Viral vectors encoding recombinant fviii variants with increased expression for gene therapy of hemophilia a

Abstract

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 .- 74 . (canceled) 
     
     
         75 . 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 to SEQ ID NO: 3;   the light chain of the Factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 99% identity to SEQ ID NO: 4; and   wherein the polypeptide linker comprises a furin cleavage site.   
     
     
         76 . The polynucleotide of  claim 75 , wherein the encoded polypeptide linker comprises a glycosylation peptide having an amino acid sequence selected from the group of SEQ ID NOs: 51, 53, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75. 
     
     
         77 . The polynucleotide of  claim 75 , wherein the encoded polypeptide linker has an amino acid sequence selected from the group of SEQ ID NOs: 110-112 and 114-123. 
     
     
         78 . The polynucleotide of  claim 75 , wherein the polypeptide linker is encoded by a third nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 7, 36, 37, and 39-48. 
     
     
         79 . The polynucleotide of  claim 75 , wherein the polypeptide linker is encoded by a third nucleotide sequence of SEQ ID NO: 6. 
     
     
         80 . The polynucleotide according to  claim 75 , wherein:
 the first nucleotide sequence is SEQ ID NO: 3; and   the second nucleotide sequence is SEQ ID NO: 4.   
     
     
         81 . The polynucleotide according to  claim 76 , wherein:
 the first nucleotide sequence is SEQ ID NO: 3; and   the second nucleotide sequence is SEQ ID NO: 4.   
     
     
         82 . The polynucleotide according to  claim 77 , wherein:
 the first nucleotide sequence is SEQ ID NO: 3; and   the second nucleotide sequence is SEQ ID NO: 4.   
     
     
         83 . The polynucleotide according to  claim 78 , wherein:
 the first nucleotide sequence is SEQ ID NO: 3; and   the second nucleotide sequence is SEQ ID NO: 4.   
     
     
         84 . The polynucleotide according to  claim 75 , wherein the polynucleotide encodes a Factor VIII polypeptide having I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO: 19. 
     
     
         85 . The polynucleotide according to  claim 76 , wherein the polynucleotide encodes a Factor VIII polypeptide having I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO: 19. 
     
     
         86 . The polynucleotide according to  claim 77 , wherein the polynucleotide encodes a Factor VIII polypeptide having I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO: 19. 
     
     
         87 . The polynucleotide according to  claim 78 , wherein the polynucleotide encodes a Factor VIII polypeptide having I105V, A127S, G151K, M166T, and L171P amino acid substitutions, relative to SEQ ID NO: 19. 
     
     
         88 . The polynucleotide of  claim 75 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         89 . The polynucleotide of  claim 76 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         90 . The polynucleotide of  claim 77 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         91 . The polynucleotide of  claim 78 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         92 . The polynucleotide of  claim 79 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         93 . The polynucleotide of  claim 80 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         94 . The polynucleotide of  claim 81 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         95 . The polynucleotide of  claim 82 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         96 . The polynucleotide of  claim 83 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         97 . The polynucleotide of  claim 84 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         98 . The polynucleotide of  claim 85 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         99 . The polynucleotide of  claim 86 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide. 
     
     
         100 . The polynucleotide of  claim 87 , further comprising a liver-specific promoter element operably linked to the polynucleotide encoding the Factor VIII polypeptide.