Vectors for Liver-Directed Gene Therapy of Hemophilia and Methods and Use Thereof

Abstract

The present invention relates to vectors containing liver-specific regulatory sequences and codon-optimized factor IX or factor VIII genes, methods employing these vectors and uses of these vectors. Expression cassettes and vectors containing these liver-specific regulatory elements and codon-optimized factor IX or factor VIII genes are also disclosed. The present invention is particularly useful for applications using gene therapy, in particular for the treatment of hemophilia A and B.

1 . A method for expressing a protein in a liver cell, the method comprising:
 introducing into the liver cell a vector comprising a nucleic acid expression cassette comprising a liver-specific regulatory element consisting of the Serpin enhancer having the nucleic acid sequence of SEQ ID NO:8, a promoter, a minute virus of mice (MVM) intron, a codon-optimized transgene, and a transcriptional termination signal; and   expressing the transgene protein product in the liver cell.   
     
     
         2 . The method according to  claim 1 , wherein the transgene encodes coagulation factor VIII (FVIII) or coagulation factor IX (FIX). 
     
     
         3 . The method according to  claim 2 , wherein the B domain of the FVIII is replaced by a linker having the sequence of SEQ ID NO:16. 
     
     
         4 . The method according to  claim 2 , wherein the FIX contains a hyper-activating mutation. 
     
     
         5 . The method according to  claim 4 , wherein the hyper-activating mutation is an R338L amino acid substitution. 
     
     
         6 . The method according to  claim 1 , wherein the promoter is a liver-specific promoter. 
     
     
         7 . The method according to  claim 1 , wherein the promoter is the transthyretin (TTR) promoter. 
     
     
         8 . The method according to  claim 1 , wherein the promoter is a minimal promoter. 
     
     
         9 . A method for obtaining levels of a coagulation factor equal to or higher than a therapeutic threshold concentration of 10 mU/ml in plasma of a subject, wherein the coagulation factor is coagulation factor VIII (FVIII) or coagulation factor IX (FIX), the method comprising transducing or transfecting a vector into the subject, said vector comprising a nucleic acid expression cassette comprising a liver-specific regulatory element consisting of the Serpin enhancer having the nucleic acid sequence of SEQ ID NO:8, a promoter, a minute virus of mice (MVM) intron, a codon-optimized transgene, and a transcriptional termination signal, wherein the transgene encodes FVIII when the coagulation factor is FVIII and wherein the transgene encodes FIX when the coagulation factor is FIX. 
     
     
         10 . The method according to  claim 9 , wherein the coagulation factor is coagulation factor IX, and wherein the therapeutic threshold concentration is 50 mU/ml, 100 mU/ml, 150 mU/ml or 200 mU/ml in the plasma of the subject. 
     
     
         11 . The method according to  claim 9 , wherein the vector is an adeno-associated virus (AAV) vector. 
     
     
         12 . The method according to  claim 10 , wherein the transduction of the vector into the subject is done at a dose lower than 2×10 12  vg/kg. 
     
     
         13 . The method according to  claim 9 , wherein the vector is a transposon-based vector selected from the group consisting of a PiggyBac-based vector and a Sleeping Beauty-based vector. 
     
     
         14 . The method according to  claim 13 , wherein the transposon-based vector is administered in combination with a vector encoding a transposase. 
     
     
         15 . A method of treating hemophilia in a subject in need thereof, wherein the hemophilia is hemophilia A or hemophilia B, said method comprising performing the method according to  claim 9 , wherein the coagulation factor is coagulation factor VIII when the hemophilia is hemophilia A, and wherein the coagulation factor is coagulation factor IX when the hemophilia is hemophilia B. 
     
     
         16 . A method of treating hemophilia in a subject in need thereof, wherein the hemophilia is hemophilia A or hemophilia B, the method comprising administering to the subject:
 (A) a vector, said vector comprising a nucleic acid expression cassette comprising a liver-specific regulatory element consisting of the Serpin enhancer having the nucleic acid sequence of SEQ ID NO:8, a promoter, a minute virus of mice (MVM) intron, a codon-optimized transgene, and a transcriptional termination signal, wherein said transgene encodes coagulation factor VIII (FVIII) when the hemophilia is hemophilia A or encodes coagulation factor IX (FIX) when the hemophilia is hemophilia B; or   (B) a pharmaceutical composition comprising the vector of (A) and a pharmaceutically acceptable carrier, optionally further comprising an active ingredient for treating the hemophilia.   
     
     
         17 . The method according to  claim 16 , wherein said treatment results in levels of factor VIII in plasma of the subject that are equal to or higher than a therapeutic threshold concentration of 10 mU/ml plasma when the hemophilia is hemophilia A, or in levels of factor IX in plasma of the subject that are equal to or higher than a therapeutic threshold concentration of 10 mU/ml, 50 mU/ml, 100 mU/ml, 150 mU/ml, or 200 mU/ml plasma when the hemophilia is hemophilia B. 
     
     
         18 . The method according to  claim 16 , wherein the vector is an adeno-associated virus (AAV) vector. 
     
     
         19 . The method according to  claim 16 , wherein the vector is a transposon-based vector selected from the group consisting of a PiggyBac-based vector and a Sleeping Beauty-based vector. 
     
     
         20 . The method according to  claim 19 , wherein the transposon-based vector is administered in combination with a vector encoding a transposase.