POLYNUCLEOTIDES ENCODING PROPIONYL-COA CARBOXYLASE ALPHA AND BETA SUBUNITS FOR THE TREATMENT OF PROPIONIC ACIDEMIA

Abstract

This disclosure relates to mRNA therapy for the treatment of propionic acidemia. mRNAs for use in the invention, when administered in vivo, encode propionyl-CoA carboxy lase alpha (PCCA) and propionyl-CoA carboxy lase beta (PCCB). mRNA therapies of the disclosure increase and/or restore deficient levels of PCCA and/or PCCB expression and/or activity in subjects.

Claims

1. A method of treating propionic academia in a human subject in need thereof, the method comprising administering to the human subject by intravenous infusion a pharmaceutical composition comprising lipid nanoparticles comprising: (1) a first messenger RNA (mRNA) comprising a first open reading frame (ORF) encoding the human propionyl-CoA carboxylase alpha (PCCA) polypeptide of SEQ ID NO:1, wherein the first ORF is at least 80% identical to the nucleotide sequence of SEQ ID NO:5, and (2) a second mRNA comprising a second ORF encoding the human propionyl-CoA carboxylase beta (PCCB) polypeptide of SEQ ID NO:2, wherein the second ORF is at least 80% identical to the nucleotide sequence of SEQ ID NO:6, wherein the first mRNA and the second mRNA are present in the lipid nanoparticles at a ratio of 1:1, and wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.3 mg/kg to about 1.0 mg/kg.

2. The method of claim 1, wherein the first ORF is at least 95% identical to the nucleotide sequence of SEQ ID NO:5 and the second ORF is at least 95% identical to the nucleotide sequence of SEQ ID NO:6.

3. The method of claim 1, wherein the first ORF is 100% identical to the nucleotide sequence of SEQ ID NO:5 and the second ORF is 100% identical to the nucleotide sequence of SEQ ID NO:6.

4. The method of any one of claims 1 to 3, wherein the first mRNA comprises a 5 UTR comprising the nucleic acid sequence of SEQ ID NO:55 and the second mRNA comprises a 5 UTR comprising the nucleic acid sequence of SEQ ID NO:55.

5. The method of any one of claims 1 to 4, wherein the first mRNA comprises a 3 UTR comprising the nucleic acid sequence of SEQ ID NO: 114 and the second mRNA comprises a 3 UTR comprising the nucleic acid sequence of SEQ ID NO:114.

6. The method of claim 1, wherein the first mRNA comprises the nucleic acid sequence of SEQ ID NO:7 and the second mRNA comprises the nucleic acid sequence of SEQ ID NO:8.

7. The method of any one of claims 1 to 6, wherein the first mRNA and the second mRNA each comprise a 5 terminal cap.

8. The method of claim 7, wherein the 5 terminal cap comprises a guanine cap nucleotide containing an N7 methylation and the 5-terminal nucleotide of each of the first mRNA and the second mRNA contains a 2-O-methyl.

9. The method of any one of claims 1 to 8, wherein the first mRNA and the second mRNA each comprise a poly-A region.

10. The method of claim 9, wherein the poly-A region is 100 residues in length (SEQ ID NO: 195).

11. The method of any one of claims 1 to 10, wherein all of the uracils of the first mRNA and the second mRNA are N1-methylpseudouracils.

12. The method of claim 1, wherein: the first mRNA comprises a 5 terminal cap comprising a guanine cap nucleotide containing an N7 methylation wherein the 5-terminal nucleotide of the first mRNA contains a 2-O-methyl, the nucleic acid sequence of SEQ ID NO:7, and a poly-A region 100 residues in length (SEQ ID NO: 195), wherein all of the uracils of the first mRNA are N1-methylpseudouracils; and the second mRNA comprises a 5 terminal cap comprising a guanine cap nucleotide containing an N7 methylation wherein the 5-terminal nucleotide of the second mRNA contains a 2-O-methyl, the nucleic acid sequence of SEQ ID NO:8, and a poly-A region 100 residues in length (SEQ ID NO:195), wherein all of the uracils of the second mRNA are N1-methylpseudouracils.

13. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.3 mg/kg to about 0.6 mg/kg.

14. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.3 mg/kg.

15. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.4 mg/kg.

16. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose about 0.5 mg/kg.

17. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.6 mg/kg.

18. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.7 mg/kg.

19. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.8 mg/kg.

20. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 0.9 mg/kg.

21. The method of any one of claims 1 to 12, wherein the first mRNA and the second mRNA are administered to the human subject at a combined dose of about 1.0 mg/kg.

22. The method of any one of claims 1 to 21, comprising multiple administrations of the combined dose.

23. The method of claim 22, wherein the combined dose is administered repeatedly at intervals of about once every 2 to 4 weeks.

24. The method of claim 22, wherein the combined dose is administered repeatedly at intervals of about once every 2 weeks.

25. The method of claim 22, wherein the combined dose is administered repeatedly at intervals of about once every 3 weeks.

26. The method of claim 22, wherein the combined dose is administered repeatedly at intervals of about once every 4 weeks.

27. The method of any one of claims 22 to 26, comprising at least 10 administrations of the combined dose.

28. The method of any one of claims 1 to 27, wherein the human subject has a loss of function mutation in the PCCA gene.

29. The method of any one of claims 1 to 27, wherein the human subject has a loss of function mutation in the PCCB gene.

30. The method of any one of claims 1 to 27, wherein the human subject has a loss of function mutation in the PCCA gene and a loss of function mutation in the PCCB gene.

31. The method of any one of claims 1 to 30, wherein the human subject is at least 1 year of age.

32. The method of any one of claims 1 to 31, wherein the human subject is administered at least one of an H.sub.2 blocker, an H.sub.1 blocker, or acetaminophen/paracetamol or ibuprofen prior to infusion of the pharmaceutical composition.

33. The method of any one of claims 1 to 31, wherein the human subject is administered an H.sub.2 blocker, an H.sub.1 blocker, and acetaminophen/paracetamol or ibuprofen prior to infusion of the pharmaceutical composition.

34. The method of any one of claims 1 to 33, wherein the treatment reduces 2-methylcitric acid (2-MC) levels from baseline.

35. The method of any one of claims 1 to 33, wherein the treatment reduces 3-hydroxypropionic acid (3-HP) levels from baseline.

36. The method of any one of claims 1 to 33, wherein the treatment reduces 2-MC levels and 3-HP levels from baseline.

37. The method of any one of claims 1 to 33, wherein the treatment increases PCCA and PCCB mRNA levels from baseline.

38. The method of any one of claims 1 to 33, wherein the treatment reduces the frequency and duration of clinically significant events.

39. The method of any one of claims 1 to 33, wherein the treatment reduces the frequency and duration of metabolic decompensation events.

40. The method of any one of claims 1 to 33, wherein the treatment reduces the incidence and duration of healthcare utilization visits.

41. The method of any one of claims 1 to 33, wherein the treatment increases Quality-of-Life measurements.

42. The method of any one of claims 1 to 33, wherein the treatment improves cardiac structure and/or function.

43. The method of any one of claims 1 to 33, wherein the treatment improves renal function.

44. The method of any one of claims 1 to 33, wherein the treatment reduces the C.sub.3/C.sub.2 carnitine ratio from baseline.

45. The method of any one of claims 1 to 33, wherein the treatment reduces propionylglycine levels from baseline.

46. The method of any one of claims 1 to 33, wherein the treatment reduces glycine levels from baseline.

47. The method of any one of claims 1 to 33, wherein the treatment reduces the C.sub.3/C.sub.2 carnitine ratio, propionylglycine levels, and glycine levels and from baseline.

48. The method of any one of claims 1 to 33, wherein the treatment increases height and weight growth velocity of the human subject.

49. The method of any one of claims 1 to 33, wherein the treatment reduces fibroblast growth factor 21 (FGF-21) levels from baseline.

50. The method of any one of claims 1 to 33, wherein the treatment reduces ammonia, lactate, and/or venous blood gas levels from baseline.

51. The method of any one of claims 1 to 50, wherein the lipid nanoparticles comprise a compound of Formula (I): ##STR00159## or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched, wherein R.sup.branched is: ##STR00160## wherein ##STR00161## denotes a point of attachment; wherein R.sup.a?, R.sup.a?, R.sup.a?, and R.sup.a? are each independently selected from the group consisting of H, C.sub.2-12 alkyl, and C.sub.2-12 alkenyl; R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and C.sub.2-14 alkenyl; R.sup.4 is selected from the group consisting of (CH.sub.2)OH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and ##STR00162## wherein ##STR00163## denotes a point of attachment; wherein R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R.sup.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each R.sup.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; M and M are each independently selected from the group consisting of C(O)O and OC(O); R is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; l is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

52. The method of claim 51, wherein the lipid nanoparticles further comprise a phospholipid, a structural lipid, and a PEG-lipid.

53. The method of claim 52, wherein the PEG-lipid is Compound I.

54. The method of claim 52 or 53, wherein the lipid nanoparticles comprise: (i) 40-50 mol % of the compound of Formula (I), 30-45 mol % of the structural lipid, 5-15 mol % of the phospholipid, and 1-5 mol % of the PEG-lipid; or (ii) 45-50 mol % of the compound of Formula (I), 35-45 mol % of the structural lipid, 8-12 mol % of the phospholipid, and 1.5 to 3.5 mol % of the PEG-lipid.

55. The method of any one of claims 1 to 50, wherein the lipid nanoparticles comprise: (i) Compound II, (ii) Cholesterol, and (iii) PEG-DMG or Compound I; (i) Compound VI, (ii) Cholesterol, and (iii) PEG-DMG or Compound I; (i) Compound II, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) PEG-DMG or Compound I; (i) Compound VI, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) PEG-DMG or Compound I; (i) Compound II, (ii) Cholesterol, and (iii) Compound I; (i) Compound II, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) Compound I; (i) Compound B, (ii) Cholesterol, and (iii) PEG-DMG or Compound I; (i) Compound B, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) PEG-DMG or Compound I; (i) Compound B, (ii) Cholesterol, and (iii) Compound I; (i) Compound B, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) Compound I; (i) Compound A, (ii) Cholesterol, and (iii) PEG-DMG or Compound I; (i) Compound A, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) PEG-DMG or Compound I; (i) Compound A, (ii) Cholesterol, and (iii) Compound I; or (i) Compound A, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) Compound I.

56. The method of any one of claims 1 to 50, wherein the lipid nanoparticles comprise Compound II and Compound I.

57. The method of any one of claims 1 to 50, wherein the lipid nanoparticles comprise Compound II, DSPC, Cholesterol, and Compound I.

Description

DETAILED DESCRIPTION

Propionyl-CoA Carboxylase (PCC)

[0119] Propionyl-CoA carboxylase (PCC; EC 6.4.1.3) catalyzes the carboxylation of propionyl-CoA with bicarbonate, producing methylmalonyl-CoA. Methylmalonyl-CoA is then converted to succinyl-CoA, which is an intermediate in the tricarboxylic acid cycle (TCA). In the cell, PCC exists as a heterododecamer composed of six propionyl-CoA carboxylase alpha subunits, encoded by PCCA (OMIM 232000), and 6 propionyl-CoA beta subunits, encoded by PCCB (OMIM 232050).

[0120] Propionic acidemia is an autosomal recessive metabolic disorder associated with PCC function. PA results when bi-allelic variants eliminate or reduce the function of the PCCA or PCCB subunits of PCC. Propionyl-CoA accumulates intracellularly in human subjects with PA, which has many metabolic effects, including, e.g., the inhibition of mitochondrial respiratory function and reduced synthesis of citrate, GTP and ATP. A variety of variant PCCA and PCCB proteins have different levels of activity, with the severity of PA being correlated with the severity of the enzymes' mutations. For example, PCCA null mutations, such as R288X and S537X, result in severe phenotypes, while certain splice site variants can result in milder disease. Generally, variations in the PCCA N and C terminal regions can cause PA because regions are necessary for PCC's holocarboxylase synthase interaction. PCCB mutations, e.g., A497V, R512C, L519P, and W531X, often affect the interaction between PCCA and PCCB, thereby disturbing PCC stability and function.

Polynucleotides and Open Reading Frames (ORFs)

[0121] The instant invention features mRNAs for use in treating or preventing PA. The mRNAs featured for use in the invention are administered to subjects and encode human PCCA and PCCB proteins in vivo. Accordingly, the invention relates to polynucleotides, e.g., mRNAs, comprising an open reading frame of linked nucleosides encoding human PCCA (SEQ ID NO:1) and human PCCB (SEQ ID NO:2). In particular, the invention provides sequence-optimized polynucleotides comprising nucleotides encoding the polypeptide sequence of human PCCA and PCCB, or sequence having high sequence identity with those sequence optimized polynucleotides.

[0122] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PCCA polypeptide (e.g., SEQ ID NO:1), wherein the nucleotide sequence has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5.

[0123] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PCCB polypeptide (e.g., SEQ ID NO:2), wherein the nucleotide sequence has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6.

[0124] In some embodiments, the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a PCCA or PCCB polypeptide further comprises at least one nucleic acid sequence that is noncoding, e.g., a microRNA binding site. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention further comprises a 5 UTR (e.g., SEQ ID NO:55) and a 3 UTR (e.g., SEQ ID NO:114). In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises the sequence of SEQ ID NO:7. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises the sequence of SEQ ID NO:8. In a further embodiment, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 5 terminal cap (e.g., m.sup.7Gp-ppGm-A, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5 methylG cap, or an analog thereof) and a poly-A-tail region (e.g., about 100 nucleotides in length). In some embodiments, the mRNA comprises a poly A tail. In some instances, the poly A tail is 50-150 (SEQ ID NO:197), 75-150 (SEQ ID NO:198), 85-150 (SEQ ID NO:199), 90-120 (SEQ ID NO: 193), 90-130 (SEQ ID NO:194), or 90-150 (SEQ ID NO:192) nucleotides in length. In some instances, the poly A tail is 100 nucleotides in length (SEQ ID NO: 195). In some instances, the poly A tail is protected (e.g., with an inverted deoxy-thymidine). In some instances, the poly A tail comprises A100-UCUAG-A20-inverted deoxy-thymidine. In some instances, the poly A tail is A100-UCUAG-A20-inverted deoxy-thymidine.

[0125] In some embodiments, the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a PCCA or PCCB polypeptide is single stranded or double stranded.

[0126] In some embodiments, the polynucleotide of the invention comprising a nucleotide sequence (e.g., an ORF) encoding a PCCA or PCCB polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof) is DNA or RNA. In some embodiments, the polynucleotide of the invention is RNA. In some embodiments, the polynucleotide of the invention is, or functions as, an mRNA. In some embodiments, the mRNA comprises a nucleotide sequence (e.g., an ORF) that encodes at least one PCCA or PCCB polypeptide, and is capable of being translated to produce the encoded PCCA or PCCB polypeptide in vitro. in vivo, in situ or ex vivo.

[0127] In some embodiments, the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a PCCA or PCCB (e.g., sequence of SEQ ID NO: 1 or SEQ ID NO:2), wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil. In certain embodiments, all uracils in the polynucleotide are N1-methylpseudouracils. In other embodiments, all uracils in the polynucleotide are 5-methoxyuracils. In some embodiments, the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-142 and/or a miRNA binding site that binds to miR-126.

[0128] In some embodiments, the polynucleotide (e.g., a RNA, e.g., a mRNA) disclosed herein is formulated with a delivery agent comprising, e.g., a compound having the Formula (I), e.g., Compound II or Compound B; a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound VI or Compound I, or any combination thereof. In some embodiments, the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG-DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol % ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol % ionizable amino lipid, for example, 45-46 mol %, 46-47 mol %, 47-48 mol %, 48-49 mol %, or 49-50 mol % for example about 45 mol %, 45.5 mol %, 46 mol %, 46.5 mol %, 47 mol %, 47.5 mol %, 48 mol %, 48.5 mol %, 49 mol %, or 49.5 mol %; (ii) 30-45 mol % sterol (e.g., cholesterol), optionally 35-42 mol % sterol, for example, 30-31 mol %, 31-32 mol %, 32-33 mol %, 33-34 mol %, 35-35 mol %, 35-36 mol %, 36-37 mol %, 37-38 mol %, 38-39 mol %, or 39-40 mol %, or 40-42 mol % sterol; (iii) 5-15 mol % helper lipid (e.g., DSPC), optionally 10-15 mol % helper lipid, for example, 5-6 mol %, 6-7 mol %, 7-8 mol %, 8-9 mol %, 9-10 mol %, 10-11 mol %, 11-12 mol %, 12-13 mol %, 13-14 mol %, or 14-15 mol % helper lipid; and (iv) 1-5% PEG lipid (e.g., Compound I or PEG-DMG), optionally 1-5 mol % PEG lipid, for example 1.5 to 2.5 mol %, 1-2 mol %, 2-3 mol %, 3-4 mol %, or 4-5 mol % PEG lipid.

[0129] In some embodiments, the polynucleotide of the disclosure is an mRNA that comprises a 5-terminal cap (e.g., Cap1, e.g., m.sup.7Gp-ppGm-A), a 5UTR (e.g., SEQ ID NO:55), the PCCA ORF sequence of SEQ ID NO:5, a 3UTR (e.g., SEQ ID NO:114), and a poly A tail (e.g., about 100 nucleotides in length), wherein all uracils in the polynucleotide are N1-methylpseudouracils. In some embodiments, the delivery agent comprises Compound II or Compound VI as the ionizable lipid and PEG-DMG or Compound I as the PEG lipid.

[0130] In some embodiments, the polynucleotide of the disclosure is an mRNA that comprises a 5-terminal cap (e.g., Cap1, e.g., m.sup.7Gp-ppGm-A), a 5UTR (e.g., SEQ ID NO:55), the PCCB ORF sequence of SEQ ID NO:6, a 3UTR (e.g., SEQ ID NO: 114), and a poly A tail (e.g., about 100 nucleotides in length), wherein all uracils in the polynucleotide are N1-methylpseudouracils. In some embodiments, the delivery agent comprises Compound II or Compound VI as the ionizable lipid and PEG-DMG or Compound I as the PEG lipid.

[0131] In some embodiments, the polynucleotide of the disclosure is an mRNA that comprises a 5-terminal cap (e.g., Cap1, e.g., m.sup.7Gp-ppGm-A), a 5UTR (e.g., any one of SEQ ID NOs:50-79), the PCCA ORF sequence of SEQ ID NO:5, a 3UTR (e.g., any one of SEQ ID NOs: 100-136), and a poly A tail (e.g., about 100 nucleotides in length), wherein all uracils in the polynucleotide are N1-methylpseudouracils. In some embodiments, the delivery agent comprises Compound II or Compound VI as the ionizable lipid and PEG-DMG or Compound I as the PEG lipid.

[0132] In some embodiments, the polynucleotide of the disclosure is an mRNA that comprises a 5-terminal cap (e.g., Cap1, e.g., m.sup.7Gp-ppGm-A), a 5UTR (e.g., any one of SEQ ID NOs:50-79), the PCCB ORF sequence of SEQ ID NO:6, a 3UTR (e.g., any one of SEQ ID NOs: 100-136), and a poly A tail (e.g., about 100 nucleotides in length), wherein all uracils in the polynucleotide are N1-methylpseudouracils. In some embodiments, the delivery agent comprises Compound II or Compound VI as the ionizable lipid and PEG-DMG or Compound I as the PEG lipid.

Signal Sequences

[0133] The polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention can also comprise nucleotide sequences that encode additional features that facilitate trafficking of the encoded polypeptides to therapeutically relevant sites. One such feature that aids in protein trafficking is the signal sequence, or targeting sequence. The peptides encoded by these signal sequences are known by a variety of names, including targeting peptides, transit peptides, and signal peptides. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) that encodes a signal peptide operably linked to a nucleotide sequence that encodes a PCCA or PCCB polypeptide described herein.

[0134] In some embodiments, the signal sequence or signal peptide is a polynucleotide or polypeptide, respectively, which is from about 30-210, e.g., about 45-80 or 15-60 nucleotides (e.g., about 20, 30, 40, 50, 60, or 70 amino acids) in length that, optionally, is incorporated at the 5 (or N-terminus) of the coding region or the polypeptide, respectively. Addition of these sequences results in trafficking the encoded polypeptide to a desired site, such as the endoplasmic reticulum or the mitochondria through one or more targeting pathways. Some signal peptides are cleaved from the protein, for example by a signal peptidase after the proteins are transported to the desired site.

[0135] In some embodiments, the polynucleotide of the invention comprises a nucleotide sequence encoding a PCCA or PCCB polypeptide, wherein the nucleotide sequence further comprises a 5 nucleic acid sequence encoding a heterologous signal peptide.

Sequence-Optimized Nucleotide Sequences Encoding PCCA or PCCB Polypeptides

[0136] In some embodiments, the polynucleotide of the invention comprises a sequence-optimized nucleotide sequence encoding a PCCA or PCCB polypeptide disclosed herein. In some embodiments, the polynucleotide of the invention comprises an open reading frame (ORF) encoding a PCCA or PCCB polypeptide, wherein the ORF has been sequence optimized.

[0137] An exemplary sequence-optimized nucleotide sequence encoding human full length PCCA is set forth as SEQ ID NO:5. An exemplary sequence-optimized nucleotide sequence encoding human full length PCCB is set forth as SEQ ID NO:6. In some embodiments, the sequence optimized PCCA and PCCB sequences, fragments, and variants thereof are used to practice the methods disclosed herein.

[0138] In some embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a PCCA polypeptide, comprises from 5 to 3 end: [0139] (i) a 5 cap provided herein, for example, Cap1; [0140] (ii) a 5 UTR, such as the sequences provided herein, for example, SEQ ID NO:55; [0141] (iii) an open reading frame encoding a PCCA polypeptide, e.g., a sequence optimized nucleic acid sequence encoding PCCA set forth as SEQ ID NO:5; [0142] (iv) at least one stop codon (if not present at 5 terminus of 3UTR); [0143] (v) a 3 UTR, such as the sequences provided herein, for example, SEQ ID NO:114; and [0144] (vi) a poly-A tail provided above.

[0145] In some embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a PCCB polypeptide, comprises from 5 to 3 end: [0146] (i) a 5 cap provided herein, for example, Cap1; [0147] (ii) a 5 UTR, such as the sequences provided herein, for example, SEQ ID NO:55; [0148] (iii) an open reading frame encoding a PCCB polypeptide, e.g., a sequence optimized nucleic acid sequence encoding PCCB set forth as SEQ ID NO:6; [0149] (iv) at least one stop codon (if not present at 5 terminus of 3UTR); [0150] (v) a 3 UTR, such as the sequences provided herein, for example, SEQ ID NO:114; and [0151] (vi) a poly-A tail provided above.

[0152] In certain embodiments, all uracils in the polynucleotide are N1-methylpseudouracil. In certain embodiments, all uracils in the polynucleotide are 5-methoxyuracil.

[0153] The sequence-optimized nucleotide sequences disclosed herein are distinct from the corresponding wild type nucleotide acid sequences and from other known sequence-optimized nucleotide sequences, e.g., these sequence-optimized nucleic acids have unique compositional characteristics.

[0154] In some embodiments, the percentage of uracil or thymine nucleobases in a sequence-optimized nucleotide sequence (e.g., encoding a PCCA or PCCB polypeptide, a functional fragment, or a variant thereof) is modified (e.g., reduced) with respect to the percentage of uracil or thymine nucleobases in the reference wild-type nucleotide sequence. Such a sequence is referred to as a uracil-modified or thymine-modified sequence. The percentage of uracil or thymine content in a nucleotide sequence can be determined by dividing the number of uracils or thymines in a sequence by the total number of nucleotides and multiplying by 100. In some embodiments, the sequence-optimized nucleotide sequence has a lower uracil or thymine content than the uracil or thymine content in the reference wild-type sequence. In some embodiments, the uracil or thymine content in a sequence-optimized nucleotide sequence of the invention is greater than the uracil or thymine content in the reference wild-type sequence and still maintain beneficial effects, e.g., increased expression and/or reduced Toll-Like Receptor (TLR) response when compared to the reference wild-type sequence.

[0155] Methods for optimizing codon usage are known in the art. For example, an ORF of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art-non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

Modified Nucleotide Sequences Encoding PCCA and PCCB Polypeptides

[0156] In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil, N1-methylpseudouracil, 5-methoxyuracil, or the like. In some embodiments, the mRNA is a uracil-modified sequence comprising an ORF encoding a PCCA or PCCB polypeptide, wherein the mRNA comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil, N1-methylpseudouracil, or 5-methoxyuracil.

[0157] In certain aspects of the invention, when the modified uracil base is connected to a ribose sugar, as it is in polynucleotides, the resulting modified nucleoside or nucleotide is referred to as modified uridine. In some embodiments, uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% modified uracil. In one embodiment, uracil in the polynucleotide is at least 95% modified uracil. In another embodiment, uracil in the polynucleotide is 100% modified uracil.

[0158] In embodiments where uracil in the polynucleotide is at least 95% modified uracil overall uracil content can be adjusted such that an mRNA provides suitable protein expression levels while inducing little to no immune response. In some embodiments, the uracil content of the ORF is between about 100% and about 150%, between about 100% and about 110%, between about 105% and about 115%, between about 110% and about 120%, between about 115% and about 125%, between about 120% and about 130%, between about 125% and about 135%, between about 130% and about 140%, between about 135% and about 145%, between about 140% and about 150% of the theoretical minimum uracil content in the corresponding wild-type ORF (% U.sub.TM). In other embodiments, the uracil content of the ORF is between about 121% and about 136% or between 123% and 134% of the % U.sub.TM. In some embodiments, the uracil content of the ORF encoding a PCCA or PCCB polypeptide is about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% of the % U.sub.TM. In this context, the term uracil can refer to modified uracil and/or naturally occurring uracil.

[0159] In some embodiments, the uracil content in the ORF of the mRNA encoding a PCCA or PCCB polypeptide of the invention is less than about 30%, about 25%, about 20%, about 15%, or about 10% of the total nucleobase content in the ORF. In some embodiments, the uracil content in the ORF is between about 10% and about 20% of the total nucleobase content in the ORF. In other embodiments, the uracil content in the ORF is between about 10% and about 25% of the total nucleobase content in the ORF. In one embodiment, the uracil content in the ORF of the mRNA encoding a PCCA or PCCB polypeptide is less than about 20% of the total nucleobase content in the open reading frame. In this context, the term uracil can refer to modified uracil and/or naturally occurring uracil.

[0160] In further embodiments, the ORF of the mRNA encoding a PCCA or PCCB polypeptide having modified uracil and adjusted uracil content has increased Cytosine (C), Guanine (G), or Guanine/Cytosine (G/C) content (absolute or relative). In some embodiments, the overall increase in C, G, or G/C content (absolute or relative) of the ORF is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the wild-type ORF. In some embodiments, the G, the C, or the G/C content in the ORF is less than about 100%, less than about 90%, less than about 85%, or less than about 80% of the theoretical maximum G, C, or G/C content of the corresponding wild type nucleotide sequence encoding the PCCA or PCCB polypeptide (% G.sub.TMX; % C.sub.TMX, or % G/C.sub.TMX). In some embodiments, the increases in G and/or C content (absolute or relative) described herein can be conducted by replacing synonymous codons with low G, C, or G/C content with synonymous codons having higher G, C, or G/C content. In other embodiments, the increase in G and/or C content (absolute or relative) is conducted by replacing a codon ending with U with a synonymous codon ending with G or C.

[0161] In further embodiments, the ORF of the mRNA encoding a PCCA or PCCB polypeptide of the invention comprises modified uracil and has an adjusted uracil content containing less uracil pairs (UU) and/or uracil triplets (UUU) and/or uracil quadruplets (UUUU) than the corresponding wild-type nucleotide sequence encoding the PCCA or PCCB polypeptide. In some embodiments, the ORF of the mRNA encoding a PCCA or PCCB polypeptide of the invention contains no uracil pairs and/or uracil triplets and/or uracil quadruplets. In some embodiments, uracil pairs and/or uracil triplets and/or uracil quadruplets are reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the ORF of the mRNA encoding the PCCA or PCCB polypeptide. In a particular embodiment, the ORF of the mRNA encoding the PCCA or PCCB polypeptide of the invention contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-phenylalanine uracil pairs and/or triplets. In another embodiment, the ORF of the mRNA encoding the PCCA or PCCB polypeptide contains no non-phenylalanine uracil pairs and/or triplets.

[0162] In further embodiments, the ORF of the mRNA encoding a PCCA or PCCB polypeptide of the invention comprises modified uracil and has an adjusted uracil content containing less uracil-rich clusters than the corresponding wild-type nucleotide sequence encoding the PCCA or PCCB polypeptide. In some embodiments, the ORF of the mRNA encoding the PCCA or PCCB polypeptide of the invention contains uracil-rich clusters that are shorter in length than corresponding uracil-rich clusters in the corresponding wild-type nucleotide sequence encoding the PCCA or PCCB polypeptide.

[0163] In further embodiments, alternative lower frequency codons are employed. At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in the PCCA or PCCB polypeptide-encoding ORF of the modified uracil-comprising mRNA are substituted with alternative codons, each alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set. The ORF also has adjusted uracil content, as described above. In some embodiments, at least one codon in the ORF of the mRNA encoding the PCCA or PCCB polypeptide is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.

[0164] In some embodiments, the adjusted uracil content, PCCA or PCCB polypeptide-encoding ORF of the modified uracil-comprising mRNA exhibits expression levels of PCCA or PCCB when administered to a mammalian cell that are higher than expression levels of PCCA or PCCB from the corresponding wild-type mRNA. In some embodiments, the mammalian cell is a mouse cell, a rat cell, or a rabbit cell. In other embodiments, the mammalian cell is a monkey cell or a human cell. In some embodiments, the human cell is a HeLa cell, a BJ fibroblast cell, or a peripheral blood mononuclear cell (PBMC). In some embodiments, PCCA or PCCB is expressed at a level higher than expression levels of PCCA or PCCB from the corresponding wild-type mRNA when the mRNA is administered to a mammalian cell in vivo. In some embodiments, the mRNA is administered to mice, rabbits, rats, monkeys, or humans. In one embodiment, mice are null mice. In some embodiments, the mRNA is administered intravenously or intramuscularly. In other embodiments, the PCCA or PCCB polypeptide is expressed when the mRNA is administered to a mammalian cell in vitro. In some embodiments, the expression is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 500-fold, at least about 1500-fold, or at least about 3000-fold. In other embodiments, the expression is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about 100%.

[0165] In some embodiments, adjusted uracil content, PCCA or PCCB polypeptide-encoding ORF of the modified uracil-comprising mRNA exhibits increased stability. In some embodiments, the mRNA exhibits increased stability in a cell relative to the stability of a corresponding wild-type mRNA under the same conditions. In some embodiments, the mRNA exhibits increased stability including resistance to nucleases, thermal stability, and/or increased stabilization of secondary structure. In some embodiments, increased stability exhibited by the mRNA is measured by determining the half-life of the mRNA (e.g., in a plasma, serum, cell, or tissue sample) and/or determining the area under the curve (AUC) of the protein expression by the mRNA over time (e.g., in vitro or in vivo). An mRNA is identified as having increased stability if the half-life and/or the AUC is greater than the half-life and/or the AUC of a corresponding wild-type mRNA under the same conditions.

[0166] In some embodiments, the mRNA of the present invention induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by a corresponding wild-type mRNA under the same conditions. In other embodiments, the mRNA of the present disclosure induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by an mRNA that encodes for a PCCA or PCCB polypeptide but does not comprise modified uracil under the same conditions, or relative to the immune response induced by an mRNA that encodes for a PCCA or PCCB polypeptide and that comprises modified uracil but that does not have adjusted uracil content under the same conditions. The innate immune response can be manifested by increased expression of pro-inflammatory cytokines, activation of intracellular PRRs (RIG-I, MDA5, etc.), cell death, and/or termination or reduction in protein translation. In some embodiments, a reduction in the innate immune response can be measured by expression or activity level of Type 1 interferons (e.g., IFN-?, IFN-?, IFN-?, IFN-?, IFN-?, IFN-?, IFN-?, and IFN-?) or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8), and/or by decreased cell death following one or more administrations of the mRNA of the invention into a cell.

[0167] In some embodiments, the expression of Type-1 interferons by a mammalian cell in response to the mRNA of the present disclosure is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% relative to a corresponding wild-type mRNA, to an mRNA that encodes a PCCA or PCCB polypeptide but does not comprise modified uracil, or to an mRNA that encodes a PCCA or PCCB polypeptide and that comprises modified uracil but that does not have adjusted uracil content. In some embodiments, the interferon is IFN-?. In some embodiments, cell death frequency caused by administration of mRNA of the present disclosure to a mammalian cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding wild-type mRNA, an mRNA that encodes for a PCCA or PCCB polypeptide but does not comprise modified uracil, or an mRNA that encodes for a PCCA or PCCB polypeptide and that comprises modified uracil but that does not have adjusted uracil content. In some embodiments, the mammalian cell is a BJ fibroblast cell. In other embodiments, the mammalian cell is a splenocyte. In some embodiments, the mammalian cell is that of a mouse or a rat. In other embodiments, the mammalian cell is that of a human. In one embodiment, the mRNA of the present disclosure does not substantially induce an innate immune response of a mammalian cell into which the mRNA is introduced.

Methods for Modifying Polynucleotides

[0168] The disclosure includes modified polynucleotides comprising a polynucleotide described herein (e.g., a polynucleotide, e.g. mRNA, comprising a nucleotide sequence encoding a PCCA or PCCB polypeptide). The modified polynucleotides can be chemically modified and/or structurally modified. When the polynucleotides of the present invention are chemically and/or structurally modified the polynucleotides can be referred to as modified polynucleotides.

[0169] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides) encoding a PCCA or PCCB polypeptide. A nucleoside refers to 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). A nucleotide refers to a nucleoside including a phosphate group. Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[0170] The modified polynucleotides disclosed herein can comprise various distinct modifications. In some embodiments, the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide, introduced to a cell can exhibit one or more desirable properties, e.g., improved protein expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodified polynucleotide. In some embodiments, a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA or PCCB polypeptide) is structurally 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 can be chemically modified to AT-5meC-G. The same polynucleotide can be structurally modified from ATCG to ATCCCG. Here, the dinucleotide CC has been inserted, resulting in a structural modification to the polynucleotide.

[0171] Therapeutic compositions of the present disclosure comprise, in some embodiments, at least one nucleic acid (e.g., RNA) having an open reading frame encoding PCCA or PCCB (e.g., SEQ ID NO:1 or SEQ ID NO:2), wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.

[0172] In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0173] In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; or PCT/IB2017/051367 all of which are incorporated by reference herein.

[0174] In some embodiments, at least one RNA (e.g., mRNA) of the present disclosure is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).

[0175] Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.

[0176] Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.

[0177] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0178] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0179] Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

[0180] The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A nucleoside refers to 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). A nucleotide refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.

[0181] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into nucleic acids of the present disclosure.

[0182] In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise N1-methyl-pseudouridine (m1?), 1-ethyl-pseudouridine (e1?), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (?). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxy methyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

[0183] In some embodiments, a RNA nucleic acid of the disclosure comprises N1-methyl-pseudouridine (m1?) substitutions at one or more or all uridine positions of the nucleic acid.

[0184] In some embodiments, a RNA nucleic acid of the disclosure comprises N1-methyl-pseudouridine (m1?) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0185] In some embodiments, a RNA nucleic acid of the disclosure comprises pseudouridine (?) substitutions at one or more or all uridine positions of the nucleic acid.

[0186] In some embodiments, a RNA nucleic acid of the disclosure comprises pseudouridine (?) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0187] In some embodiments, a RNA nucleic acid of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

[0188] In some embodiments, nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with N1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with N1-methyl-pseudouridine. Similarly, a nucleic acid 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.

[0189] The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly A tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0190] The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0191] The nucleic acids may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

Untranslated Regions (UTRs)

[0192] Untranslated regions (UTRs) are nucleic acid sections of a polynucleotide before a start codon (5 UTR) and after a stop codon (3 UTR) that are not translated. In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the invention comprising an open reading frame (ORF) encoding a PCCA or PCCB polypeptide further comprises UTR (e.g., a 5 UTR or functional fragment thereof, a 3 UTR or functional fragment thereof, or a combination thereof).

[0193] A UTR (e.g., 5 UTR or 3 UTR) can be homologous or heterologous to the coding region in a polynucleotide. In some embodiments, the UTR is homologous to the ORF encoding the PCCA or PCCB polypeptide. In some embodiments, the UTR is heterologous to the ORF encoding the PCCA or PCCB polypeptide.

[0194] In some embodiments, the polynucleotide comprises two or more 5 UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences. In some embodiments, the polynucleotide comprises two or more 3 UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.

[0195] In some embodiments, the 5 UTR or functional fragment thereof, 3 UTR or functional fragment thereof, or any combination thereof is sequence optimized.

[0196] In some embodiments, the 5UTR or functional fragment thereof, 3 UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil. UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency. A polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods. In some embodiments, a functional fragment of a 5 UTR or 3 UTR comprises one or more regulatory features of a full length 5 or 3 UTR, respectively.

[0197] Natural 5UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that 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 (SEQ ID NO: 214), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another G. 5 UTRs also have been known to form secondary structures that are involved in elongation factor binding.

[0198] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of a polynucleotide. 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, can enhance expression of polynucleotides in hepatic cell lines or liver. Likewise, use of 5UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).

[0199] In some embodiments, UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.

[0200] In some embodiments, the 5 UTR and the 3 UTR can be heterologous. In some embodiments, the 5 UTR can be derived from a different species than the 3 UTR. In some embodiments, the 3 UTR can be derived from a different species than the 5 UTR.

[0201] Co-owned International Patent Application No. PCT/US2014/021522 (Publ. No. WO/2014/164253, incorporated herein by reference in its entirety) provides a listing of exemplary UTRs that can be utilized in the polynucleotide of the present invention as flanking regions to an ORF.

[0202] Additional exemplary UTRs of the application include, but are not limited to, one or more 5UTR and/or 3UTR derived from the nucleic acid sequence of: a globin, such as an ?- or ?-globin (e.g., a Xenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 a polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17-?) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a sindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein (e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucose transporter (e.g., hGLUT1 (human glucose transporter 1)); an actin (e.g., human a or ? actin); a GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a topoisomerase (e.g., a 5UTR of a TOP gene lacking the 5 TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32 (L32); a ribosomal protein (e.g., human or mouse ribosomal protein, such as, for example, rps9); an ATP synthase (e.g., ATP5A1 or the ? subunit of mitochondrial H.sup.+-ATP synthase); a growth hormone e (e.g., bovine (bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1 al (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyte enhancer factor 2A (MEF2A); a ?-F1-ATPase, a creatine kinase, a myoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen (e.g., collagen type I, alpha 2 (Col1A2), collagen type I, alpha 1 (Col1A1), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1 (Col6A1)); a ribophorin (e.g., ribophorin I (RPNI)); a low density lipoprotein receptor-related protein (e.g., LRP1); a cardiotrophin-like cytokine factor (e.g., Nnt1); calreticulin (Calr); a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1); and a nucleobindin (e.g., Nucb1).

[0203] In some embodiments, the 5 UTR is selected from the group consisting of a ?-globin 5 UTR; a 5UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 a polypeptide (CYBA) 5 UTR; a hydroxysteroid (17-?) dehydrogenase (HSD17B4) 5 UTR; a Tobacco etch virus (TEV) 5 UTR; a Venezuelen equine encephalitis virus (TEEV) 5 UTR; a 5 proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5 UTR; a heat shock protein 70 (Hsp70) 5 UTR; a eIF4G 5 UTR; a GLUT1 5 UTR; functional fragments thereof and any combination thereof.

[0204] In some embodiments, the 3 UTR is selected from the group consisting of a ?-globin 3 UTR; a CYBA 3 UTR; an albumin 3 UTR; a growth hormone (GH) 3 UTR; a VEEV 3 UTR; a hepatitis B virus (HBV) 3 UTR; ?-globin 3UTR; a DEN 3 UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3 UTR; an elongation factor 1 al (EEF1A1) 3 UTR; a manganese superoxide dismutase (MnSOD) 3 UTR; a ? subunit of mitochondrial H(+)-ATP synthase (?-mRNA) 3 UTR; a GLUT1 3 UTR; a MEF2A 3 UTR; a ?-F1-ATPase 3 UTR; functional fragments thereof and combinations thereof.

[0205] Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the invention. In some embodiments, a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. In some embodiments, variants of 5 or 3 UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.

[0206] Additionally, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety.

[0207] UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5 and/or 3 UTR can be inverted, shortened, lengthened, or combined with one or more other 5 UTRs or 3 UTRs.

[0208] In some embodiments, the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5 UTR or 3 UTR. For example, a double UTR comprises two copies of the same UTR either in series or substantially in series. For example, a double beta-globin 3UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).

[0209] The polynucleotides of the invention can comprise combinations of features. For example, the ORF can be flanked by a 5UTR that comprises a strong Kozak translational initiation signal and/or a 3UTR comprising an oligo(dT) sequence for templated addition of a poly-A tail. A 5UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different UTRs (see, e.g., US2010/0293625, herein incorporated by reference in its entirety).

[0210] Other non-UTR sequences can be used as regions or subregions within the polynucleotides of the invention. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of the invention. Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels. In some embodiments, the polynucleotide of the invention comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1): 189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the polynucleotide comprises an IRES instead of a 5 UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5 UTR in combination with a non-synthetic 3 UTR.

[0211] In some embodiments, the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, TEE, which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide. As a non-limiting example, the TEE can be located between the transcription promoter and the start codon. In some embodiments, the 5 UTR comprises a TEE.

[0212] In one aspect, a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.

a. 5 UTR Sequences

[0213] 5 UTR sequences are important for ribosome recruitment to the mRNA and have been reported to play a role in translation (Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6).

[0214] Disclosed herein, inter alia, is a polynucleotide, e.g., mRNA, comprising an open reading frame encoding a PCCA and/or PCCB polypeptide (e.g., SEQ ID NO:1 or SEQ ID NO:2), which polynucleotide has a 5 UTR that confers an increased half-life, increased expression and/or increased activity of the polypeptide encoded by said polynucleotide, or of the polynucleotide itself. In an embodiment, a polynucleotide disclosed herein comprises: (a) a 5-UTR (e.g., as provided in Table 1 or a variant or fragment thereof); (b) a coding region comprising a stop element (e.g., as described herein); and (c) a 3-UTR (e.g., as described herein), and LNP compositions comprising the same. In an embodiment, the polynucleotide comprises a 5-UTR comprising a sequence provided in Table 1 or a variant or fragment thereof (e.g., a functional variant or fragment thereof).

[0215] In an embodiment, the polynucleotide having a 5 UTR sequence provided in Table 1 or a variant or fragment thereof, has an increase in the half-life of the polynucleotide, e.g., about 1.5-20-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-fold, or more. In an embodiment, the increase in half life is about 1.5-fold or more. In an embodiment, the increase in half life is about 2-fold or more. In an embodiment, the increase in half life is about 3-fold or more. In an embodiment, the increase in half life is about 4-fold or more. In an embodiment, the increase in half life is about 5-fold or more.

[0216] In an embodiment, the polynucleotide having a 5 UTR sequence provided in Table 1 or a variant or fragment thereof, results in an increased level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide. In an embodiment, the 5UTR results in about 1.5-20-fold increase in level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide. In an embodiment, the increase in level and/or activity is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-fold, or more. In an embodiment, the increase in level and/or activity is about 1.5-fold or more. In an embodiment, the increase in level and/or activity is about 2-fold or more. In an embodiment, the increase in level and/or activity is about 3-fold or more. In an embodiment, the increase in level and/or activity is about 4-fold or more. In an embodiment, the increase in level and/or activity is about 5-fold or more.

[0217] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 5 UTR, has a different 5 UTR, or does not have a 5 UTR described in Table 1 or a variant or fragment thereof.

[0218] In an embodiment, the increase in half-life of the polynucleotide is measured according to an assay that measures the half-life of a polynucleotide.

[0219] In an embodiment, the increase in level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide is measured according to an assay that measures the level and/or activity of a polypeptide.

[0220] In an embodiment, the 5 UTR comprises a sequence provided in Table 1 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 5 UTR sequence provided in Table 1, or a variant or a fragment thereof. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 78.

[0221] In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 50. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 51. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 52. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 53. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 54. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 55. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 56. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 57. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 58. In an embodiment, the 5 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 78.

[0222] In an embodiment, the 5 UTR comprises the sequence of SEQ ID NO:78. In an embodiment, the 5 UTR consists of the sequence of SEQ ID NO: 78.

[0223] In an embodiment, the 5 UTR comprises the sequence of SEQ ID NO:55. In an embodiment, the 5 UTR consists of the sequence of SEQ ID NO:55.

[0224] In an embodiment, a 5 UTR sequence provided in Table 1 has a first nucleotide which is an A. In an embodiment, a 5 UTR sequence provided in Table 1 has a first nucleotide which is a G.

TABLE-US-00001 TABLE1 5UTRsequences SEQID Sequence NO: name Sequence 50 A1 GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCC 51 A5 GGAAAUCCCCACAACCGCCUCAUAUCCAGGCUCAAGAAUAG AGCUCAGUGUUUUGUUGUUUAAUCAUUCCGACGUGUUUUGC GAUAUUCGCGCAAAGCAGCCAGUCGCGCGCUUGCUUUUAAG UAGAGUUGUUUUUCCACCCGUUUGCCAGGCAUCUUUAAUUU AACAUAUUUUUAUUUUUCAGGCUAACCUACGCCGCCACC 52 A6 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAU CUCCCUGAGCUUCAGGGAGCCCCGGCGCCGCCACC 53 A7 GGAAACCCCCCACCCCCGUAAGAGAGAAAAGAAGAGUAAGA AGAAAUAUAAGAUCUCCCUGAGCUUCAGGGAGCCCCGGCGC CGCCACC 54 A8 GGAGAACUUCCGCUUCCGUUGGCGCAAGCGCUUUCAUUUUU UCUGCUACCGUGACUAAG 55 A9 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG CCACC 56 A11 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAC (Refer- CCCGGCGCCGCCACC ence) 57 A2 GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAG UUUUCUCGCAACUAGCAAGCUUUUUGUUCUCGCCGCCGCC 58 A3 GGAAAUCGCAAAAUUUUCUUUUCGCGUUAGAUUUCUUUUAG UUUUCUUUCAACUAGCAAGCUUUUUGUUCUCGCCGCCGCC 59 A4 GGAAAUCGCAAAA(N.sub.2).sub.x(N.sub.3).sub.xCU (N.sub.4).sub.x(N.sub.5).sub.xCGCGUUAGAUUUCUUU UAGUUUUCUN.sub.6N.sub.7CAACUAGCA AGCUUUUUGUUCUCGCC(N.sub.8C C)x (N.sub.2).sub.xisauracilandxisaninteger from0to5,e.g.,whereinx=3or4; (N.sub.3).sub.xisaguanineandxisanintegerfrom0to1; (N.sub.4).sub.xisacytosineandxisanintegerfrom0to1; (N.sub.5).sub.xisauracilandxisanintegerfrom0to5, e.g.,whereinx=2or3; N.sub.6isauracilorcytosine; N.sub.7isauracilorguanine; N.sub.8isadenineorguanine andxisanintegerfrom0to1. 60 A27 GGAAAAUUUUAGCCUGGAACGUUAGAUAACUGUCCUGUUGU CUUUAUAUACUUGGUCCCCAAGUAGUUUGUCUUCCAAA 61 A12 GGAAACUUUAUUUAGUGUUACUUUAUUUUCUGUUUAUUUGU GUUUCUUCAGUGGGUUUGUUCUAAUUUCCUUGGCCGCC 62 A13 GGAAAAUCUGUAUUAGGUUGGCGUGUUCUUUGGUCGGUUGU UAGUAUUGUUGUUGAUUCGUUUGUGGUCGGUUGCCGCC 63 A14 GGAAAAUUAUUAACAUCUUGGUAUUCUCGAUAACCAUUCGU UGGAUUUUAUUGUAUUCGUAGUUUGGGUUCCUGCCGCC 64 A15 GGAAAUUAUUAUUAUUUCUAGCUACAAUUUAUCAUUGUAUU AUUUUAGCUAUUCAUCAUUAUUUACUUGGUGAUCAACA 65 A16 GGAAAUAGGUUGUUAACCAAGUUCAAGCCUAAUAAGCUUGG AUUCUGGUGACUUGCUUCACCGUUGGCGGGCACCGAUC 66 A17 GGAAAUCGUAGAGAGUCGUACUUAGUACAUAUCGACUAUCG GUGGACACCAUCAAGAUUAUAAACCAGGCCAGA 67 A18 GGAAACCCGCCCAAGCGACCCCAACAUAUCAGCAGUUGCCC AAUCCCAACUCCCAACACAAUCCCCAAGCAACGCCGCC 68 A19 GGAAAGCGAUUGAAGGCGUCUUUUCAACUACUCGAUUAAGG UUGGGUAUCGUCGUGGGACUUGGAAAUUUGUUGUUUCC 69 A20 GGAAACUAAUCGAAAUAAAAGAGCCCCGUACUCUUUUAUUU CUAUUAGGUUAGGAGCCUUAGCAUUUGUAUCUUAGGUA 70 A21 GGAAAUGUGAUUUCCAGCAACUUCUUUUGAAUAUAUUGAAU UCCUAAUUCAAAGCGAACAAAUCUACAAGCCAUAUACC 71 A22 GGAAAUCGUAGAGAGUCGUACUUACGUGGUCGCCAUUGCAU AGCGCGCGAAAGCAACAGGAACAAGAACGCGCC 72 A23 GGAAAUCGUAGAGAGUCGUACUUAGAAUAAACAGAGUCGGG UCGACUUGUCUCUGAUACUACGACGUCACAAUC 73 A24 GGAAAAUUUGCCUUCGGAGUUGCGUAUCCUGAACUGCCCAG CCUCCUGAUAUACAACUGUUCCGCUUAUUCGGGCCGCC 74 A25 GGAAAUCUGAGCAGGAAUCCUUUGUGCAUUGAAGACUUUAG AUUCCUCUCUGCGGUAGACGUGCACUUAUAAGUAUUUG 75 A26 GGAAAGCGAUUGAAGGCGUCUUUUCAACUACUCGAUUAAGG UUGGGUAUCGUCGUGGGACUUGGAAAUUUGUUGCCACC 76 A28 GGAAAUUUUUUUUUGAUAUUAUAAGAGUUUUUUUUUGAUAU UAAGAAAAUUUUUUUUUGAUAUUAGAAGAGUAAGAAGAAAU AUAAGACCCCGGCGCCGCCACC 77 A29 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG CCAAAAAAAAAAAACC 78 A30 GGAAAUCUCCCUGAGCUUCAGGGAGUAAGAGAGAAAAGAAG AGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC 79 A31 GCCRCC,whereinR=AorG

[0225] In an embodiment, the 5 UTR comprises a variant of SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a nucleic acid sequence of Formula A:

TABLE-US-00002 (SEQIDNO:59) GGAAAUCGCAAAA(N.sub.2).sub.x(N.sub.3).sub.xCU (N.sub.4).sub.x(N.sub.5).sub.xCGCGUUAGAUUUCUUU UAGUUUUCUN.sub.6N.sub.7CAACUAGCA AGCUUUUUGUUCUCGCC(N.sub.8C C)x
wherein: [0226] (N.sub.2).sub.x is a uracil and x is an integer from 0 to 5, e.g., wherein x-3 or 4; [0227] (N.sub.3).sub.x is a guanine and x is an integer from 0 to 1; [0228] (N.sub.4).sub.x is a cytosine and x is an integer from 0 to 1; [0229] (N.sub.5).sub.x is a uracil and x is an integer from 0 to 5, e.g., wherein x=2 or 3; [0230] N.sub.6 is a uracil or cytosine; [0231] N.sub.7 is a uracil or guanine; [0232] N.sub.8 is adenine or guanine and x is an integer from 0 to 1.

[0233] In an embodiment (N.sub.2).sub.x is a uracil and x is 0. In an embodiment (N.sub.2).sub.x is a uracil and x is 1. In an embodiment (N.sub.2).sub.x is a uracil and x is 2. In an embodiment (N.sub.2).sub.x is a uracil and x is 3. In an embodiment, (N.sub.2).sub.x is a uracil and x is 4. In an embodiment (N.sub.2).sub.x is a uracil and x is 5.

[0234] In an embodiment, (N.sub.3).sub.x is a guanine and x is 0. In an embodiment, (N.sub.3).sub.x is a guanine and x is 1.

[0235] In an embodiment, (N.sub.4).sub.x is a cytosine and x is 0. In an embodiment, (N.sub.4).sub.x is a cytosine and x is 1.

[0236] In an embodiment (N.sub.5).sub.x is a uracil and x is 0. In an embodiment (N.sub.5).sub.x is a uracil and x is 1. In an embodiment (N.sub.5).sub.x is a uracil and x is 2. In an embodiment (N.sub.5).sub.x is a uracil and x is 3. In an embodiment, (N.sub.5).sub.x is a uracil and x is 4. In an embodiment (N.sub.5).sub.x is a uracil and x is 5.

[0237] In an embodiment, N6 is a uracil. In an embodiment, N6 is a cytosine.

[0238] In an embodiment, N7 is a uracil. In an embodiment, N7 is a guanine.

[0239] In an embodiment, N8 is an adenine and x is 0. In an embodiment, N8 is an adenine and x is 1.

[0240] In an embodiment, N8 is a guanine and x is 0. In an embodiment, N8 is a guanine and x is 1.

[0241] In an embodiment, the 5 UTR comprises a variant of SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 50% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 60% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 70% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 80% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 90% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 95% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 96% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 97% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 98% identity to SEQ ID NO: 50. In an embodiment, the variant of SEQ ID NO: 50 comprises a sequence with at least 99% identity to SEQ ID NO: 50.

[0242] In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 5%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 10%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 20%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 30%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 40%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 50%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 60%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 70%. In an embodiment, the variant of SEQ ID NO: 50 comprises a uridine content of at least 80%.

[0243] In an embodiment, the variant of SEQ ID NO: 50 comprises at least 2, 3, 4, 5, 6 or 7 consecutive uridines (e.g., a polyuridine tract). In an embodiment, the polyuridine tract in the variant of SEQ ID NO: 50 comprises at least 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, or 3-5 consecutive uridines. In an embodiment, the polyuridine tract in the variant of SEQ ID NO: 50 comprises 4 consecutive uridines. In an embodiment, the polyuridine tract in the variant of SEQ ID NO: 50 comprises 5 consecutive uridines.

[0244] In an embodiment, the variant of SEQ ID NO: 50 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 polyuridine tracts. In an embodiment, the variant of SEQ ID NO: 50 comprises 3 polyuridine tracts. In an embodiment, the variant of SEQ ID NO: 50 comprises 4 polyuridine tracts. In an embodiment, the variant of SEQ ID NO: 50 comprises 5 polyuridine tracts.

[0245] In an embodiment, one or more of the polyuridine tracts are adjacent to a different polyuridine tract. In an embodiment, each of, e.g., all, the polyuridine tracts are adjacent to each other, e.g., all of the polyuridine tracts are contiguous.

[0246] In an embodiment, one or more of the polyuridine tracts are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18. 19, 20, 30, 40, 50 or 60 nucleotides.

[0247] In an embodiment, each of, e.g., all of, the polyuridine tracts are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18. 19, 20, 30, 40, 50 or 60 nucleotides.

[0248] In an embodiment, a first polyuridine tract and a second polyuridine tract are adjacent to each other.

[0249] In an embodiment, a subsequent, e.g., third, fourth, fifth, sixth or seventh, eighth, ninth, or tenth, polyuridine tract is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18. 19, 20, 30, 40, 50 or 60 nucleotides from the first polyuridine tract, the second polyuridine tract, or any one of the subsequent polyuridine tracts.

[0250] In an embodiment, a first polyuridine tract is separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, 15, 16, 17, 18. 19, 20, 30, 40, 50 or 60 nucleotides from a subsequent polyuridine tract, e.g., a second, third, fourth, fifth, sixth or seventh, eighth, ninth, or tenth polyuridine tract. In an embodiment, one or more of the subsequent polyuridine tracts are adjacent to a different polyuridine tract.

[0251] In an embodiment, the 5 UTR comprises a Kozak sequence, e.g., a GCCRCC nucleotide sequence (SEQ ID NO: 79) wherein R is an adenine or guanine. In an embodiment, the Kozak sequence is disposed at the 3 end of the 5UTR sequence.

[0252] In an aspect, the polynucleotide (e.g., mRNA) comprising an open reading frame encoding a PCCA or PCCB polypeptide (e.g., SEQ ID NO: 1 or SEQ ID NO:2) and comprising a 5 UTR sequence disclosed herein is formulated as an LNP. In an embodiment, the LNP composition comprises: (i) an ionizable lipid, e.g., an amino lipid; (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-lipid.

[0253] In another aspect, the LNP compositions of the disclosure are used in a method of treating PA in a subject.

[0254] In an aspect, an LNP composition comprising a polynucleotide disclosed herein encoding a PCCA or PCCB polypeptide, e.g., as described herein, can be administered with an additional agent, e.g., as described herein.

b. 3 UTR Sequences

[0255] 3UTR sequences have been shown to influence translation, half-life, and subcellular localization of mRNAs (Mayr C., Cold Spring Harb Persp Biol 2019 Oct. 1:11(10):a034728).

[0256] Disclosed herein, inter alia, is a polynucleotide, e.g., mRNA, comprising an open reading frame encoding a PCCA or PCCB polypeptide (e.g., SEQ ID NO: 1 or SEQ ID NO:2), which polynucleotide has a 3 UTR that confers an increased half-life, increased expression and/or increased activity of the polypeptide encoded by said polynucleotide, or of the polynucleotide itself. In an embodiment, a polynucleotide disclosed herein comprises: (a) a 5-UTR (e.g., as described herein); (b) a coding region comprising a stop element (e.g., as described herein); and (c) a 3-UTR (e.g., as provided in Table 2 or a variant or fragment thereof), and LNP compositions comprising the same. In an embodiment, the polynucleotide comprises a 3-UTR comprising a sequence provided in Table 2 or a variant or fragment thereof.

[0257] In an embodiment, the polynucleotide having a 3 UTR sequence provided in Table 2 or a variant or fragment thereof, results in an increased half-life of the polynucleotide, e.g., about 1.5-10-fold increase in half-life of the polynucleotide. In an embodiment, the increase in half-life is about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, or more. In an embodiment, the increase in half-life is about 1.5-fold or more. In an embodiment, the increase in half-life is about 2-fold or more. In an embodiment, the increase in half-life is about 3-fold or more. In an embodiment, the increase in half-life is about 4-fold or more. In an embodiment, the increase in half-life is about 5-fold or more. In an embodiment, the increase in half-life is about 6-fold or more. In an embodiment, the increase in half-life is about 7-fold or more. In an embodiment, the increase in half-life is about 8-fold. In an embodiment, the increase in half-life is about 9-fold or more. In an embodiment, the increase in half-life is about 10-fold or more.

[0258] In an embodiment, the polynucleotide having a 3 UTR sequence provided in Table 2 or a variant or fragment thereof, results in a polynucleotide with a mean half-life score of greater than 10.

[0259] In an embodiment, the polynucleotide having a 3 UTR sequence provided in Table 2 or a variant or fragment thereof, results in an increased level and/or activity, e.g., output, of the polypeptide encoded by the polynucleotide.

[0260] In an embodiment, the increase is compared to an otherwise similar polynucleotide which does not have a 3 UTR, has a different 3 UTR, or does not have a 3 UTR of Table 2 or a variant or fragment thereof.

[0261] In an embodiment, the polynucleotide comprises a 3 UTR sequence provided in Table 2 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a 3 UTR sequence provided in Table 2, or a fragment thereof. In an embodiment, the 3 UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO:115, or SEQ ID NO:136.

[0262] In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 100, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 100. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 101, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 101. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 102, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 102. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 103, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 103. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 104, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 104. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 105, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 105. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 106, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 106. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 107, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 107. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 108, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 108. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 109, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 109. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 110, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 110. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 111, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 111. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 112, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 112. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 113, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 113. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 114, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 114. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 115, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 115. In an embodiment, the 3 UTR comprises the sequence of SEQ ID NO: 136, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 136.

TABLE-US-00003 TABLE2 3UTRsequences SEQ Sequence IDNO information Sequence 100 B1 UAAUAGUAAGCUGGAGCCUCCUGAGAGACCUGUGUGAACUAUUG AGAAGAUCGGAACAGCUCCUUACUCUGAGGAAGUUGGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 101 B3 UAAUAGUAAGCUGGAGCCUCACUCUCCUCUCCAUCCCGUAUCCA GGCUGUGAAUUUUUCAAGGAAUAUAAAGAUCGGGAUGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 102 B4 UGAUAGUAAGCUGGAGCCUCUAGUGACGGCAACAGGGCUUGGUU UUUCCUUGUUGUGAAAUCGACAUCUCUGAAGACAGGGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 103 B5 UGAUAGUAAGCUGGAGCCUCCUUCCAUCUAGUCACAAAGACUCC UUCGUCCCCAGUUGCCGUCUAGGAUUGGGCCUCCCAGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 104 B6 UGAUAGUAAGCUGGAGCCUCCCAUAACAUGACAUAUCUGGAUUU UGUGCUUAGAACCUUAAAUUGGAAGCAUUCUUAAUUGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 105 B7 UAAUAGUAAGCUGGAGCCUCCGGAAAACUAAAAUAGAGAUAUUU CAAGAUUUUAUAAUUUUCAAAGACCUUUGAAAUAUUGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 106 B8 UAAUAGUAAGCUGGAGCCUCUACACAUUGCUUCUAGUUGGCAGA AAUAAUUGAUUAAAAGACCAGAAACUGUGAUAACUGGUACCCCC GUGGUCUUUAAAUAAAGUCUAAGUGGGCGGC 107 B9 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGGGC 108 B10 UGAUAAUAGGCUGGAGCCUCGGUGGCCUJAGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC GUGGUCUUUGAAUAAAGUCUGAGUGGGGGC 109 B11 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC CAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC 110 B12 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC CAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC 111 B13 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC UCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUG AGUGGGCGGC 112 B14 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC CGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC 113 B15 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCG GUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCU CCCCUUCCUGCACCCGUACCCCCCGCAUUAUUACUCACGGUACG AGUGGUCUUUGAAUAAAGUCUGAGUGGGGGGC 114 B16 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCG GUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAAC ACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCC CCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUC UGAGUGGGCGGC 115 B2 CUGAGAGACCUGUGUGAACUAUUGAGAAGAUCGGAACAGCUCCU UACUCUGAGGAAGUUG 116 B17 UGAUAAUAGGCUGGAGCCUCUCACACACCUCUGCCCCUUGGGCC UCCCACUCCCAUGGCUCUGGGCGGUCCAGAAGGAGCGUACCCCC GUGGUCUIJUGAAUAAAGUCUGAGUGGGGGGC 117 B18 UGAUAAUAGGCUGGAGCCUCCACCGCGUUAUCCGUUCCUCGUAG GCUGGUCCUGGGGAACGGGUCGGCGGGUACCCCCGUGGUCUUUG AAUAAAGUCUGAGUGGGGGGC 118 B19 UGAUAAUAGGCUGGAGCCUCUGCCCGGCAACGGCCAGGUCUGUG CCAAGUGUUUGCUGACGCAACCCCCACUGGCUGGGGCUUGGUCA UGGGCCAUCAGOGCGUGCGUGGAACCUUUUCGGCUCCUCUGCCG AUCCAUACUGCGGAACUCCUAGCCGCUUGUUUUGCUCGCAGCAG GUCUGGAGCAAACAUUAUCGGGACUGAUAACUCUGUUGUCCUGU ACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 119 B20 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCUU UUUCUUUCUUUUUUUCUUUUUUUUUCUUUUUUUUUUCUUUUUU UUUUUUUUUUCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 120 B21 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUG GGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCUU UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU UUUUUUUUUUCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 133 B22 UAAGUCUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUG GCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACU ACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCU CCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC 134 B23 UAAAGCGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGC CUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCC AUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGU GGGCGGC 135 B24 UAAAGCUCCCCGGGGUCCAUAAAGUAGGAAACACUACAGCUGGA GCCUCCUGAGAGACCUGUGUGAACUAUUGAGAAGAUCGGAACAG CUCCUUACUCUGAGGAAGUUGUCCAUAAAGUAGGAAACACUACA GUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAU AAAGUCUGAGUGGGCGGC 136 B25 UAAUAGUAAACCUCACUCACGGCCACAUUGAGUGCCAGGCUCCG GGCUGGUUUAUAGUAGUGUAGAGCAUUGCAGCACUUAGACUGGG GUGCUGUAGUCUUUAUUGUAGUCUUUCCACAUACCUGAUAAUUC UUAGAUAAUUUCUUAUUUUAAUUCCAUAAAGUAGGAAACACUAC AUAAAUCUCCAUAAAGUAGGAAACACUACAUAUUCUUCCAUAAA GUAGGAAACACUACAUAGGCU

[0263] In an embodiment, the 3 UTR comprises a micro RNA (miRNA) binding site, e.g., as described herein, which binds to a miR present in a human cell. In an embodiment, the 3 UTR comprises a miRNA binding site of SEQ ID NO: 212, SEQ ID NO: 174, SEQ ID NO: 152 or a combination thereof. In an embodiment, the 3 UTR comprises a plurality of miRNA binding sites, e.g., 2, 3, 4, 5, 6, 7 or 8 miRNA binding sites. In an embodiment, the plurality of miRNA binding sites comprises the same or different miRNA binding sites.

TABLE-US-00004 (SEQIDNO:212) miR122bs=CAAACACCAUUGUCACACUCCA (SEQIDNO:174) miR-142-3pbs=UCCAUAAAGUAGGAAACACUACA (SEQIDNO:152) miR-126bs=CGCAUUAUUACUCACGGUACGA

Regions Having a 5 Cap

[0264] The disclosure also includes a polynucleotide that comprises both a 5 Cap and a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA or PCCB polypeptide to be expressed).

[0265] The 5 cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5 proximal introns during mRNA splicing.

[0266] Endogenous mRNA molecules can be 5-end capped generating a 5-ppp-5-triphosphate linkage between a terminal guanosine cap residue and the 5-terminal transcribed sense nucleotide of the mRNA molecule. This 5-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5 end of the mRNA can optionally also be 2-O-methylated. 5-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0267] In some embodiments, the polynucleotides of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA or PCCB polypeptide) incorporate a cap moiety.

[0268] In some embodiments, polynucleotides of the present invention comprise a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5-ppp-5 phosphorodiester linkages, modified nucleotides can be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with ?-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5-ppp-5 cap. Additional modified guanosine nucleotides can be used such as ?-methyl-phosphonate and seleno-phosphate nucleotides.

[0269] Additional modifications include, but are not limited to, 2-O-methylation of the ribose sugars of 5-terminal and/or 5-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2-hydroxyl group of the sugar ring. Multiple distinct 5-cap structures can be used to generate the 5-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5-caps in their chemical structure, while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the invention.

[0270] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5-5-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3-O-methyl group (i.e., N7,3-O-dimethyl-guanosine-5-triphosphate-5-guanosine (m.sup.7G-3mppp-G; which can equivalently be designated 3 O-Me-m.sup.7G(5)ppp(5)G). The 3-O atom of the other, unmodified, guanine becomes linked to the 5-terminal nucleotide of the capped polynucleotide. The N7- and 3-O-methlyated guanine provides the terminal moiety of the capped polynucleotide.

[0271] Another exemplary cap is mCAP, which is similar to ARCA but has a 2-O-methyl group on guanosine (i.e., N7,2-O-dimethyl-guanosine-5-triphosphate-5-guanosine, m.sup.7Gm-ppp-G).

[0272] Another exemplary cap is m.sup.7G-ppp-Gm-A (i.e., N7,guanosine-5-triphosphate-2-O-dimethyl-guanosine-adenosine).

[0273] In some embodiments, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No. U.S. Pat. No. 8,519,110, the contents of which are herein incorporated by reference in its entirety.

[0274] In another embodiment, the cap is a cap analog is a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and/or described herein. Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5)ppp(5)G and a N7-(4-chlorophenoxyethyl)-m.sup.3OG(5)ppp(5)G cap analog (See, e.g. the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574; the contents of which are herein incorporated by reference in its entirety). In another embodiment, a cap analog of the present invention is a 4-chloro/bromophenoxyethyl analog.

[0275] Polynucleotides of the invention can also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes, in order to generate more authentic 5-cap structures. As used herein, the phrase more authentic refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a more authentic feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5cap structures of the present invention are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5 endonucleases and/or reduced 5decapping, as compared to synthetic 5cap structures known in the art (or to a wild-type, natural or physiological 5cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2-O-methyltransferase enzyme can create a canonical 5-5-triphosphate linkage between the 5-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5-terminal nucleotide of the mRNA contains a 2-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5cap analog structures known in the art. Cap structures include, but are not limited to, 7 mG(5)ppp(5)N1pN2p (cap 0), 7 mG(5)ppp(5)N1mpNp (cap 1), and 7 mG(5)-ppp(5)N1mpN2mp (cap 2).

[0276] As a non-limiting example, capping chimeric polynucleotides post-manufacture can be more efficient as nearly 100% of the chimeric polynucleotides can be capped. This is in contrast to ?80% when a cap analog is linked to a chimeric polynucleotide in the course of an in vitro transcription reaction.

[0277] According to the present invention, 5 terminal caps can include endogenous caps or cap analogs. According to the present invention, a 5 terminal cap can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0278] Also provided herein are exemplary caps including those that can be used in co-transcriptional capping methods for ribonucleic acid (RNA) synthesis, using RNA polymerase, e.g., wild type RNA polymerase or variants thereof, e.g., such as those variants described herein. In one embodiment, caps can be added when RNA is produced in a one-pot reaction, without the need for a separate capping reaction.

[0279] Thus, the methods, in some embodiments, comprise reacting a polynucleotide template with an RNA polymerase variant, nucleoside triphosphates, and a cap analog under in vitro transcription reaction conditions to produce RNA transcript.

[0280] As used here the term cap includes the inverted G nucleotide and can comprise one or more additional nucleotides 3 of the inverted G nucleotide, e.g., 1, 2, 3, or more nucleotides 3 of the inverted G nucleotide and 5 to the 5 UTR, e.g., a 5 UTR described herein.

[0281] Exemplary caps comprise a sequence of GG, GA, or GGA, wherein the underlined, italicized G is an in inverted G nucleotide followed by a 5-5-triphosphate group.

[0282] In one embodiment, a cap comprises a compound of formula (I)

##STR00006##

or a stereoisomer, tautomer or salt thereof, wherein

##STR00007## [0283] ring B.sub.1 is a modified or unmodified Guanine; [0284] ring B.sub.2 and ring B3 each independently is a nucleobase or a modified nucleobase; [0285] X.sub.2 is O, S(O).sub.p, NR.sub.24 or CR.sub.25R.sub.26 in which p is 0, 1, or 2; [0286] Y.sub.0 is O or CR.sub.6R.sub.7; [0287] Y1 is O, S(O).sub.n, CR.sub.6R.sub.7, or NR.sub.8, in which n is 0, 1, or 2; [0288] each is a single bond or absent, wherein when each is a single bond, Yi is O, S(O).sub.n, CR.sub.6R.sub.7, or NR.sub.8; and when each is absent, Y.sub.1 is void; [0289] Y.sub.2 is (OP(O)R.sub.4).sub.m in which m is 0, 1, or 2, or O(CR.sub.40R.sub.41)u-Q.sub.0-(CR.sub.42R.sub.43)v-, in which Q.sub.0 is a bond, O, S(O).sub.r, NR.sub.44, or CR.sub.45R.sub.46, r is 0, 1, or 2, and each of u and v independently is 1, 2, 3 or 4; [0290] each R.sub.2 and R.sub.2 independently is halo, LNA, or OR.sub.3; [0291] each R.sub.3 independently is H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6 alkynyl and R.sub.3, when being C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6 alkynyl, is optionally substituted with one or more of halo, OH and C.sub.1-C.sub.6 alkoxyl that is optionally substituted with one or more OH or OC(O)C.sub.1-C.sub.6 alkyl; [0292] each R.sub.4 and R.sub.4 independently is H, halo, C.sub.1-C.sub.6 alkyl, OH, SH, SeH, or BH.sub.3; [0293] each of R.sub.6, R.sub.7, and R.sub.8, independently, is -Q.sub.1-T.sub.1, in which Q.sub.1 is a bond or C.sub.1-C.sub.3 alkyl linker optionally substituted with one or more of halo, cyano, OH and C.sub.1-C.sub.6 alkoxy, and T.sub.1 is H, halo, OH, COOH, cyano, or R.sub.s1, in which R.sub.s1 is C.sub.1-C.sub.3 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, C(O)OC.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, NR.sub.31R.sub.32, (NR.sub.31R.sub.32R.sub.33).sup.+, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and R.sub.s1 is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C.sub.1-C.sub.6 alkyl, COOH, C(O)OC.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl, NR.sub.31R.sub.32, (NR.sub.31R.sub.32R.sub.33).sup.+, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; [0294] each of R.sub.10, R.sub.11, R.sub.12, R.sub.13 R.sub.14, and R.sub.15, independently, is -Q.sub.2-T.sub.2, in which Q.sub.2 is a bond or C.sub.1-C.sub.3 alkyl linker optionally substituted with one or more of halo, cyano, OH and C.sub.1-C.sub.6 alkoxy, and T2 is H, halo, OH, NH.sub.2, cyano, NO.sub.2, N.sub.3, R.sub.s2, or OR.sub.s2, in which R.sub.s2 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, NHC(O)C.sub.1-C.sub.6 alkyl, NR.sub.31R.sub.32, (NR.sub.31R.sub.32R.sub.33).sup.+, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and R.sub.s2 is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C.sub.1-C.sub.6 alkyl, COOH, C(O)OC.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl, NR.sub.31R.sub.32, (NR.sub.31R.sub.32R.sub.33).sup.+, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; or alternatively R.sup.12 together with R.sup.14 is oxo, or R.sup.13 together with R.sub.15 is oxo, [0295] each of R.sub.20, R.sub.21, R.sub.22, and R.sub.23 independently is -Q.sub.3-T.sub.3, in which Q.sub.3 is a bond or C.sub.1-C.sub.3 alkyl linker optionally substituted with one or more of halo, cyano, OH and C.sub.1-C.sub.6 alkoxy, and T.sub.3 is H, halo, OH, NH.sub.2, cyano, NO.sub.2, N.sub.3, R.sub.s3, or OR.sub.S3, in which R.sub.S3 is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, NHC(O)C.sub.1-C.sub.6 alkyl, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and R.sub.S3 is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C.sub.1-C.sub.6 alkyl, COOH, C(O)OC.sub.1-C.sub.6 alkyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; [0296] each of R.sub.24, R.sub.25, and R.sup.26 independently is H or C.sub.1-C.sub.6 alkyl; each of R.sub.27 and R.sub.28 independently is H or OR.sub.29; or R.sub.27 and R.sub.28 together form OR.sub.30O; each R.sub.29 independently is H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6 alkynyl and R.sub.29, when being C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6 alkynyl, is optionally substituted with one or more of halo, OH and C.sub.1-C.sub.6 alkoxyl that is optionally substituted with one or more OH or OC(O)C.sub.1-C.sub.6 alkyl; [0297] R.sub.30 is C.sub.1-C.sub.6 alkylene optionally substituted with one or more of halo, OH and C.sub.1-C.sub.6 alkoxyl; [0298] each of R.sub.31, R.sub.32, and R.sub.33, independently is H, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; [0299] each of R.sub.40, R.sub.41, R.sub.42, and R.sub.43 independently is H, halo, OH, cyano, N3, OP(O)R.sub.47R.sub.48, or C.sub.1-C.sub.6 alkyl optionally substituted with one or more OP(O)R.sub.47R.sub.48, or one R.sub.41 and one R.sub.43, together with the carbon atoms to which they are attached and Q.sub.0, form C.sub.4-C.sub.10 cycloalkyl, 4- to 14-membered heterocycloalkyl, C.sub.6-C.sub.10 aryl, or 5- to 14-membered heteroaryl, and each of the cycloalkyl, heterocycloalkyl, phenyl, or 5- to 6-membered heteroaryl is optionally substituted with one or more of OH, halo, cyano, N.sub.3, OxO, OP(O)R.sub.47R.sub.48, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, COOH, C(O)OC.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxyl, C.sub.1-C.sub.6 haloalkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino, and di-C.sub.1-C.sub.6 alkylamino; [0300] R.sub.44 is H, C.sub.1-C.sub.6 alkyl, or an amine protecting group; [0301] each of R.sub.45 and R.sub.46 independently is H, OP(O)R.sub.47R.sub.48, or C.sub.1-C.sub.6 alkyl optionally substituted with one or more OP(O)R.sub.47R.sub.48, and [0302] each of R.sub.47 and R.sub.48, independently is H, halo, C.sub.1-C.sub.6 alkyl, OH, SH, SeH, or BH.sub.3.sup.?.

[0303] It should be understood that a cap analog, as provided herein, may include any of the cap analogs described in international publication WO 2017/066797, published on 20 Apr. 2017, incorporated by reference herein in its entirety.

[0304] In some embodiments, the B2 middle position can be a non-ribose molecule, such as arabinose.

[0305] In some embodiments R.sub.2 is ethyl-based.

[0306] Thus, in some embodiments, a cap comprises the following structure:

##STR00008##

[0307] In other embodiments, a cap comprises the following structure:

##STR00009##

[0308] In yet other embodiments, a cap comprises the following structure:

##STR00010##

[0309] In still other embodiments, a cap comprises the following structure:

##STR00011##

[0310] In some embodiments, R is an alkyl (e.g., C.sub.1-C.sub.6 alkyl). In some embodiments, R is a methyl group (e.g., C.sub.1 alkyl). In some embodiments, R is an ethyl group (e.g., C.sub.2 alkyl).

[0311] In some embodiments, a cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, a cap comprises GAA. In some embodiments, a cap comprises GAC. In some embodiments, a cap comprises GAG. In some embodiments, a cap comprises GAU. In some embodiments, a cap comprises GCA. In some embodiments, a cap comprises GCC. In some embodiments, a cap comprises GCG. In some embodiments, a cap comprises GCU. In some embodiments, a cap comprises GGA. In some embodiments, a cap comprises GGC. In some embodiments, a cap comprises GGG. In some embodiments, a cap comprises GGU. In some embodiments, a cap comprises GUA. In some embodiments, a cap comprises GUC. In some embodiments, a cap comprises GUG. In some embodiments, a cap comprises GUU.

[0312] In some embodiments, a cap comprises a sequence selected from the following sequences: m.sup.7GpppApA, m.sup.7GpppApC, m.sup.7GpppApG, m.sup.7GpppApU, m.sup.7GpppCpA, m.sup.7GpppCpC, m.sup.7GpppCpG, m.sup.7GpppCpU, m.sup.7GpppGpA, m.sup.7GpppGpC, m.sup.7GpppGpG, m.sup.7GpppGpU, m.sup.7GpppUpA, m.sup.7GpppUpC, m.sup.7GpppUpG, and m.sup.7GpppUpU.

[0313] In some embodiments, a cap comprises m.sup.7GpppApA. In some embodiments, a cap comprises m.sup.7GpppApC. In some embodiments, a cap comprises m.sup.7GpppApG. In some embodiments, a cap comprises m.sup.7GpppApU. In some embodiments, a cap comprises m.sup.7GpppCpA. In some embodiments, a cap comprises m.sup.7GpppCpC. In some embodiments, a cap comprises m.sup.7GpppCpG. In some embodiments, a cap comprises m.sup.7GpppCpU. In some embodiments, a cap comprises m.sup.7GpppGpA. In some embodiments, a cap comprises m.sup.7GpppGpC. In some embodiments, a cap comprises m.sup.7GpppGpG. In some embodiments, a cap comprises m.sup.7GpppGpU. In some embodiments, a cap comprises m.sup.7GpppUpA. In some embodiments, a cap comprises m.sup.7GpppUpC. In some embodiments, a cap comprises m.sup.7GpppUpG. In some embodiments, a cap comprises m.sup.7GpppUpU.

[0314] A cap, in some embodiments, comprises a sequence selected from the following sequences: m.sup.7G.sub.3OMepppApA, m.sup.7G.sub.3OMepppApC, m.sup.7G.sub.3OMepppApG, m.sup.7G.sub.3OMeppApU, m.sup.7G.sub.3OMepppCpA, m.sup.7G.sub.3OMepppCpC, m.sup.7G.sub.3OMepppCpG, m.sup.7G.sub.3OMepppCpU, m.sup.7G.sub.3OMepppGpA, m.sup.7G.sub.3OMepppGpC, m.sup.7G.sub.3OMepppGpG, m.sup.7G.sub.3OMepppGpU, m.sup.7G.sub.3OMepppUpA, m.sup.7G.sub.3OMepppUpC, m.sup.7G.sub.3OMepppUpG, and m.sup.7G.sub.3OMepppUpU.

[0315] In some embodiments, a cap comprises m.sup.7G.sub.3OMepppApA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppApC. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppApG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppApU. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppCpA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppCpC. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppCpG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppCpU. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppGpA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppGpC. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppGpG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppGpU. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppUpA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppUpC. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppUpG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppUpU.

[0316] A cap, in other embodiments, comprises a sequence selected from the following sequences: m.sup.7G.sub.3OMepppA.sub.2OMepA, m.sup.7G.sub.3OMepppA.sub.2OMepC, m.sup.7G.sub.3OMepA.sub.2OMepG, m.sup.7G.sub.3OMepA.sub.2OMepU, m.sup.7G.sub.2OMeppC.sub.2OMepA, m.sup.7G.sub.3OMepppC.sub.2OMepC, m.sup.7G.sub.3OMepppC.sub.2OMepG, m.sup.7G.sub.3OMepppC.sub.2OMepU, m.sup.7G.sub.3OMeppG.sub.2OMepA, m.sup.7G.sub.3OMepG.sub.2OMepC, m.sup.7G.sub.3OMeppG.sub.2OMepG, m.sup.7G.sub.3OMepppG.sub.2OMepU, m.sup.7G.sub.3OMepppU.sub.2OMepA, m.sup.7G.sub.3OMepppU.sub.2OMepC, m.sup.7G.sub.3OMepppU.sub.2OMepG, and m.sup.7G.sub.3OMepppU.sub.2OMepU.

[0317] In some embodiments, a cap comprises m.sup.7G.sub.3OMepppA.sub.2OMepA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppA.sub.2OMepC. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppA.sub.2OMepG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppA.sub.2OMepU. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppC.sub.2OMepA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppC.sub.2OMepC.

[0318] In some embodiments, a cap comprises m.sup.7G.sub.3OMepppC.sub.2OMepG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppC.sub.2OMepU. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppG.sub.2OMepA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppG.sub.2OMepC. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppG.sub.2OMepG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppG.sub.2OMepU. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppU.sub.2OMepA. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppU.sub.2OMepC. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppU.sub.2OMepG. In some embodiments, a cap comprises m.sup.7G.sub.3OMepppU.sub.2OMepU.

[0319] A cap, in still other embodiments, comprises a sequence selected from the following sequences: m.sup.7GpppA.sub.2OMepA, m.sup.7GpppA.sub.2OMepC, m.sup.7GpppA.sub.2OMepG, m.sup.7GppA.sub.2OMepU, m.sup.7GpC.sub.2OMepA, m.sup.7GppC.sub.2OMepC, m.sup.7GppC.sub.2OMepG, m.sup.7GpppC.sub.2OMepU, m.sup.7GpppG.sub.2OMepA, m.sup.7GpppG.sub.2OMepC, m.sup.7GpppG.sub.2OMepG, m.sup.7GpppG.sub.2OMepU, m.sup.7GpppU.sub.2OMepA, m.sup.7GpppU.sub.2OMepC, m.sup.7GpppU.sub.2OMepG, and m.sup.7GpppU.sub.2OMepU.

[0320] In some embodiments, a cap comprises m.sup.7GpppA.sub.2OMepA. In some embodiments, a cap comprises m.sup.7GpppA.sub.2OMepC. In some embodiments, a cap comprises m.sup.7GpppA.sub.2OMepG. In some embodiments, a cap comprises m.sup.7GpppA.sub.2OMepU. In some embodiments, a cap comprises m.sup.7GpppC.sub.2OMepA. In some embodiments, a cap comprises m.sup.7GpppC.sub.2OMepC. In some embodiments, a cap comprises m.sup.7GpppC.sub.2OMepG. In some embodiments, a trinucleotide cap comprises m.sup.7GpppC.sub.2OMepU. In some embodiments, a cap comprises m.sup.7GpppG.sub.2OMepA. In some embodiments, a cap comprises m.sup.7GpppG.sub.2OMepC. In some embodiments, a cap comprises m.sup.7GpppG.sub.2OMepG. In some embodiments, a cap comprises m.sup.7GpppG.sub.2OMepU. In some embodiments, a cap comprises m.sup.7GpppU.sub.2OMepA. In some embodiments, a cap comprises m.sup.7GpppU.sub.2OMepC. In some embodiments, a cap comprises m.sup.7GpppU.sub.2OMepG. In some embodiments, a cap comprises m.sup.7GpppU.sub.2OMepU.

[0321] In some embodiments, a cap comprises m.sup.7Gpppm.sup.6A.sub.2OmepG. In some embodiments, a cap comprises m.sup.7Gpppe.sup.6A.sub.2OmepG.

[0322] In some embodiments, a cap comprises GAG. In some embodiments, a cap comprises GCG. In some embodiments, a cap comprises GUG. In some embodiments, a cap comprises GGG.

[0323] In some embodiments, a cap comprises any one of the following structures:

##STR00012##

[0324] In some embodiments, the cap comprises .sup.m7GpppN.sub.1N.sub.2N.sub.3, where N.sub.1, N.sub.2, and N.sub.3 are optional (i.e., can be absent or one or more can be present) and are independently a natural, a modified, or an unnatural nucleoside base. In some embodiments, .sup.m7G is further methylated, e.g., at the 3 position. In some embodiments, the .sup.m7G comprises an O-methyl at the 3 position. In some embodiments N.sub.1, N.sub.2, and N.sub.3 if present, optionally, are independently an adenine, a uracil, a guanidine, a thymine, or a cytosine. In some embodiments, one or more (or all) of N.sub.1, N.sub.2, and N.sub.3, if present, are methylated, e.g., at the 2 position. In some embodiments, one or more (or all) of N.sub.1, N.sub.2, and N.sub.3, if present have an O-methyl at the 2 position.

[0325] In some embodiments, the cap comprises the following structure:

##STR00013## [0326] wherein B.sub.1, B.sub.2, and B.sub.3 are independently a natural, a modified, or an unnatural nucleoside based; and R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently OH or O-methyl. In some embodiments, R.sub.3 is O-methyl and R.sub.4 is OH. In some embodiments, R.sub.3 and R.sub.4 are O-methyl. In some embodiments, R.sub.4 is O-methyl. In some embodiments, R.sub.1 is OH, R.sub.2 is OH, R.sub.3 is O-methyl, and R.sub.4 is OH. In some embodiments, R.sub.1 is OH, R.sub.2 is OH, R.sub.3 is O-methyl, and R.sub.4 is O-methyl. In some embodiments, at least one of R.sub.1 and R.sub.2 is O-methyl, R.sub.3 is O-methyl, and R.sub.4 is OH. In some embodiments, at least one of R.sub.1 and R.sub.2 is O-methyl, R.sub.3 is O-methyl, and R.sub.4 is O-methyl.

[0327] In some embodiments, B.sub.1, B.sub.3, and B.sub.3 are natural nucleoside bases. In some embodiments, at least one of B.sub.1, B.sub.2, and B.sub.3 is a modified or unnatural base. In some embodiments, at least one of B.sub.1, B.sub.2, and B.sub.3 is N6-methyladenine. In some embodiments, B.sub.1 is adenine, cytosine, thymine, or uracil. In some embodiments, B.sub.1 is adenine, B.sub.2 is uracil, and B.sub.3 is adenine. In some embodiments, R.sub.1 and R.sub.2 are OH, R.sub.3 and R.sub.4 are O-methyl, B.sub.1 is adenine, B.sub.2 is uracil, and B.sub.3 is adenine.

[0328] In some embodiments the cap comprises a sequence selected from the following sequences: GAAA, GACA, GAGA, GAUA, GCAA, GCCA, GCGA, GCUA, GGAA, GGCA, GGGA, GGUA, GUCA, and GUUA. In some embodiments the cap comprises a sequence selected from the following sequences: GAAG, GACG, GAGG, GAUG, GCAG, GCCG, GCGG, GCUG, GGAG, GGCG, GGGG, GGUG, GUCG, GUGG, and GUUG. In some embodiments the cap comprises a sequence selected from the following sequences: GAAU, GACU, GAGU, GAUU, GCAU, GCCU, GCGU, GCUU, GGAU, GGCU, GGGU, GGUU, GUAU, GUCU, GUGU, and GUUU. In some embodiments the cap comprises a sequence selected from the following sequences: GAAC, GACC, GAGC, GAUC, GCAC, GCCC, GCGC, GCUC, GGAC, GGCC, GGGC, GGUC, GUAC, GUCC, GUGC, and GUUC.

[0329] A cap, in some embodiments, comprises a sequence selected from the following sequences: m.sup.7G.sub.3OMepppApApN, m.sup.7G.sub.3OMepppApCpN, m.sup.7G.sub.3OMepApGpN, m.sup.7G.sub.3OMepApUpN, m.sup.7G.sub.3OMeppCpApN, m.sup.7G.sub.3OMepppCpCpN, m.sup.7G.sub.3OMepppCpGpN, m.sup.7G.sub.3OMepppCpUpN, m.sup.7G.sub.3OMepppGpApN, m.sup.7G.sub.3OMepppGpCpN, m.sup.7G.sub.3OMepppGpGpN, m.sup.7G.sub.3OMepppGpUpN, m.sup.7G.sub.3OMepppUpApN, m.sup.7G.sub.3OMepppUpCpN, m.sup.7G.sub.3OMepppUpGpN, and m.sup.7G.sub.3OMepppUpUpN, where N is a natural, a modified, or an unnatural nucleoside base.

[0330] A cap, in other embodiments, comprises a sequence selected from the following sequences: m.sup.7G.sub.3OMepppA.sub.2OMepApN, m.sup.7G.sub.3OMeppp A.sub.2OMepCpN, m.sup.7G.sub.3OMeppA.sub.2OMepGpN, m.sup.7G.sub.3OMepA.sub.2OMepUpN, m.sup.7G.sub.3OMeppC.sub.2OMepApN, m.sup.7G.sub.3OMeppC.sub.2OMepCpN, m.sup.7G.sub.3OMepC.sub.2OMepGpN, m.sup.7G.sub.3OMeppC.sub.2OMepUpN, m.sup.7G.sub.3OMepG.sub.2OMepApN, m.sup.7G.sub.3OMepG.sub.2OMepCpN, m2G.sub.3OMeppG.sub.2OMepGpN, m.sup.7G.sub.3OMeppG.sub.2OMepUpN, m.sup.7G.sub.3OMeppU.sub.2OMepApN, m.sup.7G.sub.3OMeppU.sub.2OMepCpN, m.sup.7G.sub.3OMepppU.sub.2OMepGpN, and m.sup.7G.sub.3OMepppU.sub.2OMepUpN, where N is a natural, a modified, or an unnatural nucleoside base.

[0331] A cap, in still other embodiments, comprises a sequence selected from the following sequences: m.sup.7GpppA.sub.2OMepApN, m.sup.7GpppA.sub.2OMepCpN, m.sup.7GpppA.sub.2OMepGpN, m.sup.7GpppA.sub.2OMepUpN, m.sup.7GpppC.sub.2OMepApN, m.sup.7GpppC.sub.2OMepCpN, m.sup.7GpppC.sub.2OMepGpN, m.sup.7GpppC.sub.2OMepUpN, m.sup.7GpppG.sub.2OMepApN, m.sup.7GpppG.sub.2OMepCpN, m.sup.7GpppG.sub.2OMepGpN, m.sup.7m.sup.7GpppG.sub.2OMepUpN, m.sup.7GpppU.sub.2OMepApN, m.sup.7GpppU.sub.2OMepCpN, m.sup.7GpppU.sub.2OMepGpN, and m.sup.7GpppU.sub.2OMepUpN, where N is a natural, a modified, or an unnatural nucleoside base.

[0332] A cap, in other embodiments, comprises a sequence selected from the following sequences: m.sup.7G.sub.3OMepppA.sub.2OMepA.sub.2OMepN, m.sup.7G.sub.3OMepppA.sub.2OMepC.sub.2OMepN, m.sup.7G.sub.3OMepppA.sub.2OMepG.sub.2OMepN, m.sup.7G.sub.3OMepppA.sub.2OMepU.sub.2OMepN, m.sup.7G.sub.3OMepppC.sub.2OMePA.sub.2OMepN, m.sup.7G.sub.3OMeppPC.sub.2OMePC.sub.2OMepN, m.sup.7G.sub.3OMepG.sub.2OMepA.sub.2OMepN, m.sup.7G.sub.3OMepG.sub.2OMepC.sub.2OMepN, m.sup.7G.sub.3OMepU.sub.2OMepA.sub.2OMepN, m.sup.7G.sub.3OMepppU.sub.2OMepC.sub.2OMepN, m.sup.7G.sub.3OMepU.sub.2OMepG.sub.2OMepN, and m.sup.7G.sub.3OMeppU.sub.2OMepU.sub.2OMepN, where N is a natural, a modified, or an unnatural nucleoside base.

[0333] A cap, in still other embodiments, comprises a sequence selected from the following sequences: m.sup.7GpppA.sub.2OMepA.sub.2OMepN, m.sup.7GpppA.sub.2OMepC.sub.2OMepN, m.sup.7GppA.sub.2OMepG.sub.2OMepN, m.sup.7GpA.sub.2OMepU.sub.2OMepN, m.sup.7GppC.sub.2OMepA.sub.2OMepN, m.sup.7GpppC.sub.2OMepC.sub.2OMepN, m.sup.7GppC.sub.2OMepG.sub.2OMepN, m.sup.7GpppC.sub.2OMepU.sub.2OMepN, m.sup.7GppG.sub.2OMepA.sub.2OMepN, m.sup.7GppG.sub.2OMepC.sub.2OMepN, m.sup.7GppG.sub.2OMepG.sub.2OMepN, m.sup.7GppG.sub.2OMepU.sub.2OMepN, m.sup.7GppU.sub.2OMepA.sub.2OMepN, m.sup.7GpppU.sub.2OMepC.sub.2OMepN, m.sup.7GpppU.sub.2OMepG.sub.2OMepN, and m.sup.7GpppU.sub.2OMepU.sub.2OMepN, where N is a natural, a modified, or an unnatural nucleoside base.

[0334] In some embodiments, a cap comprises GGAG. In some embodiments, a cap comprises the following structure:

##STR00014##

Poly-A Tails

[0335] In some embodiments, the polynucleotides of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA and/or PCCB polypeptide) further comprise a poly-A tail. In further embodiments, terminal groups on the poly-A tail can be incorporated for stabilization. In other embodiments, a poly-A tail comprises des-3 hydroxyl tails.

[0336] During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3 end of the transcript can be cleaved to free a 3 hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long. In one embodiment, the poly-A tail is 100 nucleotides in length (SEQ ID NO:195).

[0337] Poly A tails can also be added after the construct is exported from the nucleus. According to the present invention, terminal groups on the poly A tail can be incorporated for stabilization. Polynucleotides of the present invention can include des-3 hydroxyl tails. They can also include structural moieties or 2-Omethyl modifications as taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507 Aug. 23, 2005, the contents of which are incorporated herein by reference in its entirety).

[0338] The polynucleotides of the present invention can be designed to encode transcripts with alternative poly A tail structures including histone mRNA. According to Norbury, Terminal uridylation has also been detected on human replication-dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of chromosomal DNA replication. These mRNAs are distinguished by their lack of a 3 poly(A) tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs (Norbury, Cytoplasmic RNA: a case of the tail wagging the dog, Nature Reviews Molecular Cell Biology:AOP, published online 29 Aug. 2013; doi: 10.1038/nrm3645) the contents of which are incorporated herein by reference in its entirety.

[0339] Unique poly-A tail lengths provide certain advantages to the polynucleotides of the present invention. Generally, the length of a poly-A tail, when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 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 nucleotides).

[0340] In some embodiments, the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0341] In some embodiments, the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.

[0342] In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A tail can also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of polynucleotides for Poly-A binding protein can enhance expression.

[0343] Additionally, multiple distinct polynucleotides can be linked together via the PABP (Poly-A binding protein) through the 3-end using modified nucleotides at the 3-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection.

[0344] In some embodiments, the polynucleotides of the present invention are designed to include a polyA-G Quartet region. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the poly A-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone (SEQ ID NO:196).

[0345] In some embodiments, the poly A tail comprises an alternative nucleoside, e.g., inverted thymidine. Poly A tails comprising an alternative nucleoside, e.g., inverted thymidine, may be generated as described herein. For instance, mRNA constructs may be modified by ligation to stabilize the poly(A) tail. Ligation may be performed using 0.5-1.5 mg/ml mRNA (5 Cap1, 3 A100), 50 mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2, 1 mM TCEP, 1000 units/mL T4 RNA Ligase 1, 1 mM ATP, 20% w/v polyethylene glycol 8000, and 5:1 molar ratio of modifying oligo to mRNA. Modifying oligo has a sequence of 5-phosphate-AAAAAAAAAAAAAAAAAAAA-(inverted deoxy thymidine (idT) (SEQ ID NO:209)) (see below). Ligation reactions are mixed and incubated at room temperature (?22? C.) for, e.g., 4 hours. Stable tail mRNA are purified by, e.g., dT purification, reverse phase purification, hydroxyapatite purification, ultrafiltration into water, and sterile filtration. The resulting stable tail-containing mRNAs contain the following structure at the 3end, starting with the poly A region: A100-UCUAGAAAAAAAAAAAAAAAAAAAA-inverted deoxy thymidine (SEQ ID NO:211).

[0346] Modifying oligo to stabilize tail (5-phosphate-AAAAAAAAAAAAAAAAAAAA-(inverted deoxythymidine)(SEQ ID NO:209)):

##STR00015##

[0347] In some instances, the poly A tail comprises A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211). In some instances, the poly A tail consists of A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211).

Start Codon Region

[0348] The invention also includes a polynucleotide that comprises both a start codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA or PCCB polypeptide). In some embodiments, the polynucleotides of the present invention can have regions that are analogous to or function like a start codon region.

[0349] In some embodiments, the translation of a polynucleotide can initiate on a codon that is not the start codon AUG. Translation of the polynucleotide can initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLOS ONE, 2010 5:11; the contents of each of which are herein incorporated by reference in its entirety).

[0350] As a non-limiting example, the translation of a polynucleotide begins on the alternative start codon ACG. As another non-limiting example, polynucleotide translation begins on the alternative start codon CTG or CUG. As yet another non-limiting example, the translation of a polynucleotide begins on the alternative start codon GTG or GUG.

[0351] Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See, e.g., Matsuda and Mauro PLOS ONE, 2010 5:11: the contents of which are herein incorporated by reference in its entirety). Masking any of the nucleotides flanking a codon that initiates translation can be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.

[0352] In some embodiments, a masking agent can be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acids (LNA) polynucleotides and exon-junction complexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agents LNA polynucleotides and EJCs (PLOS ONE, 2010 5:11); the contents of which are herein incorporated by reference in its entirety).

[0353] In another embodiment, a masking agent can be used to mask a start codon of a polynucleotide in order to increase the likelihood that translation will initiate on an alternative start codon. In some embodiments, a masking agent can be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.

[0354] In some embodiments, a start codon or alternative start codon can be located within a perfect complement for a miRNA binding site. The perfect complement of a miRNA binding site can help control the translation, length and/or structure of the polynucleotide similar to a masking agent. As a non-limiting example, the start codon or alternative start codon can be located in the middle of a perfect complement for a miRNA binding site. The start codon or alternative start codon can be located after the first nucleotide, second nucleotide, third nucleotide, fourth nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide, twentieth nucleotide or twenty-first nucleotide.

[0355] In another embodiment, the start codon of a polynucleotide can be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon that is not the start codon. Translation of the polynucleotide can begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon. In a non-limiting example, the start codon ATG or AUG is removed as the first 3 nucleotides of the polynucleotide sequence in order to have translation initiate on a downstream start codon or alternative start codon. The polynucleotide sequence where the start codon was removed can further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide.

Stop Codon Region

[0356] The invention also includes a polynucleotide that comprises both a stop codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA and/or PCCB polypeptide). In some embodiments, the polynucleotides of the present invention can include at least two stop codons before the 3 untranslated region (UTR). The stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA. In some embodiments, the polynucleotides of the present invention include the stop codon TGA in the case or DNA, or the stop codon UGA in the case of RNA, and one additional stop codon. In a further embodiment the addition stop codon can be TAA or UAA. In another embodiment, the polynucleotides of the present invention include three consecutive stop codons, four stop codons, or more.

Combination of mRNA Elements

[0357] Any of the polynucleotides disclosed herein can comprise one, two, three, or all of the following elements: (a) a 5-UTR, e.g., as described herein; (b) a coding region comprising a stop element (e.g., as described herein); (c) a 3-UTR (e.g., as described herein) and; optionally (d) a 3 stabilizing region, e.g., as described herein. Also disclosed herein are LNP compositions comprising the same.

[0358] In an embodiment, a polynucleotide of the disclosure comprises (a) a 5 UTR described in Table 1 or a variant or fragment thereof and (b) a coding region comprising a stop element provided herein. In an embodiment, the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as described herein. In an embodiment, the polynucleotide further comprises a 3 stabilizing region, e.g., as described herein.

[0359] In an embodiment, a polynucleotide of the disclosure comprises (a) a 5 UTR described in Table 1 or a variant or fragment thereof and (c) a 3 UTR described in Table 2 or a variant or fragment thereof. In an embodiment, the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as described herein. In an embodiment, the polynucleotide further comprises a 3 stabilizing region, e.g., as described herein.

[0360] In an embodiment, a polynucleotide of the disclosure comprises (c) a 3 UTR described in Table 2 or a variant or fragment thereof and (b) a coding region comprising a stop element provided herein. In an embodiment, the polynucleotide comprises a sequence provided in Table 3. In an embodiment, the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as described herein. In an embodiment, the polynucleotide further comprises a 3 stabilizing region, e.g., as described herein.

[0361] In an embodiment, a polynucleotide of the disclosure comprises (a) a 5 UTR described in Table 1 or a variant or fragment thereof; (b) a coding region comprising a stop element provided herein; and (c) a 3 UTR described in Table 2 or a variant or fragment thereof. In an embodiment, the polynucleotide further comprises a cap structure, e.g., as described herein, or a poly A tail, e.g., as described herein. In an embodiment, the polynucleotide further comprises a 3 stabilizing region, e.g., as described herein.

TABLE-US-00005 TABLE3 Exemplary3UTRandstopelementsequences SEQID Sequence NO information Sequence 121 3UTRwithstop UAGGGUUAAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C11(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 122 3UTRwithstop UAAAGCUCCGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C10(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 123 3UTRwithstop UAAGCCCCUGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C9(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 124 3UTRwithstop UAAGCACCCGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C8(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 125 3UTRwithstop UAAGCCCCUCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCC C7(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 126 3UTRwithstop UAAGGCUAAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C6(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 127 3UTRwithstop UAAGUCUCCGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C5(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 128 3UTRwithstop UAAAGCUAAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C4(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 129 3UTRwithstop UAAGUCUAAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCC C3(underlined) CUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCC GUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC 130 3UTRwithC10 UAAAGCUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUC stop(underlined) GGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAG GAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCA CCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUC UUUGAAUAAAGUCUGAGUGGGCGGC 131 3UTRwithC7 UAAGCCCCUCCGGGGUCCAUAAAGUAGGAAACACUACAGC stop(underlined) CUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAG UAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCU GCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC 132 3UTRwithC8 UAAAGCUCCCCGGGGUCCAUAAAGUAGGAAACACUACAGC stop(underlined) CUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAG UAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCU GCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC
Polynucleotide Comprising an mRNA Encoding a PCCA and/or PCCB Polypeptide

[0362] In certain embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a PCCA polypeptide, comprises from 5 to 3 end: [0363] (i) a 5 cap provided above; [0364] (ii) a 5 UTR, such as the sequences provided above; [0365] (iii) an ORF encoding a human PCCA polypeptide, wherein the ORF has at least 79%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:5; [0366] (iv) at least one stop codon; [0367] (v) a 3 UTR, such as the sequences provided above; and [0368] (vi) a poly-A tail provided above.

[0369] In certain embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a PCCB polypeptide, comprises from 5 to 3 end: [0370] (i) a 5 cap provided above; [0371] (ii) a 5 UTR, such as the sequences provided above; [0372] (iii) an ORF encoding a human PCCB polypeptide, wherein the ORF has at least 79%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:6; [0373] (iv) at least one stop codon; [0374] (v) a 3 UTR, such as the sequences provided above; and [0375] (vi) a poly-A tail provided above.

[0376] In some embodiments, the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miRNA-142. In some embodiments, the 5 UTR comprises the miRNA binding site. In some embodiments, the 3 UTR comprises the miRNA binding site.

[0377] In some embodiments, a polynucleotide of the present disclosure comprises a nucleotide sequence encoding a polypeptide sequence at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the protein sequence of human PCCA (SEQ ID NO:1).

[0378] In some embodiments, a polynucleotide of the present disclosure comprises a nucleotide sequence encoding a polypeptide sequence at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the protein sequence of human PCCB (SEQ ID NO:2).

[0379] In some embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a polypeptide, comprises (1) a 5 cap such as provided above, for example, m.sup.7Gp-ppGm-A, (2) a 5 UTR, (3) a nucleotide sequence ORF of SEQ ID NO:5, (3) a stop codon, (4) a 3UTR, and (5) a poly-A tail provided above, for example, a poly-A tail of SEQ ID NO:195 or A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211).

[0380] In some embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a polypeptide, comprises (1) a 5 cap such as provided above, for example, m.sup.7Gp-ppGm-A, (2) a 5 UTR, (3) a nucleotide sequence ORF of SEQ ID NO:6, (3) a stop codon, (4) a 3UTR, and (5) a poly-A tail provided above, for example, a poly-A tail of SEQ ID NO:195 or A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211).

[0381] An exemplary PCCA nucleotide construct is described: SEQ ID NO:7 consists from 5 to 3 end; 5 UTR of SEQ ID NO:55, ORF Sequence of SEQ ID NO:5, and 3 UTR of SEQ ID NO: 114.

[0382] In certain embodiments, in a construct with SEQ ID NO:7, all uracils therein are replaced by N1-methylpseudouracil.

[0383] An exemplary PCCB nucleotide construct is described: SEQ ID NO:8 consists from 5 to 3 end: 5 UTR of SEQ ID NO:55, ORF Sequence of SEQ ID NO:6, and 3 UTR of SEQ ID NO: 114.

[0384] In certain embodiments, in a construct with SEQ ID NO:8, all uracils therein are replaced by N1-methylpseudouracil.

[0385] In some embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a PCCA polypeptide, comprises (1) a 5 cap such as provided above, for example, m.sup.7Gp-ppGm-A, (2) a nucleotide sequence of SEQ ID NO:7, and (3) a poly-A tail provided above, for example, a poly A tail of ?100 residues, e.g., SEQ ID NO: 195 or A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211). In certain embodiments, in constructs with SEQ ID NO:7, all uracils therein are replaced by N1-methylpseudouracil.

[0386] In some embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding a PCCB polypeptide, comprises (1) a 5 cap such as provided above, for example, m.sup.7Gp-ppGm-A, (2) a nucleotide sequence of SEQ ID NO:8, and (3) a poly-A tail provided above, for example, a poly A tail of ?100 residues, e.g., SEQ ID NO:195 or A100-UCUAG-A20-inverted deoxy-thymidine (SEQ ID NO:211). In certain embodiments, in constructs with SEQ ID NO:8, all uracils therein are replaced by N1-methylpseudouracil.

Methods of Making Polynucleotides

[0387] The present disclosure also provides methods for making a polynucleotide of the invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA or PCCB polypeptide) or a complement thereof.

[0388] In some aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding a PCCA or PCCB polypeptide, can be constructed using in vitro transcription (IVT). In other aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding a PCCA or PCCB polypeptide, can be constructed by chemical synthesis using an oligonucleotide synthesizer.

[0389] In other aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding a PCCA or PCCB polypeptide is made by using a host cell. In certain aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding a PCCA or PCCB polypeptide is made by one or more combination of the IVT, chemical synthesis, host cell expression, or any other methods known in the art.

[0390] Naturally occurring nucleosides, non-naturally occurring nucleosides, or combinations thereof, can totally or partially naturally replace occurring nucleosides present in the candidate nucleotide sequence and can be incorporated into a sequence-optimized nucleotide sequence (e.g., a RNA, e.g., an mRNA) encoding a PCCA or PCCB polypeptide. The resultant polynucleotides, e.g., mRNAs, can then be examined for their ability to produce protein and/or produce a therapeutic outcome.

Pharmaceutical Compositions and Formulations

[0391] The present invention provides pharmaceutical compositions and formulations that comprise any of the polynucleotides described above. In some embodiments, the composition or formulation further comprises a delivery agent.

[0392] In some embodiments, the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a PCCA or PCCB polypeptide. In some embodiments, the composition or formulation can contain a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes a PCCA or PCCB polypeptide. In some embodiments, the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27 and miR-26a.

[0393] Pharmaceutical compositions or formulation can optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances. Pharmaceutical compositions or formulation of the present invention can be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents can 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). 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 polynucleotides to be delivered as described herein.

[0394] Formulations and pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient 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.

[0395] A pharmaceutical composition or formulation in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a unit dose refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. 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 can 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.

[0396] In some embodiments, the compositions and formulations described herein can contain at least one polynucleotide of the invention. As a non-limiting example, the composition or formulation can contain 1, 2, 3, 4 or 5 polynucleotides of the invention. In some embodiments, the compositions or formulations described herein can comprise more than one type of polynucleotide. In some embodiments, the composition or formulation can comprise a polynucleotide in linear and circular form. In another embodiment, the composition or formulation can comprise a circular polynucleotide and an in vitro transcribed (IVT) polynucleotide. In yet another embodiment, the composition or formulation can comprise an IVT polynucleotide, a chimeric polynucleotide and a circular polynucleotide.

[0397] Although the descriptions of pharmaceutical compositions and formulations provided herein are principally directed to pharmaceutical compositions and formulations that 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.

[0398] The present invention provides pharmaceutical formulations that comprise a polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a PCCA or PCCB polypeptide). The polynucleotides described herein 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 of the polynucleotide); (4) alter the biodistribution (e.g., target the polynucleotide 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 some embodiments, the pharmaceutical formulation further comprises a delivery agent comprising, e.g., a compound having the Formula (I), e.g., Compound II or Compound B; or a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound I or VI, or any combination thereof. In some embodiments, the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG-DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol % ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol % ionizable amino lipid, for example, 45-46 mol %, 46-47 mol %, 47-48 mol %, 48-49 mol %, or 49-50 mol % for example about 45 mol %, 45.5 mol %, 46 mol %, 46.5 mol %, 47 mol %, 47.5 mol %, 48 mol %, 48.5 mol %, 49 mol %, or 49.5 mol %; (ii) 30-45 mol % sterol (e.g., cholesterol), optionally 35-42 mol % sterol, for example, 30-31 mol %, 31-32 mol %, 32-33 mol %, 33-34 mol %, 35-35 mol %, 35-36 mol %, 36-37 mol %, 37-38 mol %, 38-39 mol %, or 39-40 mol %, or 40-42 mol % sterol; (iii) 5-15 mol % helper lipid (e.g., DSPC), optionally 10-15 mol % helper lipid, for example, 5-6 mol %, 6-7 mol %, 7-8 mol %, 8-9 mol %, 9-10 mol %, 10-11 mol %, 11-12 mol %, 12-13 mol %, 13-14 mol %, or 14-15 mol % helper lipid; and (iv) 1-5% PEG lipid (e.g., Compound I or PEG-DMG), optionally 1-5 mol % PEG lipid, for example 1.5 to 2.5 mol %, 1-2 mol %, 2-3 mol %, 3-4 mol %, or 4-5 mol % PEG lipid.

[0399] A pharmaceutically acceptable excipient, as used herein, includes, but are not limited to, any and all solvents, dispersion media, or other liquid vehicles, dispersion or suspension aids, diluents, granulating and/or dispersing agents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, binders, lubricants or oil, coloring, sweetening or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelants, cyoprotectants, and/or bulking agents, as suited to the particular dosage form desired. Various excipients for Formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, M D, 2006; incorporated herein by reference in its entirety).

[0400] Exemplary diluents include, but are not limited to, calcium or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, etc., and/or combinations thereof.

[0401] Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEENR80], sorbitan monopalmitate [SPAN?40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR?), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ?30]), PLUORINCRF 68, POLOXAMER?188, etc. and/or combinations thereof.

[0402] Exemplary binding agents include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.

[0403] Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. In order to prevent oxidation, antioxidants can be added to the formulations. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.

[0404] Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, etc., and combinations thereof.

[0405] Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzoic acid, hydroxy benzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof.

[0406] Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisol, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and combinations thereof.

[0407] In some embodiments, the pH of polynucleotide solutions is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH can include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc., and/or combinations thereof.

[0408] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, etc., and combinations thereof.

[0409] The pharmaceutical composition or formulation described here can contain a cryoprotectant to stabilize a polynucleotide described herein during freezing. Exemplary cryoprotectants include, but are not limited to mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., and combinations thereof.

[0410] The pharmaceutical composition or formulation described here can contain a bulking agent in lyophilized polynucleotide formulations to yield a pharmaceutically elegant cake, stabilize the lyophilized polynucleotides during long term (e.g., 36 month) storage. Exemplary bulking agents of the present invention can include, but are not limited to sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof.

[0411] In some embodiments, the pharmaceutical composition or formulation further comprises a delivery agent. The delivery agent of the present disclosure can include, without limitation, liposomes, lipid nanoparticles, lipidoids, polymers, lipoplexes, microvesicles, exosomes, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics, nanotubes, conjugates, and combinations thereof.

Delivery Agents

Lipid Compound

[0412] The present disclosure provides pharmaceutical compositions with advantageous properties. The lipid compositions described herein may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents, e.g., mRNAs, to mammalian cells or organs. For example, the lipids described herein have little or no immunogenicity. For example, the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA, has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0413] In certain embodiments, the present application provides pharmaceutical compositions comprising: [0414] (a) a polynucleotide comprising a nucleotide sequence encoding a PCCA and/or PCCB polypeptide; and [0415] (b) a delivery agent.

Lipid Nanoparticle Formulations

[0416] In some embodiments, nucleic acids of the invention (e.g., a PCCA and/or PCCB 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.

[0417] Nucleic acids of the present disclosure (e.g., a PCCA and/or PCCB 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.

[0418] 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 40-50 mol %, optionally 45-50 mol %, for example, 45-46 mol %, 46-47 mol %, 47-48 mol %, 48-49 mol %, or 49-50 mol %, for example about 45 mol %, 45.5 mol %, 46 mol %, 46.5 mol %, 47 mol %, 47.5 mol %, 48 mol %, 48.5 mol %, 49 mol %, or 49.5 mol % ionizable cationic lipid.

[0419] 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 5-15 mol %, optionally 10-12 mol %, for example, 5-6 mol %, 6-7 mol %, 7-8 mol %, 8-9 mol %, 9-10 mol %, 10-11 mol %, 11-12 mol %, 12-13 mol %, 13-14 mol %, or 14-15 mol % non-cationic lipid.

[0420] 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 30-45 mol %, optionally 35-40 mol %, for example, 30-31 mol %, 31-32 mol %, 32-33 mol %, 33-34 mol %, 35-35 mol %, 35-36 mol %, 36-37 mol %, 38-38 mol %, 38-39 mol %, or 39-40 mol % sterol.

[0421] 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 1-5%, optionally 1-3 mol %, for example 1.5 to 2.5 mol %, 1-2 mol %, 2-3 mol %, 3-4 mol %, or 4-5 mol % PEG-modified lipid.

[0422] 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.

[0423] In some embodiments, the lipid nanoparticle comprises a molar ratio of 40-50% ionizable cationic lipid, 5-15% non-cationic lipid, 30-45% sterol, and 1-5% PEG-modified lipid.

[0424] In some embodiments, the lipid nanoparticle comprises a molar ratio of 45-50% ionizable cationic lipid, 10-12% non-cationic lipid, 35-40% sterol, and 1-3% PEG-modified lipid.

[0425] In some embodiments, the lipid nanoparticle comprises a molar ratio of 45-50% ionizable cationic lipid, 10-12% non-cationic lipid, 35-40% sterol, and 1.5-2.5% PEG-modified lipid.

Ionizable Amino Lipids

[0426] In some aspects, the disclosure relates to a compound of Formula (I):

##STR00016##

or its N-oxide, or a salt or isomer thereof,
wherein R.sup.a is R.sup.branched, wherein [0427] R.sup.branched is:

##STR00017##

wherein

##STR00018##

denotes a point of attachment; [0428] wherein R.sup.a?, R.sup.a?, R.sup.a?, and R.sup.a? are each independently selected from the group consisting of H, C.sub.2-12 alkyl, and C.sub.2-12 alkenyl; [0429] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl; [0430] R.sup.4 is selected from the group consisting of (CH.sub.2)OH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and

##STR00019## [0431] wherein

##STR00020##

denotes a point of attachment; wherein [0432] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0433] each R.sup.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; [0434] each R.sup.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; [0435] M and M are each independently selected from the group consisting of C(O)O and

OC(O);

[0436] R is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0437] l is selected from the group consisting of 1, 2, 3, 4, and 5; and [0438] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0439] In some embodiments of the compounds of Formula (I), R.sup.a is R.sup.branched; [0440] R.sup.branched is

##STR00021##

denotes a point of attachment; R.sup.a?, R.sup.a?, R.sup.a?, and R.sup.a? are each H; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is (CH.sub.2).sub.nOH; n is 2; each R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; 1 is 5; and m is 7.

[0441] In some embodiments of the compounds of Formula (I), R.sup.a is R.sup.branched; [0442] R.sup.branched is

##STR00022##

denotes a point of attachment; R.sup.a?, R.sup.a?, R.sup.a?, and R.sup.a? are each H; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is (CH.sub.2).sub.nOH; n is 2; each R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; 1 is 3; and m is 7.

[0443] In some embodiments of the compounds of Formula (I), R.sup.a is R.sup.branched; [0444] R.sup.branched is

##STR00023##

denotes a point of attachment; R.sup.a? is C.sub.2-12 alkyl; R.sup.a?, R.sup.a?, and R.sup.a? are each H; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is

##STR00024##

R.sup.10 NH(C.sub.1-6 alkyl); n2 is 2; R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; l is 5; and m is 7.

[0445] In some embodiments of the compounds of Formula (I), R.sup.a is R.sup.branched; [0446] R.sup.branched is

##STR00025##

denotes a point of attachment; R.sup.a?, R.sup.a?, and R.sup.a? are each H; R.sup.a? is C.sub.2-12 alkyl; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is (CH.sub.2).sub.nOH; n is 2; each R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; l is 5; and m is 7.

[0447] In some embodiments, the compound of Formula (I) is selected from:

##STR00026##

[0448] In some embodiments, the compound of Formula (I) is:

##STR00027##

[0449] In some embodiments, the compound of Formula (I) is:

##STR00028##

[0450] In some embodiments, the compound of Formula (I) is:

##STR00029##

[0451] In some embodiments, the compound of Formula (I) is:

##STR00030##

[0452] In some aspects, the disclosure relates to a compound of Formula (Ia):

##STR00031##

or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched, wherein [0453] R.sup.branched is:

##STR00032##

wherein

##STR00033##

denotes a point of attachment; [0454] wherein R.sup.a?, R.sup.a?, and R.sup.a? are each independently selected from the group consisting of H, C.sub.2-12 alkyl, and C.sub.2-12 alkenyl; [0455] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl;

[0456] R.sup.4 is selected from the group consisting of (CH.sub.2).sub.nOH wherein n is selected

##STR00034##

from the group consisting of 1, 2, 3, 4, and 5, and [0457] wherein

##STR00035##

denotes a point of attachment; wherein [0458] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0459] each R.sup.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; [0460] each R.sup.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; [0461] M and M are each independently selected from the group consisting of C(O)O and OC(O); [0462] R is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0463] l is selected from the group consisting of 1, 2, 3, 4, and 5; and [0464] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0465] In some aspects, the disclosure relates to a compound of Formula (Ib):

##STR00036##

or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched, wherein [0466] R.sup.branched is:

##STR00037##

wherein

##STR00038##

denotes a point of attachment; [0467] wherein R.sup.a?, R.sup.a?, R.sup.a?, and R.sup.a? are each independently selected from the group consisting of H, C.sub.2-12 alkyl, and C.sub.2-12 alkenyl; [0468] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl; [0469] R.sup.4 is (CH.sub.2).sub.nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; [0470] each R.sup.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; [0471] each R.sup.6 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; [0472] M and M are each independently selected from the group consisting of C(O)O and

OC(O);

[0473] R is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0474] l is selected from the group consisting of 1, 2, 3, 4, and 5; and [0475] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0476] In some embodiments of Formula (I) or (Ib), R.sup.a is R.sup.branched; R.sup.branched is

##STR00039##

denotes a point of attachment; R.sup.a?, R.sup.a?, and R.sup.a? are each H; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is (CH.sub.2).sub.nOH; n is 2; each R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; I is 5; and m is 7.

[0477] In some embodiments of Formula (I) or (Ib), R.sup.a is R.sup.branched; R.sup.branched is

##STR00040##

denotes a point of attachment; R.sup.a?, R.sup.a?, and R.sup.a? are each H; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is (CH.sub.2)OH; n is 2; each R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; l is 3; and m is 7.

[0478] In some embodiments of Formula (I) or (Ib), R.sup.a is R.sup.branched; R.sup.branched is

##STR00041##

denotes a point of attachment; R.sup.a? and R.sup.a? are each H; R.sup.a? is C.sub.2-12 alkyl; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is (CH.sub.2).sub.nOH; n is 2; each R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; l is 5; and m is 7.

[0479] In some aspects, the disclosure relates to a compound of Formula (Ic):

##STR00042##

or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched, wherein [0480] R.sup.branched is:

##STR00043##

wherein

##STR00044##

denotes a point of attachment; [0481] wherein R.sup.a?, R.sup.a?, R.sup.a?, and R.sup.a? are each independently selected from the group consisting of H, C.sub.2-12 alkyl, and C.sub.2-12 alkenyl; [0482] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl; [0483] R.sup.4 is

##STR00045## [0484] wherein

##STR00046##

denotes a point of attachment; wherein [0485] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0486] each R.sup.5 is independently selected from the group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; [0487] each R.sup.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 each independently selected from the group consisting of C(O)O and

OC(O);

[0489] R is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0490] l is selected from the group consisting of 1, 2, 3, 4, and 5; and [0491] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

##STR00047##

[0492] In some embodiments, R.sup.a is R.sup.branched; R.sup.branched is denotes a point of attachment; R.sup.a?, R.sup.a?, and R.sup.a? are each H; R.sup.a? is C.sub.2-12 alkyl; R.sup.2 and R.sup.3 are each C.sub.1-14 alkyl; R.sup.4 is

##STR00048##

denotes a point of attachment; R.sup.10 is NH(C.sub.1-6 alkyl); n2 is 2; each R.sup.5 is H; each R.sup.6 is H; M and M are each C(O)O; R is a C.sub.1-12 alkyl; l is 5; and m is 7.

[0493] In some embodiments, the compound of Formula (Ic) is:

##STR00049##

[0494] In some aspects, the disclosure relates to a compound of Formula (II):

##STR00050##

or its N-oxide, or a salt or isomer thereof, [0495] wherein R.sup.a is R.sup.branched or R.sup.cyclic, wherein [0496] R.sup.branched is

##STR00051##

and Rcyclic is:

[0497] ##STR00052##

and [0498] R.sup.b is:

##STR00053## [0499] wherein

##STR00054##

denotes a point of attachment; [0500] R.sup.a? and R.sup.a? are each independently selected from the group consisting of H, C.sub.1-12 alkyl, and C.sub.2-12 alkenyl, wherein at least one of R.sup.a? and R.sup.a? is selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0501] R.sup.b? and R.sup.b? are each independently selected from the group consisting of H, C.sub.1-12 alkyl, and C.sub.2-12 alkenyl, wherein at least one of R.sup.b? and R.sup.b? is selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0502] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl; [0503] R.sup.4 is selected from the group consisting of (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and

##STR00055## [0504] wherein

##STR00056##

denotes a point of attachment; wherein [0505] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0506] each R independently is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0507] Y.sup.a is a C.sub.3-6 carbocycle; [0508] R*.sup.a is selected from the group consisting of C.sub.1-15 alkyl and C.sub.2-15 alkenyl; and [0509] s is 2 or 3; [0510] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; [0511] l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0512] In some aspects, the disclosure relates to a compound of Formula (II-a):

##STR00057##

or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched or R.sup.cyclic, wherein [0513] R.sup.branched is:

##STR00058##

and R.SUP.b .is:

[0514] ##STR00059## [0515] wherein

##STR00060##

denotes a point of attachment; [0516] R.sup.a? and R.sup.a? are each independently selected from the group consisting of H, C.sub.1-12 alkyl, and C.sub.2-12 alkenyl, wherein at least one of R.sup.a? and R.sup.a? is selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0517] R.sup.b? and R.sup.b? are each independently selected from the group consisting of H, C.sub.1-12 alkyl, and C.sub.2-12 alkenyl, wherein at least one of R.sup.b? and R.sup.b? is selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0518] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl; [0519] R.sup.4 is selected from the group consisting of (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and

##STR00061## [0520] wherein

##STR00062##

denotes a point of attachment; wherein [0521] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0522] each R independently is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0523] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; [0524] l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0525] In some aspects, the disclosure relates to a compound of Formula (II-b):

##STR00063##

or its N-oxide, or a salt or isomer thereof, [0526] wherein R.sup.a is R.sup.branched or R.sup.cyclic, wherein [0527] R.sup.branched is:

##STR00064##

and R.SUP.b .is:

[0528] ##STR00065## [0529] wherein

##STR00066##

denotes a point of attachment; [0530] R.sup.a? and R.sup.b? are each independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0531] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and C.sub.2-14 alkenyl; [0532] R.sup.4 is selected from the group consisting of (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and

##STR00067## [0533] wherein

##STR00068##

denotes a point of attachment; wherein [0534] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0535] each R independently is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0536] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; [0537] l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0538] In some aspects, the disclosure relates to a compound of Formula (II-c):

##STR00069## [0539] or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched or R.sup.cyclic, wherein [0540] R.sup.branched is:

##STR00070##

and R.SUP.b .is:

[0541] ##STR00071## [0542] wherein

##STR00072##

denotes a point of attachment; [0543] wherein R.sup.a? is selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0544] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl; [0545] R.sup.4 is selected from the group consisting of (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and

##STR00073## [0546] wherein

##STR00074##

denotes a point of attachment; wherein [0547] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0548] R is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0549] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; [0550] l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0551] In some aspects, the disclosure relates to a compound of Formula (II-d):

##STR00075##

or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched or R.sup.cyclic, wherein [0552] R.sup.branched is:

##STR00076##

and R.SUP.b .is:

[0553] ##STR00077## [0554] wherein

##STR00078##

denotes a point of attachment; [0555] wherein R.sup.a? and R.sup.b? are each independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0556] R.sup.4 is selected from the group consisting of (CH.sub.2)OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and

##STR00079## [0557] wherein

##STR00080##

denotes a point of attachment; wherein [0558] R.sup.10 is N(R).sub.2; each R is independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; [0559] each R independently is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0560] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; [0561] l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0562] In some aspects, the disclosure relates to a compound of Formula (II-e):

##STR00081##

or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched or R.sup.cyclic, wherein [0563] R.sup.branched is:

##STR00082##

and R.SUP.b .is:

[0564] ##STR00083## [0565] wherein

##STR00084##

denotes a point of attachment: [0566] wherein R.sup.a? is selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0567] R.sup.2 and R.sup.3 are each independently selected from the group consisting of C.sub.1-14 alkyl and
C.sub.2-14 alkenyl; [0568] R.sup.4 is (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; [0569] R is a C.sub.1-12 alkyl or C.sub.2-12 alkenyl; [0570] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; [0571] l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0572] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each independently selected from 4, 5, and 6. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and I are each 5.

[0573] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R independently is a C.sub.1-12 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R independently is a C.sub.2-5 alkyl.

[0574] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.b is:

##STR00085##

and R.sup.2 and R.sup.3 are each independently a C.sub.1-14 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.b is:

##STR00086##

and R.sup.2 and R.sup.3 are each independently a C.sub.6-10 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.b is:

##STR00087##

and R.sup.2 and R.sup.3 are each a C.sub.8 alkyl.

[0575] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00088##

and R.SUP.b .is:

[0576] ##STR00089##

R.sup.a? is a C.sub.1-12 alkyl and R.sub.2 and R.sub.3 are each independently a C.sub.6-10 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00090##

and R.SUP.b .is:

[0577] ##STR00091##

R.sup.a? is a C.sub.2-6 alkyl and R.sup.2 and R.sup.3 are each independently a C.sub.6-10 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00092##

and R.SUP.b .is:

[0578] ##STR00093##

R.sup.a? is a C.sub.2-6 alkyl, and R.sup.2 and R.sup.3 are each a C.sub.8 alkyl.

[0579] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00094##

R.SUP.b .is:

[0580] ##STR00095##

and R.sup.a? and R.sup.b? are each a C.sub.1-12 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00096##

R.SUP.b .is:

[0581] ##STR00097##

and R.sup.a? and R.sup.b? are each a C.sub.2-6 alkyl.

[0582] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each independently selected from 4, 5, and 6 and each R independently is a C.sub.1-12 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each 5 and each R independently is a C.sub.2-5 alkyl.

[0583] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00098##

R.SUP.b .is:

[0584] ##STR00099##

m and l are each independently selected from 4, 5, and 6, each R independently is a C.sub.1-12 alkyl, and R.sup.a? and R.sup.b? are each a C.sub.1-12 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00100##

R.SUP.b .is:

[0585] ##STR00101##

m and l are each 5, each R independently is a C.sub.2-5 alkyl, and R.sup.a? and R.sup.b? are each a C.sub.2-6 alkyl.

[0586] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00102##

and R.SUP.b .is:

[0587] ##STR00103##

m and l are each independently selected from 4, 5, and 6, R is a C.sub.1-12 alkyl, R.sup.a? is a C.sub.1-12 alkyl and R.sup.2 and R.sup.3 are each independently a C.sub.6-10 alkyl. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00104##

and R.SUP.b .is:

[0588] ##STR00105##

m and l are each 5, R is a C.sub.2-5 alkyl, R.sup.a? is a C.sub.2-6 alkyl, and R.sup.2 and R.sup.3 are each a C.sub.8 alkyl.

[0589] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.4 is

##STR00106##

wherein R.sup.10 is NH(C.sub.1-6 alkyl) and n2 is 2. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.4 is

##STR00107##

wherein R.sup.10 is NH(CH.sub.3) and n2 is 2.

[0590] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00108##

R.SUP.b .is:

[0591] ##STR00109##

m and l are each independently selected from 4, 5, and 6, each R independently is a C.sub.1-12 alkyl, R.sup.a? and R.sup.b? are each a C.sub.1-12 alkyl, and R.sup.4 is

##STR00110##

wherein R.sup.10 is NH(C.sub.1-6 alkyl), and n2 is 2. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00111##

R.SUP.b .is:

[0592] ##STR00112##

m and l are each .sub.5, each R independently is a C.sub.2-5 alkyl, R.sup.a? and R.sup.b? are each a C.sub.2-6 alkyl, and R.sup.4 is

##STR00113##

wherein R.sup.10 is NH(CH.sub.3) and n2 is 2.

[0593] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00114##

and R.SUP.b .is:

[0594] ##STR00115##

m and l are each independently selected from 4, 5, and 6, R is a C.sub.1-12 alkyl, R.sup.2 and R.sup.3 are each independently a C.sub.6-10 alkyl, R.sup.a? is a C.sub.1-12 alkyl, and R.sup.4 is

##STR00116##

wherein R.sup.10 is NH(C.sub.1-6 alkyl) and n2 is 2. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00117##

and R.SUP.b .is:

[0595] ##STR00118##

m and l are each 5, R is a C.sub.2-5 alkyl, R.sup.a? is a C.sub.2-6 alkyl, R.sup.2 and R.sup.3 are each a C.sub.8 alkyl, and R.sup.4 is

##STR00119##

wherein R.sup.10 is NH(CH.sub.3) and n2 is 2.

[0596] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.4 is (CH.sub.2).sub.nOH and n is 2, 3, or 4. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.4 is (CH.sub.2).sub.nOH and n is 2.

[0597] In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00120##

R.SUP.b .is:

[0598] ##STR00121##

m and l are each independently selected from 4, 5, and 6, each R independently is a C.sub.1-12 alkyl, R.sup.a? and R.sup.b? are each a C.sub.1-12 alkyl, R.sup.4 is (CH.sub.2).sub.nOH, and n is 2, 3, or 4. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R.sup.branched is:

##STR00122##

R.SUP.b .is:

[0599] ##STR00123##

m and l are each 5, each R independently is a C.sub.2-5 alkyl, R.sup.a? and R.sup.b? are each a C.sub.2-6 alkyl, R.sup.4 is (CH.sub.2).sub.nOH, and n is 2.

[0600] In some aspects, the disclosure relates to a compound of Formula (II-f):

##STR00124##

or its N-oxide, or a salt or isomer thereof, wherein R.sup.a is R.sup.branched or Rcyclic, wherein [0601] R.sup.branched is:

##STR00125##

and R.SUP.b .is:

[0602] ##STR00126## [0603] wherein

##STR00127##

denotes a point of attachment; [0604] R.sup.a? is a C.sub.1-12 alkyl; [0605] R.sup.2 and R.sup.3 are each independently a C.sub.1-14 alkyl; [0606] R.sup.4 is (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; [0607] R is a C.sub.1-12 alkyl; [0608] m is selected from 4, 5, and 6; and [0609] l is selected from 4, 5, and 6.

[0610] In some embodiments of the compound of Formula (II-f), m and l are each 5, and n is 2, 3, or 4.

[0611] In some embodiments of the compound of Formula (II-f) R is a C.sub.2-5 alkyl, R.sup.a? is a C.sub.2-6 alkyl, and R.sup.2 and R.sup.3 are each a C.sub.6-10 alkyl.

[0612] In some embodiments of the compound of Formula (II-f), m and l are each 5, [0613] n is 2, 3, or 4, R is a C.sub.2-5 alkyl, R.sup.a? is a C.sub.2-6 alkyl, and R.sup.2 and R.sup.3 are each a C.sub.6-10 alkyl.

[0614] In some aspects, the disclosure relates to a compound of Formula (II-g):

##STR00128##

wherein [0615] R.sup.a? is a C.sub.2-6 alkyl; [0616] R is a C.sub.2-5 alkyl; and [0617] R.sup.4 is selected from the group consisting of (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 3, 4, and 5, and

##STR00129## [0618] wherein

##STR00130##

denotes a point of attachment, R.sup.10 is NH(C.sub.1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.

[0619] In some aspects, the disclosure relates to a compound of Formula (II-h):

##STR00131##

wherein [0620] R.sup.a? and R.sup.b? are each independently a C.sub.2-6 alkyl; [0621] each R independently is a C.sub.2-5 alkyl; and [0622] R.sup.4 is selected from the group consisting of (CH.sub.2).sub.nOH wherein n is selected from the group consisting of 3, 4, and 5, and

##STR00132##

wherein

##STR00133##

denotes a point of attachment, R.sup.10 is NH(C.sub.1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.

[0623] In some embodiments of the compound of Formula (II-g) or (II-h), R.sup.4 is

##STR00134##

wherein [0624] R.sup.10 is NH(CH.sub.3) and n2 is 2.

[0625] In some embodiments of the compound of Formula (II-g) or (II-h), R.sup.4 is (CH.sub.2).sub.2OH.

[0626] In some aspects, the disclosure relates to a compound having the Formula (III):

##STR00135##

or a salt or isomer thereof, wherein [0627] 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, C.sub.5-20 alkenyl, RMR, R*YR, YR, and R*OR; [0628] 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).sub.2, an aryl group, and a heteroaryl group; [0629] 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), C(O)CH.sub.2, CH.sub.2C(O), C(O)OCH.sub.2, OC(O)CH.sub.2, CH.sub.2C(O)O, CH.sub.2OC(O), CH(OH), C(S), and CH(SH); [0630] each Y is independently a C.sub.3-6 carbocycle; [0631] each R* is independently selected from the group consisting of C.sub.1-12 alkyl and C.sub.2-12 alkenyl; [0632] each R is independently selected from the group consisting of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle; [0633] each R is independently selected from the group consisting of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H; and [0634] each R is independently selected from the group consisting of C.sub.3-12 alkyl and C.sub.3-12 alkenyl, and wherein; [0635] i) at least one of X.sup.1, X.sup.2, and X.sup.3 is not CH.sub.2; and/or [0636] ii) at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is RMR.

[0637] In some embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are each C.sub.5-20 alkyl; X.sup.1 is CH.sub.2; and X.sup.2 and X.sup.3 are each C(O).

[0638] In some embodiments, the compound of Formula (III) is:

##STR00136##

or a salt or isomer thereof.

Phospholipids

[0639] 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.

[0640] 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.

[0641] 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, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0642] 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.

[0643] 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).

[0644] 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.

[0645] 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.

[0646] 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):

##STR00137##

or a salt thereof, wherein: [0647] 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; [0648] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0649] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0650] A is of the Formula:

##STR00138## [0651] 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); [0652] 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, 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; [0653] each instance of R.sup.N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; [0654] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and [0655] p is 1 or 2; [0656] provided that the compound is not of the Formula:

##STR00139## [0657] wherein each instance of R.sup.2 is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0658] In some embodiments, the phospholipids may be one or more of the phospholipids described in U.S. Application No. 62/520,530.

Phospholipid Head Modifications

[0659] 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 R.sup.1 is not methyl. In certain embodiments, at least one of R.sup.1 is not hydrogen or methyl. In certain embodiments, the compound of Formula (IV) is of one of the following Formulae:

##STR00140##

or a salt thereof, wherein: [0660] each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0661] each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and [0662] each v is independently 1, 2, or 3.

[0663] In certain embodiments, a compound of Formula (IV) is of Formula (IV-a):

##STR00141##

or a salt thereof.

[0664] 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):

##STR00142##

or a salt thereof.

Phospholipid Tail Modifications

[0665] 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 R.sup.2 is each instance of R.sup.2 is optionally substituted C.sub.1-30 alkyl, 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, 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.

[0666] In certain embodiments, the compound of Formula (IV) is of Formula (IV-c):

##STR00143##

or a salt thereof, wherein: [0667] each x is independently an integer between 0-30, inclusive; and [0668] 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(R.sup.N), O, S, C(O), C(O)N(R.sup.N), NR.sup.NC(O), NR.sup.NC(O)N(R.sup.N), C(O)O, OC(O), OC(O)O, OC(O)N(R.sup.N), NR.sup.NC(O)O, C(O)S, SC(O), C(?NR.sup.N), C(?NR.sup.N)N(R.sup.N), NR.sup.NC(?NR.sup.N), NR.sup.NC(?NR.sup.N)N(R.sup.N), C(S), C(S)N(R.sup.N), NR.sup.NC(S), NR.sup.NC(S)N(R.sup.N), S(O), OS(O), S(O)O, OS(O)O, OS(O).sub.2, S(O).sub.2O, OS(O).sub.2O, N(R.sup.N)S(O), S(O)N(R.sup.N), N(R.sup.N)S(O)N(R.sup.N), OS(O)N(R.sup.N), N(R.sup.N)S(O)O, S(O).sub.2, N(R.sup.N)S(O).sub.2, S(O).sub.2N(R.sup.N), N(R.sup.N)S(O).sub.2N(R.sup.N), OS(O).sub.2N(R.sup.N), or N(R.sup.N)S(O).sub.2O. Each possibility represents a separate embodiment of the present invention.

[0669] 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:

##STR00144##

or a salt thereof.

Alternative Lipids

[0670] 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.

[0671] In certain embodiments, an alternative lipid is used in place of a phospholipid of the present disclosure.

[0672] In certain embodiments, an alternative lipid of the invention is oleic acid.

[0673] In certain embodiments, the alternative lipid is one of the following:

##STR00145##

Structural Lipids

[0674] 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.

[0675] 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.

[0676] In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No. 62/520,530.

Polyethylene Glycol (PEG)-Lipids

[0677] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.

[0678] 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.

[0679] 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).

[0680] 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.

[0681] In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C.sub.14 to about C.sub.22, preferably from about C.sub.14 to about C.sub.16. In some embodiments, a PEG moiety, for example an mPEG-NH.sub.2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG-lipid is PEG.sub.2k-DMG.

[0682] 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.

[0683] 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.

[0684] 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.

[0685] 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.

[0686] In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:

##STR00146##

[0687] 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.

[0688] In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (V). Provided herein are compounds of Formula (V):

##STR00147##

or salts thereof, wherein: [0689] R.sup.3 is OR.sup.O; [0690] R.sup.O is hydrogen, optionally substituted alkyl, or an oxygen protecting group; [0691] r is an integer between 1 and 100, inclusive; [0692] 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); [0693] D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; [0694] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; [0695] A is of the Formula:

##STR00148## [0696] 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); [0697] 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, 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; [0698] each instance of R.sup.N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; [0699] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and [0700] p is 1 or 2.

[0701] In certain embodiments, the compound of Formula (V) is a PEG-OH lipid (i.e., R.sup.3 is OR.sup.O, and R.sup.O is hydrogen). In certain embodiments, the compound of Formula (V) is of Formula (V-OH):

##STR00149##

or a salt thereof.

[0702] 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):

##STR00150##

or a salts thereof, wherein: [0703] R.sup.3 is OR.sup.O. [0704] R.sup.O is hydrogen, optionally substituted alkyl or an oxygen protecting group; [0705] r is an integer between 1 and 100, inclusive; [0706] 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, SC(O), C(?NR.sup.N), C(?NR.sup.N)N(R.sup.N), NR.sup.NC(?NR.sup.N), NR.sup.NC(?NR.sup.N)N(R.sup.N), C(S), C(S)N(R.sup.N), NR.sup.NC(S), NR.sup.NC(S)N(R.sup.N), S(O), OS(O), S(O)O, OS(O)O, OS(O).sub.2, S(O).sub.2O, OS(O).sub.2O, N(R.sup.N)S(O), S(O)N(R.sup.N), N(R.sup.N)S(O)N(R.sup.N), OS(O)N(R.sup.N), N(R.sup.N)S(O)O, S(O).sub.2, N(R.sup.N)S(O).sub.2, S(O).sub.2N(R.sup.N), N(R.sup.N)S(O).sub.2N(R.sup.N), OS(O).sub.2N(R.sup.N), or N(R.sup.N)S(O).sub.2O; and [0707] each instance of R.sup.N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0708] In certain embodiments, the compound of Formula (VI) is of Formula (VI-OH):

##STR00151##

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

[0709] In yet other embodiments the compound of Formula (VI) is:

##STR00152##

or a salt thereof.

[0710] In one embodiment, the compound of Formula (VI) is

##STR00153##

[0711] In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

[0712] In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. 62/520,530.

[0713] 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.

[0714] 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.

[0715] 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.

[0716] 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.

[0717] 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.

[0718] 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.

[0719] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00154##

and a PEG lipid comprising Formula VI.

[0720] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00155##

and an alternative lipid comprising oleic acid.

[0721] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00156##

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

[0722] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00157##

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

[0723] In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

##STR00158## [0724] a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.

[0725] In some embodiments, a LNP of the invention comprises an N:P ratio of from about 2:1 to about 30:1.

[0726] In some embodiments, a LNP of the invention comprises an N:P ratio of about 6:1.

[0727] In some embodiments, a LNP of the invention comprises an N:P ratio of about 3:1.

[0728] 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.

[0729] 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.

[0730] 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.

[0731] In some embodiments, a LNP of the invention has a mean diameter from about 50 nm to about 150 nm.

[0732] In some embodiments, a LNP of the invention has a mean diameter from about 70 nm to about 120 nm.

[0733] As used herein, the term alkyl, alkyl group, or alkylene means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation C.sub.1-14 alkyl means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.

[0734] As used herein, the term alkenyl, alkenyl group, or alkenylene means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation C.sub.2-14 alkenyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, C.sub.18 alkenyl may include one or more double bonds. A C.sub.18 alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

[0735] As used herein, the term alkynyl, alkynyl group, or alkynylene means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted. The notation C.sub.2-14 alkynyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. For example, C.sub.18 alkynyl may include one or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

[0736] As used herein, the term carbocycle or carbocyclic group means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. The notation C.sub.3-6 carbocycle means a carbocycle including a single ring having 3-6 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2 dihydronaphthyl groups. The term cycloalkyl as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.

[0737] As used herein, the term heterocycle or heterocyclic group means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings. Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. The term heterocycloalkyl as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.

[0738] As used herein, the term heteroalkyl, heteroalkenyl, or heteroalkynyl, refers respectively to an alkyl, alkenyl, alkynyl group, as defined herein, which further comprises one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus) wherein the one or more heteroatoms is inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms is inserted between a carbon atom and the parent molecule, i.e., between the point of attachment. Unless otherwise specified, heteroalkyls, heteroalkenyls, or heteroalkynyls described herein refers to both unsubstituted and substituted heteroalkyls, heteroalkenyls, or heteroalkynyls, i.e., optionally substituted heteroalkyls, heteroalkenyls, or heteroalkynyls.

[0739] As used herein, a biodegradable group is a group that may facilitate faster metabolism of a lipid in a mammalian entity. A biodegradable group may be selected from the group consisting of, but is not limited to, 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, an aryl group, and a heteroaryl group. As used herein, an aryl group is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a heteroaryl group is an optionally substituted heterocyclic group including one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. For example, M and M can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the Formulas herein, M and M can be independently selected from the list of biodegradable groups above. Unless otherwise specified, aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0740] Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Optional substituents may be selected from the group consisting of, but are not limited to, a halogen atom (e.g., a chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., C(O)OH), an alcohol (e.g., a hydroxyl, OH), an ester (e.g., C(O)OR OC(O)R), an aldehyde (e.g., C(O)H), a carbonyl (e.g., C(O)R, alternatively represented by C?O), an acyl halide (e.g., C(O)X, in which X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., OC(O)OR), an alkoxy (e.g., OR), an acetal (e.g., C(OR).sub.2R, in which each OR are alkoxy groups that can be the same or different and R is an alkyl or alkenyl group), a phosphate (e.g., P(O).sub.4.sup.3?), a thiol (e.g., SH), a sulfoxide (e.g., S(O)R), a sulfinic acid (e.g., S(O)OH), a sulfonic acid (e.g., S(O).sub.2OH), a thial (e.g., C(S)H), a sulfate (e.g., S(O).sub.4.sup.2?), a sulfonyl (e.g., S(O).sub.2), an amide (e.g., C(O)NR.sub.2, or N(R)C(O)R), an azido (e.g., N.sub.3), a nitro (e.g., NO.sub.2), a cyano (e.g., CN), an isocyano (e.g., NC), an acyloxy (e.g., OC(O)R), an amino (e.g., NR.sub.2, NRH, or NH.sub.2), a carbamoyl (e.g., OC(O)NR.sub.2, OC(O)NRH, or OC(O)NH.sub.2), a sulfonamide (e.g., S(O).sub.2NR.sub.2, S(O).sub.2NRH, S(O).sub.2NH.sub.2, N(R)S(O).sub.2R, N(H)S(O).sub.2R, N(R)S(O).sub.2H, or N(H)S(O).sub.2H), an alkyl group, an alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R is an alkyl or alkenyl group, as defined herein. In some embodiments, the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. For example, a C.sub.1-6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

[0741] Compounds of the disclosure that contain nitrogens can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxy benzoic acid (mCPBA) and/or hydrogen peroxides) to afford other compounds of the disclosure. Thus, all shown and claimed nitrogen-containing compounds are considered, when allowed by valency and structure, to include both the compound as shown and its N-oxide derivative (which can be designated as N.fwdarw.O or N+O). Furthermore, in other instances, the nitrogens in the compounds of the disclosure can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m CPBA. All shown and claimed nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as shown and its N-hydroxy (i.e., NOH) and N-alkoxy (i.e., NOR, wherein R is substituted or unsubstituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

Other Lipid Composition Components

[0742] The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components. For example, a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No. 2005/0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[0743] A polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and/or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[0744] The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt).

[0745] In some embodiments, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In some embodiments, the wt/wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

[0746] In some embodiments, the pharmaceutical composition disclosed herein can contain more than one polypeptides. For example, a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA).

[0747] In one embodiment, the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1 to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about 20:1, from about 15:1 to about 25:1, from about 15:1 to about 30:1, from about 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

[0748] In one embodiment, the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

Nanoparticle Compositions

[0749] In some embodiments, the pharmaceutical compositions disclosed herein are Formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding a PCCA and/or PCCB polypeptide. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding a PCCA and/or PCCB polypeptide.

[0750] Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0751] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and/or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

[0752] In one embodiment, a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid. In some embodiments, the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG-modified lipid.

[0753] In some embodiments, the LNP has a polydispersity value of less than 0.4. In some embodiments, the LNP has a net neutral charge at a neutral pH. In some embodiments, the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.

[0754] As generally defined herein, the term lipid refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids leads them to form liposomes, vesicles, or membranes in aqueous media.

[0755] In some embodiments, a lipid nanoparticle (LNP) may comprise an ionizable amino lipid. As used herein, the term ionizable amino lipid has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable amino lipid may be positively charged or negatively charged. An ionizable amino lipid may be positively charged, in which case it can be referred to as cationic lipid. In certain embodiments, an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid. As used herein, a charged moiety is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or ?1), divalent (+2, or ?2), trivalent (+3, or ?3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively-charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

[0756] It should be understood that the terms charged or charged moiety does not refer to a partial negative charge or partial positive charge on a molecule. The terms partial negative charge and partial positive charge are given its ordinary meaning in the art. A partial negative charge may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

[0757] The ionizable amino lipid is sometimes referred to in the art as an ionizable cationic lipid. In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

[0758] In addition to these, an ionizable amino lipid may also be a lipid including a cyclic amine group.

[0759] In one embodiment, the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos. WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety.

[0760] In yet another embodiment, the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969; each of which is herein incorporated by reference in their entirety.

[0761] In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety. In one embodiment, the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety. Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0762] The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.

[0763] As used herein, size or mean size in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

[0764] In one embodiment, the polynucleotide encoding a PCCA and/or PCCB polypeptide are formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm.

[0765] In one embodiment, the nanoparticles have a diameter from about 10 to 500 nm. In one embodiment, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[0766] In some embodiments, the largest dimension of a nanoparticle composition is 1 ?m or shorter (e.g., 1 ?m, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).

[0767] A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) poly dispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.

[0768] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition disclosed herein can be from about ?10 mV to about +20 mV, from about ?10 mV to about +15 mV, from about 10 mV to about +10 mV, from about ?10 mV to about +5 mV, from about ?10 mV to about 0 mV, from about ?10 mV to about ?5 mV, from about ?5 mV to about +20 mV, from about ?5 mV to about +15 mV, from about ?5 mV to about +10 mV, from about ?5 mV to about +5 mV, from about ?5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0769] In some embodiments, the zeta potential of the lipid nanoparticles can be from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, from about 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mV to about 30 mV, from about 10 mV to about 20 mV, from about 20 mV to about 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about 80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV, from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, from about 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about 30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mV to about 70 mV, from about 30 mV to about 60 mV, from about 30 mV to about 50 mV, from about 30 mV to about 40 mV, from about 40 mV to about 100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80 mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV, and from about 40 mV to about 50 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be from about 10 mV to about 50 mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and from about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.

[0770] The term encapsulation efficiency of a polynucleotide describes the amount of the polynucleotide that is encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. As used herein, encapsulation can refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[0771] Encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the polynucleotide in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents.

[0772] Fluorescence can be used to measure the amount of free polynucleotide in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a polynucleotide can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0773] The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.

[0774] For example, the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary.

[0775] The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability. For compositions including an mRNA as a polynucleotide, the N:P ratio can serve as a useful metric.

[0776] As the N:P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable. N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.

[0777] In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. In certain embodiments, the N:P ratio is between 5:1 and 6:1. In one specific aspect, the N:P ratio is about is about 5.67:1.

[0778] In addition to providing nanoparticle compositions, the present disclosure also provides methods of producing lipid nanoparticles comprising encapsulating a polynucleotide. Such method comprises using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles in accordance with methods of production of lipid nanoparticles known in the art. See, e.g., Wang et al. (2015) Delivery of oligonucleotides with lipid nanoparticles Adv. Drug Deliv. Rev. 87:68-80; Silva et al. (2015) Delivery Systems for Biopharmaceuticals. Part I; Nanoparticles and Microparticles Curr. Pharm. Technol. 16: 940-954: Naseri et al. (2015) Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application Adv. Pharm. Bull. 5:305-13: Silva et al. (2015) Lipid nanoparticles for the delivery of biopharmaceuticals Curr. Pharm. Biotechnol. 16:291-302, and references cited therein.

Methods of Use

[0779] The polynucleotides, pharmaceutical compositions and formulations described above are used in the preparation, manufacture and therapeutic use of to treat and/or prevent PCC-related diseases, disorders or conditions. In some embodiments, the polynucleotides, compositions and formulations of the present disclosure are used to treat and/or prevent propionic acidemia.

[0780] In some embodiments, the polynucleotides, pharmaceutical compositions and formulations of the present disclosure are used in methods for reducing the levels of ammonia in a subject in need thereof. For instance, one aspect of the present disclosure provides a method of alleviating the signs and symptoms of propionic acidemia in a subject comprising the administration of a composition or formulation comprising a polynucleotide encoding PCCA and/or PCCB to that subject (e.g., mRNAs encoding an PCCA and/or PCCB polypeptide).

[0781] In some embodiments, the administration of an effective amount of a polynucleotide, pharmaceutical composition or formulation of the invention reduces the levels of a biomarker of propionic acidemia, e.g., propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (30HPA), propionylglycine, glycine, lactate and/or ammonia. In some embodiments, the administration of the polynucleotide, pharmaceutical composition or formulation of the invention results in reduction in the level of one or more biomarkers of propionic acidemia, e.g., propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (3OHPA), propionylglycine, glycine, lactate and/or ammonia, within a short period of time (e.g., within about 6 hours, within about 8 hours, within about 12 hours, within about 16 hours, within about 20 hours, or within about 24 hours) after administration of the polynucleotide, pharmaceutical composition or formulation of the invention.

[0782] In some embodiments, the administration of an effective amount of a polynucleotide, pharmaceutical composition or formulation of the invention increases body weight of a human subject. In some embodiments, the administration of the polynucleotide, pharmaceutical composition or formulation of the invention results in an increase in body weight within a short period of time (e.g., within about 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 7 days, 14 days, 24 days, 48 days, or 60 days) after administration of the polynucleotide, pharmaceutical composition or formulation of the invention.

[0783] In some embodiments, the administration of an effective amount of a polynucleotide, pharmaceutical composition or formulation of the invention maintains body weight of a human subject.

[0784] Replacement therapy is a potential treatment for propionic acidemia. Thus, in certain aspects of the present disclosure, the polynucleotides, e.g., mRNA, disclosed herein comprise one or more sequences encoding an PCCA and/or PCCB polypeptide that is suitable for use in gene replacement therapy for propionic acidemia. In some embodiments, the present disclosure treats a lack of PCC or PCC activity, or decreased or abnormal PCC activity in a subject by providing a polynucleotide, e.g., mRNA, that encodes an PCCA and/or PCCB polypeptide to the subject. In some embodiments, the polynucleotide is sequence-optimized. In some embodiments, the polynucleotide (e.g., an mRNA) comprises a nucleic acid sequence (e.g., an ORF) encoding an PCCA and/or PCCB polypeptide, wherein the nucleic acid is sequence-optimized, e.g., by modifying its G/C, uridine, or thymidine content, and/or the polynucleotide comprises at least one chemically modified nucleoside. In some embodiments, the polynucleotide comprises a miRNA binding site, e.g., a miRNA binding site that binds miRNA-142.

[0785] In some embodiments, the administration of a composition or formulation comprising polynucleotide, pharmaceutical composition or formulation of the present disclosure to a subject results in a decrease in ammonia in cells to a level at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or to 100% lower than the level observed prior to the administration of the composition or formulation.

[0786] In some embodiments, the administration of the polynucleotide, pharmaceutical composition or formulation of the present disclosure results in expression of PCCA and/or PCCB in cells of the subject. In some embodiments, administering the polynucleotide, pharmaceutical composition or formulation of the present disclosure results in an increase of PCC enzymatic activity in the subject. For example, in some embodiments, the polynucleotides of the present disclosure are used in methods of administering a composition or formulation comprising an mRNA encoding an PCCA and/or PCCB polypeptide to a subject, wherein the method results in an increase of PCC enzymatic activity in at least some cells of a subject.

[0787] In some embodiments, the administration of a composition or formulation comprising an mRNA encoding an PCCA and/or PCCB polypeptide to a subject results in an increase of PCC enzymatic activity in cells subject to a level at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or to 100% or more of the activity level expected in a normal subject, e.g., a human not suffering from propionic acidemia.

[0788] In another embodiment, the polynucleotides, pharmaceutical compositions, or formulations of the present disclosure can be repeatedly administered such that PCCA and/or PCCB protein is expressed at a therapeutic level for a period of time sufficient to have a beneficial biological effect as described herein.

[0789] In some embodiments, the expression of the encoded polypeptide is increased. In some embodiments, the polynucleotide increases PCCA and/or PCCB expression levels in cells when introduced into those cells, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or to 100% with respect to the PCC expression level in the cells before the polypeptide is introduced in the cells.

[0790] In some embodiments, the method or use comprises administering a polynucleotide, e.g., mRNA, comprising a nucleotide sequence having sequence similarity to a polynucleotide of SEQ ID NO:5, wherein the polynucleotide encodes a PCCA polypeptide.

[0791] In some embodiments, the method or use comprises administering a polynucleotide, e.g., mRNA, comprising a nucleotide sequence having sequence similarity to a polynucleotide of SEQ ID NO:6, wherein the polynucleotide encodes a PCCB polypeptide.

[0792] Other aspects of the present disclosure relate to transplantation of cells containing polynucleotides to a mammalian subject. Administration of cells to mammalian subjects is known to those of ordinary skill in the art, and includes, but is not limited to, local implantation (e.g., topical or subcutaneous administration), organ delivery or systemic injection (e.g., intravenous injection or inhalation), and the formulation of cells in pharmaceutically acceptable carriers.

[0793] The present disclosure also provides methods to increase PCC activity in a subject in need thereof, e.g., a subject with PA, comprising administering to the subject a therapeutically effective amount of a composition or formulation comprising mRNA encoding a PCCA and/or PCCB polypeptide disclosed herein, e.g., a human PCCA and/or PCCB polypeptide, a mutant thereof, or a fusion protein comprising a human PCCA and/or PCCB.

[0794] In some aspects, the PCC activity measured after administration to a subject in need thereof, e.g., a subject with propionic acidemia, is at least the normal PCC activity level observed in healthy human subjects. In some aspects, the PCC activity measured after administration is at higher than the PCC activity level observed in propionic acidemia patients, e.g., untreated propionic acidemia patients. In some aspects, the increase in PCC activity in a subject in need thereof, e.g., a subject with propionic acidemia, after administering to the subject a therapeutically effective amount of a composition or formulation comprising mRNA encoding an PCCA and/or PCCB polypeptide disclosed herein is at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, or greater than 100 percent of the normal PCC activity level observed in healthy human subjects. In some aspects, the increase in PCC activity above the PCC activity level observed in propionic acidemia patients after administering to the subject a composition or formulation comprising an mRNA encoding an PCCA and/or PCCB polypeptide disclosed herein (e.g., after a single dose administration) is maintained for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 21 days, or at least 28 days.

[0795] The present disclosure also provides a method to treat, prevent, or ameliorate the symptoms of propionic acidemia in an propionic acidemia patient comprising administering to the subject a therapeutically effective amount of a composition or formulation comprising mRNA encoding an PCCA and/or PCCB polypeptide disclosed herein. In some aspects, the administration of a therapeutically effective amount of a composition or formulation comprising mRNA encoding an PCCA and/or PCCB polypeptide disclosed herein to subject in need of treatment for propionic acidemia results in reducing the symptoms of propionic acidemia.

[0796] In some embodiments, the polynucleotides (e.g., mRNA), pharmaceutical compositions and formulations used in the methods of the invention comprise a uracil-modified sequence encoding a PCCA and/or PCCB polypeptide disclosed herein and a miRNA binding site disclosed herein, e.g., a miRNA binding site that binds to miR-142 and/or a miRNA binding site that binds to miR-126. In some embodiments, the uracil-modified sequence encoding a PCCA and/or PCCB polypeptide comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil. In some embodiments, at least 95% of a type of nucleobase (e.g., uracil) in a uracil-modified sequence encoding a PCCA and/or PCCB polypeptide of the invention are modified nucleobases. In some embodiments, the polynucleotide (e.g., a RNA, e.g., a mRNA) disclosed herein is Formulated with a delivery agent comprising, e.g., a compound having the Formula (I), e.g., Compound II or Compound B; or a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound I or Compound VI, or any combination thereof. In some embodiments, the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG-DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol % ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol % ionizable amino lipid, for example, 45-46 mol %, 46-47 mol %, 47-48 mol %, 48-49 mol %, or 49-50 mol % for example about 45 mol %, 45.5 mol %, 46 mol %, 46.5 mol %, 47 mol %, 47.5 mol %, 48 mol %, 48.5 mol %, 49 mol %, or 49.5 mol %; (ii) 30-45 mol % sterol (e.g., cholesterol), optionally 35-42 mol % sterol, for example, 30-31 mol %, 31-32 mol %, 32-33 mol %, 33-34 mol %, 35-35 mol %, 35-36 mol %, 36-37 mol %, 37-38 mol %, 38-39 mol %, or 39-40 mol %, or 40-42 mol % sterol; (iii) 5-15 mol % helper lipid (e.g., DSPC), optionally 10-15 mol % helper lipid, for example, 5-6 mol %, 6-7 mol %, 7-8 mol %, 8-9 mol %, 9-10 mol %, 10-11 mol %, 11-12 mol %, 12-13 mol %, 13-14 mol %, or 14-15 mol % helper lipid; and (iv) 1-5% PEG lipid (e.g., Compound I or PEG-DMG), optionally 1-5 mol % PEG lipid, for example 1.5 to 2.5 mol %, 1-2 mol %, 2-3 mol %, 3-4 mol %, or 4-5 mol % PEG lipid.

[0797] The skilled artisan will appreciate that the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of expression of an encoded protein (e.g., enzyme) in a sample or in samples taken from a subject (e.g., from a preclinical test subject (rodent, primate, etc.) or from a clinical subject (human). Likewise, the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of activity of an encoded protein (e.g., enzyme) in a sample or in samples taken from a subject (e.g., from a preclinical test subject (rodent, primate, etc.) or from a clinical subject (human). Furthermore, the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of an appropriate biomarker in sample(s) taken from a subject. Levels of protein and/or biomarkers can be determined post-administration with a single dose of an mRNA therapeutic of the invention or can be determined and/or monitored at several time points following administration with a single dose or can be determined and/or monitored throughout a course of treatment, e.g., a multi-dose treatment.

PCCA and or PCCB Protein Expression Levels

[0798] Certain aspects of the invention feature measurement, determination and/or monitoring of the expression level or levels of PCCA and/or PCCB protein in a subject, for example, in an animal (e.g., rodents, primates, and the like) or in a human subject. Animals include normal, healthy or wild type animals, as well as animal models for use in understanding propionic acidemia and treatments thereof. Exemplary animal models include rodent models, for example, PCCA and/or PCCB deficient mice also referred to as PCCA and/or PCCB mice.

[0799] PCCA and/or PCCB protein expression levels can be measured or determined by any art-recognized method for determining protein levels in biological samples, e.g., from blood samples or a needle biopsy. The term level or level of a protein as used herein, preferably means the weight, mass or concentration of the protein within a sample or a subject. It will be understood by the skilled artisan that in certain embodiments the sample may be subjected, e.g., to any of the following: purification, precipitation, separation, e.g. centrifugation and/or HPLC, and subsequently subjected to determining the level of the protein, e.g., using mass and/or spectrometric analysis. In exemplary embodiments, enzyme-linked immunosorbent assay (ELISA) can be used to determine protein expression levels. In other exemplary embodiments, protein purification, separation and LC-MS can be used as a means for determining the level of a protein according to the invention. In some embodiments, an mRNA therapy of the invention (e.g., a single intravenous dose) results in increased PCCA and/or PCCB protein expression levels in the tissue (e.g., heart, liver, brain, or skeletal muscle) of the subject (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold increase and/or increased to at least 50%, at least 60%, at least 70%, at least 75%, 80%, at least 85%, at least 90%, at least 95%, or at least 100% of normal levels) for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 122 hours after administration of a single dose of the mRNA therapy.

PCC Activity

[0800] In propionic acidemia patients, PCC enzymatic activity is reduced compared to a normal physiological activity level. Further aspects of the invention feature measurement, determination and/or monitoring of the activity level(s) (i.e., enzymatic activity level(s)) of PCCA and/or PCCB protein in a subject, for example, in an animal (e.g., rodent, primate, and the like) or in a human subject. Activity levels can be measured or determined by any art-recognized method for determining enzymatic activity levels in biological samples. The term activity level or enzymatic activity level as used herein, preferably means the activity of the enzyme per volume, mass or weight of sample or total protein within a sample. In exemplary embodiments, the activity level or enzymatic activity level is described in terms of units per milliliter of fluid (e.g., bodily fluid, e.g., serum, plasma, urine and the like) or is described in terms of units per weight of tissue or per weight of protein (e.g., total protein) within a sample. Units (U) of enzyme activity can be described in terms of weight or mass of substrate hydrolyzed per unit time. In certain embodiments of the invention feature PCC activity described in terms of U/ml plasma or U/mg protein (tissue), where units (U) are described in terms of nmol substrate hydrolyzed per hour (or nmol/hr).

[0801] In certain embodiments, an mRNA therapy of the invention features a pharmaceutical composition comprising a dose of mRNA effective to result in at least 5 U/mg, at least 10 U/mg, at least 20 U/mg, at least 30 U/mg, at least 40 U/mg, at least 50 U/mg, at least 60 U/mg, at least 70 U/mg, at least 80 U/mg, at least 90 U/mg, at least 100 U/mg, or at least 150 U/mg of PCC activity in tissue (e.g., liver) between 6 and 12 hours, or between 12 and 24, between 24 and 48, or between 48 and 72 hours post administration (e.g., at 48 or at 72 hours post administration).

[0802] In some embodiments, an mRNA therapy of the invention (e.g., a single intravenous dose) results in increased PCC activity levels in the liver tissue of the subject (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold increase and/or increased to at least 50%, at least 60%, at least 70%, at least 75%, 80%, at least 85%, at least 90%, at least 95%, or at least 100% of normal levels) for at least 6 hours, at least 12 hours, at least 24 hours, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more days after administration of a single dose of the mRNA therapy.

[0803] In exemplary embodiments, an mRNA therapy of the invention features a pharmaceutical composition comprising a single intravenous dose of mRNA that results in the above-described levels of activity. In another embodiment, an mRNA therapy of the invention features a pharmaceutical composition which can be administered in multiple single unit intravenous doses of mRNA that maintain the above-described levels of activity.

PCC Biomarkers

[0804] In some embodiments, the administration of an effective amount of a polynucleotide, pharmaceutical composition or formulation of the invention reduces the levels of a biomarker of PCC, e.g., propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (30HPA), propionylglycine, glycine, lactate and/or ammonia levels. In some embodiments, the administration of the polynucleotide, pharmaceutical composition or formulation of the invention results in reduction in the level of one or more biomarkers of PCC, e.g., propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (3OHPA), propionylglycine, glycine, lactate and/or ammonia levels, within a short period of time after administration of the polynucleotide, pharmaceutical composition or formulation of the invention.

[0805] Further aspects of the invention feature determining the level (or levels) of a biomarker determined in a sample as compared to a level (e.g., a reference level) of the same or another biomarker in another sample, e.g., from the same patient, from another patient, from a control and/or from the same or different time points, and/or a physiologic level, and/or an elevated level, and/or a supraphysiologic level, and/or a level of a control. The skilled artisan will be familiar with physiologic levels of biomarkers, for example, levels in normal or wild type animals, normal or healthy subjects, and the like, in particular, the level or levels characteristic of subjects who are healthy and/or normal functioning. As used herein, the phrase elevated level means amounts greater than normally found in a normal or wild type preclinical animal or in a normal or healthy subject, e.g. a human subject. As used herein, the term supraphysiologic means amounts greater than normally found in a normal or wild type preclinical animal or in a normal or healthy subject, e.g. a human subject, optionally producing a significantly enhanced physiologic response. As used herein, the term comparing or compared to preferably means the mathematical comparison of the two or more values, e.g., of the levels of the biomarker(s). It will thus be readily apparent to the skilled artisan whether one of the values is higher, lower or identical to another value or group of values if at least two of such values are compared with each other. Comparing or comparison to can be in the context, for example, of comparing to a control value, e.g., as compared to a reference blood, serum, plasma, and/or tissue (e.g., liver) propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (30HPA), propionylglycine, glycine, lactate and/or ammonia level, in said subject prior to administration (e.g., in a person suffering from propionic acidemia) or in a normal or healthy subject. Comparing or comparison to can also be in the context, for example, of comparing to a control value, e.g., as compared to a reference blood, serum, plasma and/or tissue (e.g., liver) propionyl-L-carnitine (C3), 2-methylcitric acid (2-MC), 3-hydroxypropionic acid, (30HPA), propionylglycine, glycine, lactate and/or ammonia level in said subject prior to administration (e.g., in a person suffering from propionic acidemia) or in a normal or healthy subject.

[0806] As used herein, a control is preferably a sample from a subject wherein the propionic acidemia status of said subject is known. In one embodiment, a control is a sample of a healthy patient. In another embodiment, the control is a sample from at least one subject having a known propionic acidemia status, for example, a severe, mild, or healthy propionic acidemia status, e.g. a control patient. In another embodiment, the control is a sample from a subject not being treated for propionic acidemia. In a still further embodiment, the control is a sample from a single subject or a pool of samples from different subjects and/or samples taken from the subject(s) at different time points.

[0807] The term level or level of a biomarker as used herein, preferably means the mass, weight or concentration of a biomarker of the invention within a sample or a subject. It will be understood by the skilled artisan that in certain embodiments the sample may be subjected to, e.g., one or more of the following: substance purification, precipitation, separation, e.g. centrifugation and/or HPLC and subsequently subjected to determining the level of the biomarker, e.g. using mass spectrometric analysis. In certain embodiments, LC-MS can be used as a means for determining the level of a biomarker according to the invention.

[0808] The term determining the level of a biomarker as used herein can mean methods which include quantifying an amount of at least one substance in a sample from a subject, for example, in a bodily fluid from the subject (e.g., serum, plasma, urine, lymph, etc.) or in a tissue of the subject (e.g., liver, etc.).

[0809] The term reference level as used herein can refer to levels (e.g., of a biomarker) in a subject prior to administration of an mRNA therapy of the invention (e.g., in a person suffering from propionic acidemia) or in a normal or healthy subject.

[0810] As used herein, the term normal subject or healthy subject refers to a subject not suffering from symptoms associated with propionic acidemia. Moreover, a subject will be considered to be normal (or healthy) if it has no mutation of the functional portions or domains of the PCCA and or PCCB gene and/or no mutation of the PCCA and or PCCB gene resulting in a reduction of or deficiency of the enzyme PCC or the activity thereof, resulting in symptoms associated with propionic acidemia. Said mutations will be detected if a sample from the subject is subjected to a genetic testing for such PCCA and or PCCB mutations. In certain embodiments of the present invention, a sample from a healthy subject is used as a control sample, or the known or standardized value for the level of biomarker from samples of healthy or normal subjects is used as a control.

[0811] In some embodiments, comparing the level of the biomarker in a sample from a subject in need of treatment for propionic acidemia or in a subject being treated for propionic acidemia to a control level of the biomarker comprises comparing the level of the biomarker in the sample from the subject (in need of treatment or being treated for propionic acidemia) to a baseline or reference level, wherein if a level of the biomarker in the sample from the subject (in need of treatment or being treated for propionic acidemia) is elevated, increased or higher compared to the baseline or reference level, this is indicative that the subject is suffering from propionic acidemia and/or is in need of treatment; and/or wherein if a level of the biomarker in the sample from the subject (in need of treatment or being treated for propionic acidemia) is decreased or lower compared to the baseline level this is indicative that the subject is not suffering from, is successfully being treated for propionic acidemia, or is not in need of treatment for propionic acidemia. The stronger the reduction (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at least 20-fold, at least-30 fold, at least 40-fold, at least 50-fold reduction and/or at least 10%, at least 20%, at least 30% at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% reduction) of the level of a biomarker, within a certain time period, e.g., within 6 hours, within 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours, and/or for a certain duration of time, e.g., 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, etc. the more successful is a therapy, such as for example an mRNA therapy of the invention (e.g., a single dose or a multiple regimen).

[0812] A reduction of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 100% or more of the level of biomarker, in particular, in bodily fluid (e.g., plasma, serum, urine, e.g., urinary sediment) or in tissue(s) in a subject (e.g., liver), within 1, 2, 3, 4, 5, 6 or more days following administration is indicative of a dose suitable for successful treatment propionic acidemia, wherein reduction as used herein, preferably means that the level of biomarker determined at the end of a specified time period (e.g., post-administration, for example, of a single intravenous dose) is compared to the level of the same biomarker determined at the beginning of said time period (e.g., pre-administration of said dose). Exemplary time periods include 12, 24, 48, 72, 96, 120 or 144 hours post administration, in particular 24, 48, 72 or 96 hours post administration.

[0813] A sustained reduction in substrate levels (e.g., biomarkers) is particularly indicative of mRNA therapeutic dosing and/or administration regimens successful for treatment of propionic acidemia. Such sustained reduction can be referred to herein as duration of effect. In exemplary embodiments, a reduction of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100% or more of the level of biomarker, in particular, in a bodily fluid (e.g., plasma, serum, urine, e.g., urinary sediment) or in tissue(s) in a subject (e.g., liver), within 1, 2, 3, 4, 5, 6, 7, 8 or more days following administration is indicative of a successful therapeutic approach. In exemplary embodiments, sustained reduction in substrate (e.g., biomarker) levels in one or more samples (e.g., fluids and/or tissues) is preferred. For example, mRNA therapies resulting in sustained reduction in a biomarker, optionally in combination with sustained reduction of said biomarker in at least one tissue, preferably two, three, four, five or more tissues, is indicative of successful treatment.

Compositions and Formulations for Use

[0814] Certain aspects of the invention are directed to compositions or formulations comprising any of the polynucleotides disclosed above.

[0815] In some embodiments, the composition or formulation comprises: [0816] (i) a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a PCCA polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil (e.g., wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1-methylpseudouracils or 5-methoxyuracils), and wherein the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-142 (e.g., a miR-142-3p or miR-142-5p binding site) and/or a miRNA binding site that binds to miR-126 (e.g., a miR-126-3p or miR-126-5p binding site); [0817] (ii) a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a PCCB polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., N1 methylpseudouracil or 5-methoxyuracil (e.g., wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1 methylpseudouracils or 5-methoxyuracils), and wherein the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-142 (e.g., a miR-142-3p or miR-142-5p binding site) and/or a miRNA binding site that binds to miR-126 (e.g., a miR-126-3p or miR-126-5p binding site); and [0818] (iii) a delivery agent comprising, e.g., a compound having the Formula (I), e.g., Compound II or Compound B: a compound having the Formula (III), (IV), (V), or (VI), e.g., Compound I or Compound VI, or any combination thereof. In some embodiments, the delivery agent is a lipid nanoparticle comprising Compound II, Compound VI, a salt or a stereoisomer thereof, or any combination thereof. In some embodiments, the delivery agent comprises an ionizable amino lipid (e.g., Compound II, VI, or B), a helper lipid (e.g., DSPC), a sterol (e.g., Cholesterol), and a PEG lipid (e.g., Compound I or PEG-DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol % ionizable amino lipid (e.g., Compound II, VI, or B), optionally 45-50 mol % ionizable amino lipid, for example, 45-46 mol %, 46-47 mol %, 47-48 mol %, 48-49 mol %, or 49-50 mol % for example about 45 mol %, 45.5 mol %, 46 mol %, 46.5 mol %, 47 mol %, 47.5 mol %, 48 mol %, 48.5 mol %, 49 mol %, or 49.5 mol %; (ii) 30-45 mol % sterol (e.g., cholesterol), optionally 35-42 mol % sterol, for example, 30-31 mol %, 31-32 mol %, 32-33 mol %, 33-34 mol %, 35-35 mol %, 35-36 mol %, 36-37 mol %, 37-38 mol %, 38-39 mol %, or 39-40 mol %, or 40-42 mol % sterol; (iii) 5-15 mol % helper lipid (e.g., DSPC), optionally 10-15 mol % helper lipid, for example, 5-6 mol %, 6-7 mol %, 7-8 mol %, 8-9 mol %, 9-10 mol %, 10-11 mol %, 11-12 mol %, 12-13 mol %, 13-14 mol %, or 14-15 mol % helper lipid; and (iv) 1-5% PEG lipid (e.g., Compound I or PEG-DMG), optionally 1-5 mol % PEG lipid, for example 1.5 to 2.5 mol %, 1-2 mol %, 2-3 mol %, 3-4 mol %, or 4-5 mol % PEG lipid.

[0819] In some embodiments, the uracil or thymine content of the ORF relative to the theoretical minimum uracil or thymine content of a nucleotide sequence encoding the a PCCA and/or PCCB polypeptide (% U.sub.TM or % T.sub.TM), is between about 100% and about 150%.

[0820] In some embodiments, the polynucleotides, compositions or formulations above are used to treat and/or prevent PCC-related diseases, disorders or conditions, e.g., PA.

Definitions

[0821] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0822] 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.

[0823] In this specification and the appended claims, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise. The terms a (or an), as well as the terms one or more, and at least one can be used interchangeably herein. In certain aspects, the term a or an means single. In other aspects, the term a or an includes two or more or multiple.

[0824] Furthermore, and/or where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or as used in a phrase such as A and/or B herein is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term and/or as used in a phrase such as A, B, and/or C is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0825] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press: The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0826] Wherever aspects are described herein with the language comprising, otherwise analogous aspects described in terms of consisting of and/or consisting essentially of are also provided.

[0827] Units, prefixes, and symbols are denoted in their Syst?me International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0828] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5 to 3 orientation. Nucleobases are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.

[0829] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0830] About: The term about as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art, such interval of accuracy is ?10%.

[0831] Where ranges are given, endpoints are included. Furthermore, 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.

[0832] Approximately: As used herein, the term approximately, as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term approximately refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0833] Dosing regimen: As used herein, a dosing regimen or a dosing regimen is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.

[0834] Effective Amount: As used herein, the term effective amount of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an effective amount depends upon the context in which it is being applied. For example, in the context of administering an agent that treats a protein deficiency (e.g., an PCC deficiency), an effective amount of an agent is, for example, an amount of mRNA expressing sufficient PCCA and/or PCCB to ameliorate, reduce, eliminate, or prevent the symptoms associated with the PCC deficiency, as compared to the severity of the symptom observed without administration of the agent. The term effective amount can be used interchangeably with effective dose, therapeutically effective amount, or therapeutically effective dose.

[0835] PCC Associated Disease: As use herein the terms PCC-associated disease or PCC-associated disorder refer to diseases or disorders, respectively, which result from aberrant PCC activity (e.g., decreased activity or increased activity). As a non-limiting example, propionic acidemia is an PCC-associated disease.

[0836] The terms PCC enzymatic activity and PCC activity, are used interchangeably in the present disclosure and refer to PCC's ability to catalyze the carboxylation of propionyl-CoA to methylmalonyl-CoA. Accordingly, a fragment or variant retaining or having PCC enzymatic activity or PC activity refers to a fragment or variant that has measurable enzymatic activity in catalyzing the carboxylation of propionyl-CoA to methylmalonyl-CoA.

[0837] Ionizable amino lipid: The term ionizable amino lipid includes those lipids having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group). An ionizable amino lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the amino head group and is substantially not charged at a pH above the pKa. Such ionizable amino lipids include, but are not limited to DLin-MC3-DMA (MC3) and (13Z,165Z)N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608).

[0838] Methods of Administration: As used herein, methods of administration can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. A method of administration can be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body.

[0839] Nanoparticle Composition: As used herein, a nanoparticle composition is a composition comprising one or more lipids. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0840] The phrase nucleotide sequence encoding refers to the nucleic acid (e.g., an mRNA or DNA molecule) coding sequence which encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further include sequences that encode signal peptides.

[0841] Patient: As used herein, patient refers to a subject who can seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition. In some embodiments, the treatment is needed, required, or received to prevent or decrease the risk of developing acute disease, i.e., it is a prophylactic treatment.

[0842] Pseudouridine: As used herein, pseudouridine (w) refers to the C-glycoside isomer of the nucleoside uridine. A pseudouridine analog is any modification, variant, isoform or derivative of pseudouridine. For example, pseudouridine analogs include but are not limited to 1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine, 1-methylpseudouridine (m.sup.1?) (also known as N1-methyl-pseudouridine), 1-methyl-4-thio-pseudouridine (m.sup.1s.sup.4?), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m.sup.3?), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 1-methyl-3-(3-amino-3-carboxy propyl)pseudouridine (acp.sup.3?), and 2-O-methyl-pseudouridine (?m).

[0843] Subject: By subject or individual or animal or patient or mammal, is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. In other embodiments, a subject is a human patient. In a particular embodiment, a subject is a human patient in need of treatment.

[0844] Therapeutically effective amount: As used herein, the term therapeutically effective amount means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

[0845] Uracil: Uracil is one of the four nucleobases in the nucleic acid of RNA, and it is represented by the letter U. Uracil can be attached to a ribose ring, or more specifically, a ribofuranose via a ?-N.sub.1-glycosidic bond to yield the nucleoside uridine. The nucleoside uridine is also commonly abbreviated according to the one letter code of its nucleobase, i.e., U. Thus, in the context of the present disclosure, when a monomer in a polynucleotide sequence is U, such U is designated interchangeably as a uracil or a uridine.

[0846] Uridine Content: The terms uridine content or uracil content are interchangeable and refer to the amount of uracil or uridine present in a certain nucleic acid sequence. Uridine content or uracil content can be expressed as an absolute value (total number of uridine or uracil in the sequence) or relative (uridine or uracil percentage respect to the total number of nucleobases in the nucleic acid sequence).

[0847] Uridine-Modified Sequence: The terms uridine-modified sequence refers to a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with a different overall or local uridine content (higher or lower uridine content) or with different uridine patterns (e.g., gradient distribution or clustering) with respect to the uridine content and/or uridine patterns of a candidate nucleic acid sequence. In the content of the present disclosure, the terms uridine-modified sequence and uracil-modified sequence are considered equivalent and interchangeable.

[0848] Nucleobase: As used herein, the term nucleobase (alternatively nucleotide base or nitrogenous base) refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivatives or analogs of the naturally occurring purines and pyrimidines that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the nucleobases predominately found in natural nucleic acids. Other natural, non-natural, and/or synthetic nucleobases, as known in the art and/or described herein, can be incorporated into nucleic acids. Unless otherwise specified, the nucleobase sequence of a SEQ ID NO described herein encompasses both natural nucleobases and chemically modified nucleobases (e.g., a U designation in a SEQ ID NO encompasses both uracil and chemically modified uracil).

[0849] Nucleoside Nucleotide: As used herein, the term nucleoside refers to a compound containing a sugar molecule (e.g., a ribose in RNA or a deoxyribose in DNA), or derivative or analog thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as nucleobase), but lacking an internucleoside linking group (e.g., a phosphate group). As used herein, the term nucleotide refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and/or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

[0850] Nucleic acid: As used herein, the term nucleic acid is used in its broadest sense and encompasses any compound and/or substance that includes a polymer of nucleotides, or derivatives or analogs thereof. These polymers are often referred to as polynucleotides. Accordingly, as used herein the terms nucleic acid and polynucleotide are equivalent and are used interchangeably. Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, mRNAs, modified mRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ?-D-ribo configuration, ?-LNA having an ?-L-ribo configuration (a diastereomer of LNA), 2-amino-LNA having a 2-amino functionalization, and 2-amino-?-LNA having a 2-amino functionalization) or hybrids thereof.

[0851] Open Reading Frame: As used herein, the term open reading frame, abbreviated as ORF, refers to a segment or region of an mRNA molecule that encodes a polypeptide. The ORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

EQUIVALENTS AND SCOPE

[0852] 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.

[0853] In the claims, articles such as a, an, and the can 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.

[0854] It is also noted that the term comprising is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term comprising is used herein, the term consisting of is thus also encompassed and disclosed.

[0855] 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.

[0856] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art can 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 can be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded thereby: any method of production: any method of use: etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0857] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control. Section and table headings are not intended to be limiting.

EXAMPLES

Example 1: Synthesis of mRNAs Encoding PCCA and PCCB

[0858] An mRNA encoding human PCCA is constructed by using the ORF sequence (nucleotide) provided in SEQ ID NO:5. The mRNA sequence includes both 5 and 3 UTR regions flanking the ORF sequence. The 5 UTR and 3 UTR sequences of the mRNA are SEQ ID NO:55 and SEQ ID NO: 114, respectively.

[0859] An mRNA encoding human PCCB is constructed by using the ORF sequence (nucleotide) provided in SEQ ID NO:6. The mRNA sequence includes both 5 and 3 UTR regions flanking the ORF sequence. The 5 UTR and 3 UTR sequences of the mRNA are SEQ ID NO:55 and SEQ ID NO:114, respectively.

[0860] The PCCA and PCCB mRNA sequences are prepared as modified mRNAs. Specifically, during in vitro transcription, modified mRNAs can be generated using N1-methylpseudouridine-5-triphosphate to ensure that the mRNAs contain 100% N1-methylpseudouridine instead of uridine. Alternatively, during in vitro transcription, modified mRNA can be generated using N1-methoxyuridine-5-Triphosphate to ensure that the mRNAs contain 100%5-methoxyuridine instead of uridine. Further, PCCA-mRNA and PCCB-mRNA can be synthesized with a primer that introduces a polyA-tail, and a cap structure is generated on both mRNAs using co-transcriptional capping via m.sup.7Gp-ppGm-pA-pG tetranucleotide to incorporate a m.sup.7Gp-ppGm-pA-pG 5 cap. Alternatively, mRNA can be synthesized and the poly A-tail introduced during Gibson assembly of the DNA template.

Example 2: Production of PCCA/PCCB mRNA Nanoparticle Compositions

A. Production of Nanoparticle Compositions

[0861] Nanoparticles can be made with mixing processes such as microfluidics and T-junction mixing of two fluid streams, one of which contains the polynucleotide and the other has the lipid components.

[0862] Lipid compositions are prepared by combining an ionizable amino lipid disclosed herein, e.g., a lipid according to Formula (I) such as Compound II or a lipid according to Formula (II) such as Compound B, a phospholipid (such as Compound I or 1,2 dimyristoyl sn glycerol methoxypolyethylene glycol, also known as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, AL), a PEG lipid (such as 1,2 dimyristoyl sn glycerol methoxypolyethylene glycol, also known as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, AL), and a structural lipid (such as cholesterol, obtainable from Sigma Aldrich, Taufkirchen, Germany, or a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof) at concentrations of about 50 mM in ethanol. Solutions should be refrigerated for storage at, for example, ?20? C. Lipids are combined to yield desired molar ratios and diluted with water and ethanol to a final lipid concentration of between about 5.5 mM and about 25 mM.

[0863] Nanoparticle compositions including a polynucleotide and a lipid composition are prepared by combining the lipid solution with a solution including the a polynucleotide at lipid composition to polynucleotide wt:wt ratios between about 5:1 and about 50:1. The lipid solution is rapidly injected using a NanoAssemblr microfluidic based system at flow rates between about 10 ml/min and about 18 ml/min into the polynucleotide solution to produce a suspension with a water to ethanol ratio between about 1:1 and about 4:1.

[0864] For nanoparticle compositions including an RNA, solutions of the RNA at concentrations of 0.1 mg/ml in deionized water are diluted in 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution.

[0865] Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange. Formulations are dialyzed twice against phosphate buffered saline (PBS), pH 7.4, at volumes 200 times that of the primary product using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, IL) with a molecular weight cutoff of 10 kD. The first dialysis is carried out at room temperature for 3 hours. The formulations are then dialyzed overnight at 4? C. The resulting nanoparticle suspension is filtered through 0.2 ?m sterile filters (Sarstedt, N?mbrecht, Germany) into glass vials and sealed with crimp closures. Nanoparticle composition solutions of 0.01 mg/ml to 0.10 mg/ml are generally obtained.

[0866] The method described above induces nano-precipitation and particle formation. Alternative processes including, but not limited to, T-junction and direct injection, can be used to achieve the same nano-precipitation.

B. Characterization of Nanoparticle Compositions

[0867] A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, the poly dispersity index (PDI) and the zeta potential of the nanoparticle compositions in 1?PBS in determining particle size and 15 mM PBS in determining zeta potential.

[0868] Ultraviolet-visible spectroscopy can be used to determine the concentration of a polynucleotide (e.g., RNA) in nanoparticle compositions. 100 ?L of the diluted formulation in 1?PBS is added to 900 ?L of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of polynucleotide in the nanoparticle composition can be calculated based on the extinction coefficient of the polynucleotide used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.

[0869] For nanoparticle compositions including an RNA, a QUANT-IT? RIBOGREENR RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluate the encapsulation of an RNA by the nanoparticle composition. The samples are diluted to a concentration of approximately 5 ?g/mL in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 ?L of the diluted samples are transferred to a polystyrene 96 well plate and either 50 ?L of TE buffer or 50 ?L of a 2% Triton X-100 solution is added to the wells. The plate is incubated at a temperature of 37? C. for 15 minutes. The RIBOGREENR reagent is diluted 1:100 in TE buffer, and 100 ?L of this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, MA) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample 5 (caused by the addition of Triton X-100).

[0870] Exemplary formulations of the nanoparticle compositions are presented in the Table 4 below. The term Compound refers to an ionizable amino lipid such as MC3, Compound II, Compound VI, Compound A, or Compound B. Phospholipid can be DSPC or DOPE. PEG-lipid can be PEG-DMG or Compound I.

TABLE-US-00006 TABLE 4 Exemplary Formulations of Nanoparticles Composition (mol %) Components 47:11:39:3 Compound:Phospholipid:Chol:PEG-lipid 40:20:38.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 45:15:38.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 50:10:38.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 45:20:33.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 50:20:28.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 40:15:43.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 50:15:33.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 40:10:48.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 45:10:43.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 40:5:53.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 45:5:48.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 50:5:43.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 40:20:40:0 Compound:Phospholipid:Chol:PEG-lipid 45:20:35:0 Compound:Phospholipid:Chol:PEG-lipid 50:20:30:0 Compound:Phospholipid:Chol:PEG-lipid 40:15:45:0 Compound:Phospholipid:Chol:PEG-lipid 45:15:40:0 Compound:Phospholipid:Chol:PEG-lipid 50:15:35:0 Compound:Phospholipid:Chol:PEG-lipid 40:10:50:0 Compound:Phospholipid:Chol:PEG-lipid 45:10:45:0 Compound:Phospholipid:Chol:PEG-lipid 50:10:40:0 Compound:Phospholipid:Chol:PEG-lipid 47.5:10.5:39:3 Compound:Phospholipid:Chol:PEG-lipid 47.5:10:39.5:3 Compound:Phospholipid:Chol:PEG-lipid 47.5:11:39.5:2 Compound:Phospholipid:Chol:PEG-lipid 47.5:10.5:39.5:2.5 Compound:Phospholipid:Chol:PEG-lipid 47.5:11:39:2.5 Compound:Phospholipid:Chol:PEG-lipid 48.5:10:38.5:3 Compound:Phospholipid:Chol:PEG-lipid 48.5:10.5:39:2 Compound:Phospholipid:Chol:PEG-lipid 48.5:10.5:38.5:2.5 Compound:Phospholipid:Chol:PEG-lipid 48.5:10.5:39.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 48.5:10.5:38.0:3 Compound:Phospholipid:Chol:PEG-lipid 47:10.5:39.5:3 Compound:Phospholipid:Chol:PEG-lipid 47:10:40.5:2.5 Compound:Phospholipid:Chol:PEG-lipid 47:11:40:2 Compound:Phospholipid:Chol:PEG-lipid 47:10.5:39.5:3 Compound:Phospholipid:Chol:PEG-lipid 48:10.5:38.5:3 Compound:Phospholipid:Chol:PEG-lipid 48:10:39.5:2.5 Compound:Phospholipid:Chol:PEG-lipid 48:11:39:2 Compound:Phospholipid:Chol:PEG-lipid 48:10.5:38.5:3 Compound:Phospholipid:Chol:PEG-lipid

Example 3: Phase 1/2 Clinical Trial

[0871] This study evaluates the safety, pharmacodynamics, and pharmacokinetics of mRNA-3927 in participants greater than or equal to 1 year of age who have propionic academia with mutations in PCCA and/or PCCB genes. This study is designed to characterize the safety, tolerability, and pharmacological activity (as assessed by biomarker measurements) and to determine the optimal dose of mRNA 3927 in participants with propionic academia.

[0872] mRNA-3927 is a lipid nanoparticle containing two mRNAs (one encoding a human PCCA subunit and the other encoding a human PCCB subunit), Compound II, Compound I (a polyethylene glycol-lipid conjugate), DSPC (1,2-distearoyl-SN-glycero-3-phosphocholine), and cholesterol. The components of the PCCA mRNA and the PCCB mRNA are described in detail below. The PCCA mRNA and the PCCB mRNA are encapsulated together in lipid nanoparticles at a ratio of 1:1.

PCCA mRNA

TABLE-US-00007 Componentsof DescriptionofPCCA PCCAmRNA mRNAComponent 5-terminalcap Cap1 Chemistry alluracilsinthemRNA 5untranslated areN1-methylpseudouracils region GGAAAUAAGAGAGAAAAGAA (UTR) GAGUAAGAAGAAAUAUAAGA GCCACC (SEQIDNO:55) openreading AUGGCCGGCUUCUGGGUCGGCACAGCCCCU frame CUGGUGGCAGCCGGCAGAAGAGGACGGUGG (ORF) CCUCCCCAGCAACUGAUGCUGAGCGCCGCC CUGAGAACCCUGAAGCACGUGCUGUACUAC AGCAGACAGUGCCUGAUGGUGAGCAGAAAU CUGGGCAGCGUGGGGUACGAUCCCAACGAG AAGACCUUCGAUAAGAUUCUGGUCGCGAAU AGAGGCGAGAUCGCCUGCAGGGUGAUCAGA ACCUGCAAGAAGAUGGGCAUCAAGACCGUG GCCAUCCAUUCGGACGUCGACGCGAGCAGC GUUCACGUGAAGAUGGCAGACGAGGCCGUG UGCGUGGGACCCGCCCCGACCAGCAAGAGC UACCUGAACAUGGACGCCAUCAUGGAGGCC AUCAAGAAGACCCGCGCUCAAGCCGUGCAC CCGGGCUACGGCUUUCUGAGCGAGAACAAG GAAUUCGCCAGGUGUCUCGCCGCCGAGGAC GUAGUCUUCAUCGGCCCUGAUACGCACGCG AUCCAGGCCAUGGGCGACAAGAUCGAGAGC AAACUGCUGGCCAAGAAAGCAGAAGUCAAC ACCAUCCCCGGCUUCGACGGCGUGGUGAAG GACGCCGAAGAGGCUGUCCGCAUCGCCAGA GAGAUCGGCUACCCUGUGAUGAUAAAGGCU AGCGCUGGAGGUGGCGGAAAGGGCAUGAGA AUCGCCUGGGACGACGAGGAGACUAGAGAC GGCUUCAGACUGUCCUCCCAGGAGGCCGCC AGCUCCUUCGGAGACGACAGACUGCUGAUC GAGAAGUUCAUCGACAACCCCAGACACAUC GAAAUCCAGGUGCUCGGUGACAAGCACGGG AACGCCCUGUGGCUGAACGAGAGAGAGUGC AGCAUCCAGAGAAGAAACCAGAAGGUGGUG GAGGAGGCGCCGAGCAUCUUUCUGGACGCG GAGACAAGGAGAGCGAUGGGCGAACAGGCC GUCGCCCUAGCAAGAGCCGUGAAGUACUCC AGUGCCGGAACCGUCGAGUUUCUUGUCGAC AGCAAGAAGAAUUUCUACUUCCUGGAGAUG AACACCAGGCUGCAGGUGGAGCAUCCCGUG ACAGAGUGCAUCACUGGACUGGAUCUGGUG CAGGAGAUGAUCAGGGUGGCCAAGGGCUAU CCCCUGAGACACAAGCAGGCCGACAUCAGA AUCAACGGCUGGGCCGUGGAGUGCAGAGUG UACGCCGAGGACCCCUACAAGAGCUUCGGC CUGCCCAGCAUCGGCAGACUGAGCCAGUAC CAGGAGCCCCUGCACCUGCCCGGCGUGAGA GUGGACAGCGGCAUCCAACCGGGGAGCGAU AUCAGCAUCUACUACGACCCCAUGAUCAGC AAGCUGAUAACCUACGGCAGCGACAGAACC GAGGCCCUGAAGAGAAUGGCCGACGCCCUG GACAACUACGUGAUCAGAGGCGUGACCCAC AACAUCGCCCUGCUGAGAGAGGUGAUCAUC AACUCGAGGUUCGUGAAAGGCGACAUCAGC ACCAAGUUCCUGAGCGACGUGUAUCCCGAC GGAUUCAAAGGUCACAUGCUGACCAAGAGC GAGAAGAACCAGCUGCUGGCCAUCGCCUCA UCCCUGUUCGUGGCCUUCCAGCUGAGAGCC CAGCACUUCCAGGAGAACAGCAGAAUGCCC GUGAUCAAGCCCGACAUCGCCAACUGGGAG CUGAGCGUGAAGCUGCACGACAAGGUGCAC ACUGUCGUUGCCAGCAACAACGGCUCCGUG UUCAGCGUAGAGGUGGACGGAUCUAAGCUG AACGUGACCUCCACCUGGAACCUGGCAAGC CCUCUCCUGUCAGUGAGCGUGGACGGCACC CAGAGAACCGUGCAGUGUCUGUCCCGCGAG GCCGGCGGAAACAUGAGCAUCCAGUUCCUG GGCACCGUGUACAAGGUGAACAUCCUGACC AGACUGGCCGCCGAGCUGAACAAGUUCAUG CUGGAGAAAGUGACGGAGGAUACCAGCUCC GUGCUGAGAAGCCCCAUGCCCGGAGUGGUG GUGGCCGUUUCCGUGAAACCUGGUGACGCC GUGGCCGAGGGGCAAGAGAUCUGCGUGAUC GAGGCCAUGAAGAUGCAGAAUUCCAUGACC GCCGGAAAGACCGGCACCGUCAAAUCAGUG CACUGCCAGGCGGGCGACACAGUGGGUGAG GGCGACCUGCUGGUGGAGCUGGAG (SEQIDNO:5) 3UTR UGAUAAUAGUCCAUAAAGUAGGAAACACUA CAGCUGGAGCCUCGGUGGCCUAGCUUCUUG CCCCUUGGGCCUCCAUAAAGUAGGAAACAC UACAUCCCCCCAGCCCCUCCUCCCCUUCCU GCACCCGUACCCCCUCCAUAAAGUAGGAAA CACUACAGUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC (SEQIDNO:114) poly-Atail 100residuesinlength(SEQID NO:195) EncodedPCCA MAGFWVGTAPLVAAGRRGRWPPQQLMLSAA protein LRTLKHVLYYSRQCLMVSRNLGSVGYDPNE KTFDKILVANRGEIACRVIRTCKKMGIKTV AIHSDVDASSVHVKMADEAVCVGPAPTSKS YLNMDAIMEAIKKTRAQAVHPGYGFLSENK EFARCLAAEDVVFIGPDTHAIQAMGDKIES KLLAKKAEVNTIPGFDGVVKDAEEAVRIAR EIGYPVMIKASAGGGGKGMRIAWDDEETRD GFRLSSQEAASSFGDDRLLIEKFIDNPRHI EIQVLGDKHGNALWLNERECSIQRRNQKVV EEAPSIFLDAETRRAMGEQAVALARAVKYS SAGTVEFLVDSKKNFYFLEMNTRLQVEHPV TECITGLDLVQEMIRVAKGYPLRHKQADIR INGWAVECRVYAEDPYKSFGLPSIGRLSQY QEPLHLPGVRVDSGIQPGSDISIYYDPMIS KLITYGSDRTEALKRMADALDNYVIRGVTH NIALLREVIINSRFVKGDISTKFLSDVYPD GFKGHMLTKSEKNQLLAIASSLFVAFQLRA QHFQENSRMPVIKPDIANWELSVKLHDKVH TVVASNNGSVFSVEVDGSKLNVTSTWNLAS PLLSVSVDGTQRTVQCLSREAGGNMSIQFL GTVYKVNILTRLAAELNKFMLEKVTEDTSS VLRSPMPGVVVAVSVKPGDAVAEGQEICVI EAMKMQNSMTAGKTGTVKSVHCQAGDTVGE GDLLVELE (SEQIDNO:1) Construct SEQIDNO:7consistsfrom Sequence 5to3end:5UTRofSEQID NO:55,ORFSequenceofSEQID NO:5,and3UTRofSEQID NO:114
PCCB mRNA

TABLE-US-00008 Componentsof DescriptionofPCCB PCCBmRNA mRNAComponent 5-terminalcap Cap1 Chemistry alluracilsinthemRNA areN1-methylpseudouracils 5 GGAAAUAAGAGAGAAAAGAA untranslated GAGUAAGAAGAAAUAUAAGA region GCCACC (UTR) (SEQIDNO:55) openreading AUGGCGGCGGCAUUACGGGUGGCGGCGGUC frame GGGGCAAGGCUCAGCGUGCUGGCCAGCGGC (ORF) CUGAGAGCCGCCGUGAGAAGCCUGUGCAGC CAGGCCACCAGCGUGAACGAGAGAAUCGAG AACAAGAGAAGAACCGCCCUGCUGGGCGGC GGCCAGAGAAGAAUCGACGCCCAGCACAAG AGAGGCAAGCUGACCGCCAGAGAGAGAAUC AGCCUGCUGCUGGACCCCGGCAGCUUCGUG GAGAGCGACAUGUUCGUGGAGCACAGGUGC GCCGACUUCGGCAUGGCCGCCGACAAGAAC AAGUUCCCCGGCGACAGCGUGGUGACCGGC AGAGGCAGAAUCAACGGCAGACUGGUGUAC GUGUUCAGCCAGGACUUCACCGUGUUCGGC GGCAGCCUGAGCGGCGCCCACGCCCAGAAG AUCUGCAAGAUCAUGGACCAGGCCAUCACC GUGGGCGCGCCCGUGAUCGGCCUGAACGAC AGCGGCGGCGCCAGAAUCCAGGAGGGCGUG GAGAGCCUGGCCGGCUACGCCGACAUCUUC CUGAGAAACGUGACCGCCAGCGGCGUGAUC CCACAGAUCAGCCUGAUCAUGGGCCCCUGC GCCGGCGGCGCCGUGUACAGCCCCGCCCUG ACCGACUUCACCUUCAUGGUGAAGGACACC AGCUACCUGUUCAUCACCGGCCCCGACGUG GUGAAGAGCGUGACCAACGAGGACGUGACC CAGGAGGAGCUGGGCGGCGCCAAGACCCAC ACCACCAUGAGCGGCGUGGCCCACAGAGCC UUCGAGAACGACGUGGACGCCCUGUGCAAC CUGAGAGACUUCUUCAACUACCUGCCCCUG AGCAGCCAGGACCCCGCGCCCGUGAGAGAG UGCCACGACCCCAGCGACAGACUGGUGCCC GAGCUGGACACCAUCGUGCCCCUGGAGAGC ACCAAGGCCUACAACAUGGUGGACAUCAUC CACAGCGUGGUGGACGAGAGAGAGUUCUUC GAGAUCAUGCCCAACUACGCCAAGAACAUC AUCGUGGGCUUCGCCAGAAUGAACGGCAGA ACCGUGGGCAUCGUGGGCAACCAGCCCAAG GUGGCCAGCGGCUGCCUGGACAUCAACAGC AGCGUGAAGGGCGCCAGAUUCGUGAGAUUC UGCGACGCCUUCAACAUCCCUCUGAUCACC UUCGUGGACGUGCCCGGCUUCCUGCCCGGC ACCGCCCAGGAGUACGGCGGCAUCAUCAGA CACGGCGCCAAGCUGCUGUACGCCUUCGCC GAGGCCACCGUGCCCAAGGUGACCGUGAUC ACCAGAAAGGCCUACGGCGGCGCCUACGAC GUGAUGAGCAGCAAGCACCUGUGCGGCGAC ACCAACUACGCCUGGCCCACCGCCGAGAUC GCCGUGAUGGGCGCCAAGGGCGCCGUGGAG AUCAUCUUCAAGGGCCACGAGAACGUGGAG GCCGCCCAGGCCGAGUACAUCGAGAAGUUC GCCAACCCCUUCCCCGCCGCCGUGAGAGGC UUCGUGGACGACAUCAUCCAGCCCAGCAGC ACCAGAGCCAGAAUCUGCUGCGACCUGGAC GUGCUGGCCAGCAAGAAGGUGCAGAGACCC UGGAGAAAGCACGCCAACAUCCCUCUG (SEQIDNO:6) 3UTR UGAUAAUAGUCCAUAAAGUAGGAAACACUA CAGCUGGAGCCUCGGUGGCCUAGCUUCUUG CCCCUUGGGCCUCCAUAAAGUAGGAAACAC UACAUCCCCCCAGCCCCUCCUCCCCUUCCU GCACCCGUACCCCCUCCAUAAAGUAGGAAA CACUACAGUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC (SEQIDNO:114) poly-Atail 100residuesinlength (SEQIDNO:195) EncodedPCCB MAAALRVAAVGARLSVLASGLRAAVRSLCS protein QATSVNERIENKRRTALLGGGQRRIDAQHK RGKLTARERISLLLDPGSFVESDMFVEHRC ADFGMAADKNKFPGDSVVTGRGRINGRLVY VFSQDFTVFGGSLSGAHAQKICKIMDQAIT VGAPVIGLNDSGGARIQEGVESLAGYADI FLRNVTASGVIPQISLIMGPCAGGAVYSPA LTDFTFMVKDTSYLFITGPDVVKSVTNEDV TQEELGGAKTHTTMSGVAHRAFENDVDALC NLRDFFNYLPLSSQDPAPVRECHDPSDRLV PELDTIVPLESTKAYNMVDIIHSVVDEREF FEIMPNYAKNIIVGFARMNGRTVGIVGNQP KVASGCLDINSSVKGARFVRFCDAFNIPLI TFVDVPGFLPGTAQEYGGIIRHGAKLLYAF AEATVPKVTVITRKAYGGAYDVMSSKHLCG DTNYAWPTAEIAVMGAKGAVEIIFKGHENV EAAQAEYIEKFANPFPAAVRGFVDDIIQPS STRARICCDLDVLASKKVQRPWRKHANIPL (SEQIDNO:2) Construct SEQIDNO:8consistsfrom Sequence 5to3end:5UTRofSEQID NO:55,ORFSequenceofSEQID NO:6,and3UTRofSEQID NO:114

[0873] This study comprises two stages: a Dose Optimization Stage followed by a Dose Expansion Stage. The first 2 participants enrolled in the Dose Optimization Stage will be greater than or equal to 8 years of age. The study is designed to evaluate multiple doses and dosing intervals of mRNA 3927, optimized based on the safety and pharmacodynamics of the preceding cohort, and to characterize the safety, tolerability, and pharmacological activity (as assessed by biomarker measurements) of mRNA-3927 in participants with propionic acidemia.

[0874] In both stages, after confirmation of eligibility within the Screening Period, participants will enter the Observation Period, followed by the Treatment Period (an optional Prescreening Period for molecular genetic testing may be done, in which participants in whom the diagnosis of propionic acidemia has not been confirmed by molecular genetic testing, may have the testing performed when the prescreening consent is signed or during the Screening Period). The Observation Period will include safety, biomarker, health-related quality-of-life (HRQOL), health utilization, and health economics outcome assessments for approximately 48 to 72 hours, including inpatient admission for additional safety and biomarker assessments for 24 hours before entering the Treatment Period.

[0875] In the Treatment Period, all participants will receive up to 10 doses of study drug. After which, they may either enter the 2-year Follow-up Period of the study or roll over into an extension study.

[0876] During the study, participants will attend inpatient and outpatient visits, with the option for home health care services for some visits, as per the Schedule of Assessments.

Dose Optimization Stage

[0877] Each dose cohort will consist of 3 participants, unless 1 of the 3 participants meets the dose limiting toxicity (DLT) criteria, in which case an additional 3 participants will be enrolled to further characterize the safety and tolerability at that dose.

[0878] After each dose cohort is fully enrolled, and the DLT Observation Window of at least 14 days is complete for the final participant in that cohort, the Sponsor will review the totality of available safety data in conjunction with all available PK/PD data. Based on this review, the Sponsor will recommend a revised dose and/or dosing interval. The Sponsor will abide by predefined constraints as to the maximum percentage change in dose and dose interval.

[0879] The role of the independent Safety Monitoring Committee (SMC) will be to ensure that there are no safety concerns before moving between dose cohorts or at any stage of the study.

[0880] A maximum of 5 cohorts will be enrolled into the study.

Dose Expansion Stage

[0881] A minimum of 4, and a maximum of 6, additional participants (including at least 2 participants with propionyl CoA carboxylase subunit a [PCCA] deficiency and 2 participants with propionyl CoA carboxylase subunit ? [PCCB] deficiency) will be enrolled to confirm the selected dose and dose interval in both propionic acidemia subtypes.

Dosage and Mode of Administration

[0882] In the Treatment Period, participants will be treated with mRNA-3927 once every 2 to 4 weeks (q2W to q4W) by intravenous (IV) infusion over 1 to 4 hours. Participants will receive up to 10 doses of mRNA-3927.

[0883] The starting dose and interval for the study is 0.3 mg/kg every 3 weeks (q3W).

[0884] For participants in the Dose Expansion Stage, the study drug assigned will be mRNA-3927 at a dose and interval selected, following data review by the SMC in the Dose Optimization Stage.

Dose Optimization

[0885] Circulating levels of 2-methylcitric acid (2-MC) and 3-hydroxypropionic acid (3-HP) are many orders of magnitude higher in patients with PA than in healthy individuals. Although 2-MC and 3-HP are just two of many metabolites that are elevated in propionic acidemia, both are known to be toxins and are reported to be significantly reduced in patients with propionic acidemia following liver transplantation, although they do not fall to normal levels.

[0886] The aims of the Dose Optimization Stage are to strive for a large relative reduction in 2-MC and 3-HP levels while appreciating that complete normalization is not achievable. Targets of ?50% reduction in plasma levels would be desirable. Furthermore, repeat dosing with mRNA 3927 is expected to lead to sustained reductions in circulating 2 MC and 3-HP levels and inhibit predose rises in these toxins.

Dose Optimization Rules

[0887] The maximum increase in dose allowed is an increase of 50% from 1 cohort to the next (for example from 0.3 mg/kg to 0.45 mg/kg). The total maximum dose is 0.6 mg/kg.

[0888] The maximum reduction in the dosing interval is 1 week from 1 dose cohort to the next.

[0889] The minimum dosing interval is q2W.

[0890] If supported by the PK/PD data, it is permitted to simultaneously increase the dose and reduce the dosing interval (e.g., suboptimal reduction in plasma biomarkers and rapid return to peak levels seen in the previous cohort).

[0891] Reduction in dose and/or dosing frequency is allowed if the PK/PD modeling of data from preceding cohorts suggests that no further substantial decrease in PD biomarkers (2-MC and 3-HP) is expected upon an increase in dose or reduction in dosing frequency.

[0892] If the dose is reduced to optimize the pharmacological profile, and not because of a DLT or ADR mandating a dose reduction, a sentinel dosing strategy will not be required for the subsequent cohort

Objectives and Endpoints

[0893] The primary objective of the study is to evaluate the safety and tolerability of mRNA-3927 in participants with propionic acidemia. The primary objective is evaluated by measuring the incidence and severity of adverse events (including study drug-related and not related adverse events), serious adverse events, and adverse events leading to treatment discontinuation.

[0894] The secondary objectives of the study are (1) to characterize the pharmacodynamic (PD) responses of mRNA 3927 as determined by changes in plasma 2-MC and 3-HP after single and repeated administrations of mRNA, (2) to characterize the single dose and repeated-dose pharmacokinetic (PK) of mRNA-3927, and (3) to assess for the presence of anti-PEG (a component of the lipid nanoparticle) antibodies.

[0895] The secondary objectives are evaluated by measuring the following endpoints; (1) change in plasma 2-MC and 3-HP levels from baseline (predose levels) to postdose levels measured after single and repeated administrations of mRNA 3927; and estimation of PD parameters from baseline after single and repeated administration of mRNA-3927, including E.sub.max, AUEC, and duration of response, (2) estimation of PK parameters of PCCA and PCCB mRNAs and Compound II, including C.sub.max, t.sub.max, AUC, t.sub.1/2, CL, V.sub.z, and V.sub.ss, and (3) presence and titers of anti-PEG antibodies.

[0896] The exploratory objectives of the study are (1) to characterize the PD response to mRNA-3927 as determined by other biomarkers after single and repeated administrations, (2) to assess the relationships between PK, PD, and disease related parameters, (3) to assess for the presence of antibodies to PCC, (4) to evaluate pretreatment and post-treatment adverse events in participants with propionic acidemia, (5) to evaluate changes in clinically significant events, (6) to evaluate changes in metabolic decompensation events, (7) to evaluate changes in health care resource utilization, (8) to characterize changes in disease impact on missed school/workdays, (9) to evaluate changes in ammonia, lactate, and venous blood gas in participants with propionic acidemia, (10) to evaluate cardiac and renal function in participants with propionic acidemia, (11) to assess HRQOL measures, (12) to evaluate observer reported outcomes, and (13) to assess impact of diet on PD biomarkers.

[0897] The exploratory objectives are evaluated by measuring the following endpoints: (1) changes in other biomarkers, including total, free, and acyl carnitines (C2 and C3), propionylglycine, glycine, and FGF-21; and estimation of PD parameters for these other biomarkers after single and repeated administration of mRNA-3927, including to, E.sub.max, AUEC, and duration of response, (2) association of C.sub.max and AUC and E.sub.max and AUEC of biomarkers across dose levels with propionic acidemia-related clinical events, (3) antibodies to PCC, (4) incidence and severity of pretreatment and post-treatment adverse events and serious adverse events, (5) changes in clinically significant events pretreatment and post-treatment (clinically significant event is defined as a composite of the following: hospitalization, excluding hospitalizations for chronic diseases not related to propionic acidemia or elective hospitalizations for conditions not related to propionic acidemia: emergency room visits: emergency interventions outside of health care settings to prevent a metabolic decompensation event (sick-day diets and fluid resuscitation at home); and complications of cardiomyopathy treated outside of hospital (documented arrhythmia, emergent changes in diuretic therapy due to increasing fluid overload)), (6) changes in metabolic decompensation events pretreatment and post-treatment, (7) changes in health care resource utilization pretreatment and post-treatment, (8) changes in disease impact on missed school and workdays pretreatment and post-treatment, (9) ammonia, lactate and venous blood gas levels in participants with propionic academia, (10) cardiac parameters evaluating for cardiomyopathy, including ECHO and NT-BNP; eGFR, (11) changes in Pediatric Quality of Life Inventory (PedsQL?) and PedsQL Family Impact Module?, (12) changes in Methylmalonic Acidemia and Propionic Acidemia Questionnaire (MMAPAQ) Symptom Score, Caregiver Report of Global Impression of Improvement (CrGI-I), Caregiver Report of Global Impression of Severity (CrGI-S), and Investigator Global Assessment of Improvement (IGA-I), and (13) relationship between dietary changes and PD biomarkers.

Premedication Before Infusion

[0898] Participants will receive each of the following medications 50 to 90 minutes before each infusion of study drug: (1) acetaminophen/paracetamol or ibuprofen with age and/or weight-appropriate dosing, given orally or via feeding tube, (2) H.sub.1-receptor blocker; diphenhydramine, hydroxyzine, cetirizine, fexofenadine, or equivalent H.sub.1-receptor blocker with age and/or weight-appropriate dosing, given intravenously, orally, or via feeding tube, and (3) H.sub.2-receptor blocker: famotidine, or equivalent H.sub.2-receptor blocker with age and/or weight appropriate dosing, given intravenously, orally, or via feeding tube. Further premedication, including corticosteroids, can be administered if considered appropriate by the Investigator.

Example 4: Justification for Dose

[0899] The justification for the starting dose and clinical dosing regimen of 0.3 mg/kg q3W was based on results from nonclinical pharmacology studies and was further supported by an interspecies population PK/PD model, as well as Good Laboratory Practice (GLP)-compliant toxicology studies.

[0900] An 8-week, single-dose, dose range-finding study in propionic acidemia hypomorphic mice identified 0.5 mg/kg as the minimum effective dose that resulted in significant reductions in plasma disease biomarkers (2-MC, C3/C2 ratio, and 3-HP) that were sustained for a duration of 3 weeks before rebounding to pretreatment concentrations. A 12-week repeat-dose pharmacology study in Peca?/?(A138T) mice demonstrated that a q3W dosing regimen of 0.5 mg/kg mRNA-3927 led to significant and sustained reductions of disease biomarkers throughout the study and produced functional PCC enzyme in the liver, thus correcting the underlying metabolic defect in the liver.

[0901] An interspecies PK/PD model demonstrated predictability of PCCA/PCCB mRNA PK between mice, rats, and monkeys (based on allometric scaling), which provided a rationale for the allometric prediction of human PK. Assuming the concentrations of PCC protein subunits needed for a 50% reduction in plasma 2-MC from baseline are similar between Pcca?/?(A138T) mice and humans, predictions in patients with propionic acidemia suggest that a greater than 50% reduction of 2-MC will be observed for mRNA-3927 doses of greater than or equal to 0.2 mg/kg administered q3W. Therefore, the starting dose of 0.3 mg/kg administered q3W was anticipated to yield meaningful reductions in disease biomarkers.

[0902] Furthermore, GLP-compliant, repeat-dose toxicology studies in juvenile Sprague Dawley rats (up to 26 weeks in duration) and immature cynomolgus monkeys (up to 4 weeks in duration) showed that the rat was the most sensitive toxicological species with a no observed adverse effect level (NOAEL) of 3.0 mg/kg/dose. A starting dose of 0.3 mg/kg provided a 10-fold safety margin (human equivalent dose [HED] based on body weight). Scaling the NOAEL from the toxicology studies to a HED based on body weight (mg/kg basis) is appropriate for mRNA-3927. This is supported by the similar toxicity profile and NOAELs observed in the GLP-compliant juvenile rat (3 mg/kg/dose) and immature cynomolgus monkey (5 mg/kg/dose) studies. The primary toxicity observed in both studies is consistent with a transient and reversible systemic inflammatory response, which is believed to be maximum observed concentration (C.sub.max) driven and for which there is a robust correlation across species on a mg/kg basis. For example, at the end of the dosing phase of the juvenile rat and immature nonhuman primate studies, animals administered 3 mg/kg/dose had similar mRNA C.sub.max concentrations (males, 8.8 and 11.6 ?g/mL; females, 10 and 9.58 ?g/mL for rats and nonhuman primates, respectively).

[0903] An initial dosing interval of q3W is proposed on the basis of the duration of biomarker response observed in pharmacology studies in Peca?/?(A138T) mice. Specifically, at the minimum efficacious dose level of 0.5 mg/kg, decreases in plasma disease biomarkers were sustained for 3 weeks before rebounding toward pretreatment levels in propionic acidemia mice. The potential for drug product accumulation is considered low, with the planned clinical dosing regimen based on the kinetics of the components of the drug product. The maximum mean tissue terminal-phase elimination half-life (t.sub.1/2) values of Compound II and mRNA in rodents were 54.4 hours and 57.6 hours, respectively. The dosing interval is further supported by the interspecies PK/PD model, which demonstrated that a q3W dosing interval would be sufficient to yield meaningful reductions in disease biomarkers.

[0904] A 10-dose treatment period was decided upon to ensure steady-state pharmacology and thus allow sufficient collection of PK/PD data to support dose selection in future studies. The impact of longer-term dosing may be characterized in the proposed extension study and later clinical studies.

[0905] The maximum clinical dose for this study is 0.6 mg/kg administered q2W. Based on the PK/PD modeling data, increasing the dose beyond 0.6 mg/kg q2W was not expected to lead to significant further reduction in plasma PD biomarkers. As such, no additional potential benefit was expected. However, based on the observed PD in the first cohort of this study, it is expected that larger PD effects may be seen at dose levels up to 1 mg/kg.