Adeno-associated virus factor viii vectors

Abstract

The invention provides improved adeno-associated virus (AAV) Factor VIII (FVIII) vectors, including AAV FVIII vectors that produce a functional Factor VIII polypeptide and AAV FVIII vectors with high expression activity.

1 - 8 . (canceled) 
     
     
         9 . An adeno-associated virus (AAV) vector, comprising an AAV2 5′ inverted terminal repeat (ITR), a liver-specific transcriptional regulatory region, a nucleotide sequence that is at least 85% identical to a codon optimized Factor VIII nucleic acid sequence (SEQ ID NO: 46) from which the B domain has been replaced by 14 amino acid SQ sequence, a polyadenylation sequence, an AAV2 3′ ITR, and optionally one or more introns, wherein said AAV vector is less than 5.0 kb in length. 
     
     
         10 . The AAV vector of  claim 9 , wherein said liver-specific transcriptional regulatory region is located immediately 3′ to said AAV2 5′ ITR. 
     
     
         11 . The AAV vector of  claim 10 , wherein said liver-specific regulatory region comprises nucleotides 146-397 of SEQ ID NO: 1. 
     
     
         12 . The AAV vector of  claim 9 , which comprises nucleotides 1-397 of SEQ ID NO: 1. 
     
     
         13 . A method of producing a recombinant adeno-associated virus (AAV) particle comprising (a) culturing a host cell that has been transfected with the AAV vector of  claim 9  to provide a cell culture, and (b) recovering recombinant AAV particle from the supernatant of said cell culture. 
     
     
         14 . A viral particle comprising the AAV vector of  claim 9 . 
     
     
         15 . A composition comprising the viral particle of  claim 14  for the treatment of hemophilia A. 
     
     
         16 . A method of treating a patient suffering from hemophilia A comprising administering to the patient an effective amount of the viral particle of  claim 14 . 
     
     
         17 . An adeno-associated virus (AAV) vector, comprising an AAV2 5′ inverted terminal repeat (ITR), a liver-specific transcriptional regulatory region, a nucleotide sequence that is at least 90% identical to nucleotides 923-5296 of SEQ ID NO: 10, and optionally one or more introns, wherein said AAV vector is less than 5.0 kb in length. 
     
     
         18 . The AAV vector of  claim 17 , wherein said liver-specific transcriptional regulatory region is located immediately 3′ to said AAV2 5′ ITR. 
     
     
         19 . The AAV vector of  claim 18 , wherein said liver-specific regulatory region comprises nucleotides 146-397 of SEQ ID NO: 1. 
     
     
         20 . The AAV vector of  claim 17 , which comprises nucleotides 1-397 of SEQ ID NO: 1. 
     
     
         21 . A method of producing a recombinant adeno-associated virus (AAV) particle comprising (a) culturing a host cell that has been transfected with the AAV vector of  claim 9  to provide a cell culture, and (b) recovering recombinant AAV particle from the supernatant of said cell culture. 
     
     
         22 . A viral particle comprising the AAV vector of  claim 21 . 
     
     
         23 . A composition comprising the viral particle of  claim 22  for the treatment of hemophilia A. 
     
     
         24 . A method of treating a patient suffering from hemophilia A comprising administering to the patient an effective amount of the viral particle of  claim 22 .