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 - 26 . (canceled) 
     
     
         27 . A method for treating hemophilia A comprising administering, to a patient with hemophilia A, a nucleic acid composition comprising a Factor VIII polynucleotide encoding a Factor VIII protein, the Factor VIII polynucleotide having the nucleic acid sequence of SEQ ID NO:1. 
     
     
         28 . The method of  claim 27 , wherein the nucleic acid composition further comprises a promoter having the nucleic acid sequence of SEQ ID NO:6 operatively linked to the Factor VIII polynucleotide. 
     
     
         29 . The method of  claim 28 , wherein there are no nucleotides between the promoter and the Factor VIII polynucleotide in the nucleic acid composition. 
     
     
         30 . The method of  claim 27 , wherein the nucleic acid composition further comprises a liver-specific element having the nucleic acid sequence of SEQ ID NO:5 operatively linked to the Factor VIII polynucleotide. 
     
     
         31 . The method of claim  26 , wherein the nucleic acid composition further comprises a second liver-specific element operatively linked to the Factor VIII polynucleotide. 
     
     
         32 . The method of  claim 31 , wherein the nucleic acid composition further comprises a promoter operatively linked to the Factor VIII polynucleotide, wherein:
 the liver-specific element is positioned upstream of the promoter,   the promoter is positioned upstream of the Factor VIII polynucleotide, and   there are no nucleotides between the liver-specific element and the promoter.   
     
     
         33 . The method of  claim 32 , wherein the nucleic acid composition has a nucleic acid sequence comprising SEQ ID NO: 3. 
     
     
         34 . The method of  claim 27 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         35 . The method of  claim 28 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         36 . The method of  claim 29 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         37 . The method of  claim 30 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         38 . The method of  claim 31 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         39 . The method of  claim 32 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         40 . The method of  claim 33 , wherein the nucleic acid composition comprises a mammalian gene therapy vector. 
     
     
         41 . The method of  claim 27 , wherein the nucleic acid composition comprises an adeno-associated virus (AAV) particle. 
     
     
         42 . The method of  claim 41 , wherein the AAV particle is a serotype 1 adeno-associated virus (AAV-1), a serotype 2 adeno-associated virus (AAV-2), a serotype 3 adeno-associated virus (AAV-3), a serotype 4 adeno-associated virus (AAV-4), a serotype 6 adeno-associated virus (AAV-5), a serotype 6 adeno-associated virus (AAV-6), a serotype 7 adeno-associated virus (AAV-7), serotype 8 adeno-associated virus (AAV-8), or a serotype 9 adeno-associated virus (AAV-9). 
     
     
         43 . The method of  claim 41 , wherein the AAV particle is a serotype 8 adeno-associated virus (AAV-8). 
     
     
         44 . The method of  claim 41 , wherein the AAV particle is a serotype 9 adeno-associated virus (AAV-9). 
     
     
         45 . The method of  claim 41 , wherein the Factor VIII polynucleotide encoding the Factor VIII protein is a single-stranded polynucleotide. 
     
     
         46 . The method of  claim 42 , wherein the Factor VIII polynucleotide encoding the Factor VIII protein is a single-stranded polynucleotide.