Claims for Patent: 10,537,617

Summary for Patent: 10,537,617

Title:	Modified factor IX, and compositions, methods and uses for gene transfer to cells, organs, and tissues
Abstract:	The invention relates to modified Factor IX coding sequence, expression cassette, vectors such as viral (e.g., lenti- or adeno-associated viral) vectors, and gene transfer methods and uses. In particular, to target Factor IX nucleic acid to cells, tissues or organs for expression (transcription) of Factor IX.
Inventor(s):	High; Katherine A. (Merion Station, PA), Anguela; Xavier (Philadelphia, PA)
Assignee:	THE CHILDREN\'S HOSPITAL OF PHILADELPHIA (Philadelphia, PA)
Application Number:	15/191,357
Patent Claims:	1. A recombinant AAV (rAAV) vector comprising a capsid and a genome, wherein said capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:4, and wherein said genome comprises a nucleic acid comprising a sequence encoding human Factor IX (FIX) protein, wherein the coding sequence of said nucleic acid is not naturally occurring, is at least 90% identical to SEQ ID NO:10, encodes the same human Factor IX protein encoded by SEQ ID NO:10, has a reduced number of CpG di-nucleotides compared to a wild-type sequence encoding human Factor IX protein and optionally is interrupted by an intron. 2. The rAAV vector of claim 1, wherein said genome further comprises an additional sequence selected from the group consisting of an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) and a filler polynucleotide sequence, wherein said additional sequence is optionally modified to have a reduced number of CpG di-nucleotides compared to the unmodified sequence. 3. The rAAV vector of claim 2, wherein the intron is within the sequence encoding human Factor IX protein, or wherein the expression control element is operably linked to the sequence encoding human Factor IX protein, or wherein the AAV ITR(s) flanks the 5' or 3'end of the sequence encoding human Factor IX protein, or wherein the filler polynucleotide sequence flanks the 5' or 3'end of the sequence encoding human Factor IX protein. 4. The rAAV vector of claim 2, wherein the expression control element comprises an enhancer sequence comprising the sequence set forth as SEQ ID NO:14. 5. The rAAV vector of claim 2, wherein the expression control element comprises a promoter sequence comprising the sequence set forth as SEQ ID NO:15. 6. The rAAV vector of claim 2, wherein the sequence of the one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs) comprises the sequence set forth as SEQ ID NO:13 or SEQ ID NO:20. 7. The rAAV vector of claim 2, wherein the filler polynucleotide sequence comprises a sequence set forth as SEQ ID NO:21. 8. The rAAV vector of claim 2, wherein the expression control element comprises an element that confers expression in liver. 9. The rAAV vector of claim 2, wherein the expression control element comprises a human al-anti-trypsin (hAAT) promoter, or apolipoprotein E (ApoE) HCR-1 or HCR-2 enhancer. 10. The rAAV vector of claim 1, wherein the sequence encoding human FIX protein has 1-55 fewer CpG di-nucleotides than native sequence encoding human Factor IX. 11. The rAAV vector of claim 1, wherein the sequence encoding human FIX protein is devoid of any CpG di-nucleotides. 12. The rAAV vector of claim 1, wherein the sequence encoding human FIX protein is interrupted by an intron. 13. The rAAV vector of claim 1, wherein the sequence encoding human FIX protein comprises a sequence with 95% or more identity to SEQ ID NO:10. 14. A recombinant AAV (rAAV) vector comprising a capsid and a genome, wherein said capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:4, and wherein said genome comprises a nucleic acid comprising a sequence encoding human FIX protein, wherein the coding sequence of said nucleic acid is not naturally occurring, is at least 90% identical to SEQ ID NO:10, encodes the same human FIX protein encoded by SEQ ID NO:10, and optionally is interrupted by an intron. 15. The rAAV vector of claim 1 or 14, wherein the intron comprises the sequence set forth in SEQ ID NO:17. 16. The rAAV vector of claim 1 or 14, further comprising a poly-adenylation sequence located 3' of the nucleic acid sequence encoding human Factor IX. 17. The rAAV vector of claim 16, wherein the poly-adenylation sequence located 3' of the nucleic acid sequence encoding human Factor IX comprises a poly-adenylation sequence having all CpG di-nucleotides removed therefrom. 18. The rAAV vector of claim 1 or 14, wherein said genome further comprises an ITR sequence of any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8 AAV serotypes. 19. A pharmaceutical composition comprising the rAAV vector of claim 1 or 14. 20. A pharmaceutical composition according to claim 19, further comprising empty capsid AAV. 21. A pharmaceutical composition according to claim 20 wherein said empty capsid AAV is selected from serotype AAV AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. 22. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 95% identical to SEQ ID NO:10. 23. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 96% identical to SEQ ID NO:10. 24. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 97% identical to SEQ ID NO:10. 25. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 98% identical to SEQ ID NO:10. 26. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 99% identical to SEQ ID NO:10. 27. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 91% identical to SEQ ID NO:10. 28. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 92% identical to SEQ ID NO:10. 29. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 93% identical to SEQ ID NO:10. 30. The rAAV vector of claim 1 or 14, wherein the sequence of said nucleic acid is at least 94% identical to SEQ ID NO:10. 31. A method for delivering or transferring a nucleic acid sequence into a mammal or a cell of a mammal, comprising administering the rAAV vector of claim 1 or 14 to said mammal or a cell of said mammal, thereby delivering or transferring the nucleic acid sequence into the mammal or cell of the mammal. 32. A method of treating a mammal in need of Factor IX protein, comprising: (a) providing a rAAV vector of claim 1 or 14; and (b) administering an amount of the rAAV vector of claim 1 or 14 to the mammal wherein said Factor IX is expressed in the mammal. 33. The method of claim 32, wherein said Factor IX protein is expressed in a cell, tissue or organ of said mammal. 34. A method of treating a human subject with hemophilia B comprising administering to said subject a therapeutically effective amount of the rAAV vector of claim 1 or 14. 35. A recombinant AAV (rAAV) vector for treating hemophilia B, comprising a capsid and a genome, wherein said capsid comprises a VP1 protein having the amino acid sequence of SEQ ID NO:4; and wherein said genome is single-stranded and comprises the following elements in 5' to 3' order: (a) a first AAV2 ITR, (b) an ApoE HCR-1 enhancer, (c) an AAT promoter, (d) a nucleic acid comprising a sequence encoding human FIX Padua variant, wherein the coding sequence of said nucleic acid is not naturally occurring, is at least 90% identical to SEQ ID NO:10, encodes the same human FIX protein encoded by SEQ ID NO:10, and optionally is interrupted by an intron, (e) a polyadenylation sequence; and (f) a second AAV2 ITR. 36. The rAAV vector of claim 35, wherein (a) the nucleic acid sequence of the first AAV2 ITR consists of nucleotides 1-141 of SEQ ID NO:12, (b) the nucleic acid sequence of said ApoE HCR-1 enhancer consists of nucleotides 152-472 of SEQ ID NO:12, (c) the nucleic acid sequence of said ATT promoter consists of nucleotides 482-878 of SEQ ID NO:12, (d) the nucleic acid sequence of said nucleic acid encoding human Factor IX Padua variant consists of nucleotides 908-3731 of SEQ ID NO:12, (e) the nucleic acid sequence of said polyadenylation sequence consists of nucleotides 3820-4047 of SEQ ID NO:12; and (f) the nucleic acid sequence of said second AAV2 ITR consists of nucleotides 4097-4204 of SEQ ID NO:12. 37. The rAAV vector of claim 36, wherein the genome comprises a nucleic acid sequence corresponding to nucleotides 1-4204 of SEQ ID NO:12. 38. A method for treating a human subject with hemophilia B comprising administering to said subject a therapeutically effective amount of the rAAV vector of claim 35. 39. The method of claim 38, wherein said subject has severe hemophilia B, and wherein said treatment is effective to reduce the hemophilia symptoms from severe to those of moderate or mild hemophilia B. 40. A packaging cell comprising (i) a nucleic acid comprising a sequence encoding human FIX protein, wherein the coding sequence of said nucleic acid is not naturally occurring, is at least 90% identical to SEQ ID NO:10, encodes the same human FIX protein encoded by SEQ ID NO:10, and optionally is interrupted by an intron, (ii) AAV rep protein, (iii) AAV capsid protein comprising the amino acid sequence of SEQ ID NO:4, and (iv) adenovirus helper protein(s). 41. A rAAV vector produced by culturing the packaging cell of claim 40 under conditions allowing packaging of a nucleic acid comprising the sequence from 1-4204 of SEQ ID NO:12 into AAV particles thereby producing rAAV vector. 42. The rAAV vector produced by the method of claim 41, wherein the rAAV vector is isolated or purified. 43. A recombinant AAV (rAAV) vector comprising a capsid and a genome, wherein said capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:4, and wherein said genome comprises at least one AAV ITR and a nucleic acid comprising a sequence encoding human FIX protein, wherein the coding sequence of said nucleic acid is not naturally occurring, is at least 90% identical to SEQ ID NO:10, encodes the same human FIX protein encoded by SEQ ID NO:10, and optionally is interrupted by an intron. 44. The rAAV vector of claim 43, said genome further comprising an expression control element operably linked with said nucleic acid sequence encoding human FIX protein. 45. The rAAV vector of claim 44, said expression control element comprising a promoter and optionally an enhancer. 46. The rAAV vector of claim 45, wherein said expression control element is tissue specific. 47. The rAAV vector of claim 46, wherein said expression control element is liver specific. 48. The rAAV vector of claim 45, wherein said genome further comprises a polynucleotide stuffer. 49. The rAAV vector of claim 45, wherein said genome further comprises a transcription terminator. 50. A recombinant AAV (rAAV) vector comprising a capsid and a genome, wherein said capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:4, and wherein said genome comprises an AAV2 ITR, a liver-specific enhancer and promoter in operable linkage with a nucleic acid comprising a sequence encoding human FIX protein, wherein the coding sequence of said nucleic acid is not naturally occurring, is at least 90% identical to SEQ ID NO:10, encodes the same human FIX protein encoded by SEQ ID NO:10, and optionally is interrupted by an intron, a transcription terminator, and optionally a second AAV2 ITR. 51. The rAAV vector of claim 50, wherein said first AAV2 ITR is positioned at the 5' or 3' end of the genome, and said second AAV2 ITR, if present, is positioned at the opposite end of the genome. 52. The rAAV vector of claim 51, said genome further comprising an intron or a polynucleotide stuffer. 53. The rAAV vector of claim 52, wherein said genome is single stranded DNA. 54. The rAAV vector of claim 52, wherein said intron comprises the nucleic acid sequence of SEQ ID NO:17 and said polynucleotide stuffer comprises the nucleic acid sequence of SEQ ID NO:21. 55. The rAAV vector of claim 52, wherein the sequence of said nucleic acid encoding human FIX protein and intron comprises the nucleic acid sequence of SEQ ID NO:25. 56. The rAAV vector of claim 50, wherein said promoter is a human alphal-antitrysin (AAT) promoter and said enhancer is an apolipoprotein E (ApoE) HCR-1 or HCR-2 enhancer. 57. The rAAV vector of claim 56, wherein said enhancer comprises the nucleic acid sequence of SEQ ID NO:14. 58. The rAAV vector of claim 56, wherein said promoter comprises the nucleic acid sequence of SEQ ID NO:15. 59. The rAAV of claim 50, wherein said AAV2 ITR comprises the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:20. 60. A recombinant AAV (rAAV) vector for treating hemophilia B, comprising a capsid and a genome, wherein said capsid comprises a VP1 protein having the amino acid sequence of SEQ ID NO:4; and wherein said genome is comprises the following elements: (a) a first AAV2 ITR, (b) a liver specific enhancer and promoter, (c) a nucleic acid sequence encoding human FIX Padua variant, wherein the coding sequence of said nucleic acid is not naturally occurring, is at least 90% identical to SEQ ID NO:10, encodes the same human FIX protein encoded by SEQ ID NO:10, and is interrupted by an intron, (d) a polyadenylation sequence; and (e) a second AAV2 ITR. 61. The rAAV vector of claim 60, wherein said enhancer is an ApoE HCR-1 enhancer, said promoter is an hAAT promoter, said intron is a hFIX Intron A, and said polyadenylation sequence is a bGH polyA sequence. 62. The rAAV vector of claim 60, wherein (a) the nucleic acid sequence of the first AAV2 ITR consists of nucleotides 1-141 of SEQ ID NO:12, (b) the nucleic acid sequence of the enhancer comprises nucleotides 152-472 of SEQ ID NO:12, (c) the nucleic acid sequence of the promoter comprises nucleotides 482-878 of SEQ ID NO:12, (d) the nucleic acid sequence encoding human Factor IX consists of nucleotides 908-995 and 2434-3731 of SEQ ID NO:12, (e) the nucleic acid sequence of the intron comprises nucleotides 996-2433 of SEQ ID NO:12, (e) the nucleic acid sequence of the polyadenylation sequence comprises nucleotides 3820-4047 of SEQ ID NO:12; and (f) the nucleic acid sequence of the second AAV2 ITR consists of nucleotides 4097-4204 of SEQ ID NO:12. 63. The rAAV vector of claim 60, wherein said genome further comprises 5' UTR positioned after the promoter and before the coding sequence, and 3' UTR positioned after the coding sequence and before the polyadenylation sequence. 64. The rAAV vector of claim 63, wherein the nucleic acid sequence of the 5' UTR comprises nucleotides 879-907 of SEQ ID NO:12 and the 3' UTR comprises nucleotides 3732-3779 of SEQ ID NO:12. 65. The rAAV vector of claim 64, wherein the genome is single-stranded DNA the nucleic acid sequence of which comprises nucleotides 1-4204 of SEQ ID NO:12.

Details for Patent 10,537,617

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2035-06-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2035-06-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2035-06-23