Gene therapy for treating hemophilia a

Abstract

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) useful as a liver-directed therapeutic for hemophilia A, said rAAV comprising an AAV capsid, and a vector genome packaged therein, said vector genome comprising:
 (a) an AAV 5′ inverted terminal repeat (ITR) sequence;   (b) a transthyretin enhancer (enTTR);   (c) a transthyretin (TTR) promoter;   (d) a coding sequence encoding a human Factor VIII having coagulation function;   (e) an AAV 3′ ITR.   
     
     
         2 . The rAAV according to  claim 1 , wherein the human Factor VIII is a B-domain deleted factor VIII SQ which is about 1457 amino acid residues in length. 
     
     
         3 . The rAAV according to  claim 1 , wherein the coding sequence of (d) is selected from SEQ ID NO: 1 and SEQ ID NO: 2. 
     
     
         4 . The rAAV according to  claim 1 , wherein the rAAV capsid is a hu37 capsid. 
     
     
         5 . The rAAV according to  claim 1 , wherein the AAV 5′ ITR and/or AAV3′ ITR is from AAV2. 
     
     
         6 . The rAAV according to  claim 1 , wherein the vector genome further comprises a polyA which is about 75 bp in size. 
     
     
         7 . The rAAV according to  claim 1 , wherein the vector genome is about 5 kilobases to about 5.5 kilobases in size. 
     
     
         8 . An aqueous suspension suitable for administration to a hemophilia A patient, said suspension comprising an aqueous suspending liquid and about 1×10 12  GC/mL to about 1×10 14  GC/mL of a recombinant adeno-associated virus (rAAV) useful as a liver-directed therapeutic for hemophilia A, said rAAV having an AAV capsid, and having packaged therein a vector genome comprising:
 (a) an AAV 5′ inverted terminal repeat (ITR) sequence; 
 (b) a transthyretin enhancer (enTTR); 
 (c) a transthyretin (TTR) promoter; 
 (d) a coding sequence encoding a human Factor VIII having coagulation function; 
 (e) an AAV 3′ ITR. 
 
     
     
         9 . The suspension according to  claim 8 , wherein the suspension is suitable for intravenous injection. 
     
     
         10 . The suspension according to  claim 8 , wherein the suspension further comprises a surfactant, preservative, and/or buffer dissolved in the aqueous suspending liquid. 
     
     
         11 . A method of treating a patient having hemophilia A with an rAAV according to  claim 1 , wherein the rAAV is delivered about 1×10 12  to about 1×10 14  genome copies (GC)/kg in an aqueous suspension, wherein the GC are calculated as determined based on oqPCR. 
     
     
         12 . The method according to  claim 11 , wherein the rAAV is readministered at a later time point. 
     
     
         13 . The rAAV according to  claim 1 , wherein the vector genome comprises nt 1-5110 of SEQ ID NO: 13. 
     
     
         14 . The rAAV according to  claim 13 , wherein the rAAV capsid is an hu37 capsid. 
     
     
         15 . The rAAV according to  claim 1 , wherein
 the enTTR is SEQ ID NO: 5;   the TTR promoter is SEQ ID NO: 7;   the coding sequence is SED ID NO: 2.   
     
     
         16 . The rAAV according to  claim 15  wherein the AAV 5′ ITR is SEQ ID NO: 11 and the AAV 3′ ITR is SEQ ID NO: 12. 
     
     
         17 . The rAAV according to either of  claim 15 , further comprising a polyA sequence of SEQ ID NO: 10. 
     
     
         18 . The rAAV according to  claim 15 , wherein the rAAV capsid is an hu37 capsid.