Treatment of retinitis pigmentosa using engineered meganucleases

Abstract

Disclosed are recombinant meganucleases engineered to recognize and cleave recognition sequences present in a mutant RHO P23H allele. The invention further relates to the use of such recombinant meganucleases in methods for treating retinitis pigmentosa, wherein the mutant RHO P23H allele is preferentially targeted, cleaved, and inactivated.

1 .- 35 . (canceled) 
     
     
         36 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease that recognizes and cleaves a P23H recognition sequence consisting of SEQ ID NO: 1, wherein said engineered meganuclease comprises a first subunit and a second subunit, and wherein said first subunit binds to a first recognition half-site of said P23H recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said P23H recognition sequence and comprises a second hypervariable (HVR2) region, and wherein said engineered meganuclease comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:7. 
     
     
         37 . The pharmaceutical composition of  claim 36 , wherein said pharmaceutical composition comprises a recombinant adeno-associated viral (AAV) vector comprising said polynucleotide, wherein said nucleic acid sequence encoding said engineered meganuclease is operably linked to a promoter. 
     
     
         38 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is serotype 5. 
     
     
         39 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is serotype 2. 
     
     
         40 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is a self-complementary AAV vector. 
     
     
         41 . The pharmaceutical composition of  claim 36 , wherein said first subunit comprises an amino acid sequence having at least 97% sequence identity to residues 198-344 of SEQ ID NO: 7 and wherein said second subunit comprises an amino acid sequence having at least 97% sequence identity to residues 7-153 of SEQ ID NO: 7. 
     
     
         42 . The pharmaceutical composition of  claim 36 , wherein said first subunit comprises residues 198-344 of SEQ ID NO: 7. 
     
     
         43 . The pharmaceutical composition of  claim 36 , wherein said second subunit comprises residues 7-153 of SEQ ID NO: 7. 
     
     
         44 . The pharmaceutical composition of  claim 36 , wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 7. 
     
     
         45 . The pharmaceutical composition of  claim 36 , wherein said engineered meganuclease preferentially recognizes and cleaves a P23H recognition sequence consisting of SEQ ID NO: 1 relative to a recognition sequence consisting of SEQ ID NO: 5. 
     
     
         46 . The pharmaceutical composition of  claim 37 , wherein said promoter is a retina cell-specific promoter. 
     
     
         47 . The pharmaceutical composition of  claim 37 , wherein said promoter is a rod photoreceptor cell-specific promoter. 
     
     
         48 . The pharmaceutical composition of  claim 37 , wherein said promoter is a human G-protein-coupled receptor protein kinase 1 (GRK1) promoter. 
     
     
         49 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is serotype 5 and wherein said recombinant AAV vector is a self-complementary AAV vector. 
     
     
         50 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is serotype 5 and wherein said nucleic acid sequence encoding said engineered meganuclease is operably linked to a human GRK1 promoter. 
     
     
         51 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is serotype 5, and wherein said nucleic acid sequence encoding said engineered meganuclease is operably linked to a human GRK1 promoter, and wherein said recombinant AAV vector is a self-complementary AAV vector. 
     
     
         52 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is serotype 5, wherein said promoter is a human GRK1 promoter, and wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 7. 
     
     
         53 . The pharmaceutical composition of  claim 37 , wherein said recombinant AAV vector is serotype 5, wherein said promoter is a human GRK1 promoter, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 7, and wherein said recombinant AAV vector is a self-complementary AAV vector.