique IRES were able to increase the frequency that the T-cells expressed the CAR protein and level of CAR expression on the surface of the cells. The increase frequency of expression of the CAR protein and level of CAR expression on the surface of cells lead to an improved anti-tumor response.

Example 63

Cytosolic and Surface Proteins Expressed from Circular RNA Construct in Primary Human T-Cells

[1493] Circular RNA construct contained either a sequence encoding for a fluorescent cytosolic reporter or a surface antigen reporter. Fluorescent reporters included green fluorescent protein, mCitrine, mWasabi, Tsapphire. Surface reporters included CD52 and Thy1.1.sup.bio Primary human T-cells were activated with an anti-CD3/anti-CD28 antibody and electroporated 6 days post activation of the circular RNA containing a reporter sequence. T-cells were harvested and analyzed via flow cytometry 24 hours post electroporation. Surface antigens were stained with commercially available antibodies (e.g., Biolegend, Miltenyi, and BD).

[1494] As seen in FIG. 60A and FIG. 60B, cytosolic and surface proteins can be expressed from circular RNA encoding the proteins in primary human T-cells.

Example 64

Circular RNAs Containing Unique IRES Sequences have Improved Translation Expression Over Linear mRNA

[1495] Circular RNA constructs contained a unique IRES along with an expression sequence for Firefly luciferase (FLuc).

[1496] Human T-cells from 2 donors were enriched and stimulated with anti-CD3/anti-CD28 antibodies. After several days of proliferation, activated T cells were harvested and electroporated with equal molar of either mRNA or circular RNA expressing FLuc payloads. Various IRES sequences, including those derived from Caprine Kobuvirus, Apodemus Picornavirus, and Parabovirus, were studied to evaluate expression level and durability of the payload expression across 7 days. Across the 7 days, the T-cells were lysed with Promega Brightglo to evaluate for bioluminsences.

[1497] As shown in FIGS. 61C, 61D, 61E, 61F, and 61G, the presence of an IRES within a circular RNA can increase translation and expression of a cytosolic protein by orders of magnitude and can improve expression compared to linear mRNA. This was found consistent across multiple human T-cell donors.

Example 65

Example 65A: LNP-Circular RNA Encoding Anti-CD19 Mediates Human T-Cell Killing of K562 Cells

[1498] Circular RNA constructs contained a sequence encoding for anti-CD19 antibodies. Circular RNA constructs were then encapsulated within a lipid nanoparticle (LNP).

[1499] Human T-cells were stimulated with anti-CD3/anti-CD28 and left to proliferate up to 6 says. At day 6, LNP-circular RNA and ApoE3 (1 ?g/mL) were co-cultured with the T-cells to mediate transfection. 24 hours later, Fluc+ K562 cells were electroporated with 200 ng of circular RNA encoding anti-CD19 antibodies and were later co-cultured at day 7. 48 hours post co-culture, the assay was assessed for CAR expression and cytotoxic killing of K562 cells through Fluc expression.

[1500] As shown in FIG. 62A and FIG. 62B, there is T-cell expression of anti-CD19 CAR from the LNP-mediated delivery of a CAR in vitro to T-cells and its capability to lyse tumor cells in a specific, antigen dependent manner in engineered K562 cells.

Example 65B: LNP-Circular RNA Encoding Anti-BCMA Antibody Mediates Human T-Cell Killing of K562 Cells

[1501] Circular RNA constructs contained a sequence encoding for anti-BCMA antibodies. Circular RNA constructs were then encapsulated within a lipid nanoparticle (LNP).

[1502] Human T-cells were stimulated with anti-CD3/anti-CD28 and left to proliferate up to 6 says. At day 6, LNP-circular RNA and ApoE3 (1 ?g/mL) were co-cultured with the T-cells to mediate transfection. 24 hours later, Fluc+ K562 cells were electroporated with 200 ng of circular RNA encoding anti-BCMA antibodies and were later co-cultured at day 7. 48 hours post co-culture, the assay was assessed for CAR expression and cytotoxic killing of K562 cells through Fluc expression.

[1503] As shown in FIG. 62B, there is T-cell expression of BCMA CAR from the LNP-mediated delivery of a CAR in vitro to T-cells and its capability to lyse tumor cells in a specific, antigen dependent manner in engineered K562 cells.

Example 66

Anti-CD19 CAR T-Cells Exhibit Anti-Tumor Activity In Vitro

[1504] Human T-cells were activated with anti-CD3/anti-CD28 and electroporated once with 200 ng of anti-CD19 CAR-expressing circular RNA. Electroporated T-cells were co-cultured with FLuc+ Nalm6 target cells and non-target Fluc+K562 cells to evaluate CAR-mediated killing. After 24 hours post co-culture, the T-cells were lysed and examined for remanent FLuc expression by target and non-target cells to evaluate expression and stability of expression across 8 days total.

[1505] As shown in FIGS. 63A and 63B, T-cells express circular RNA CAR constructs in specific, antigen-dependent manner. Results also shows improved cytotoxicity of circular RNAs encoding CARs compared to linear mRNA encoding CARs and delivery of a functional surface receptor.

Example 67

Effective LNP Transfection of Circular RNA Mediated with ApoE3

[1506] Human T-cells were stimulated with anti-CD3/anti-CD28 and left to proliferate up to 6 days. At day 6, lipid nanoparticle (LNP) was and circular RNA expressing green fluorescence protein solution with or without ApoE3 (1 ?g/mL) were co-cultured with the T-cells. 24 hours later, the T-cells were stained for live/dead T-cells and the live T-cells were analyzed for GFP expression on a flow cytometer.

[1507] As shown by FIGS. 64A, 64B, 61C, 61D, and 64E, efficient LNP transfection can be mediated by ApoE3 on activated T-cells, followed by significant payload expression. These results were exhibited across multiple donors.

INCORPORATION BY REFERENCE

[1508] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated by reference herein.