Phenylalanine hydroxylase variants and their use

Abstract

Variant phenylalanine hydroxylase polypeptides having substitutions at predetermined amino acid residues are disclosed. Methods of treating disorders such as phenylketonuria using variant phenylalanine hydroxylase polypeptides, or polynucleotides encoding the variant phenylalanine hydroxylase polypeptides, are also disclosed.

Claims

1. A polypeptide comprising the amino acid sequence shown in Sequence ID No.

4.

2. A polynucleotide comprising an open reading frame (ORF) encoding the polypeptide described in claim 1.

3. A messenger RNA (mRNA) comprising a polynucleotide comprising an open reading frame (ORF) encoding a polypeptide comprising the amino acid sequence shown in Sequence ID No. 3, wherein the mRNA comprises a polyA region, the polyA region is at least 10 nucleotides long, and the mRNA comprises an inverted deoxythymidine at its 3' end.

4. Messenger RNA (mRNA) comprising polynucleotides as described in claim 2.

5. The mRNA according to claim 3, wherein the ORF is at least 90% identical to the nucleotide sequence shown in Sequence ID No.

22.

6. The mRNA according to claim 3, wherein the ORF is at least 97% identical to the nucleotide sequence shown in Sequence ID No.

22.

7. The mRNA according to claim 3, wherein the ORF is 100% identical to the nucleotide sequence shown in Sequence ID No.

22.

8. The mRNA according to claim 4, wherein the ORF is at least 90% identical to the nucleotide sequence shown in Sequence ID No.

23.

9. The mRNA according to claim 4, wherein the ORF is at least 97% identical to the nucleotide sequence shown in Sequence ID No.

23.

10. The mRNA according to claim 4, wherein the ORF is 100% identical to the nucleotide sequence shown in Sequence ID No.

23.

11. The mRNA according to any one of claims 3 to 10, comprising a 5' UTR containing a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 55 or SEQ ID NO:

56.

12. The mRNA according to any one of claims 3 to 10, comprising a 5' UTR containing a nucleic acid sequence that is 100% identical to the nucleic acid sequence shown in Sequence ID No.

56.

13. The mRNA according to any one of claims 3 to 12, comprising a 3'UTR containing a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence shown in SEQ ID NO: 113, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO:

111.

14. The mRNA according to any one of claims 3 to 12, comprising a 3'UTR containing a nucleic acid sequence that is 100% identical to the nucleic acid sequence shown in Sequence ID No.

108.

15. The mRNA according to claim 3, comprising the nucleotide sequence shown in Sequence ID No.

42.

16. The mRNA according to claim 4, comprising the nucleotide sequence shown in Sequence ID No.

43.

17. mRNA according to any one of claims 3 to 16, comprising a 5' terminal cap.

18. The mRNA according to claim 17, wherein the 5' terminal cap includes the caps 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, or 5'methyl G.

19. mRNA according to any one of claims 3 to 18, comprising a poly-A region.

20. The mRNA according to claim 19, wherein the polyA region is at least 10 nucleotides long.

21. The mRNA according to claim 20, wherein the polyA region is at least 100 nucleotides long.

22. The mRNA according to claim 20 or 21, comprising the nucleotide sequence UCUAGAAA

23. The mRNA according to any one of claims 3 to 22, wherein the 3' end of the mRNA contains an inverted deoxythymidine.

24. The mRNA according to any one of claims 3 to 23, wherein all of the uracil in the mRNA is N1-methylpseudolacil.

25. The mRNA according to claim 3, wherein the ORF is 100% identical to the nucleic acid sequence shown in SEQ ID NO: 22, the mRNA includes a poly-A region of at least 100 nucleotides in length, and all of the uracil in the mRNA is N1-methylpseudolacil.

26. The mRNA according to claim 4, wherein the ORF is 100% identical to the nucleic acid sequence shown in SEQ ID NO: 23, the mRNA includes a polyA region of at least 100 nucleotides in length, and all of the uracil in the mRNA is N1-methylpseudolacil.

27. The mRNA according to claim 3, wherein the mRNA comprises a 5' terminal cap containing a guanine cap nucleotide containing N7 methylation, the 5' terminal nucleotide of the mRNA comprises 2'-O-methyl, the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 42, the mRNA comprises a poly(A) region of at least 100 nucleotides in length, and all of the uracil in the mRNA is N1-methylpseudolacil.

28. The mRNA according to claim 4, wherein the mRNA comprises a 5' terminal cap containing a guanine cap nucleotide containing N7 methylation, the 5' terminal nucleotide of the mRNA comprises 2'-O-methyl, the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 43, the mRNA comprises a poly(A) region of at least 100 nucleotides in length, and all of the uracil in the mRNA is N1-methylpseudolacil.

29. The mRNA according to any one of claims 25 to 28, comprising the nucleotide sequence UCUAGAAA

30. The mRNA according to any one of claims 25 to 29, wherein the 3' end of the mRNA contains an inverted deoxythymidine.

31. An expression vector comprising the polynucleotide described in claim 2.

32. The expression vector according to claim 31, wherein the ORF is at least 90% identical to the nucleotide sequence shown in SEQ ID NO:

23.

33. The expression vector according to claim 31, wherein the ORF is 100% identical to the nucleotide sequence shown in SEQ ID NO:

23.

34. An expression vector according to any one of claims 31 to 33, which is a plasmid, a minimally immunologically defined gene expression (MIDGE) vector, a terminally closed linear double-stranded DNA (ceDNA), or a viral vector.

35. An expression vector according to any one of claims 31 to 34, comprising a transcriptional regulatory element.

36. The expression vector according to claim 35, wherein the transcriptional regulatory element is a promoter, enhancer, or stop sequence.

37. Lipid nanoparticles comprising the polynucleotide described in claim 2, the mRNA described in any one of claims 3 to 30, or the expression vector described in any one of claims 31 to 36.

38. (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, or (i) Compound II, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv) Compound I It contains, and compound I, compound II, and compound VI have the following structures: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 Lipid nanoparticles according to claim 37.

39. A pharmaceutical composition comprising a polypeptide according to claim 1, a polynucleotide according to claim 2, mRNA according to any one of claims 3 to 30, or an expression vector according to any one of claims 31 to 36, and a pharmaceutically acceptable excipient.

40. A pharmaceutical composition comprising lipid nanoparticles according to claim 37 or 38.

41. A polypeptide according to claim 1, a polynucleotide according to claim 2, an mRNA according to any one of claims 3 to 30, an expression vector according to any one of claims 31 to 36, a lipid nanoparticle according to claim 37 or 38, or a pharmaceutical composition according to claim 39 or 40, for use in a method for expressing a phenylalanine hydroxylase polypeptide.

42. A polypeptide according to claim 1, a polynucleotide according to claim 2, an mRNA according to any one of claims 3 to 30, an expression vector according to any one of claims 31 to 36, a lipid nanoparticle according to claim 37 or 38, or a pharmaceutical composition according to claim 39 or 40, for use in a method for improving phenylalanine hydroxylase activity.

43. A polypeptide according to claim 1, a polynucleotide according to claim 2, an mRNA according to any one of claims 3 to 30, an expression vector according to any one of claims 31 to 36, a lipid nanoparticle according to claim 37 or 38, or a pharmaceutical composition according to claim 39 or 40, for use in a method for treating, preventing or delaying the onset and / or progression of phenylketonuria.

44. A polypeptide according to claim 1, a polynucleotide according to claim 2, an mRNA according to any one of claims 3 to 30, an expression vector according to any one of claims 31 to 36, a lipid nanoparticle according to claim 37 or 38, or a pharmaceutical composition according to claim 39 or 40, for use in a method for reducing phenylalanine levels.

45. The polypeptide, polynucleotide, mRNA, expression vector, lipid nanoparticle, or pharmaceutical composition according to claim 44, wherein the phenylalanine level is the phenylalanine level in blood, plasma, serum, liver, and / or urine.

46. A polypeptide, polynucleotide, mRNA, expression vector, lipid nanoparticle, or pharmaceutical composition according to any one of claims 41 to 45, for intravenous administration.

47. A polypeptide, polynucleotide, mRNA, expression vector, lipid nanoparticle, or pharmaceutical composition according to any one of claims 41 to 45, for subcutaneous administration.

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