Engineered nucleases that target human and canine factor viii genes as a treatment for 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 . An engineered meganuclease that recognizes and cleaves a recognition sequence positioned within an int22h-1 sequence of said Factor VIII gene, wherein said recognition sequence does not comprise a CpG site and is at least 95% identical between the human genome and the canine genome, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region. 
     
     
         2 . The engineered meganuclease of  claim 1 , wherein said int22h-1 sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. 
     
     
         3 . The engineered meganuclease of  claim 1  or  2 , wherein said recognition sequence is within an F8A1 coding sequence of said Factor VIII gene. 
     
     
         4 . The engineered meganuclease of  claim 3 , wherein said F8A1 coding sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         5 . The engineered meganuclease of any one of  claims 1 - 4 , wherein said recognition sequence comprises SEQ ID NO: 7. 
     
     
         6 . The engineered meganuclease of any one of  claims 1 - 5 , wherein said recognition sequence comprises SEQ ID NO: 9 or SEQ ID NO: 11. 
     
     
         7 . The engineered meganuclease of  claim 5  or  claim 6 , wherein said HVR1 region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 13-21. 
     
     
         8 . The engineered meganuclease of any one of  claims 5 - 7 , wherein said HVR1 region comprises residues corresponding to residues 24, 26, 28, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 13-21. 
     
     
         9 . The engineered meganuclease of any one of  claims 5 - 8 , wherein said HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 13-21. 
     
     
         10 . The engineered meganuclease of any one of  claims 5 - 9 , wherein said HVR2 region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 13-21. 
     
     
         11 . The engineered meganuclease of  claim 5 - 10 , wherein said HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 13-21. 
     
     
         12 . The engineered meganuclease of any one of  claims 5 - 11 , wherein said HVR2 region further comprises a residue corresponding to residue 245 of SEQ ID NO: 16. 
     
     
         13 . The engineered meganuclease of any one of  claims 5 - 12 , wherein said HVR2 region further comprises a residue corresponding to residue 262 of SEQ ID NO: 19. 
     
     
         14 . The engineered meganuclease of any one of  claims 5 - 13 , wherein said HVR2 region further comprises residues corresponding to residues 262, 263, 264, and 265 of SEQ ID NO: 19 or SEQ ID NO: 21. 
     
     
         15 . The engineered meganuclease of any one of  claims 5 - 14 , wherein said HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 13-21. 
     
     
         16 . The engineered meganuclease of any one of  claims 5 - 15 , wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of any one of SEQ ID NOs: 13-21, and wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of any one of SEQ ID NOs: 13-21. 
     
     
         17 . The engineered meganuclease of any one of  claims 5 - 16 , wherein said second subunit further comprises a residue corresponding to residue 210 of any one of SEQ ID NOs: 13-21. 
     
     
         18 . The engineered meganuclease of any one of  claims 5 - 17 , wherein said first subunit comprises residues 7-153 of any one of SEQ ID NOs: 13-21. 
     
     
         19 . The engineered meganuclease of any one of  claims 5 - 18 , wherein said second subunit comprises residues 198-344 of any one of SEQ ID NOs: 13-21. 
     
     
         20 . The engineered meganuclease of any one of  claims 5 - 19 , wherein said engineered meganuclease is a single-chain meganuclease comprising a linker, wherein said linker covalently joins said first subunit and said second subunit. 
     
     
         21 . The engineered meganuclease of any one of  claims 5 - 20 , wherein said engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs: 13-21. 
     
     
         22 . A polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         23 . The polynucleotide of  claim 22 , wherein said polynucleotide is an mRNA. 
     
     
         24 . A recombinant DNA construct comprising a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         25 . The recombinant DNA construct of  claim 24 , wherein said recombinant DNA construct encodes a viral vector comprising said nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         26 . The recombinant DNA construct of  claim 24  or  claim 25 , wherein said viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral (AAV) vector. 
     
     
         27 . The recombinant DNA construct of  claim 26 , wherein said viral vector is a recombinant AAV vector. 
     
     
         28 . A viral vector comprising a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         29 . The viral vector of  claim 28 , wherein said viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral (AAV) vector. 
     
     
         30 . The viral vector of  claim 29 , wherein said viral vector is a recombinant AAV vector. 
     
     
         31 . 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.   
     
     
         32 . The pharmaceutical composition of  claim 31 , wherein said int22h-1 sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. 
     
     
         33 . The pharmaceutical composition of  claim 31  or  claim 32 , wherein said first recognition sequence is within an F8A1 coding sequence of said Factor VIII gene. 
     
     
         34 . The pharmaceutical composition of any one of  claims 31 - 33 , wherein said F8A1 coding sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         35 . The pharmaceutical composition of any one of  claims 31 - 34 , wherein said engineered nuclease has specificity for a second recognition sequence that is identical to said first recognition sequence, wherein said second recognition sequence is positioned in a repeat sequence telomeric to said Factor VIII gene in the X chromosome, and wherein said repeat sequence is identical to said int22h-1 sequence except that said repeat sequence is in reverse orientation relative to said int22h-1 sequence. 
     
     
         36 . The pharmaceutical composition of any one of  claims 31 - 35 , wherein said nucleic acid is an mRNA. 
     
     
         37 . The pharmaceutical composition of  claim 36 , wherein said mRNA is encapsulated in a lipid nanoparticle. 
     
     
         38 . The pharmaceutical composition of any one of  claims 31 - 35 , wherein said pharmaceutical composition comprises a recombinant DNA construct comprising said nucleic acid. 
     
     
         39 . The pharmaceutical composition of any one of  claims 31 - 35 , wherein said pharmaceutical composition comprises a viral vector comprising said nucleic acid. 
     
     
         40 . The pharmaceutical composition of  claim 39 , wherein said viral vector is a recombinant AAV vector. 
     
     
         41 . The pharmaceutical composition of any one of  claims 31 - 40 , wherein said engineered nuclease is an engineered meganuclease, a TALEN, a zinc finger nuclease, a compact TALEN, a CRISPR, or a megaTAL. 
     
     
         42 . The pharmaceutical composition of  claim 41 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         43 . The pharmaceutical composition of  claim 42 , wherein said first recognition sequence comprises SEQ ID NO: 7. 
     
     
         44 . The pharmaceutical composition of  claim 42  or  claim 43 , wherein said first recognition sequence comprises SEQ ID NO: 9 or SEQ ID NO: 11. 
     
     
         45 . The pharmaceutical composition of  claim 43  or  claim 44 , wherein said nucleic acid encodes said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         46 . The pharmaceutical composition of  claim 43  or  claim 44 , wherein said engineered meganuclease is said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         47 . A method for treating a subject having hemophilia A characterized by an inversion of exons 1-22 of the Factor VIII gene, said method comprising administering to said subject said pharmaceutical composition of any one of  claims 31 - 46 . 
     
     
         48 . The method of  claim 47 , wherein said engineered nuclease, or said nucleic acid encoding said engineered nuclease, is delivered to cells which express Factor VIII in a wild-type subject, or progenitor cells which differentiate into cells which express Factor VIII in a wild-type subject. 
     
     
         49 . The method of  claim 47  or  claim 48 , wherein said cells are hepatic sinusoidal endothelial cells. 
     
     
         50 . The method of  claim 47  or  claim 48 , wherein said cells are progenitor cells which differentiate into hepatic sinusoidal endothelial cells. 
     
     
         51 . The method of any one of  claims 47 - 50 , wherein said engineered nuclease recognizes and cleaves said first recognition sequence to promote recombination between said int22h-1 sequence and said repeat sequence, resulting in reversion of exons 1-22 to generate a Factor VIII gene having a wild-type orientation. 
     
     
         52 . The method of  claim 51 , wherein said engineered nuclease further recognizes and cleaves said second recognition sequence in said repeat sequence. 
     
     
         53 . The method of any one of  claims 47 - 52 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         54 . The method of  claim 53 , wherein said pharmaceutical composition comprises a nucleic acid encoding said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         55 . The method of any one of  claims 47 - 54 , wherein said subject is a human. 
     
     
         56 . The method of any one of  claims 47 - 54 , wherein said subject is a canine. 
     
     
         57 . 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.   
     
     
         58 . The method of  claim 57 , wherein said int22h-1 sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4. 
     
     
         59 . The method of  claim 57  or  claim 58 , wherein said first recognition sequence is within an F8A1 coding sequence of said Factor VIII gene. 
     
     
         60 . The method of  claim 59 , wherein said F8A1 coding sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         61 . The method of any one of  claims 57 - 60 , wherein said engineered nuclease has specificity for a second recognition sequence that is identical to said first recognition sequence, wherein said second recognition sequence is positioned in a repeat sequence telomeric to said Factor VIII gene in the X chromosome, and wherein said repeat sequence is identical to said int22h-1 sequence except that said repeat sequence is in reverse orientation relative to said int22h-1 sequence. 
     
     
         62 . The method of any one of  claims 57 - 61 , wherein said nucleic acid is an mRNA. 
     
     
         63 . The method of  claim 62 , wherein said mRNA is encapsulated in a lipid nanoparticle. 
     
     
         64 . The method of any one of  claims 57 - 61 , wherein said nucleic acid is introduced using a recombinant DNA construct comprising said nucleic acid. 
     
     
         65 . The method of any one of  claims 57 - 61 , wherein said nucleic acid is introduced using a viral vector comprising said nucleic acid. 
     
     
         66 . The method of  claim 65 , wherein said viral vector is a recombinant AAV vector. 
     
     
         67 . The method of any one of  claims 57 - 66 , wherein said engineered nuclease is an engineered meganuclease, a TALEN, a zinc finger nuclease, a compact TALEN, a CRISPR, or a megaTAL. 
     
     
         68 . The method of any one of  claims 57 - 67 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         69 . The method of  claim 68 , wherein said first recognition sequence comprises SEQ ID NO: 7. 
     
     
         70 . The method of  claim 68  or  claim 69 , wherein said first recognition sequence comprises SEQ ID NO: 9 or SEQ ID NO: 11. 
     
     
         71 . The method of any one of  claims 68 - 70 , wherein said nucleic acid encodes said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         72 . The method of any one of  claims 68 - 70 , wherein said engineered meganuclease is said engineered meganuclease of any one of  claims 1 - 21 . 
     
     
         73 . The method of any one of  claims 57 - 72 , wherein said mammalian cell can express Factor VIII following reversion of exons 1-22 to a wild-type orientation. 
     
     
         74 . The method of  claim 73 , wherein said mammalian cell is a progenitor cell which can differentiate into a cell which can express Factor VIII following reversion of exons 1-22 to a wild-type orientation. 
     
     
         75 . The method of  claim 73 , wherein said mammalian cell is a hepatic cell. 
     
     
         76 . The method of  claim 75 , wherein said mammalian cell is a hepatic sinusoidal endothelial cell. 
     
     
         77 . The method of  claim 74 , wherein said mammalian cell is a progenitor cell capable of differentiating into a hepatic sinusoidal endothelial cell. 
     
     
         78 . The method of  claim 77 , wherein said progenitor cell is a hepatic stem cell. 
     
     
         79 . The method of  claim 73 , wherein said mammalian cell is a hematopoietic endothelial cell. 
     
     
         80 . The method of  claim 74 , wherein said mammalian cell is a progenitor cell capable of differentiating into a hematopoietic endothelial cell. 
     
     
         81 . The method of any one of  claims 57 - 80 , wherein said method is performed in a subject in vivo. 
     
     
         82 . The method of  claim 81 , wherein said method comprises administering to said subject said pharmaceutical composition of any one of  claims 31 - 46 . 
     
     
         83 . The method of  claim 81  or  claim 82 , wherein said subject is a human. 
     
     
         84 . The method of  claim 81  or  claim 82 , wherein said subject is a canine. 
     
     
         85 . The method of any one of  claims 57 - 80 , wherein said method is performed in vitro. 
     
     
         86 . The method of  claim 85 , wherein said mammalian cell is a human cell. 
     
     
         87 . The method of  claim 86 , wherein said mammalian cell is a canine cell. 
     
     
         88 . The method of any one of  claims 85 - 87 , wherein said mammalian cell is a pluripotent cell. 
     
     
         89 . A genetically-modified cell made by the method of any one of  claims 57 - 88 .