Adeno-associated virus factor VIII vectors
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. An adeno-associated virus (AAV) vector, comprising an AAV2 5′ inverted terminal repeat (ITR), a liver-specific transcriptional regulatory region, a functionally active FVIII coding region, 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.
2. The AAV vector of claim 1 , wherein said liver-specific transcriptional regulatory region is located immediately 3′ to said AAV2 5′ ITR.
3. The AAV vector of claim 2 , wherein said liver-specific regulatory region comprises nucleotides 146-397 of SEQ ID NO:1.
4. The AAV vector of claim 1 , which comprises nucleotides 1-397 of SEQ ID NO:1.
5. The AAV vector of claim 1 , wherein said functionally active FVIII coding region comprises nucleotides 923-5296 of SEQ ID NO: 9.
6. 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 1 to provide a cell culture, and (b) recovering recombinant AAV particle from the supernatant of said cell culture.
7. A viral particle comprising the AAV vector of claim 1 .
8. A composition comprising the viral particle of claim 7 for the treatment of hemophilia A.
9. A method of treating a patient suffering from hemophilia A comprising intravenously administering to the patient an effective amount of the viral particle of claim 7 .
10. The AAV vector of claim 1 wherein the functionally active FVIII coding region encodes the FVIII-SQ variant.