Engineered nucleases useful for treatment of hemophilia a

Abstract

The present invention encompasses engineered nucleases which recognize and cleave a recognition sequence within the int22h-1 sequence of a Factor VIII gene. The present invention also encompasses methods of using such engineered nucleases to make genetically-modified cells, and the use of such cells in a pharmaceutical composition and in methods for treating hemophilia A. Further, the invention encompasses pharmaceutical compositions comprising engineered nuclease proteins, nucleic acids encoding engineered nucleases, or genetically-modified cells of the invention, and the use of such compositions for treating of hemophilia A.

1 . A method for genetically-modifying a Factor VIII gene in the genome of a mammalian cell to generate a reversion of exons 1-22, wherein said mammalian cell comprises an inversion of exons 1-22 in the Factor VIII gene compared to a wild-type Factor VIE gene, said method comprising delivering to said mammalian cell a lipid nanoparticle composition comprising a nucleic acid encoding an engineered nuclease having specificity for a recognition sequence positioned within an int22h-1 sequence of a Factor VIII gene, wherein said engineered nuclease is expressed in said mammalian cell;
 wherein said engineered nuclease cleaves said recognition sequence and generates a reversion of exons 1-22 to a wild-type orientation in said genetically-modified mammalian cell.   
     
     
         2 . The method of  claim 1 , wherein said genetically-modified cell produces a functional Factor VIII protein following said reversion of exons 1-22 to a wild-type orientation. 
     
     
         3 . The method of  claim 1 , wherein said recognition sequence is within an F8A1 coding sequence of said Factor VIII gene. 
     
     
         4 . The method of  claim 1 , wherein said F8A1 coding sequence has at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         5 . The method of  claim 1 , wherein said nucleic acid is an mRNA. 
     
     
         6 . The method of  claim 1 , wherein said engineered nuclease is an engineered mega nuclease, a TALEN, a zinc finger nuclease, a compact TALEN, a CRISPR, or a megaTAL. 
     
     
         7 . The method of  claim 1 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         8 . The method of  claim 1 , wherein said mammalian cell is a hepatic sinusoidal endothelial cell or a progenitor cell capable of differentiating into a hepatic sinusoidal endothelial cell. 
     
     
         9 . The method of  claim 1 , wherein said mammalian cell is a human cell. 
     
     
         10 . The method of  claim 1 , wherein said int22h-1 sequence of said Factor VIII gene has at least 95% sequence identity to SEQ ID NO: 3. 
     
     
         11 . The method of  claim 1 , wherein said mammalian cell is a canine cell. 
     
     
         12 . The method of  claim 1 , wherein said int22h-1 sequence of said Factor VIII gene has at least 95% sequence identity to SEQ H) NO: 4. 
     
     
         13 . The method of  claim 1 , wherein said recognition sequence comprises the nucleic acid sequence of SEQ ID NO: 9. 
     
     
         14 . A method for treating a subject having Hemophilia A comprising genetically-modifying a Factor VIII gene in the genome of a target cell within said subject to generate a reversion of exons 1-22, wherein said target cell comprises an inversion of exons 1-22 in the Factor VIII gene compared to a wild-type Factor VIII gene, said method comprising delivering to said target cell a lipid nanoparticle composition comprising a nucleic acid encoding an engineered nuclease having specificity for a recognition sequence positioned within an int22h-1 sequence of a Factor VIII gene, wherein said engineered nuclease is expressed in said target cell;
 wherein said engineered nuclease cleaves said recognition sequence and generates a reversion of exons 1-22 to a wild-type orientation in said target cell, thereby treating said subject having Hemophilia A.