Engineered nucleases that target human and canine factor viii genes as a treatment for hemophilia a

A pharmaceutical composition for treatment of a subject having hemophilia A characterized by an inversion of exons 1-22 in a Factor VIII gene, said pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of:

• (a) a nucleic acid encoding an engineered nuclease, wherein said engineered nuclease is expressed in a target cell in vivo;
• or
• (b) an engineered nuclease protein;
• wherein said engineered nuclease has specificity for a first recognition sequence positioned within an int22h-1 sequence of said Factor VIII gene;
• wherein said first recognition sequence does not comprise a CpG site;
• and wherein said first recognition sequence is at least 95% identical between the human genome and in the canine genome.

A method for genetically modifying the Factor VIII gene in the genome of a mammalian cell, wherein said mammalian cell comprises an inversion of exons 1-22 in the Factor VIII gene compared to a wild-type Factor VIII gene, said method comprising introducing into said mammalian cell:

• (a) an engineered nuclease having specificity for a first recognition sequence positioned within an int22h-1 sequence of said Factor VIII gene;
• or
• (b) a nucleic acid encoding said engineered nuclease, wherein said engineered nuclease is expressed in said mammalian cell;
• wherein said engineered nuclease cleaves said first recognition sequence and causes a reversion of exons 1-22 to a wild-type orientation in said genetically modified mammalian cell;
• and wherein said recognition sequence does not comprise a CpG site and is at least 95% identical between the human genome and the canine genome.