Viral vectors encoding recombinant FVIII variants with increased expression for gene therapy of hemophilia A
The present disclosure provides, among other aspects, codon-altered polynucleotides encoding Factor VIII variants for expression in mammalian cells. In some embodiments, the disclosure also provides mammalian gene therapy vectors and methods for treating hemophilia A.
1. A polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain,
wherein the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity over the entire length of SEQ ID NO: 3;
wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity over the entire length of SEQ ID NO: 4; and
wherein the polypeptide linker comprises a furin cleavage site and a glycosylation peptide having an amino acid sequence of SEQ ID NO:55.
2. The polynucleotide of claim 1 , wherein the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity over the entire length of SEQ ID NO:38.
3. The polynucleotide of claim 1 , wherein the polypeptide linker is encoded by a third nucleotide sequence of SEQ ID NO:38.
4. The polynucleotide of claim 1 , wherein:
the first nucleotide sequence is SEQ ID NO: 3; and
the second nucleotide sequence is SEQ ID NO: 4.
5. The polynucleotide of claim 4 , wherein the polypeptide linker is encoded by a third nucleotide sequence of SEQ ID NO:38.
6. The polynucleotide of claim 1 , further comprising a promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide.
7. The polynucleotide of claim 6 , wherein the promoter element is a liver-specific promoter sequence upstream of the nucleotide sequence encoding the Factor VIII polypeptide.
8. The polynucleotide of claim 7 , further comprising an intron sequence positioned between the liver-specific promoter sequence and the nucleotide sequence encoding the Factor VIII polypeptide.
9. An adeno-associated virus (AAV) vector comprising a polynucleotide according to claim 1 .
10. An adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 1 .
11. A host cell infected with an adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 1 .
12. A method for producing an adeno-associated virus (AAV) particle comprising introducing a polynucleotide according to claim 1 into a mammalian host cell, wherein the polynucleotide is competent for replication in the mammalian host cell.
13. A method for treating hemophilia A comprising administering, to a patient in need thereof, an adeno-associated virus (AAV) particle according to claim 10 .
14. A method for transducing a host cell comprising contacting the host cell with an adeno-associated virus (AAV) particle according to claim 10 .
15. A polynucleotide comprising a nucleotide sequence having at least 99% identity over the entire length of SEQ ID NO:1, wherein the polynucleotide encodes a Factor VIII polypeptide having I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO: 19.
16. The polynucleotide of claim 15 , wherein:
the Factor VIII polypeptide comprises a light chain, a heavy chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain; and
the polypeptide linker includes a glycosylation peptide having an amino acid sequence of SEQ ID NO:55 inserted between the heavy chain and the light chain.
17. The polynucleotide of claim 15 , comprising the sequence of SEQ ID NO:94.
18. The polynucleotide of claim 15 , further comprising a promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide.
19. The polynucleotide of claim 18 , wherein the promoter element is a liver-specific promoter sequence upstream of the nucleotide sequence encoding the Factor VIII polypeptide.
20. The polynucleotide of claim 19 , further comprising an intron sequence positioned between the liver-specific promoter sequence and the nucleotide sequence encoding the Factor VIII polypeptide.
21. An adeno-associated virus (AAV) vector comprising a polynucleotide according to claim 15 .
22. An adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 15 .
23. A host cell infected with an adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 15 .
24. A method for producing an adeno-associated virus (AAV) particle comprising introducing a polynucleotide according to claim 15 into a mammalian host cell, wherein the polynucleotide is competent for replication in the mammalian host cell.
25. A method for treating hemophilia A comprising administering, to a patient in need thereof, an adeno-associated virus (AAV) particle according to claim 22 .
26. A method for transducing a host cell comprising contacting the host cell with an adeno-associated virus (AAV) particle according to claim 22 .
27. A polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide, the Factor VIII polypeptide comprising a light chain, a heavy chain, and a polypeptide linker joining the C-terminus of the heavy chain to the N-terminus of the light chain, the Factor VIII polypeptide having I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO: 19,
wherein the heavy chain of the Factor VIII polypeptide is encoded by a first nucleotide sequence having at least 99% identity over the entire length of SEQ ID NO: 3;
wherein the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity over the entire length of SEQ ID NO: 4; and
wherein the polypeptide linker comprises a furin cleavage site and a glycosylation peptide having an amino acid sequence of SEQ ID NO:55.
28. The polynucleotide of claim 27 , wherein the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity over the entire length of SEQ ID NO:38.
29. The polynucleotide of claim 27 , wherein the polypeptide linker is encoded by a third nucleotide sequence of SEQ ID NO:38.
30. The polynucleotide of claim 27 , further comprising a promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide.
31. The polynucleotide of claim 30 , wherein the promoter element is a liver-specific promoter sequence upstream of the nucleotide sequence encoding the Factor VIII polypeptide.
32. The polynucleotide of claim 31 , further comprising an intron sequence positioned between the liver-specific promoter sequence and the nucleotide sequence encoding the Factor VIII polypeptide.
33. An adeno-associated virus (AAV) vector comprising a polynucleotide according to claim 27 .
34. An adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 27 .
35. A host cell infected with an adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 27 .
36. A method for producing an adeno-associated virus (AAV) particle comprising introducing a polynucleotide according to claim 27 into a mammalian host cell, wherein the polynucleotide is competent for replication in the mammalian host cell.
37. A method for treating hemophilia A comprising administering, to a patient in need thereof, an adeno-associated virus (AAV) particle according to claim 34 .
38. A method for transducing a host cell comprising contacting the host cell with an adeno-associated virus (AAV) particle according to claim 34 .