MULTIMERIC POLYNUCLEOTIDES AND USES THEREOF

Abstract

Aspects of the disclosure relate to multimeric molecules and methods of producing the same. In some embodiments, the multimeric molecules comprise at least two nucleic acid molecules (e.g., mRNA molecules) joined by covalent bonds between non-coding regions.

Claims

1. A composition comprising polynucleotides encoding one or more polypeptides of interest, the composition comprising a compound of formula I:
[(A).sub.m-L.sup.1-B].sub.n-L.sup.2 Formula I wherein n is 1, 2, or 3; each m is, independently, 1 or 2; each A and B is, independently, a polynucleotide comprising: (i) at least one 5-cap structure; (ii) a 5-untranslated region (5-UTR); (iii) an open reading frame encoding one of the polypeptides of interest; and (iv) a 3-untranslated region (3-UTR); L.sup.1 is a branched or unbranched linker; and L.sup.2 is absent or a branched linker, wherein each A is attached at the 3-terminus to an L.sup.1, each B is attached at the 5- or 3-terminus to an L.sup.1, and, when L.sup.2 is present, each B is attached at the 3-terminus to L.sup.2, and wherein if n is 1 and m is 1, then at least one of A and B comprises an open reading frame encoding one of the polypeptides of interest consisting of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine.

2. A composition comprising a plurality of lipid nanoparticles wherein the plurality of lipid nanoparticles has a mean particle size of between 70 nm and 100 nm and a mean PDI of between 0.1 and 0.25; and wherein at least 90% of the lipid nanoparticles comprise a compound of Formula I:
[(A).sub.m-L.sup.1-B].sub.n-L.sup.2 Formula I wherein n is 1, 2, or 3; each m is, independently, 1 or 2; each A and B is, independently, a polynucleotide comprising: (i) at least one 5-cap structure; (ii) a 5-UTR; (iii) an open reading frame encoding one of the polypeptides of interest; and (iv) a 3-UTR; L.sup.1 is a branched or unbranched linker; and L.sup.2 is absent or a branched linker, wherein each A is attached at the 3-terminus to an L.sup.1, each B is attached at the 5- or 3-terminus to an L.sup.1, and, when L.sup.2 is present, each B is attached at the 3-terminus to L.sup.2.

3. The composition of claim 1 or 2, wherein each B is attached at the 3-terminus to an L.sup.1.

4. The composition of claim 1 or 2, wherein each B is attached at the 5-terminus to an L.sup.1.

5. The composition of any one of claims 1 to 4, wherein the coding region of each A and each B encode the same polypeptide of interest.

6. The composition of any one of claims 1 to 4, wherein the coding region of each A and each B encode different polypeptides of interest.

7. The composition of any one of claims 1 to 6, wherein any A and/or any B further comprise a poly-A region.

8. The composition of claim 7, wherein each A and each B comprise a poly-A region.

9. The composition of any one of claims 1 to 8, wherein any A and/or any B comprise at least one modified nucleotide.

10. The polynucleotide or composition of claim 9, wherein each A and each B comprise at least one modified nucleotide.

11. The composition of any one of claims 1 to 10, wherein, upon administration to a mammalian cell, the compound of Formula I has an increased half-life compared to the half-life of any A and/or any B.

12. The composition of any one of claims 1 to 11, wherein, upon administration to a mammalian cell, the compound of Formula I has increased protein expression compared to any A and/or any B.

13. The composition of any one of claims 1 to 12, wherein L.sup.2 and each L.sup.1, independently, has the structure of Formula II: ##STR00068## wherein o is 1 or 2; a, b, c, d, e, and f are each, independently, 0 or 1; each of R.sup.1, R.sup.3, R.sup.5, and R.sup.7, is, independently, selected from optionally substituted C.sub.1-C.sub.6 alkylene, optionally substituted C.sub.1-C.sub.6 heteroalkylene, O, S, and NR.sup.8; R.sup.2 and R.sup.6 are each, independently, selected from carbonyl, thiocarbonyl, sulfonyl, or phosphoryl; R.sup.4 is optionally substituted branched or unbranched C.sub.1-C.sub.10 alkylene, optionally substituted branched or unbranched C.sub.2-C.sub.10 alkenylene, optionally substituted C.sub.2-C.sub.10 alkynylene, optionally substituted C.sub.2-C.sub.9 heterocyclylene, optionally substituted C.sub.6-C.sub.12 arylene, optionally substituted branched or unbranched C.sub.2-C.sub.100 polyethylene glycolene, or optionally substituted branched or unbranched C.sub.1-C.sub.10 heteroalkylene, or a bond linking (R).sub.a(R.sup.2).sub.b(R.sup.3).sub.c to (R.sup.5).sub.d(R.sup.6).sub.e(R.sup.7).sub.f; and R.sup.8 is hydrogen, optionally substituted C.sub.1-C.sub.4 alkyl, optionally substituted C.sub.2-C.sub.4 alkenyl, optionally substituted C.sub.2-C.sub.4 alkynyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.12 aryl, or optionally substituted C.sub.1-C.sub.7 heteroalkyl.

14. The composition of any one of claims 1 to 13, wherein n is 1, m is 1, and L.sup.2 is absent.

15. The composition of claim 14, wherein L.sup.1 is ##STR00069##

16. The composition of any one of claims 1 to 13, wherein n is 1, m is 2, and L.sup.2 is absent.

17. The composition of claim 16, wherein L.sup.1 has the structure: ##STR00070##

18. The composition of claim 16 or 17, wherein the compound has the structure: ##STR00071##

19. The compound of claim 17 or 18, wherein R.sup.4 is optionally substituted C.sub.1-C.sub.10 alkylene.

20. The compound of any one of claims 17 to 19, wherein each R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkylene.

21. The composition of any one of claims 17 to 20, wherein each e is 0.

22. The composition of any one of claims 17 to 21, wherein each f is 0.

23. The composition of any one of claims 16 to 22, wherein the compound has the structure: ##STR00072##

24. The composition of any one of claims 1 to 13, wherein n is 2 and m is 2.

25. The composition of claim 24, wherein L.sup.1 has the structure: ##STR00073##

26. The composition of claim 24 or 25, wherein the compound has the structure: ##STR00074##

27. The composition of any one of claim 25 or 26, wherein R.sup.4 is optionally substituted C.sub.1-C.sub.10 alkylene.

28. The composition of any one of claims 25 to 27, wherein each R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkylene.

29. The composition of any one of claims 25 to 28, wherein each e is 0.

30. The composition of any one of claims 25 to 29, wherein each f is 0.

31. The composition of any one of claims 25 to 30, wherein the compound has the structure: ##STR00075##

32. The composition of any one of claims 24 to 31, wherein L.sup.2 is ##STR00076##

33. The composition of any one of claims 1 to 13, wherein n is 1, m is 3, and L.sup.2 is absent.

34. The composition of claim 33, wherein L.sup.1 has the structure: ##STR00077##

35. The composition of claim 33 or 34, wherein the compound has the structure: ##STR00078##

36. The composition of claim 34 or 35, wherein R.sup.4 is optionally substituted C.sub.1-C.sub.10 alkylene.

37. The composition of any one of claims 34 to 36, wherein each R.sup.5 is optionally substituted C.sub.1-C.sub.6 heteroalkylene.

38. The composition of any one of claims 34 to 37, wherein each e is 0.

39. The composition of any one of claims 34 to 38, wherein each f is 0.

40. The composition of any one of claims 33 to 39, wherein the compound has the structure: ##STR00079##

41. The compound of any one of claims 1 to 13, wherein n is 3 and m is 2.

42. The compound of claim 41, wherein L.sup.1 has the structure: ##STR00080##

43. The compound of claim 41 or 42, wherein the compound has the structure: ##STR00081##

44. The compound of claim 42 or 43, wherein R.sup.4 is optionally substituted C.sub.1-C.sub.10 alkylene.

45. The compound of any one of claims 42 to 44, wherein each R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkylene.

46. The compound of any one of claims 42 to 45, wherein each e is 0.

47. The compound of any one of claims 42 to 46, wherein each f is 0.

48. The compound of any one of claims 41 to 47, wherein the compound has the structure: ##STR00082##

49. The compound of any one of claims 41 to 48, wherein L.sup.2 has the structure: ##STR00083##

50. The compound of claim 49, wherein R.sup.4 is optionally substituted C.sub.1-C.sub.10 alkylene.

51. The compound of claim 49 or 50, wherein each R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkylene.

52. The compound of any one of claims 49 to 51, wherein each e is 0.

53. The compound of any one of claims 49 to 52, wherein each f is 0.

54. The compound of any one of claims 41 to 53, wherein the compound has the structure: ##STR00084##

55. The composition of any one of claims 1 to 54, wherein at least one A and/or at least one B comprises at least one inverted nucleotide.

56. A method of producing a composition of any one of claims 1 to 55, the method comprising: (a) providing a first polynucleotide comprising an inverted nucleotide at the 3-terminus; (b) phosphorylating the 5-position of the inverted nucleotide; and (c) ligating the 3-terminus of a second polynucleotide to the first polynucleotide, wherein at least one of the first polynucleotide or the second polynucleotide comprises a coding region encoding a polypeptide of interest.

57. A method of producing a composition of any one of claims 1 to 55, the method comprising: (a) providing a first polynucleotide comprising a monophosphate at the 5-terminus and an inverted nucleotide at the 3-terminus; (b) phosphorylating the 5-position of the inverted nucleotide; and (c) ligating the 3-terminus of a second polynucleotide to the 5-terminus of the first polynucleotide and the 3-terminus of a third polynucleotide to 3-terminus of the first polynucleotide, wherein at least one of the second polynucleotide or third polynucleotide comprises a coding region encoding a polypeptide of interest.

58. The method of claim 56 or 57, wherein the phosphorylating of step (b) comprises a polynucleotide kinase.

59. A method of expressing a protein in a mammalian cell, the method comprising: (i) providing a composition of any one of claims 1 to 55; and (ii) introducing the composition to the mammalian cell under conditions that permit the expression of the polypeptide of interest by the mammalian cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0173] FIG. 1 is a scheme illustrating the synthesis of an mRNA with two 5 ends.

[0174] FIG. 2 is an HPLC spectra illustrating the addition of an inverted thymidine to the 3 end of an mRNA.

[0175] FIG. 3 is an HPLC spectra illustrating the addition of a second polynucleotide at an inverted thymidine.

[0176] FIG. 4 is an image illustrating the structure of an mRNA multimer including two polynucleotides.

[0177] FIG. 5 is a spectra illustrating the synthesis of an mRNA multimer including three polynucleotides.

[0178] FIG. 6 is an image illustrating the structure of an mRNA multimer including three polynucleotides.

[0179] FIG. 7 is a spectra illustrating the synthesis of an mRNA multimer including four polynucleotides.

[0180] FIG. 8 is an image illustrating the structure of an mRNA multimer including four polynucleotides.

[0181] FIG. 9 is a spectra illustrating the synthesis of an mRNA multimer including four polynucleotides.

[0182] FIG. 10 is an image illustrating the structure of an mRNA multimer including four polynucleotides.

[0183] FIG. 11 is a graph illustrating expression of an mRNA multimer including two polynucleotides compared to a monomeric mRNA expressing the same polypeptide.

[0184] FIG. 12 is a graph illustrating expression of mRNA multimers compared to a monomeric mRNA expressing the same polypeptide.

[0185] FIG. 13 is a graph illustrating immune response to mRNA multimers compared to a monomeric mRNA expressing the same polypeptide.

[0186] FIG. 14 is a graph illustrating expression of mRNA multimers compared to a monomeric mRNA expressing the same polypeptide.

DETAILED DESCRIPTION

[0187] Some challenges exist for mRNA therapy wherein multiple mRNAs must to be administered for effective therapy, for example administration of protein complexes (e.g., multimeric polypeptides such as antibodies or receptors) or multiple genes in cancer therapy.

[0188] Current encapsulation processes use monomeric mRNAs, which result in random encapsulation of different ratios of mRNAs in lipid nanoparticles (LNPs). This presents several challenges from both manufacturing and clinical perspectives. For example, current formulation methodology is limited as to number of biopolymers (e.g., multiple mRNAs) capable of being tethered. Encapsulation efficiency for multiple biopolymers is also low and therefore insufficient for industrial scale-up. Accordingly, the discoveries described herein provide novel compositions for the delivery of multiplex biopolymers, such as multiple mRNAs and overcome prior art issues.

Polynucleotides

[0189] The instant invention is based, in part, on the discovery that formation of multimeric complexes based on covalent linkages between mRNA molecules allows for uniform distribution of the mRNA in a therapeutic composition. When multiple nucleic acids such as RNA are formulated, for instance, in a lipid based formulation, a relatively uniform distribution of the total nucleic acid through the formulation may be achieved. However, the distribution of a particular nucleic acid with respect to the other nucleic acids in the mixture is not uniform. For instance when the nucleic acid mixture is composed of two distinct mRNA sequences, some of the lipid particles or other formulatory agents will house a single mRNA sequence, while others will house the other mRNA sequence and a few will house both of the mRNA sequences. In a therapeutic context this uneven distribution of mRNA is undesirable because the dosage of the mRNA being delivered to a patient will vary from administration to administration. The methods of the invention enable the production of formulations having multiple nucleic acids wherein the nucleic acid has a uniform distribution throughout the formulation.

[0190] The methods are achieved through the use of a covalent interaction. It was surprising that a covalent interaction between the individual nucleic acids would be capable of producing such a uniform distribution of the nucleic acids in a formulation.

[0191] It was also discovered according to aspects of the invention that the multimeric nucleic acid complexes generated according to the invention did not interfere with activity such as mRNA expression activity. It was quite surprising that mRNA formed into multimeric complexes did not experience a loss of expression activity as a result of the structures.

[0192] Described herein are compositions (including pharmaceutical compositions) and methods for the delivery of multimeric nucleic acid molecules. In some embodiments the multimeric structures are uniformly distributed throughout a composition such as a lipid nanoparticle. Uniformly distributed, as used herein in the context of multiple nucleic acids (each having a unique nucleotide sequence), refers to the distribution of each of the nucleic acids relative to one another in the formulation. Distribution of the nucleic acids in a formulation may be assessed using methods known in the art. For instance, several exemplary methods are shown in the Examples below. A nucleic acid is uniformly distributed relative to another nucleic acid if the nucleic acid is associated in proximity within a particular area of the formulation to the other nucleic acid at an approximately 1:1 ratio. In some embodiments the nucleic acid is uniformly distributed relative to another nucleic acid if the nucleic acid is positioned within a particular area of the formulation to the other nucleic acid at an approximately 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2 ratio.

[0193] The multimeric structures of the invention are comprised of nucleic acid molecules, specifically polynucleotides which, in some embodiments, encode one or more peptides or polypeptides of interest. The term nucleic acid, in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides.

[0194] A multimeric structure as used herein is series of at least nucleic acids linked together to form a multimeric structure. In some embodiments a multimeric structure is composed of 2 or more, 3 or more, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more nucleic acids. In other embodiments the multimeric structure is composed of 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less nucleic acids. In yet other embodiments a multimeric structure has 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 nucleic acids.

In preferred embodiments a multimeric structure is composed of 3-5 nucleic acids.

[0195] In some embodiments the upper limit on the number of nucleic acids in a multimeric structure depends on the length of dimerizable region. A greater than 20-nucleotide space between mRNAs can provide specificity and enough force to keep the multi-mRNA complex intact for downstream processing and is thus preferred in some embodiments. In some embodiments 4-5 nucleic acids in a multimeric structure may be desirable. For instance, cell conversion/differentiation (e.g., Induced Pluripotent Stem Cells-iPS) may be achieved with four protein factors. A similar number of proteins may be effective for inhibition of tumor growth.

[0196] Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having aD-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2-amino-LNA having a 2-amino functionalization, and 2-amino-a-LNA having a 2-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.

[0197] In some aspects, the disclosure provides a multimeric molecule comprising at least two nucleic acid molecules, wherein a first nucleic acid molecule is joined to a second nucleic acid molecule by at least one covalent bond, and wherein the at least one covalent bond is located between a first non-coding region of the first nucleic acid molecule and a second non-coding region of the second nucleic acid molecule.

[0198] In addition to having at least two distinct nucleic acids with unique sequences, the multimeric molecules may comprise multiple copies of the same gene or protein (e.g., 2, 3, 4, 5, or more mRNA encoding the same protein), as long as it includes at least two distinct nucleic acids. This type of multimeric molecule may be useful for increasing expression level of a particular protein in a cell. Multimeric molecules can also comprise nucleic acids (e.g., mRNA) encoding different gene or protein (e.g., 4 mRNA molecules, wherein each mRNA molecule encodes a different subunit protein of tetrameric receptor). Multimeric molecules comprising nucleic acids encoding different genes or proteins may also be useful for delivering combination biological therapies, for example in the context of cancer chemotherapy.

[0199] In some embodiments, covalent bonds between nucleic acid molecules (e.g., mRNA molecules) are formed in a non-coding region of each molecule. As used herein, the term non-coding region refers to a location of a polynucleotide (e.g., an mRNA) that is not translated into a protein. Examples of non-coding regions include regulatory regions (e.g., DNA binding domains, promoter sequences, enhancer sequences), and untranslated regions (e.g., 5UTR, 3UTR). In some embodiments, the non-coding region is an untranslated region (UTR).

[0200] By definition, wild type untranslated regions (UTRs) of a gene are transcribed but not translated. In mRNA, the 5UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3UTR starts immediately following the stop codon and continues until the transcriptional termination signal.

[0201] Natural 5UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where Risa purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by anotherG. 5UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0202] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the polynucleotides of the invention. For example, introduction of 5 UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could be used to enhance expression of a nucleic acid molecule, such as a polynucleotides, in hepatic cell lines or liver. Likewise, use of 5 UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AMLI, G-CSF, GM-CSF, CDIIb, MSR, Fr-I, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D). Other non-UTR sequences may also be used as regions or sub regions within the polynucleotides. For example, intrans or portions of intrans sequences may be incorporated into regions of the polynucleotides of the invention. Incorporation of intronic sequences may increase protein production as well as polynucleotide levels.

[0203] Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5 UTR which may contain a strong Kozak translational initiation signal and/or a 3 UTR which may include an oligo (dT) sequence for templated addition of a poly-A tail. 5UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes.

[0204] It should be understood that any UTR from any gene may be incorporated into the regions of the polynucleotide. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5 or 3 UTR may be inverted, shortened, lengthened, made with one or more other 5 UTRs or 3 UTRs. As used herein, the term altered as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3 or 5 UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an altered UTR (whether 3 or 5) comprise a variant UTR.

[0205] In one embodiment, a double, triple or quadruple UTR such as a 5 or 3 UTR may be used. As used herein, a double UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.

[0206] It is also within the scope of the present invention to have patterned UTRs. As used herein patterned UTRs are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level

[0207] In one embodiment, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a family of proteins is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern. The untranslated region may also include translation enhancer elements (TEE).

[0208] In some embodiments, an UTR of a polynucleotide (e.g., a first nucleic acid) of the present invention is engineered or modified to have regions of complementarity with an UTR of another polynucleotide (a second nucleic acid). For example, UTR nucleotide sequences of two polynucleotides sought to be joined (e.g., in a multimeric molecule) can be modified to include a region of complementarity such that the two UTRs hybridize to form a multimeric molecule.

[0209] In some embodiments, multimeric nucleic acid molecules comprise RNA molecules. In some embodiments, the RNA molecules are mRNA molecules. As used herein, the term messenger RNA (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo. An mRNA has been transcribed from a DNA sequence by an RNA polymerase enzyme, and interacts with a ribosome synthesize genetic information encoded by DNA. Generally, mRNA are classified into two sub-classes: pre-mRNA and mature mRNA. Precursor mRNA (pre-mRNA) is mRNA that has been transcribed by RNA polymerase but has not undergone any post-transcriptional processing (e.g., 5capping, splicing, editing, and polyadenylation). Mature mRNA has been modified via post-transcriptional processing (e.g., spliced to remove intrans and polyadenylated) and is capable of interacting with ribosomes to perform protein synthesis. mRNA can be isolated from tissues or cells by a variety of methods. For example, a total RNA extraction can be performed on cells or a cell lysate and the resulting extracted total RNA can be purified (e.g., on a column comprising oligo-dT beads) to obtain extracted mRNA.

[0210] Alternatively, mRNA can be synthesized in a cell-free environment, for example by in vitro transcription (IVT). An in vitro transcription template as used herein, refers to deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some embodiments, an IVT template encodes a 5 untranslated region, contains an open reading frame, and encodes a 3 untranslated region and a polyA tail. The particular nucleotide sequence composition and length of an IVT template will depend on the mRNA of interest encoded by the template.

[0211] A 5 untranslated region (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a protein or peptide.

[0212] A 3 untranslated region (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a protein or peptide.

[0213] An open reading frame is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a protein or peptide.

[0214] A polyA tail is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3), from the 3 UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo, etc.) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus, and translation.

[0215] Thus, the polynucleotide may in some embodiments comprise (a) a first region of linked nucleosides encoding a polypeptide of interest; (b) a first terminal region located 5 relative to said first region comprising a 5 untranslated region (UTR); (c) a second terminal region located 3 relative to said first region; and (d) a tailing region. The terms poly nucleotide and nucleic acid are used interchangeably herein. In some embodiments, the first region of linked nucleosides (e.g., polypeptide encoding sequence) ranges from about 30 to about 3,000 nucleotides in length. In some embodiments, the first region of linked nucleosides (e.g., polypeptide encoding sequence) ranges from about 200 to about 3,000 nucleotides in length.

[0216] In some embodiments, the polynucleotide includes from about 30 to about 300 nucleotides (e.g., from about 30 to about 50, from about 40 to about 60, from about 50 to about 100, from about 75 to about 150, from about 125 to about 200, from about 175 to about 250, from about 225 to about 300). In some embodiments, the polynucleotide includes from about 200 to about 3,000 nucleotides (e.g., from 200 to 500, from 200 to 1,000, from 200 to 1,500, from 200 to 3,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,500 to 3,000, and from 2,000 to 3,000).

[0217] IVT mRNA may function as mRNA but are distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics. For example, IVT mRNA may be structurally modified or chemically modified. As used herein, a structural modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide ATCG may be chemically modified to AT-5meC-G. The same polynucleotide may be structurally modified from ATCG to ATCCCG. Here, the dinucleotide CC has been inserted, resulting in a structural modification to the polynucleotide.

[0218] cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate polynucleotides (e.g., modified nucleic acids).

[0219] Thus, in an exemplary aspect, polynucleotides of the invention may include at least one chemical modification. The polynucleotides can include various substitutions and/or insertions from native or naturally occurring polynucleotides. As used herein in a polynucleotide, the terms chemical modification or, as appropriate, chemically modified refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribnucleosides in one or more of their position, pattern, percent or population. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5-terminal mRNA cap moieties.

The modifications may be various distinct modifications. In some embodiments, the regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide, introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified polynucleotide.

[0220] Modifications of the polynucleotides of the multimeric structures include, but are not limited to those listed in detail below. The polynucleotide may comprise modifications which are naturally occurring, non-naturally occurring or the polynucleotide can comprise both naturally and non-naturally occurring modifications.

[0221] The polynucleotides of the multimeric structures of the invention can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present invention may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

[0222] Non-natural modified nucleotides may be introduced to polynucleotides during synthesis or post-synthesis of the chains to achieve desired functions or properties. The modifications may be on internucleotide lineage, the purine or pyrimidine bases, or sugar. The modification may be introduced at the terminal of a chain or anywhere else in the chain; with chemical synthesis or with a polymerase enzyme. Any of the regions of the polynucleotides may be chemically modified.

[0223] The present disclosure provides for multimeric structures comprised of unmodified or IO modified nucleosides and nucleotides and combinations thereof. As described herein nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as nucleobase). As described herein, nucleotide is defined as a nucleoside including a phosphate group. The modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides). The polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides. Any combination of base/sugar or linker may be incorporated into the polynucleotides of the invention. Modifications of the polynucleotides of the multimeric structures which are useful in the present invention include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2-O-dimethyladenosine; 1-methyladenosine; 2-O-methyladenosine; 2-0-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2-O-methyladenosine; 2-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2-O-dimethyladenosine; N6,2-O-dimethyladenosine; N6,N6,2-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; NI-methyl-adenosine; N6, N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2-Amino-2-deoxy-ATP; 2-Azido-2-deoxy-ATP; 2-Deoxy-2-a-aminoadenosine TP; 2-Deoxy-2-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1-Deazaadenosine TP; 2Fluoro-N6-Bz-deoxyadenosine TP; 2-OMe-2-Amino-ATP; 2O-methyl-N6-Bz-deoxyadenosine TP; 2-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2-Deoxy-2,2-difluoroadenosine TP; 2-Deoxy-2-a-mercaptoadenosine TP; 2-Deoxy-2-a-thiomethoxyadenosine TP; 2-Deoxy-2-b-aminoadenosine TP; 2-Deoxy-2-b-azidoadenosine TP; 2-Deoxy-2-b-bromoadenosine TP; 2-Deoxy-2-b-chloroadenosine TP; 2-Deoxy-2-b-fluoroadenosine TP; 2-Deoxy-2-b-iodoadenosine TP; 2-Deoxy-2-b-mercaptoadenosine TP; 2-Deoxy-2-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4-Azidoadenosine TP; 4-Carbocyclic adenosine TP; 4-Ethynyladenosine TP; 5-Homo-adenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2-O-methylcytidine; 2-O-methylcytidine; 5,2-O-dimethylcytidine; 5-formyl-2-O-methylcytidine; Lysidine; N4,2-O-dimethylcytidine; N4-acetyl-2-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; a-thio-cytidine; 2-(thio)cytosine; 2-Amino-2-deoxy-CTP; 2-Azido-2-deoxy-CTP; 2-Deoxy-2-a-aminocytidine TP; 2-Deoxy-2-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2-O-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; I-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2-anhydro-cytidine TP hydrochloride; 2Fluor-N4-Bz-cytidine TP; 2Fluoro-N4-Acetyl-cytidine TP; 2-O-Methyl-N4-Acetyl-cytidine TP; 2O-methyl-N4-Bz-cytidine TP; 2-a-Ethynylcytidine TP; 2-a-Trifluoromethylcytidine TP; 2-b-Ethynylcytidine TP; 2-b-Trifluoromethylcytidine TP; 2-Deoxy-2,2-difluorocytidine TP; 2-Deoxy-2-a-mercaptocytidine TP; 2-Deoxy-2-a-thiomethoxycytidine TP; 2-Deoxy-2-b-aminocytidine TP; 2-Deoxy-2-b-azidocytidine TP; 2-Deoxy-2-b-bromocytidine TP; 2-Deoxy-2-b-chlorocytidine TP; 2-Deoxy-2-b-fluorocytidine TP; 2-Deoxy-2-b-iodocytidine TP; 2-Deoxy-2-b-mercaptocytidine TP; 2-Deoxy-2-b-thiomethoxycytidine TP; 2-O-Methyl-5-(1-propynyl)cytidine TP; 3-Ethynylcytidine TP; 4-Azidocytidine TP; 4-Carbocyclic cytidineTP; 4-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5-Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine; N2,2-O-dimethylguanosine; N2-methylguanosine; Wyosine; 1,2-O-dimethylguanosine; 1-methylguanosine; 2-O-methylguanosine; 2-O-ribosylguanosine (phosphate); 2-O-methylguanosine; 2-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; NI-methyl-guanosine; a-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2-Amino-2-deoxy-GTP; 2-Azido-2-deoxy-GTP; 2-Deoxy-2--aminoguanosine TP; 2-Deoxy-2-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7(alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2Fluoro-N2-isobutyl-guanosine TP; 2O-methyl-N2-isobutyl-guanosine TP; 2-a-Ethynylguanosine TP; 2-a-Trifluoromethylguanosine TP; 2-b-Ethynylguanosine TP; 2-b-Trifluoromethylguanosine TP; 2-Deoxy-2,2-difluoroguanosine TP; 2-Deoxy-2-a-mercaptoguanosine TP; 2-Deoxy-2-a-thiomethoxyguanosine TP; 2-Deoxy-2-b-aminoguanosine TP; 2-Deoxy-2-b-azidoguanosine TP; 2-Deoxy-2-b-bromoguanosine TP; 2-Deoxy-2-b-chloroguanosine TP; 2-Deoxy-2-b-fluoroguanosine TP; 2-Deoxy-2-b-iodoguanosine TP; 2-Deoxy-2-b-mercaptoguanosine TP; 2-Deoxy-2-b-thiomethoxyguanosine TP; 4-Azidoguanosine TP; 4-Carbocyclic guanosine TP; 4-Ethynylguanosine TP; 5-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine; 1,2-O-dimethylinosine; 2-O-methylinosine; 7-methylinosine; 2-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine; Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; I-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-methyl-pseudouridine; 2-O-methyluridine; 2-O-methylpseudouridine; 2-O-methyluridine; 2-thio-2-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2-O-dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridinemethyl ester; 5-carboxymethylaminomethyl-2-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; NI-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)-2-thiouridine TP; 5-(iso-Pentenylaminomethyl)-2-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil; a-thio-uridine; I (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; I (aminoalkylaminocarbony lethylenyl)-2,4-(dithio)pseudouracil; I (aminoalkylaminocarbony lethylenyl)-4 (thio)pseudouracil; I (aminoalkylaminocarbonylethylenyl)-pseudouracil; I (aminocarbonylethylenyl)-2(thio)-pseudouracil; I (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; I (aminocarbon ylethylenyl)-4 (thio)pseudouracil; I (aminocarbon ylethylenyl)-pseudouracil; I substituted 2(thio)-pseudouracil; I substituted 2,4-(dithio)pseudouracil; I substituted 4(thio)pseudouracil; I substituted pseudouracil; 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil; I-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP; I-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2 (thio)pseudouracil; 2 deoxy uridine; 2 fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2 methyl, 2amino, 2azido, 2fluro-guanosine; 2-Amino-2-deoxy-UTP; 2-Azido-2-deoxy-UTP; 2-Azido-deoxyuridine TP; 2-O-methylpseudouridine; 2 deoxy uridine; 2 fluorouridine; 2-Deoxy-2--aminouridine TP; 2-Deoxy-2-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-I-alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; P seudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; I-methyl-1-deaza-pseudouridine; 1-propynyl-uridine; I-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; () 1-(2-Hydroxypropyl)pseudouridine TP; (2R)-I-(2-Hydroxypropyl)pseudouridine TP; (2S)-I-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; 1-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2-Diethoxyethyl)pseudouridine TP; 1-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1-(2,4,6-Trimethyl-benzyl)pseudo-UTP; 1-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-DTP; 1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methox yethyl)pseudouridine TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; 1-(3,4-Dimethoxybenzyl)pseudouridine TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP; 1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; 1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP; 1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP; 1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP; I(4-Nitro-phenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino-pentyl)pseudo-DTP; 1-(6-Amino-hexyl)pseudo-DTP; 1,6-Dimethyl-pseudo-DTP; -[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl} pseudouridine TP; 1-Acetylpseudouridine TP; I-Alkyl-6-(1-propynyl)-pseudo-DTP; I-Alkyl-6-(2-propynyl)-pseudo-DTP; I-Alkyl-6-allyl-pseudo-DTP; I-Alkyl-6-ethynyl-pseudo-DTP; I-Alkyl-6-homoallyl-pseudo-DTP; I-Alkyl-6-vinyl-pseudo-DTP; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudo-DTP; 1-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-DTP; I-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; I-Butyl-pseudo-DTP; 1-Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-DTP; 1-Cyclobutyl-pseudo-DTP; 1-Cycloheptylmethyl-pseudo-DTP; 1-Cycloheptyl-pseudo-DTP; 1-Cyclohex ylmethyl-pseudo-DTP; 1-Cyclohexyl-pseudo-DTP; 1-Cycloocty Imethyl-pseudo-DTP; 1-Cyclooctyl-pseudo-DTP; 1-Cyclopentylmethyl-pseudo-DTP; 1-Cyclopentyl-pseudo-DTP; 1-Cyclopropylmethyl-pseudo-DTP; 1-Cyclopropyl-pseudo-DTP; I-Ethyl-pseudo-DTP; 1-Hexyl-pseudo-DTP; 1-Homoallylpseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-DTP; I-Me-2-thio-pseudo-DTP; I-Me-4-thio-pseudo-DTP; 1-Me-alpha-thio-pseudo-DTP; 1-Methanesulfon ylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2-Trifluoroeth yl)pseudo-DTP; I-Methyl-6-(4-morpholino)-pseudo-DTP; I-Methyl-6-(4-thiomorpholino)-pseudo-DTP; I-Methyl-6-(substituted phenyl)pseudo-DTP; I-Methyl-6-amino-pseudo-DTP; I-Methyl-6-azido-pseudo-DTP; I-Methyl-6-bromo-pseudo-DTP; I-Methyl-6-butyl-pseudo-DTP; I-Methyl-6-chloro-pseudo-DTP; I-Methyl-6-cyano-pseudo-DTP; I-Methyl-6-dimethylamino-pseudo-DTP; I-Methyl-6-ethoxy-pseudo-DTP; I-Methyl-6-ethylcarboxylate-pseudo-DTP; I-Methyl-6-ethyl-pseudo-DTP; I-Methyl-6-fluoro-pseudo-DTP; I-Methyl-6-formyl-pseudo-DTP; I-Methyl-6-hydroxyamino-pseudo-DTP; I-Methyl-6-hydroxy-pseudo-DTP; I-Methyl-6-iodo-pseudo-DTP; I-Methyl-6-iso-propyl-pseudo-DTP; I-Methyl-6-methoxy-pseudo-DTP; I-Methyl-6-methylamino-pseudo-DTP; I-Methyl-6-phenyl-pseudo-DTP; I-Methyl-6-propyl-pseudo-DTP; I-Methyl-6-tert-butyl-pseudo-DTP; I-Methyl-6-trifluoromethoxy-pseudo-DTP; I-Methyl-6-trifluoromethyl-pseudo-DTP; 1-Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-DTP; I-Phenyl-pseudo-DTP; 1-Pivaloylpseudouridine TP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-DTP; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-DTP; 1-tert-Butyl-pseudo-DTP; 1-Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-DTP; 1-Vinylpseudouridine TP; 2,2-anhydro-uridine TP; 2-bromo-deoxyuridine TP; 2-F-5-Methyl-2-deoxy-DTP; 2-OMe-5-Me-DTP; 2-OMe-pseudo-DTP; 2-a-Ethynyluridine TP; 2-a-Trifluoromethyluridine TP; 2-b-Ethynyluridine TP; 2-b-Trifluoromethyluridine TP; 2-Deoxy-2,2-difluorouridineTP; 2-Deoxy-2-a-mercaptouridine TP; 2-Deoxy-2-a-thiomethoxyuridine TP; 2-Deoxy-2-b-aminouridine TP; 2-Deoxy-2-b-azidouridine TP; 2-Deoxy-2-b-bromouridine TP; 2-Deoxy-2-b-chlorouridine TP; 2-Deoxy-2-b-fluorouridine TP; 2-Deoxy-2-b-iodouridine TP; 2-Deoxy-2-b-mercaptouridine TP; 2-Deoxy-2-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-DTP; 4-Azidouridine TP; 4-Carbocyclic uridine TP; 4-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5-Homo-uridine TP; 5-iodo-2-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Dridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-DTP; 6-(4-Morpholino)-pseudo-DTP; 6-(4-Thiomorpholino)-pseudo-DTP; 6-(Substituted-Phenyl)-pseudo-DTP; 6-Amino-pseudo-DTP; 6-Azido-pseudo-DTP; 6-Bromo-pseudo-DTP; 6-Butyl-pseudo-DTP; 6-Chloro-pseudo-DTP; 6-Cyano-pseudo-DTP; 6-Dimethylamino-pseudo-DTP; 6-Ethoxy-pseudo-DTP; 6-Ethylcarboxylate-pseudo-DTP; 6-Ethyl-pseudo-DTP; 6-Fluoro-pseudo-DTP; 6-Formyl-pseudo-DTP; 6-Hydroxyamino-pseudo-DTP; 6-Hydroxy-pseudo-DTP; 6-lodo-pseudo-DTP; 6-iso-Propyl-pseudo-DTP; 6-Methoxy-pseudo-DTP; 6-Methylamino-pseudo-DTP; 6-Methyl-pseudo-DTP; 6-Phenyl-pseudo-DTP; 6-Phenyl-pseudo-DTP; 6-Propyl-pseudo-DTP; 6-tert-Butyl-pseudo-DTP; 6-Trifluoromethoxy-pseudo-DTP; 6-Trifluoromethyl-pseudo-DTP; Alpha-thio-pseudo-DTP; Pseudouridine 1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP I-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP I-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-DTP-NI-3-propionic acid; Pseudo-DTP-NI-4-butanoic acid; Pseudo-DTP-NI-5-pentanoic acid; Pseudo-DTP-NI-6-hexanoic acid; Pseudo-DTP-NI-7-heptanoic acid; Pseudo-DTP-NI-methyl-p-benzoic acid; Pseudo-DTP-NI-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine; I-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine; 2,4,5-(trimethyl)phenyl; 2 methyl, 2 amino, 2 azido, 2fluro-cytidine; 2 methyl, 2amino, 2azido, 2fluro-adenine; 2methyl, 2amino, 2azido, 2fluro-uridine; 2-amino-2-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2-azido-2-deoxyribose; 2fluoro-2-deoxyribose; 2-fluoro-modified bases; 2-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-I-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-I-(aza)-2-(thio)-3-(aza)-phenoxazin-yl; 7-(guanidiniumalkylhydroxy)-I-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-I-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted I-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin ATP; Formycin B TP; Pyrrolosine TP; 2-OH-ara-adenosine TP; 2-OH-ara-cytidine TP; 2-OH-ara-uridine TP; 2-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(19-Amino-pentaoxanonadecyl)adenosine TP.

[0224] In some embodiments, an mRNA of the invention includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0225] In some embodiments, the modified nucleobase is pseudouridine (\If), NI-methylpseudouridine (m1\If), 2-thiouridine, 4-thiouridine, 5-methylcytosine, 2-thio-1-methyl-I-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2-O-methyl uridine. In some embodiments, an mRNA of the invention includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0226] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1\If), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (\If), a-thio-guanosine, or a-thio-adenosine. In some embodiments, an mRNA of the invention includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[0227] In some embodiments, the mRNA comprises pseudouridine (\If) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1\If). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1\If) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-O-methyl uridine. In some embodiments, the mRNA comprises 2-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0228] In certain embodiments, an mRNA of the invention is uniformly modified (i.e., fully modified, modified through-out the entire sequence) for a particular modification. For example, an mRNA can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, mRNAs of the invention can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0229] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine.

[0230] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified uridine include 5-cyano uridine or 4-thio uridine.

[0231] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (mIA), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-Diaminopurine.

[0232] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (mII), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mIG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0233] In one embodiment, the polynucleotides of the present invention, such as IVT polynucleotides, may have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by mere downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine. In another embodiment, the polynucleotides may have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way). When the polynucleotides of the present invention are chemically and/or structurally modified the polynucleotides may be referred to as modified polynucleotides.

[0234] Generally, the length of the IVT polynucleotide (e.g., IVT mRNA) encoding a polypeptide of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55,60,70,80,90,100, 120,140,160,180,200,250,300,350,400,450,500, 600,700,800,900,1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).

[0235] In some embodiments, the IVT polynucleotide (e.g., IVT mRNA) includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

[0236] In some embodiments, a nucleic acid of a multimeric molecule as described herein is a chimeric polynucleotide. Chimeric polynucleotides or RNA constructs maintain a modular organization similar to IVT polynucleotides, but the chimeric polynucleotides comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide. As such, the chimeric polynucleotides which are modified mRNA molecules of the present invention are termed chimeric modified mRNA or chimeric mRNA. Chimeric polynucleotides have portions or regions which differ in size and/or chemical modification pattern, chemical modification position, chemical modification percent or chemical modification population and combinations of the foregoing.

[0237] In some embodiments, the multimeric nucleic acids are therapeutic mRNAs. As used herein, the term therapeutic mRNA refers to an mRNA that encodes a therapeutic protein. Therapeutic proteins mediate a variety of effects in a host cell or a subject in order to treat a disease or ameliorate the signs and symptoms of a disease. For example, a therapeutic protein can replace a protein that is deficient or abnormal, augment the function of an endogenous protein, provide a novel function to a cell (e.g., inhibit or activate an endogenous cellular activity, or act as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate). Therapeutic mRNA may be useful for the treatment of the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune disorders.

[0238] Thus, the multimeric structures of the invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, the mRNA of the multimeric structures described herein can be administered to a subject, wherein the polynucleotides are translated in vivo to produce a therapeutic peptide. Provided are compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agents of the invention include the multimeric structures, cells containing multimeric structures or polypeptides translated from the polynucleotides contained in the multimeric structures.

[0239] The multimeric structures may be induced for translation in a cell, tissue or organism. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell, tissue or organism is contacted with an effective amount of a composition containing a multimeric structure which contains the multiple mRNA polynucleotides each of which has at least one translatable region encoding a peptide.

[0240] An effective amount of the multimeric structures are provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the multimeric structures, and other determinants. In general, an effective amount of the multimeric structure provides an induced or boosted peptide production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same peptide or about the same or more efficient than separate mRNAs that are not part of a multimeric structure. Increased peptide production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the multimeric structures), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified polynucleotide), or altered peptide production in the host cell.

[0241] The mRNA of the present invention may be designed to encode polypeptides of interest selected from any of several target categories including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. Therapeutic protein refers to a protein that, when administered to a cell has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.

[0242] The mRNA disclosed herein, may encode one or more biologics. As used herein, a biologic is a polypeptide-based molecule produced by the methods provided herein and which may be used to treat, cure, mitigate, prevent, or diagnose a serious or life-threatening disease or medical condition. Biologics, according to the present invention include, but are not limited to, allergenic extracts (e.g., for allergy shots and tests), blood components, gene therapy products, human tissue or cellular products used in transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytics, and immunomodulators, among others.

[0243] According to the present invention, one or more biologics currently being marketed or in development may be encoded by the mRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation of the encoding polynucleotides of a known biologic into the mRNA of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and/or selectivity of the construct designs.

[0244] The mRNA disclosed herein, may encode one or more antibodies or fragments thereof. The term antibody includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fe region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments. The term immunoglobulin (Ig) is used interchangeably with antibody herein. As used herein, the term monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0245] The monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include, but are not limited to, primatized antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.

[0246] An antibody fragment comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab, F(abh and Fv fragments; diabodies; linear antibodies; nanobodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, may be encoded by the mRNA of the invention, including the heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. Also included are polynucleotide sequences encoding the subclasses, gamma and mu. Hence any of the subclasses of antibodies may be encoded in part or in whole and include the following subclasses: IgGI, IgG2, IgG3, IgG4, IgA1 and IgA2. According to the present invention, one or more antibodies or fragments currently being marketed or in development may be encoded by the mRNA of the present invention.

[0247] Antibodies encoded in the mRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, blood, cardiovascular, CNS, poisoning (including antivenoms), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, oncology, immunology, respiratory, sensory and anti-infective.

[0248] In one embodiment, mRNA disclosed herein may encode monoclonal antibodies and/or variants thereof. Variants of antibodies may also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives. In one embodiment, the mRNA disclosed herein may encode an immunoglobulin Fe region. In another embodiment, the mRNA may encode a variant immunoglobulin Fe region.

[0249] The multimeric mRNA disclosed herein, may encode one or more vaccine antigens. As used herein, a vaccine antigen is a biological preparation that improves immunity to a particular disease or infectious agent. According to the present invention, one or more vaccine antigens currently being marketed or in development may be encoded by the multimeric mRNA of the present invention.

[0250] Vaccine antigens encoded in the mRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, cancer, allergy and infectious disease.

[0251] The mRNA of the present invention may be designed to encode on or more antimicrobial peptides (AMP) or antiviral peptides (AVP). AMPs and AVPs have been isolated and described from a wide range of animals such as, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals. The anti-microbial polypeptides described herein may block cell fusion and/or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, the anti-microbial polypeptide can comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the transmembrane subunit of a viral envelope protein, e.g., HIV-1 gp120 or gp41. The amino acid and nucleotide sequences of HIV-1 gp120 or gp41 are described in, e.g., Kuiken et al., (2008). HIV Sequence Compendium, Los Alamos National Laboratory.

[0252] In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral protein sequence.

[0253] In other embodiments, the anti-microbial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a capsid binding protein. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding sequence of the capsid binding protein.

[0254] The anti-microbial polypeptides described herein may block protease dimerization and inhibit cleavage of viral proproteins (e.g., HIV Gag-pol processing) into functional proteins thereby preventing release of one or more enveloped viruses (e.g., HIV, HCV). In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence.

[0255] In other embodiments, the anti-microbial polypeptide can comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a protease binding protein. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding sequence of the protease binding protein.

[0256] A non-limiting list of infectious diseases that the mRNA vaccine antigens or anti-microbial peptides may treat is presented below: human immunodeficiency virus (HIV), HIV resulting in mycobacterial infection, AIDS related Cacheixa, AIDS related Cytomegalovirus infection, HIV-associated nephropathy, Lipodystrophy, AID related cryptococcal meningitis, AIDS related neutropaenia, Pneumocysitis jiroveci (Pneumocystis carinii) infections, AID related toxoplasmosis, hepatitis A, B, C, Dor E, herpes, herpes zoster (chicken pox), German measles (rubella virus), yellow fever, dengue fever etc. (flavi viruses), flu (influenza viruses), haemorrhagic infectious diseases (Marburg or Ebola viruses), bacterial infectious diseases such as Legionnaires' disease (Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), E. coli infections, staphylococcal infections, salmonella infections or streptococcal infections, tetanus (Clostridium tetani), protozoan infectious diseases (malaria, sleeping sickness, leishmaniasis, toxoplasmosis, i.e. infections caused by plasmodium, trypanosomes, leishmania and toxoplasma), diphtheria, leprosy, measles, pertussis, rabies, tetanus, tuberculosis, typhoid, varicella, diarrheal infections such as Amoebiasis, Clostridium difficile-associated diarrhea (CDAD), Cryptosporidiosis, Giardiasis, Cyclosporiasis and Rotaviral gastroenteritis, encephalitis such as Japanese encephalitis, Wester equine encephalitis and Tick-borne encephalitis (TBE), fungal skin diseases such as candidiasis, onychomycosis, Tinea captis/scal ringworm, Tinea corporis/body ringworm, Tinea cruris/jock itch, sporotrichosis and Tinea pedis/Athlete's foot, Meningitis such as Haemophilus influenza type b (Hib), Meningitis, viral, meningococcal infections and pneumococcal infection, neglected tropical diseases such as Argentine haemorrhagic fever, Leishmaniasis, Nematode/roundworm infections, Ross river virus infection and West Nile virus (WNV) disease, Non-HIV STDs such as Trichomoniasis, Human papillomavirus (HPV) infections, sexually transmitted chlamydia! diseases, Chancroid and Syphilis, Non-septic bacterial infections such as cellulitis, lyme disease, MRSA infection, pseudomonas, staphylococcal infections, Boutonneuse fever, Leptospirosis, Rheumatic fever, Botulism, Rickettsial disease and Mastoiditis, parasitic infections such as Cysticercosis, Echinococcosis, Trematode/Fluke infections, Trichinellosis, Babesiosis, Hypodermyiasis, Diphyllobothriasis and Trypanosomiasis, respiratory infections such as adenovirus infection, aspergillosis infections, avian (H5NI) influenza, influenza, RSV infections, severe acute respiratory syndrome (SARS), sinusitis, Legionellosis, Coccidioidomycosis and swine (HINI) influenza, sepsis such as bacteraemia, sepsis/septic shock, sepsis in premature infants, urinary tract infection such as vaginal infections (bacterial), vaginal infections (fungal) and gonococcal infection, viral skin diseases such as B19 parvovirus infections, warts, genital herpes, orofacial herpes, shingles, inner ear infections, fetal cytomegalovirus syndrome, foodborn illnesses such as brucellosis (Brucella species), Clostridium perfringens (Epsilon toxin), E. Coli O157:H7 (Escherichia coli), Salmonellosis (Salmonella species), Shingellosis (Shingella), Vibriosis and Listeriosis, bioterrorism and potential epidemic diseases such as Ebola haemorrhagic fever, Lassa fever, Marburg haemorrhagic fever, plague, Anthrax Nipah virus disease, Ranta virus, Smallpox, Glanders (Burkholderia mallei), Melioidosis (Burkholderia pseudomallei), Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), Tularemia (Fancisella tularensis), rubella, mumps and polio.

[0257] The mRNA disclosed herein, may encode one or more validated or in testing therapeutic proteins or peptides. According to the present invention, one or more therapeutic proteins or peptides currently being marketed or in development may be encoded by the mRNA of the present invention. Therapeutic proteins and peptides encoded in the mRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, blood, cardiovascular, CNS, poisoning (including antivenoms), dermatology, endocrinology, genetic, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunology, respiratory, sensory and anti-infective.

[0258] The mRNA disclosed herein, may encode one or more cell-penetrating polypeptides. As used herein, cell-penetrating polypeptide or CPP refers to a polypeptide which may facilitate the cellular uptake of molecules. A cell-penetrating polypeptide of the present invention may contain one or more detectable labels. The polypeptides may be partially labeled or completely labeled throughout. The mRNA may encode the detectable label completely, partially or not at all. The cell-penetrating peptide may also include a signal sequence. As used herein, a signal sequence refers to a sequence of amino acid residues bound at the amino terminus of a nascent protein during protein translation. The signal sequence may be used to signal the secretion of the cell-penetrating polypeptide.

[0259] In one embodiment, the mRNA may also encode a fusion protein. The fusion protein may be created by operably linking a charged protein to a therapeutic protein. As used herein, operably linked refers to the therapeutic protein and the charged protein being connected in such a way to permit the expression of the complex when introduced into the cell. As used herein, charged protein refers to a protein that carries a positive, negative or overall neutral electrical charge. Preferably, the therapeutic protein may be covalently linked to the charged protein in the formation of the fusion protein. The ratio of surface charge to total or surface amino acids may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9.

[0260] The cell-penetrating polypeptide encoded by the mRNA may form a complex after being translated. The complex may comprise a charged protein linked, e.g., covalently linked, to the cell-penetrating polypeptide.

[0261] In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, protein-binding partner includes, but is not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell where the mRNA may be introduced. The cell-penetrating polypeptide may also be capable of penetrating the first cell.

[0262] In one embodiment, the mRNA may encode a cell-penetrating polypeptide which may comprise a protein-binding partner. The protein binding partner may include, but is not limited to, an antibody, a supercharged antibody or a functional fragment. The mRNA may be introduced into the cell where a cell-penetrating polypeptide comprising the protein-binding partner is introduced.

[0263] Human and other eukaryotic cells are subdivided by membranes into many functionally distinct compartments. Each membrane-bound compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses sorting signals which are amino acid motifs located within the protein, to target proteins to particular cellular organelles. One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER).

[0264] Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a linker holding the protein to the membrane. While not wishing to be bound by theory, the molecules of the present invention may be used to exploit the cellular trafficking described above. As such, in some embodiments of the invention, mRNA are provided to express a secreted protein. In one embodiment, these may be used in the manufacture of large quantities of valuable human gene products.

[0265] In some embodiments of the invention, mRNA are provided to express a protein of the plasma membrane.

[0266] In some embodiments of the invention, mRNA are provided to express a cytoplasmic or cytoskeletal protein.

[0267] In some embodiments of the invention, mRNA are provided to express an intracellular membrane bound protein.

[0268] In some embodiments of the invention, mRNA are provided to express a nuclear protein.

[0269] In some embodiments of the invention, mRNA are provided to express a protein associated with human disease.

[0270] The mRNA may have a nucleotide sequence of a native or naturally occurring mRNA or encoding a native or naturally occurring peptide. Alternatively the mRNA may have a nucleotide sequence having a percent identity to the nucleotide sequence of a native or naturally occurring mRNA or mRNA may have a nucleotide sequence encoding a peptide having a percent identity to the nucleotide sequence of a native or naturally occurring peptide. The term identity as known in the art, refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., algorithms). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0271] Thus, in some embodiments, the peptides encoded by the mRNAs of the multimeric structure are polypeptide variants that may have the same or a similar activity as a reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide. Generally, variants of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25:3389-3402.) Other tools are described herein, specifically in the definition of Identity. Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match/Mismatch Scores 1, 2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens.

[0272] According to the present invention, the multimeric structures include mRNA to encode one or more polypeptides of interest or fragments thereof. A polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides. As used herein, the term polypeptides of interest refer to any polypeptide which is selected to be encoded in the primary construct of the present invention.

[0273] As used herein, polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0274] The term polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants will possess at least about 50% identity to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical to a native or reference sequence.

In some embodiments variant mimics are provided. As used herein, the term variant mimic is one which contains one or more amino acids which would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.

[0275] The present invention contemplates several types of compositions which are polypeptide based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. The term derivative is used synonymously with the term variant but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.

[0276] As such, mRNA encoding polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this invention. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C-terminal ends).

[0277] Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.

[0278] Substitutional variants when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0279] As used herein the term conservative amino acid substitution refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.

[0280] Insertional variants when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. Immediately adjacent to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.

[0281] Deletional variants when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.

[0282] Covalent derivatives when referring to polypeptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.

[0283] Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the polypeptides produced in accordance with the present invention.

[0284] Other post-translational modifications include hydroxylation of praline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).

[0285] As used herein when referring to polypeptides the term domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

[0286] As used herein when referring to polypeptides the terms site as it pertains to amino acid based embodiments is used synonymously with amino acid residue and amino acid side chain. A site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.

[0287] As used herein the terms termini or terminus when referring to polypeptides refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[0288] Once any of the features have been identified or defined as a desired component of a polypeptide to be encoded by the mRNA of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.

[0289] Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

[0290] The present invention provides multimeric structures and pharmaceutical compositions thereof optionally in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances. Pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0291] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase active ingredient generally refers to the multimeric structures or the polynucleotides contained therein, e.g., mRNA encoding polynucleotides to be delivered as described herein.

[0292] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0293] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

[0294] The multimeric structures of the invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with multimeric structures, hyaluronidase, nanoparticle mimics and combinations thereof.

[0295] The instant invention is based, in part, on the discovery that covalent bonding between untranslated regions of nucleic acids (e.g., mRNAs, or IVT mRNAs) allows formation of multimeric molecules and efficient encapsulation of said molecules by lipid nanoparticles (LNPs). In some embodiments, multimeric nucleic acid molecules of the invention (e.g., multimeric mRNA molecules) can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of multimeric nucleic acid molecules include lipid nanoparticles (LNPs). In some embodiments, lipid nanoparticles are MC3-based lipid nanoparticles.

Linkers

[0296] The compounds of the invention include a linker (e.g., moiety linker joining a protein binding moiety (e.g., a presenter protein binding moiety or a target protein binding moiety) to a cross-linking group or a linker joining a protein binding moiety to a protein (e.g., a presenter protein or target protein). The linker component of the invention is, at its simplest, a bond, but may also provide a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking two moieties.

[0297] In some embodiments, at least one atom of a linker participates in binding to the presenter protein and/or the target protein. In certain embodiments, at least one atom of a linker does not participate in binding to the presenter protein and/or the target protein.

[0298] Thus, a linker, when included in a compound and/or conjugate as described herein, achieves linking of two (or more) moieties by covalent means, involving bond formation with one or more functional groups located on either moiety. Examples of chemically reactive functional groups which may be employed for this purpose include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.

[0299] In some embodiments, such covalent linking of two (or more) moieties may be effected using a linker that contains reactive moieties capable of reaction with such functional groups present in either moiety. For example, an amine group of a moiety may react with a carboxyl group of the linker, or an activated derivative thereof, resulting in the formation of an amide linking the two.

[0300] Examples of moieties capable of reaction with sulfhydryl groups include a-haloacetyl compounds of the type XCH.sub.2CO (where XBr, Cl, or I), which show particular reactivity for sulfhydryl groups, but which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 11:532 (1967). N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2-iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulfide bridges.

[0301] Examples of reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include:

[0302] (i) -haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type XCH.sub.2CO (where XBr, Cl, or I), for example, as described by Wong Biochemistry 24:5337 (1979);

[0303] (ii) N-maleimide derivatives, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Biochem. J. 91:589 (1964);

[0304] (iii) aryl halides such as reactive nitrohaloaromatic compounds;

[0305] (iv) alkyl halides, as described, for example, by McKenzie et al., J. Protein Chem. 7:581 (1988);

[0306] (v) aldehydes and ketones capable of Schiff's base formation with amino groups, the adducts formed usually being stabilized through reduction to give a stable amine;

[0307] (vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups;

[0308] (vii) chlorine-containing derivatives of s-triazines, which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups;

[0309] (viii) aziridines based on s-triazine compounds detailed above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 (1954), which react with nucleophiles such as amino groups by ring opening;

[0310] (ix) squaric acid diethyl esters as described by Tietze, Chem. Ber. 124:1215 (1991); and

[0311] (x) -haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463 (1993).

[0312] Representative amino-reactive acylating agents include:

[0313] (i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively;

[0314] (ii) sulfonyl chlorides, which have been described by Herzig et al., Biopolymers 2:349 (1964);

[0315] (iii) acid halides;

[0316] (iv) active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters;

[0317] (v) acid anhydrides such as mixed, symmetrical, or N-carboxyanhydrides;

[0318] (vi) other useful reagents for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, 1984;

[0319] (vii) acylazides, e.g., wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974);

[0320] (viii) imidoesters, which form stable amidines on reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84:3491 (1962); and

[0321] (ix) haloheteroaryl groups such as halopyridine or halopyrimidine.

[0322] Aldehydes and ketones may be reacted with amines to form Schiff's bases, which may advantageously be stabilized through reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., in Bioconjugate Chem. 1:96 (1990).

[0323] Examples of reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups, for example, as described by Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modifying reagents such as carbodiimides, which react through O-acylurea formation followed by amide bond formation, may also be employed.

[0324] It will be appreciated that functional groups in either moiety may, if desired, be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.

[0325] So-called zero-length linkers, involving direct covalent joining of a reactive chemical group of one moiety with a reactive chemical group of the other without introducing additional linking material may, if desired, be used in accordance with the invention.

[0326] More commonly, however, the linker includes two or more reactive moieties, as described above, connected by a spacer element. The presence of such a spacer permits bifunctional linkers to react with specific functional groups within either moiety, resulting in a covalent linkage between the two. The reactive moieties in a linker may be the same (homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromultifunctional linker), providing a diversity of potential reagents that may bring about covalent attachment between the two moieties.

[0327] Spacer elements in the linker typically consist of linear or branched chains and may include a C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, C.sub.2-C.sub.100 polyethylene glycol, or C.sub.1-10 heteroalkyl.

[0328] In some instances, the linker is described by Formula V.

[0329] Examples of homobifunctional linkers useful in the preparation of conjugates of the invention include, without limitation, diamines and diols selected from ethylenediamine, propylenediamine and hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, cyclohexanediol, and polycaprolactone diol.

[0330] In some embodiments, the linker is a bond or a linear chain of up to 10 atoms, independently selected from carbon, nitrogen, oxygen, sulfur or phosphorous atoms, wherein each atom in the chain is optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and wherein any two atoms in the chain may be taken together with the substituents bound thereto to form a ring, wherein the ring may be further substituted and/or fused to one or more optionally substituted carbocyclic, heterocyclic, aryl, or heteroaryl rings.

[0331] In some embodiments, a linker has the structure of Formula II:

##STR00020##

[0332] where A.sup.1 is a bond between the linker and presenter protein binding moiety; A.sup.2 is a bond between the mammalian target interacting moiety and the linker; B.sup.1, B.sup.2, B.sup.3, and B.sup.4 each, independently, is selected from optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, O, S, and NR.sup.N; R.sup.N is hydrogen, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.1-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; C.sub.1 and C.sub.2 are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; a, b, c, d, e, and f are each, independently, 0 or 1; and D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.a(C.sup.1).sub.b(B.sup.2).sub.c to (B.sup.3).sub.d(C.sup.2).sub.e(B.sup.4).sub.f-A.sup.2.

Lipid Nanoparticle Formulations

[0333] In some embodiments, nucleic acids of the invention (e.g. multimeric mRNA) are formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the invention can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491. all of which are incorporated by reference herein in their entirety.

Lipid Nanoparticles Encapsulating Multimeric mRNA Encoding Therapeutic Polypeptides

[0334] Nucleic acids of the present disclosure (e.g. multimeric mRNA) are typically formulated in lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.

[0335] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% non-cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.

[0336] In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% sterol. For example, the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterol.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG-modified lipid. For example, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%,1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%, or 15% PEG-modified lipid.

[0337] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

Ionizable Lipids

[0338] In some aspects, the ionizable lipids of the present disclosure may be one or more of compounds of Formula (I):

##STR00021##

or their N-oxides, or salts or isomers thereof, wherein:
R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, R*YR, YR, and RMR;
R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, R*YR, YR, and R*OR, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
R4 is selected from the group consisting of hydrogen, a C3-6 carbocycle, (CH2)nQ, (CH2)nCHQR, CHQR, CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, OR, O(CH2)nN(R)2, C(O)OR, OC(O)R, CX3, CX2H, CXH2, CN, N(R)2, C(O)N(R)2, N(R)C(O)R, N(R)S(O)2R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(R)R8, N(R)S(O)2R8, O(CH2)nOR, N(R)C(NR9)N(R)2, N(R)C(CHR9)N(R)2, OC(O)N(R)2, N(R)C(O)OR, N(OR)C(O)R, N(OR)S(O)2R, N(OR)C(O)OR, N(OR)C(O)N(R)2, N(OR)C(S)N(R)2, N(OR)C(NR9)N(R)2, N(OR)C(CHR9)N(R)2, C(NR9)N(R)2,
C(NR9)R, C(O)N(R)OR, and C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5;
each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
M and M are independently selected from C(O)O, OC(O), OC(O)-MC(O)O, C(O)N(R), N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O)2-, SS, an aryl group, and a heteroaryl group, in which M is a bond, C1-13 alkyl or C2-13 alkenyl;
R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
R8 is selected from the group consisting of C3-6 carbocycle and heterocycle;
R9 is selected from the group consisting of H, CN, NO.sub.2, C1-6 alkyl, OR, S(O)2R, S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle;
each R is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
each R is independently selected from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, R*YR, YR, and H;
each R is independently selected from the group consisting of C.sub.3-15 alkyl and C.sub.3-15 alkenyl;
each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
each Y is independently a C.sub.3-6 carbocycle;
each X is independently selected from the group consisting of F, Cl, Br, and I; and
m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R4 is (CH.sub.2).sub.nQ, (CH.sub.2).sub.nCHQR, CHQR, or CQ(R).sub.2, then (i) Q is not N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

[0339] In certain embodiments, a subset of compounds of Formula (I) includes those of Formula (IA):

##STR00022##

or its N-oxide, or a salt or isomer thereof, wherein I is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is a bond or M; R4 is hydrogen, unsubstituted C1-3 alkyl, or (CH2)nQ, in which Q is OH, NHC(S)N(R)2, NHC(O)N(R)2, N(R)C(O)R, N(R)S(O)2R, N(R)R8,
NHC(NR9)N(R)2, NHC(CHR9)N(R)2, OC(O)N(R)2, N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M are independently selected
from C(O)O, OC(O), OC(O)-MC(O)O, C(O)N(R), P(O)(OR)O, SS, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14 alkenyl. For example, m is 5, 7, or 9. For example, Q is OH, NHC(S)N(R)2, or NHC(O)N(R)2. For example, Q is N(R)C(O)R, or N(R)S(O)2R.

[0340] In certain embodiments, a subset of compounds of Formula (I) includes those of Formula (IB):

##STR00023##

or its N-oxide, or a salt or isomer thereof in which all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9; R4 is hydrogen, unsubstituted C.sub.1-3 alkyl, or (CH2)nQ, in which Q is

OH, NHC(S)N(R)2, NHC(O)N(R)2, N(R)C(O)R, N(R)S(O)2R, N(R)R8,

[0341] NHC(NR9)N(R)2, NHC(CHR9)N(R)2, OC(O)N(R)2, N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M are independently selected
from C(O)O, OC(O), OC(O)-MC(O)O, C(O)N(R), P(O)(OR)O, SS, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, and C2-14 alkenyl. For example, m is 5, 7, or 9. For example, Q is OH, NHC(S)N(R)2, or NHC(O)N(R)2. For example, Q is N(R)C(O)R, or N(R)S(O)2R.

[0342] In certain embodiments, a subset of compounds of Formula (I) includes those of Formula (II):

##STR00024##

or its N-oxide, or a salt or isomer thereof, wherein I is selected from 1, 2, 3, 4, and 5; M1 is a bond or M; R4 is hydrogen, unsubstituted C1-3 alkyl, or (CH2)nQ, in which n is 2, 3, or 4, and Q is

OH, NHC(S)N(R)2, NHC(O)N(R)2, N(R)C(O)R, N(R)S(O)2R, N(R)R8,

[0343] NHC(NR9)N(R)2, NHC(CHR9)N(R)2, OC(O)N(R)2, N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M are independently selected
from C(O)O, OC(O), OC(O)-MC(O)O, C(O)N(R), P(O)(OR)O, SS, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl.

[0344] In one embodiment, the compounds of Formula (I) are of Formula (IIa),

##STR00025##

or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.

[0345] In another embodiment, the compounds of Formula (I) are of Formula (IIb),

##STR00026##

or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.

[0346] In another embodiment, the compounds of Formula (I) are of Formula (IIc) or (IIe):

##STR00027##

or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.

[0347] In another embodiment, the compounds of Formula (I) are of Formula (IIf):

##STR00028##

or their N-oxides, or salts or isomers thereof, wherein M is C(O)O or OC(O), M is C1-6 alkyl or C2-6 alkenyl, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl, and n is selected from 2, 3, and 4.

[0348] In a further embodiment, the compounds of Formula (I) are of Formula (IId),

##STR00029##

or their N-oxides, or salts or isomers thereof, wherein n is 2, 3, or 4; and m, R, R, and R2 through R6 are as described herein. For example, each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

[0349] In a further embodiment, the compounds of Formula (I) are of Formula (IIg),

##STR00030##

or their N-oxides, or salts or isomers thereof, wherein I is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is a bond or M; M and M are independently selected from C(O)O, OC(O), OC(O)-MC(O)O, C(O)N(R), P(O)(OR)O, SS, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. For example, M is C1-6 alkyl (e.g., C1-4 alkyl) or C2-6 alkenyl (e.g. C2-4 alkenyl). For example, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

[0350] In some embodiments, the ionizable lipids are one or more of the compounds described in U.S. Application Nos. 62/220,091, 62/252,316, 62/253,433, 62/266,460, 62/333,557, 62/382,740, 62/393,940, 62/471,937, 62/471,949, 62/475,140, and 62/475,166, and PCT Application No. PCT/US2016/052352.

[0351] In some embodiments, the ionizable lipids are selected from Compounds 1-280 described in U.S. Application No. 62/475,166.

[0352] In some embodiments, the ionizable lipid is

##STR00031##

or a salt thereof.

[0353] In some embodiments, the ionizable lipid is

##STR00032##

or a salt thereof.

[0354] In some embodiments, the ionizable lipid is

##STR00033##

or a salt thereof.

[0355] In some embodiments, the ionizable lipid is

##STR00034##

or a salt thereof.
The central amine moiety of a lipid according to Formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), or (IIg) may be protonated at a physiological pH. Thus, a lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0356] In some aspects, the ionizable lipids of the present disclosure may be one or more of compounds of formula (III),

##STR00035##

or salts or isomers thereof, wherein

[0357] W is

##STR00036##

[0358] ring A is

##STR00037##

[0359] t is 1 or 2;

[0360] A.sub.1 and A.sub.2 are each independently selected from CH or N;

[0361] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;

[0362] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are independently selected from the group consisting of C.sub.5-20 alkyl, C5-20 alkenyl, RMR, R*YR, YR, and R*OR;

[0363] R.sub.X1 and R.sub.X2 are each independently H or C.sub.1-3 alkyl;

[0364] each M is independently selected from the group consisting

of C(O)O, OC(O), OC(O)O, C(O)N(R), N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O)2-, C(O)S, SC(O), an aryl group, and a heteroaryl group;

[0365] M* is C.sub.1-C.sub.6 alkyl,

[0366] W.sup.1 and W.sup.2 are each independently selected from the group consisting of O and N(R.sub.6);

[0367] each R.sub.6 is independently selected from the group consisting of H and C.sub.1-5 alkyl;

[0368] X.sup.1, X.sup.2, and X.sup.3 are independently selected from the group consisting of a bond, CH.sub.2, (CH.sub.2).sub.2, CHR, CHY, C(O), C(O)O, OC(O), (CH.sub.2).sub.nC(O), C(O)(CH.sub.2).sub.n, (CH.sub.2).sub.nC(O)O, OC(O)(CH.sub.2).sub.n, (CH.sub.2).sub.n-OC(O), C(O)O(CH.sub.2).sub.n, CH(OH), C(S), and CH(SH); each Y is independently a C.sub.3-6 carbocycle;

[0369] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;

[0370] each R is independently selected from the group consisting of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle;

[0371] each R is independently selected from the group consisting of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H;

[0372] each R is independently selected from the group consisting of C.sub.3-12 alkyl, C.sub.3-12 alkenyl and R*MR; and

[0373] n is an integer from 1-6;

[0374] wherein when ring A is

##STR00038##

then

[0375] i) at least one of X1, X2, and X3 is not CH2-; and/or

[0376] ii) at least one of R1, R2, R3, R4, and R5 is RMR.

[0377] In some embodiments, the compound is of any of formulae (IIIa1)-(IIIa8):

##STR00039##

[0378] In some embodiments, the ionizable lipids are one or more of the compounds described in U.S. Application Nos. 62/271,146, 62/338,474, 62/413,345, and 62/519,826, and PCT Application No. PCT/US2016/068300.

[0379] In some embodiments, the ionizable lipids are selected from Compounds 1-156 described in U.S. Application No. 62/519,826.

[0380] In some embodiments, the ionizable lipids are selected from Compounds 1-16, 42-66, 68-76, and 78-156 described in U.S. Application No. 62/519,826.

[0381] In some embodiments, the ionizable lipid is

##STR00040##

or a salt thereof.

[0382] The central amine moiety of a lipid according to Formula (III), (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), or (IIIa8) may be protonated at a physiological pH. Thus, a lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0383] Phospholipids

[0384] The lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0385] 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.

[0386] 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.

[0387] Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[0388] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0389] 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. In some embodiments, a phospholipid of the invention comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0390] In certain embodiments, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC. In certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV):

##STR00041##

or a salt thereof, wherein:

[0391] each R.sup.1 is independently optionally substituted alkyl; or optionally two R.sup.1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R.sup.1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl;

[0392] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0393] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0394] A is of the formula:

##STR00042##

[0395] each instance of L.sup.2 is independently a bond or optionally substituted C.sub.1-6 alkylene, wherein one methylene unit of the optionally substituted C.sub.1-6 alkylene is optionally replaced with O, N(R.sup.N), S, C(O), C(O)N(R.sup.N), NR.sup.NC(O), C(O)O, OC(O), OC(O)O, OC(O)N(R.sup.N), NR.sup.NC(O)O, or NR.sup.NC(O)N(R.sup.N);

[0396] each instance of R.sup.2 is independently optionally substituted C.sub.1-30 alkyl, optionally substituted C.sub.1-30 alkenyl, or optionally substituted C.sub.1-30 alkynyl; optionally wherein one or more methylene units of R.sup.2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted 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, (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;

[0397] each instance of R.sup.N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

[0398] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

[0399] p is 1 or 2;

[0400] provided that the compound is not of the formula:

##STR00043##

wherein each instance of R2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0401] In some embodiments, the phospholipids may be one or more of the phospholipids described in U.S. Application No. 62/520,530.

[0402] i) Phospholipid Head Modifications

[0403] In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phospholipid head (e.g., a modified choline group). In certain embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine. For example, in embodiments of Formula (IV), at least one of R1 is not methyl. In certain embodiments, at least one of R1 is not hydrogen or methyl. In certain embodiments, the compound of Formula (IV) is of one of the following formulae:

##STR00044##

or a salt thereof, wherein:

[0404] each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0405] each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[0406] each v is independently 1, 2, or 3.

[0407] In certain embodiments, a compound of Formula (IV) is of Formula (IV-a):

##STR00045##

or a salt thereof.

[0408] In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a cyclic moiety in place of the glyceride moiety. In certain embodiments, a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety. In certain embodiments, the compound of Formula (IV) is of Formula (IV-b):

##STR00046##

or a salt thereof.

[0409] (ii) Phospholipid Tail Modifications

[0410] In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified tail. In certain embodiments, a phospholipid useful or potentially useful in the present invention is DSPC, or analog thereof, with a modified tail. As described herein, a modified tail may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof. For example, in certain embodiments, the compound of (IV) is of Formula (IV-a), or a salt thereof, wherein at least one instance of R2 is each instance of R2 is optionally substituted C1-30 alkyl, wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(NRN), C(NRN)N(RN), NRNC(NRN), NRNC(NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O.

[0411] In certain embodiments, the compound of Formula (IV) is of Formula (IV-c):

##STR00047##

or a salt thereof, wherein:

[0412] each x is independently an integer between 0-30, inclusive; and each instance is G is independently selected from the group consisting of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(NRN), C(NRN)N(RN), NRNC(NRN), NRNC(NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O. Each possibility represents a separate embodiment of the present invention.

[0413] In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, a compound of Formula (IV) is of one of the following formulae:

##STR00048##

or a salt thereof.

[0414] Alternative Lipids

[0415] In certain embodiments, an alternative lipid is used in place of a phospholipid of the present disclosure.

[0416] In certain embodiments, an alternative lipid of the invention is oleic acid.

[0417] In certain embodiments, the alternative lipid is one of the following:

##STR00049##

[0418] Structural Lipids

[0419] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term structural lipid refers to sterols and also to lipids containing sterol moieties.

Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, sterols are a subgroup of steroids consisting of steroid alcohols. 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.

[0420] In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No. 62/520,530.

[0421] Polyethylene Glycol (PEG)-Lipids

[0422] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.

[0423] As used herein, the term PEG-lipid refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0424] In some embodiments, the PEG-lipid includes, but 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).

[0425] 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.

[0426] In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG-lipid is PEG2k-DMG.

[0427] In one embodiment, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. PEG-lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.

In general, some of the other lipid components (e.g., PEG lipids) of various formulae, described herein may be synthesized as described International Patent Application No. PCT/US2016/000129, filed Dec. 10, 2016, entitled Compositions and Methods for Delivery of Therapeutic Agents, which is incorporated by reference in its entirety.
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.

[0428] In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:

##STR00050##

[0429] 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. 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. 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.

[0430] In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (V). Provided herein are compounds of Formula (V):

##STR00051##

or salts thereof, wherein:

[0431] R.sup.3 is OR.sup.O;

[0432] R.sup.O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

[0433] r is an integer between 1 and 100, inclusive;

[0434] L.sup.1 is optionally substituted C.sub.1-10 alkylene, wherein at least one methylene of the optionally substituted C.sub.1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R.sup.N), S, C(O), C(O)N(R.sup.N), NR.sup.NC(O), C(O)O, OC(O), OC(O)O, OC(O)N(R.sup.N), NR.sup.NC(O)O, or NR.sup.NC(O)N(R.sup.N);

[0435] D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;

[0436] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0437] A is of the formula:

##STR00052##

each instance of L2 is independently a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O), C(O)N(RN),NR.sup.NC(O), or NRNC(O)N(RN); each instance of R2 is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(NRN), C(NRN)N(RN), NRNC(NRN), NRNC(NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O;

[0438] each instance of R.sup.N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and [0439] p is 1 or 2.

[0440] In certain embodiments, the compound of Formula (V) is a PEG-OH lipid (i.e., R3 is ORO, and RO is hydrogen). In certain embodiments, the compound of Formula (V) is of Formula (V-OH):

##STR00053##

or a salt thereof.

[0441] In certain embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (VI). Provided herein are compounds of Formula (VI):

##STR00054##

or a salts thereof, wherein:

[0442] R.sup.3 is OR.sup.O;

[0443] R.sup.O is hydrogen, optionally substituted alkyl or an oxygen protecting group;

[0444] r is an integer between 1 and 100, inclusive; R.sup.5 is optionally substituted C.sub.10-40 alkyl, optionally substituted C.sub.10-40 alkenyl, or optionally substituted C.sub.10-40 alkynyl; and optionally one or more methylene groups of R.sup.5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted 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, S(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

[0445] each instance of R.sup.N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0446] In certain embodiments, the compound of Formula (VI) is of Formula (VI-OH):

##STR00055##

or a salt thereof. In some embodiments, r is 45.

[0447] In yet other embodiments the compound of Formula (VI) is:

##STR00056##

or a salt thereof.

[0448] In one embodiment, the compound of Formula (VI) is

##STR00057##

[0449] In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. 62/520,530.

[0450] In some embodiments, a PEG lipid of the invention comprises 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. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.

[0451] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.

[0452] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula VI.

[0453] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.

[0454] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.

[0455] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid having Formula IV, a structural lipid, and a PEG lipid comprising a compound having Formula VI.

[0456] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00058##

[0457] and a PEG lipid comprising Formula VI.

[0458] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00059##

[0459] and an alternative lipid comprising oleic acid.

[0460] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00060##

[0461] an alternative lipid comprising oleic acid, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.

[0462] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00061##

[0463] a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.

[0464] In some embodiments, a LNP of the invention comprises an N:P ratio of from about 2:1 to about 30:1.

[0465] In some embodiments, a LNP of the invention comprises an N:P ratio of about 6:1.

[0466] In some embodiments, a LNP of the invention comprises an N:P ratio of about 3:1.

[0467] In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1.

[0468] In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.

[0469] In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1.

[0470] In some embodiments, a LNP of the invention has a mean diameter from about 50 nm to about 150 nm.

[0471] In some embodiments, a LNP of the invention has a mean diameter from about 70 nm to about 120 nm.

[0472] Lipid Nanoparticles Encapsulating Multimeric mRNA Vaccines

[0473] In some embodiments, multimeric RNA (e.g., mRNA) vaccines of the invention are formulated in a lipid nanoparticle (LNP).

[0474] Vaccines of the present disclosure are typically formulated in lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.

[0475] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable cationic lipid.

[0476] In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% non-cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%,10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.

[0477] In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% sterol. For example, the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterol.

[0478] In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG-modified lipid. For example, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%,11%,12%,13%,14%, or 15% PEG-modified lipid.

[0479] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0480] In some embodiments, an ionizable cationic lipid of the invention comprises a compound of Formula (I):

##STR00062##

[0481] or a salt or isomer thereof, wherein:

[0482] R.sub.1 is selected from the group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, R*YR, YR, and RMR;

[0483] R.sub.2 and R.sub.3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl, R*YR, YR, and R*OR, or R.sub.2 and R.sub.3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0484] R.sub.4 is selected from the group consisting of a C.sub.3-6 carbocycle, (CH.sub.2).sub.nQ, (CH.sub.2).sub.nCHQR,

[0485] CHQR, CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is selected from a carbocycle, heterocycle, OR, O(CH.sub.2).sub.nN(R).sub.2, C(O)OR, OC(O)R, CX.sub.3, CX.sub.2H, CXH.sub.2, CN, N(R).sub.2, C(O)N(R).sub.2,N(R)C(O)R, N(R)S(O).sub.2R, N(R)C(O)N(R).sub.2,N(R)C(S)N(R).sub.2,N(R)R.sub.8, O(CH.sub.2).sub.nOR, N(R)C(NR.sub.9)N(R).sub.2, N(R)C(CHR.sub.9)N(R).sub.2, OC(O)N(R).sub.2, N(R)C(O)OR, N(OR)C(O)R, N(OR)S(O).sub.2R, N(OR)C(O)OR, N(OR)C(O)N(R).sub.2, N(OR)C(S)N(R).sub.2, N(OR)C(NR.sub.9)N(R).sub.2, N(OR)C(CHR.sub.9)N(R).sub.2, C(NR.sub.9)N(R).sub.2, C(NR.sub.9)R, C(O)N(R)OR, and C(R)N(R).sub.2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5;

[0486] each R.sub.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0487] each R.sub.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0488] M and M are independently selected from C(O)O, OC(O), C(O)N(R),

[0489] N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O).sub.2, SS, an aryl group, and a heteroaryl group;

[0490] R.sub.7 is selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0491] R.sub.8 is selected from the group consisting of C.sub.3-6 carbocycle and heterocycle;

[0492] R.sub.9 is selected from the group consisting of H, CN, NO.sub.2, C.sub.1-6 alkyl, OR, S(O).sub.2R, S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and heterocycle;

[0493] each R is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0494] each R is independently selected from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, R*YR, YR, and H;

[0495] each R is independently selected from the group consisting of C.sub.3-14 alkyl and

[0496] C.sub.3-14 alkenyl;

[0497] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and

[0498] C.sub.2-12 alkenyl;

[0499] each Y is independently a C.sub.3-6 carbocycle;

[0500] each X is independently selected from the group consisting of F, Cl, Br, and I; and

[0501] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0502] In some embodiments, a subset of compounds of Formula (I) includes those in which when R.sub.4 is (CH.sub.2).sub.nQ, (CH.sub.2).sub.nCHQR, CHQR, or CQ(R).sub.2, then (i) Q is not N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

[0503] In some embodiments, another subset of compounds of Formula (I) includes those in which

[0504] R.sub.1 is selected from the group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, R*YR, YR, and RMR;

[0505] R.sub.2 and R.sub.3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl, R*YR, YR, and R*OR, or R.sub.2 and R.sub.3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0506] R.sub.4 is selected from the group consisting of a C.sub.3-6 carbocycle, (CH.sub.2).sub.nQ, (CH.sub.2).sub.nCHQR, CHQR, CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is selected from a C.sub.3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, OR, O(CH.sub.2).sub.nN(R).sub.2, C(O)OR, OC(O)R, CX.sub.3, CX.sub.2H, CXH.sub.2, CN, C(O)N(R).sub.2, N(R)C(O)R, N(R)S(O).sub.2R, N(R)C(O)N(R).sub.2, N(R)C(S)N(R).sub.2, CRN(R).sub.2C(O)OR, N(R)R.sub.8, O(CH.sub.2).sub.nOR, N(R)C(NR.sub.9)N(R).sub.2, N(R)C(CHR.sub.9)N(R).sub.2, OC(O)N(R).sub.2, N(R)C(O)OR, N(OR)C(O)R, N(OR)S(O).sub.2R, N(OR)C(O)OR, N(OR)C(O)N(R).sub.2, N(OR)C(S)N(R).sub.2, N(OR)C(NR.sub.9)N(R).sub.2, N(OR)C(CHR.sub.9)N(R).sub.2, C(NR.sub.9)N(R).sub.2, C(NR.sub.9)R, C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (O), OH, amino, mono- or di-alkylamino, and C.sub.1-3 alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;

[0507] each R.sub.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0508] each R.sub.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0509] M and M are independently selected from C(O)O, OC(O), C(O)N(R), N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O).sub.2, SS, an aryl group, and a heteroaryl group;

[0510] R.sub.7 is selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0511] R.sub.8 is selected from the group consisting of C.sub.3-6 carbocycle and heterocycle;

[0512] R.sub.9 is selected from the group consisting of H, CN, NO.sub.2, C.sub.1-6 alkyl, OR, S(O).sub.2R, S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and heterocycle;

[0513] each R is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0514] each R is independently selected from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, R*YR, YR, and H;

[0515] each R is independently selected from the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;

[0516] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;

[0517] each Y is independently a C.sub.3-6 carbocycle;

[0518] each X is independently selected from the group consisting of F, Cl, Br, and I; and

[0519] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

[0520] or salts or isomers thereof.

[0521] In some embodiments, another subset of compounds of Formula (I) includes those in which

[0522] R.sub.1 is selected from the group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, R*YR, YR, and RMR;

[0523] R.sub.2 and R.sub.3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl, R*YR, YR, and R*OR, or R.sub.2 and R.sub.3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0524] R.sub.4 is selected from the group consisting of a C.sub.3-6 carbocycle, (CH.sub.2).sub.nQ, (CH.sub.2).sub.nCHQR, CHQR, CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is selected from a C.sub.3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, OR, O(CH.sub.2).sub.nN(R).sub.2, C(O)OR, OC(O)R, CX.sub.3, CX.sub.2H, CXH.sub.2, CN, C(O)N(R).sub.2, N(R)C(O)R, N(R)S(O).sub.2R, N(R)C(O)N(R).sub.2, N(R)C(S)N(R).sub.2, CRN(R).sub.2C(O)OR, N(R)R.sub.8, O(CH.sub.2).sub.nOR, N(R)C(NR.sub.9)N(R).sub.2, N(R)C(CHR.sub.9)N(R).sub.2, OC(O)N(R).sub.2, N(R)C(O)OR, N(OR)C(O)R, N(OR)S(O).sub.2R, N(OR)C(O)OR, N(OR)C(O)N(R).sub.2, N(OR)C(S)N(R).sub.2, N(OR)C(NR.sub.9)N(R).sub.2, N(OR)C(CHR.sub.9)N(R).sub.2, C(NR.sub.9)R, C(O)N(R)OR, and C(NR.sub.9)N(R).sub.2, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R.sub.4 is (CH.sub.2).sub.nQ in which n is 1 or 2, or (ii) R.sub.4 is (CH.sub.2).sub.nCHQR in which n is 1, or (iii) R.sub.4 is CHQR, and CQ(R).sub.2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;

[0525] each R.sub.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0526] each R.sub.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0527] M and M are independently selected from C(O)O, OC(O), C(O)N(R), N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O).sub.2, SS, an aryl group, and a heteroaryl group;

[0528] R.sub.7 is selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0529] R.sub.8 is selected from the group consisting of C.sub.3-6 carbocycle and heterocycle;

[0530] R.sub.9 is selected from the group consisting of H, CN, NO.sub.2, C.sub.1-6 alkyl, OR, S(O).sub.2R, S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and heterocycle;

[0531] each R is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0532] each R is independently selected from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, R*YR, YR, and H;

[0533] each R is independently selected from the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;

[0534] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;

[0535] each Y is independently a C.sub.3-6 carbocycle;

[0536] each X is independently selected from the group consisting of F, Cl, Br, and I; and

[0537] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

[0538] or salts or isomers thereof.

[0539] In some embodiments, another subset of compounds of Formula (I) includes those in which

[0540] R.sub.1 is selected from the group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, R*YR, YR, and RMR;

[0541] R.sub.2 and R.sub.3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl, R*YR, YR, and R*OR, or R.sub.2 and R.sub.3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0542] R.sub.4 is selected from the group consisting of a C.sub.3-6 carbocycle, (CH.sub.2).sub.nQ, (CH.sub.2).sub.nCHQR, CHQR, CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is selected from a C.sub.3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, OR, O(CH.sub.2).sub.nN(R).sub.2, C(O)OR, OC(O)R, CX.sub.3, CX.sub.2H, CXH.sub.2, CN, C(O)N(R).sub.2, N(R)C(O)R, N(R)S(O).sub.2R, N(R)C(O)N(R).sub.2, N(R)C(S)N(R).sub.2, CRN(R).sub.2C(O)OR, N(R)R.sub.8, O(CH.sub.2).sub.nOR, N(R)C(NR.sub.9)N(R).sub.2, N(R)C(CHR.sub.9)N(R).sub.2, OC(O)N(R).sub.2, N(R)C(O)OR, N(OR)C(O)R, N(OR)S(O).sub.2R, N(OR)C(O)OR, N(OR)C(O)N(R).sub.2, N(OR)C(S)N(R).sub.2, N(OR)C(NR.sub.9)N(R).sub.2, N(OR)C(CHR.sub.9)N(R).sub.2, C(NR.sub.9)R, C(O)N(R)OR, and C(NR.sub.9)N(R).sub.2, and each n is independently selected from 1, 2, 3, 4, and 5;

[0543] each R.sub.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0544] each R.sub.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0545] M and M are independently selected from C(O)O, OC(O), C(O)N(R), N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O).sub.2, SS, an aryl group, and a heteroaryl group;

[0546] R.sub.7 is selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0547] R.sub.8 is selected from the group consisting of C.sub.3-6 carbocycle and heterocycle;

[0548] R.sub.9 is selected from the group consisting of H, CN, NO.sub.2, C.sub.1-6 alkyl, OR, S(O).sub.2R, S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and heterocycle;

[0549] each R is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0550] each R is independently selected from the group consisting of C-18 alkyl, C.sub.2-18 alkenyl, R*YR, YR, and H;

[0551] each R is independently selected from the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;

[0552] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;

[0553] each Y is independently a C.sub.3-6 carbocycle;

[0554] each X is independently selected from the group consisting of F, Cl, Br, and I; and

[0555] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

[0556] or salts or isomers thereof.

[0557] In some embodiments, another subset of compounds of Formula (I) includes those in which

[0558] R.sub.1 is selected from the group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, R*YR, YR, and RMR;

[0559] R.sub.2 and R.sub.3 are independently selected from the group consisting of H, C.sub.2-14 alkyl, C.sub.2-14 alkenyl, R*YR, YR, and R*OR, or R.sub.2 and R.sub.3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0560] R.sub.4 is (CH.sub.2).sub.nQ or (CH.sub.2).sub.nCHQR, where Q is N(R).sub.2, and n is selected from 3, 4, and 5;

[0561] each R.sub.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0562] each R.sub.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0563] M and M are independently selected from C(O)O, OC(O), C(O)N(R), N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O).sub.2, SS, an aryl group, and a heteroaryl group;

[0564] R.sub.7 is selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0565] each R is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0566] each R is independently selected from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, R*YR, YR, and H;

[0567] each R is independently selected from the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;

[0568] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.1-12 alkenyl;

[0569] each Y is independently a C.sub.3-6 carbocycle;

[0570] each X is independently selected from the group consisting of F, Cl, Br, and I; and

[0571] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

[0572] or salts or isomers thereof.

[0573] In some embodiments, another subset of compounds of Formula (I) includes those in which

[0574] R.sub.1 is selected from the group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, R*YR, YR, and RMR;

[0575] R.sub.2 and R.sub.3 are independently selected from the group consisting of C.sub.1-14 alkyl, C.sub.2-14 alkenyl, R*YR, YR, and R*OR, or R.sub.2 and R.sub.3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0576] R.sub.4 is selected from the group consisting of (CH.sub.2).sub.nQ, (CH.sub.2).sub.nCHQR, CHQR, and CQ(R).sub.2, where Q is N(R).sub.2, and n is selected from 1, 2, 3, 4, and 5;

[0577] each R.sub.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0578] each R.sub.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0579] M and M are independently selected from C(O)O, OC(O), C(O)N(R), N(R)C(O), C(O), C(S), C(S)S, SC(S), CH(OH), P(O)(OR)O, S(O).sub.2, SS, an aryl group, and a heteroaryl group;

[0580] R.sub.7 is selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0581] each R is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;

[0582] each R is independently selected from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, R*YR, YR, and H;

[0583] each R is independently selected from the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;

[0584] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.1-12 alkenyl;

[0585] each Y is independently a C.sub.3-6 carbocycle;

[0586] each X is independently selected from the group consisting of F, Cl, Br, and I; and

[0587] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

[0588] or salts or isomers thereof.

[0589] In some embodiments, a subset of compounds of Formula (I) includes those of Formula (IA):

##STR00063##

[0590] or a salt or isomer thereof, wherein I is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M.sub.1 is a bond or M; R.sub.4 is unsubstituted C.sub.1-3 alkyl, or (CH.sub.2).sub.nQ, in which Q is OH, NHC(S)N(R).sub.2, NHC(O)N(R).sub.2, N(R)C(O)R, N(R)S(O).sub.2R, N(R)R.sub.8, NHC(NR.sub.9)N(R).sub.2, NHC(CHR.sub.9)N(R).sub.2, OC(O)N(R).sub.2, N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M are independently selected from C(O)O, OC(O), C(O)N(R), P(O)(OR)O, SS, an aryl group, and a heteroaryl group; and R.sub.2 and R.sub.3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14 alkenyl.

[0591] In some embodiments, a subset of compounds of Formula (I) includes those of Formula (I):

##STR00064##

or a salt or isomer thereof, wherein I is selected from 1, 2, 3, 4, and 5; M1 is a bond or M; R.sub.4 is unsubstituted C.sub.1-3 alkyl, or (CH.sub.2).sub.nQ, in which n is 2, 3, or 4, and Q is OH, NHC(S)N(R).sub.2, NHC(O)N(R).sub.2, N(R)C(O)R, N(R)S(O).sub.2R, N(R)R.sub.8, NHC(NR.sub.9)N(R).sub.2, NHC(CHR.sub.9)N(R).sub.2, OC(O)N(R).sub.2, N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M are independently selected from C(O)O, OC(O), C(O)N(R), P(O)(OR)O, SS, an aryl group, and a heteroaryl group; and R.sub.2 and R.sub.3 are independently selected from the group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14 alkenyl.

[0592] In some embodiments, a subset of compounds of Formula (I) includes those of Formula (IIa), (IIb), (IIc), or (IIe):

##STR00065##

[0593] or a salt or isomer thereof, wherein R.sub.4 is as described herein.

[0594] In some embodiments, a subset of compounds of Formula (1)includes those of Formula (IId):

##STR00066##

[0595] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R, R, and R.sub.2 through R.sub.6 are as described herein. For example, each of R.sub.2 and R.sub.3 may be independently selected from the group consisting of C.sub.5-14 alkyl and C.sub.5-14 alkenyl.

[0596] In some embodiments, an ionizable cationic lipid of the invention comprises a compound having structure:

##STR00067##

[0597] In some embodiments, a non-cationic lipid of the invention comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0598] In some embodiments, a PEG modified lipid of the invention comprises 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. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.

[0599] In some embodiments, a sterol of the invention comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0600] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is PEG-DMG.

[0601] In some embodiments, a LNP of the invention comprises an N:P ratio of from about 2:1 to about 30:1.

[0602] In some embodiments, a LNP of the invention comprises an N:P ratio of about 6:1.

[0603] In some embodiments, a LNP of the invention comprises an N:P ratio of about 3:1.

[0604] In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1.

[0605] In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.

[0606] In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1.

[0607] In some embodiments, a LNP of the invention has a mean diameter from about 50 nm to about 150 nm.

[0608] In some embodiments, a LNP of the invention has a mean diameter from about 70 nm to about 120 nm.

[0609] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

[0610] The multimeric structures of the present invention may be administered by any route which results in a therapeutically effective outcome. The present invention provides methods comprising administering multimeric structures and in accordance with the invention to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0611] In certain embodiments, compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.

[0612] A multimeric structure pharmaceutical composition described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0613] The present invention provides pharmaceutical compositions including multimeric molecules (e.g., multimeric mRNA molecules) and multimeric compositions and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

[0614] The present invention provides multimeric molecules (e.g., multimeric mRNA molecules) and related pharmaceutical compositions and complexes optionally in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances.

[0615] Pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0616] In some embodiments, compositions are administered to humans, human patients or subjects.

[0617] For the purposes of the present disclosure, the phrase active ingredient generally refers to the multimeric molecules (e.g., multimeric mRNA molecules), to be delivered as described herein.

[0618] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

[0619] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0620] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

Examples

Example 1: Synthesis of mRNA Multimer Including Two Polynucleotides

[0621] An oligo was purchased from Axolabs (Kulmbach, Germany) consisting of a 5 hydroxyl group, twenty adenosines, a 3-3 phosphodiester linkage, and an inverted deoxythymidine with an exposed 5 hydroxyl group. The oligo was phosphorylated using T4 polynucleotide kinase (T4 PNK) (New England Biolabs, Ipswich, Mass.) and a standard T4 PNK protocol: T4 PNK reaction buffer (10 mM MgCl.sub.2, 70 mM Tris-HCl, 5 mM DTT (pH 7.6 at 25 C.), 1 mM ATP, and 0.2 U/L PNK, incubated for 1 h at 37 C. The reaction generated a di-phosphorylated linker oligo with two 5 phosphates approaching quantitative yield. The di-phosphorylated linker oligo was ligated with mRNA at a 1:3 molar ratio using the following protocol: RNA concentration limited to <1 mg/mL, T4 RNA Ligase reaction buffer (10 mM MgCl.sub.2, 50 mM Tris-HCl, 1 mM DTT (pH 7.5 at 25 C.), 1 mM ATP, 12.5% w/v PEG 8000, 0.9 U/L T4 RNA Ligase 1 (NEB) and 1 U/L murine RNase inhibitor (NEB), incubated overnight at room temperature with gentle rotation, and purified by dT purification. The ligation product consisting of two mRNA polynucleotides covalently joined by the linker oligo was isolated by UPLC purification (FIGS. 1-4).

Example 2: Synthesis of mRNA Multimer Including Three Polynucleotides

[0622] A branched oligo was purchased from Oligo Factory (Holliston, Mass.) consisting of three polynucleotides of ten adenosines each joined by a glycerol derivative such that the branched oligo consists of two 5 hydroxyls and one 3 hydroxyls. The branched oligo was ligated with a dinucleotide (p-adenosine-p-(inverted deoxythymidine)) at a 1:10 molar ratio as in Example 1 and UPLC purified to isolate a branched oligo consisting of three 5 hydroxyls. The branched oligo was phosphorylated using T4 PNK (Example 1) to form a branched oligo featuring three 5 phosphates. The tri-phosphorylated linker oligo was ligated with mRNA at a 1:4.5 molar ratio (Example 1), and ligation product consisting of three covalently linked mRNA polynucleotides and a branched linker was isolated by UPLC purification (FIGS. 5 and 6).

Example 3: Synthesis of mRNA Multimer Including Four Polynucleotides

[0623] Branched oligo (Example 2) was ligated with di-phosphorylated linker oligo (Example 1) at a 2:1 molar ratio as in Example 1 and ligation product consisting of two covalently linked branched oligos and a linker was isolated by UPLC purification (FIGS. 7 and 8). The recovered oligo featuring four 5 hydroxyl groups was phosphorylated using T4 PNK (Example 1) to form a double-branched oligo featuring four 5 phosphates. The tetra-phosphorylated linker oligo was ligated with mRNA at a 1:6 molar ratio (Example 1), and ligation product consisting of four covalently linked mRNA polynucleotides and a branched linker was isolated by UPLC purification.

Example 4: Synthesis of mRNA Multimer Including Six Polynucleotides

[0624] Branched oligo (Example 2) was ligated with the tri-phosphorylated linker oligo (Example 2) at a 3:1 molar ratio as in Example 1 and ligation product consisting of three covalently linked branched oligos and a branched linker was isolated by UPLC purification (FIGS. 9 and 10). The recovered oligo featuring six 5 hydroxyl groups was phosphorylated using T4 PNK (Example 1) to form a triple-branched oligo featuring of six 5 phosphates. The hexa-phosphorylated linker oligo was ligated with mRNA at a 1:9 ratio (Example 1), and ligation product consisting of six covalently linked mRNA polynucleotides and a branched linker was isolated by UPLC purification.

Example 5: Expression of Multimeric mRNA

[0625] Multimeric mRNA was prepared as in Examples 1-4 using mRNA encoding enhanced green fluorescent protein (eGFP). Multimeric eGFP mRNA was transfected into BJ fibroblast or AML12 cells using lipofectamine, and eGFP protein expression was monitored (FIGS. 11 and 12) using an IncuCyte Live-Cell Analysis System (Sartorius, Ann Arbor, Mich.).

Example 6: Expression of EPO from mRNA Multimers Delivered in LNPs

[0626] Multimeric mRNA was prepared as in Examples 1-4 using mRNA encoding erythropoietin (EPO) protein. Multimeric EPO mRNA was encapsulated in LNPs. LNP-formulated multimeric EPO was used for in vivo studies in which C57BL/6 mice were dosed at 0.25 mg/kg by intravenous injection. Expression and secretion of EPO protein was assessed at assigned time points over 72 hours by sampling blood and performing ELISA assays to quantify EPO concentrations (FIGS. 13 and 14).

OTHER EMBODIMENTS

[0627] It is to be understood that while the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and alterations are within the scope of the following claims.

[0628] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0629] In the claims, articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0630] This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of including, comprising, having, containing, involving, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. These terms do not require the inclusion of additional elements or steps. When one of these terms is used herein, the term consisting of is thus also encompassed and disclosed.

[0631] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0632] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any polynucleotide or protein encoded thereby; any method of production; any method of use) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.