Gene therapy for treating hemophilia A
Compositions and regimens useful in treating hemophilia A are provided. The compositions include recombinant adeno-associated virus (rAAV) with a transthyretin enhancer and promoter driving expression of a human Factor VIII.
1. A recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, said vector genome comprising:
an AAV 5′-inverted terminal repeat (ITR) sequence;
a liver-specific promoter;
a coding sequence encoding a human Factor VIII having coagulation function; and
an AAV 3′-ITR sequence,
wherein said coding sequence comprises the nucleotide sequence as set forth in SEQ ID NO: 2.
2. The rAAV according to claim 1 , wherein the AAV capsid is a hu.37 capsid.
3. The rAAV according to claim 1 , wherein the AAV 5′-ITR is from AAV2.
4. The rAAV according to claim 3 , wherein the AAV 5′-ITR comprises the nucleotide sequence as set forth in SEQ ID NO: 11.
5. The rAAV according to claim 1 , wherein the AAV 3′-ITR is from AAV2.
6. The rAAV according to claim 5 , wherein the AAV 3′-ITR comprises the nucleotide sequence as set forth in SEQ ID NO: 12.
7. The rAAV according to claim 1 , wherein the AAV 5′-ITR and AAV 3′-ITR are from AAV2.
8. The rAAV according to claim 1 , wherein the liver-specific promoter is a transthyretin (TTR) promoter.
9. The rAAV according to claim 8 , wherein the TTR promoter comprises the nucleotide sequence as set forth in SEQ ID NO: 7.
10. The rAAV according to claim 1 , wherein the vector genome further comprises an enhancer.
11. The rAAV according to claim 10 , wherein the enhancer is a liver-specific enhancer.
12. The rAAV according to claim 11 , wherein the liver-specific enhancer is a transthyretin enhancer (enTTR).
13. The rAAV according to claim 12 , wherein the enTTR comprises the nucleotide sequence as set forth in SEQ ID NO: 5.
14. The rAAV according to claim 1 , wherein the vector genome further comprises a polyA sequence.
15. The rAAV according to claim 14 , wherein the polyA sequence comprises the nucleotide sequence as set forth in SEQ ID NO: 10.
16. The rAAV according to claim 1 , wherein the vector genome is 5 kilobases to 5.5 kilobases in size.
17. An aqueous suspension comprising an aqueous suspending liquid and 1×10 12 to 1×10 14 genome copies (GC)/kilogram of the rAAV of claim 1 , wherein the aqueous suspension is suitable for administration to a patient with hemophilia A.
18. The aqueous suspension according to claim 17 , wherein the suspension further comprises a surfactant, a preservative, and/or a buffer in the aqueous suspending liquid.
19. A recombinant adeno-associated virus (rAAV) comprising an AAVhu.37 capsid and a vector genome packaged therein, said vector genome comprising:
an AAV 5′-inverted terminal repeat (ITR) sequence;
a transthyretin (TTR) promoter;
transthyretin enhancer (enTTR);
a coding sequence encoding a human Factor VIII having coagulation function; and
an AAV 3′-ITR sequence,
wherein said coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 2.