Modified factor IX, and compositions, methods and uses for gene transfer to cells, organs, and tissues
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.
1. A method of treating a human subject with severe or moderate hemophilia B comprising:
administering to said subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector comprising a vector genome encapsidated by an AAV capsid,
wherein said vector genome comprises at least two AAV inverted terminal repeats (ITR), an expression control element conferring liver tissue-specific expression operably linked with a nucleic acid sequence encoding human Factor IX (FIX) protein, and a polyadenylation signal sequence, wherein said nucleic acid sequence encoding human FIX protein is at least 70% identical to SEQ ID NO: 10, has a reduced number of CpG di-nucleotides compared to wild-type nucleic acid sequence encoding human FIX protein and encodes the same human FIX protein encoded by SEQ ID NO:10,
wherein said therapeutically effective amount of said rAAV vector is a dose ranging from 1×10 12 to 5×10 13 vector genomes per kilogram (vg/kg) subject body weight, and is effective to reduce the severe or moderate hemophilia B to mild hemophilia B or a hemophilia B disease-free state,
wherein said AAV capsid is an AAV capsid of serotype AAV5, and
wherein said therapeutically effective amount of said rAAV vector is effective to produce 6% to 150% of normal FIX activity or is effective to produce plasma FIX levels of 0.06 to 1.50 IU/mL.
2. The method of claim 1 , wherein said nucleic acid sequence encoding human FIX protein is at least 80% identical to SEQ ID NO:10.
3. The method of claim 2 , wherein said nucleic acid sequence encoding human FIX protein is at least 85% identical to SEQ ID NO:10.
4. The method of claim 1 , wherein said vector genome further comprises an intron.
5. The method of claim 4 , wherein said intron is positioned within said nucleic acid sequence encoding human FIX protein.
6. The method of claim 1 , wherein said vector genome is linear single-stranded DNA.
7. The method of claim 1 , wherein an AAV ITR is positioned at each end of the vector genome, and said expression control element comprises an enhancer and a promoter.
8. The method of claim 7 , wherein said AAV ITRs are AAV2 ITRs, said enhancer is a human apolipoprotein HCR enhancer, and said promoter is a human alpha-1-antitrypsin gene promoter.
9. The method of claim 7 , wherein said AAV ITRs are AAV2 ITRs, said enhancer is a human apolipoprotein HCR enhancer, and said promoter is a human alpha-1-antitrypsin gene promoter.
10. The method of claim 9 , wherein said therapeutically effective amount of said rAAV vector is a dose of 2×10 13 vg/kg subject body weight.
11. The method of claim 10 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 0.50 IU/mL.
12. The method of claim 10 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 1.50 IU/mL.
13. The method of claim 9 , wherein said vector genome further comprises an intron positioned between said promoter and said nucleic acid sequence encoding human FIX protein.
14. The method of claim 13 , wherein said therapeutically effective amount of said rAAV vector is a dose of 2×10 13 vg/kg subject body weight.
15. The method of claim 7 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 0.50 IU/mL.
16. The method of claim 7 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 1.50 IU/mL.
17. The method of claim 1 , wherein said therapeutically effective amount of said rAAV vector is a dose selected from the group consisting of 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , and 5×10 13 vg/kg subject body weight.
18. The method of claim 17 , wherein said therapeutically effective amount of said rAAV vector is a dose of 2×10 13 vg/kg subject body weight.
19. The method of claim 18 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 0.50 IU/mL.
20. The method of claim 18 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 1.50 IU/mL.
21. The method of claim 1 , wherein said treatment is effective to produce an average FIX activity of less than 150% of normal for a sustained period of at least 6 months.
22. The method of claim 1 , wherein said treatment is effective to produce an average FIX activity of less than 150% of normal for a sustained period of at least 12 months.
23. The method of claim 1 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 0.50 IU/mL.
24. The method of claim 1 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 1.50 IU/mL.
25. The method of claim 1 , wherein said treatment is effective to reduce the average frequency of FIX replacement therapy needed to control bleeding by at least 80%.
26. The method of claim 1 , wherein said treatment is effective to reduce the average frequency of spontaneous bleeding episodes by at least 80%.
27. The method of claim 1 , wherein said treatment is effective to produce average FIX activity of 30% to 50% of normal 12 months after administration.
28. The method of claim 1 , wherein said treatment does not cause circulating liver enzyme levels to exceed 100% of the upper limit of normal value of liver enzymes.
29. A method of treating a human subject with severe or moderate hemophilia B comprising:
administering to said subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector comprising a vector genome encapsidated by an AAV capsid,
wherein said vector genome comprises at least two AAV inverted terminal repeats (ITR), an expression control element conferring liver tissue-specific expression operably linked with a nucleic acid sequence encoding human Factor IX (FIX) protein, and a polyadenylation signal sequence, wherein said nucleic acid sequence encoding human FIX protein is at least 70% identical to SEQ ID NO: 10, has a reduced number of CpG di-nucleotides compared to wild-type nucleic acid sequence encoding human FIX protein and encodes the same human FIX protein encoded by SEQ ID NO:10,
wherein said AAV capsid is an AAV capsid of serotype AAV5, and
wherein said therapeutically effective amount of said rAAV vector is a dose of 1×10 13 to 5×10 13 vector genomes per kilogram (vg/kg) subject body weight, and
wherein said therapeutically effective amount of said rAAV vector is effective to produce 6% to 150% of normal FIX activity or is effective to produce plasma FIX levels of about 0.06 to 1.50 IU/mL.
30. The method of claim 29 , wherein said therapeutically effective amount of said rAAV vector is effective to produce plasma FIX levels of about 0.06 to 1.50 IU/mL.
31. The method of claim 29 , wherein said therapeutically effective amount of said rAAV vector is a dose selected from the group consisting of 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , and 5×10 13 vg/kg subject body weight.
32. The method of claim 31 , wherein said therapeutically effective amount of said rAAV vector is a dose of 2×10 13 vg/kg subject body weight.
33. The method of claim 32 , wherein said treatment is effective to produce plasma FIX levels of about 0.06 to 0.50 IU/mL.