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 comprises a first polypeptide sequence having at least 95% identity to amino acids 20 to 759 of SEQ ID NO: 2;
wherein the light chain of the Factor FVIII polypeptide comprises a second polypeptide sequence having at least 95% identity to nucleic acids 774 to 1457 of SEQ ID NO: 2; 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 encoded Factor VIII polypeptide comprises 1105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO:2.
3. The polynucleotide of claim 1 , wherein the polypeptide linker comprises an amino acid sequence of SEQ ID NO:113.
4. The polynucleotide of claim 2 , wherein the polypeptide linker comprises an amino acid sequence of SEQ ID NO:113.
5. The polynucleotide of claim 1 ,
wherein the heavy chain of the Factor VIII polypeptide comprises a first polypeptide sequence having at least 99% identity to amino acids 20 to 759 of SEQ ID NO: 2;
wherein the light chain of the Factor FVIII polypeptide comprises a second polypeptide sequence having at least 99% identity to nucleic acids 774 to 1457 of SEQ ID NO: 2.
6. The polynucleotide of claim 5 , wherein the encoded Factor VIII polypeptide comprises I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO:2.
7. The polynucleotide of claim 5 , wherein the polypeptide linker comprises an amino acid sequence of SEQ ID NO:113.
8. The polynucleotide of claim 6 , wherein the polypeptide linker comprises an amino acid sequence of SEQ ID NO:113.
9. The polynucleotide of claim 1 , wherein the encoded Factor VIII polypeptide comprises a sequence having at least 99% sequence identity to SEQ ID NO: 2.
10. The polynucleotide of claim 9 , wherein the encoded Factor VIII polypeptide comprises I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO:2.
11. The polynucleotide of claim 9 , wherein the polypeptide linker comprises an amino acid sequence of SEQ ID NO:113.
12. The polynucleotide of claim 10 , wherein the polypeptide linker comprises an amino acid sequence of SEQ ID NO:113.
13. The polynucleotide of claim 1 , further comprising a promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide.
14. The polynucleotide of claim 13 , wherein the promoter element is a liver-specific promoter sequence upstream of the nucleotide sequence encoding the Factor VIII polypeptide.
15. An adeno-associated virus (AAV) vector comprising a polynucleotide according to claim 1 .
16. An adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 1 .
17. The adeno-associated virus (AAV) particle according to claim 16 , wherein the AAV particle is an AAV-8 particle.
18. A host cell infected with an adeno-associated virus (AAV) particle comprising a polynucleotide according to claim 1 .
19. A method for treating hemophilia A comprising administering, to a patient in need thereof, an adeno-associated virus (AAV) particle according to claim 16 .