Optimized Liver-Specific Expression Systems for FVIII and FIX

Abstract

The present invention relates to nucleic acid expression cassettes and vectors containing liver-specific regulatory elements and codon-optimized factor IX or factor VIII transgenes, methods employing these expression cassettes and vectors and uses thereof. The present invention is particularly useful for applications using liver-directed gene therapy, in particular for the treatment of hemophilia A and B.

1 . A nucleic acid expression cassette comprising a triple repeat of a liver-specific nucleic acid regulatory element comprising the nucleic acid fragment defined by SEQ ID NO:5 or a sequence having at least 95% identity to said sequence, wherein the liver-specific nucleic acid regulatory elements are operably linked; and a nucleic acid regulatory element comprising the nucleic acid fragment defined by SEQ ID NO: 12 or a sequence having at least 95% identity to said sequence; operably linked to a promoter and a transgene. 
     
     
         2 . The nucleic acid expression cassette according to  claim 1 , wherein the promoter is a liver-specific promoter selected from the group consisting of the minimal TTR promotor (TTRm), the AAT promoter, the albumin (ALB) promotor or minimal promoter, the apolipoprotein A1 (APOA1) promoter or minimal promoter, the complement factor B (CFB) promoter, the ketohexokinase (KHK) promoter, the hemopexin (HPX) promoter or minimal promoter, the nicotinamide N-methyltransferase (NNMT) promoter or minimal promoter, the (liver) carboxylesterase 1 (CES1) promoter or minimal promoter, the protein C (PROC) promoter or minimal promoter, the apolipoprotein C3 (APOC3) promoter or minimal promoter, the mannan-binding lectin serine protease 2 (MASP2) promoter or minimal promoter, the hepcidin antimicrobial peptide (HAMP) promoter or minimal promoter, and the serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) promoter or minimal promoter. 
     
     
         3 . The nucleic acid expression cassette according to  claim 1 , wherein said promoter is the minimal TTR promotor (TTRm) as defined by SEQ ID NO:6. 
     
     
         4 . The nucleic acid expression cassette according to  claim 1 , wherein said promoter is the AAT promoter as defined by SEQ ID NO:64. 
     
     
         5 . The nucleic acid expression cassette according to  claim 1 , wherein the liver-specific regulatory elements of the triple repeat consist of the the nucleic acid fragment defined by SEQ ID NO:5. 
     
     
         6 . The nucleic acid expression cassette according to 1, wherein said transgene is codon-optimized. 
     
     
         7 . The nucleic acid expression cassette according to  claim 1 , wherein said transgene encodes coagulation factor IX (FIX) or wherein said transgene encodes coagulation factor FIX containing a hyper-activating mutation. 
     
     
         8 . The nucleic acid expression cassette according to  claim 1 , wherein said transgene encodes coagulation factor VIII (FVIII) or wherein said transgene encodes coagulation factor VIII having a deletion of the B domain. 
     
     
         9 . The nucleic acid expression cassette according to  claim 1 , wherein the promoter is the transthyretin (TTR) promoter, thereby comprising the combination of the TTRe and TTRm nucleic acids as defined by SEQ ID NO:69. 
     
     
         10 . The nucleic acid expression cassette according to  claim 1 , further comprising a minute virus of mouse (MVM) intron. 
     
     
         11 . The nucleic acid expression cassette according to  claim 1 , further comprising a transcriptional termination signal derived from the bovine growth hormone polyadenylation signal (BGHpA) or derived from the Simian virus 40 polyadenylation signal (SV40pA), or the synthetic polyadenylation signal as defined by SEQ ID NO:56. 
     
     
         12 . A vector comprising the nucleic acid expression cassette according to  claim 1 . 
     
     
         13 . A method of treating hemophilia A or hemophilia B comprising transducing or transfecting the vector according to  claim 12  into a subject, wherein the vector comprises the FVIII transgene for use in treating hemophilia A or the vector comprises the FIX transgene for use in treating hemophilia B. 
     
     
         14 . The method according to  claim 13 , wherein after transduction or transfection of the vector according to  claim 12  into a subject, levels of factor IX or FVIII in plasma are equal to or higher than the therapeutic threshold concentration of 10 mU/ml plasma in the subject are obtained. 
     
     
         15 . The method according to  claim 14 , wherein the transduction of the viral vector into the subject is done at a dose lower than 2×10 11  vg/kg. 
     
     
         16 . The method according to  claim 15 , wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 6×10 11  vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 100 mU/ml are obtained in said subject; or wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 6×10 11  vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 50 mU/ml are obtained in said subject; or wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 2×10 12  vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 200 mU/ml are obtained in said subject; or wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 2×10 12  vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 150 mU/ml are obtained in said subject. 
     
     
         17 . A pharmaceutical composition comprising a vector according to  claim 12  and a pharmaceutically acceptable carrier, and optionally further comprising an active ingredient for treating hemophilia A when the transgene in said vector is FVIII, or an active ingredient for treating hemophilia B when the transgene in said vector is FIX. 
     
     
         18 . The nucleic acid expression cassette according to  claim 7 , wherein the hyper-activating mutation corresponds to an R338L amino acid substitution. 
     
     
         19 . The nucleic acid expression cassette according to  claim 8 , wherein the B domain of the FVIII is replaced by a linker defined by SEQ ID NO:59. 
     
     
         20 . The vector of  claim 12 , wherein the vector is a viral vector.