Optimized engineered meganucleases having specificity for a recognition sequence in the hepatitis b virus genome

Abstract

The present invention encompasses engineered nucleases which recognize and cleave a recognition sequence within a Hepatitis B virus (HBV) genome. The engineered meganucleases can exhibit at least one optimized characteristic, such as enhanced specificity and/or efficiency of indel formation, when compared to the first-generation meganuclease HBV 11-12x.26. Further, the invention encompasses pharmaceutical compositions comprising engineered meganuclease proteins, nucleic acids encoding engineered meganucleases, and the use of such compositions for treating HBV infections or hepatocellular carcinoma.

1 . An engineered meganuclease that recognizes and cleaves a recognition sequence comprising SEQ ID NO: 10 within a Hepatitis B virus 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, wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, and wherein said HVR1 region has at least 86% sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 12 or 13. 
     
     
         2 . The engineered meganuclease of  claim 1 , wherein said HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 12 or 13. 
     
     
         3 . The engineered meganuclease of  claim 1  or  claim 2 , wherein said HVR1 region comprises residues corresponding to residues 239, 241, 263, and 264 of SEQ ID NO: 12. 
     
     
         4 . The engineered meganuclease of  claim 1  or  claim 2 , wherein said HVR1 region comprises residues corresponding to residues 241, 262, 263, and 264 of SEQ ID NO: 13. 
     
     
         5 . The engineered meganuclease of any one of  claims 1 - 4 , wherein said HVR1 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 239, 241, 259, 261, 262, 263, 264, 266, and 268 of SEQ ID NO: 12 or 13. 
     
     
         6 . The engineered meganuclease of any one of  claims 1 - 5 , wherein said HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 12 or 13. 
     
     
         7 . The engineered meganuclease of any one of  claims 1 - 6 , wherein said HVR1 region comprises residues 215-270 of SEQ ID NO: 12 or 13. 
     
     
         8 . The engineered meganuclease of any one of  claims 1 - 7 , wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of SEQ ID NO: 12 or 13. 
     
     
         9 . The engineered meganuclease of any one of  claims 1 - 8 , wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of SEQ ID NO: 12 or 13, and wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of SEQ ID NO: 12 or 13. 
     
     
         10 . The engineered meganuclease of any one of  claims 1 - 9 , wherein said first subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 12 or 13. 
     
     
         11 . The engineered meganuclease of any one of  claims 1 - 10 , wherein said first subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 12 or 13. 
     
     
         12 . The engineered meganuclease of any one of  claims 1 - 11 , wherein said first subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 12 or 13. 
     
     
         13 . The engineered meganuclease of any one of  claims 1 - 12 , wherein said first subunit comprises residues 198-344 of SEQ ID NO: 12 or 13. 
     
     
         14 . The engineered meganuclease of any one of  claims 1 - 13 , wherein said HVR2 region comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 12 or 13. 
     
     
         15 . The engineered meganuclease of any one of  claims 1 - 14 , wherein said HVR2 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 12 or 13. 
     
     
         16 . The engineered meganuclease of any one of  claims 1 - 15 , wherein said HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 12 or 13. 
     
     
         17 . The engineered meganuclease of any one of  claims 1 - 16 , wherein said HVR2 region comprises residues 24-79 of SEQ ID NO: 12 or 13. 
     
     
         18 . The engineered meganuclease of any one of  claims 1 - 17 , wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of SEQ ID NO: 12 or 13. 
     
     
         19 . The engineered meganuclease of any one of  claims 1 - 18 , wherein said second subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 12 or 13. 
     
     
         20 . The engineered meganuclease of any one of  claims 1 - 19 , wherein said second subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 12 or 13. 
     
     
         21 . The engineered meganuclease of any one of  claims 1 - 20 , wherein said second subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 12 or 13. 
     
     
         22 . The engineered meganuclease of any one of  claims 1 - 21 , wherein said second subunit comprises residues 7-153 of SEQ ID NO: 12 or 13. 
     
     
         23 . The engineered meganuclease of any one of  claims 1 - 22 , wherein said engineered meganuclease comprises a linker, wherein said linker covalently joins said first subunit and said second subunit. 
     
     
         24 . The engineered meganuclease of any one of  claims 1 - 23 , wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 12 or 13. 
     
     
         25 . The engineered meganuclease of any one of  claims 1 - 24 , wherein said engineered meganuclease exhibits at least one of the following optimized characteristics as compared to the HBV 11-12x.26 meganuclease set forth as SEQ ID NO: 14: improved specificity, enhanced efficiency of cleavage, and enhanced efficiency of indel formation. 
     
     
         26 . A polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 25 . 
     
     
         27 . The polynucleotide of  claim 26 , wherein said polynucleotide is an mRNA. 
     
     
         28 . The polynucleotide of  claim 27 , wherein said mRNA is a polycistronic mRNA encoding one or more of said engineered meganucleases of any one of  claims 1 - 25 . 
     
     
         29 . The polynucleotide of  claim 28 , wherein said polycistronic mRNA encodes:
 (a) at least one of said engineered meganucleases of  claims 1 - 25  which recognizes and cleaves a recognition sequence comprising SEQ ID NO: 10; and   (b) a second engineered meganuclease which recognizes and cleaves a second recognition sequence which is present in a Hepatitis B virus genome but differs from SEQ ID NO: 10.   
     
     
         30 . The polynucleotide of  claim 29 , wherein said second recognition sequence comprises SEQ ID NO: 21. 
     
     
         31 . A recombinant DNA construct comprising said polynucleotide of  claim 26 . 
     
     
         32 . The recombinant DNA construct of  claim 31 , wherein said recombinant DNA construct comprises a cassette comprising a promoter and a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 25 . 
     
     
         33 . The recombinant DNA construct of  claim 31 , wherein said recombinant DNA construct comprises at least a first cassette and a second cassette, wherein said first cassette comprises a promoter and a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 25 , and wherein said second cassette comprises a promoter and a nucleic acid sequence encoding a second engineered meganuclease which recognizes and cleaves a second recognition sequence which is present in a Hepatitis B virus genome but differs from SEQ ID NO: 10. 
     
     
         34 . The recombinant DNA construct of  claim 31 , wherein said recombinant DNA construct comprises a cassette comprising a promoter and a polycistronic nucleic acid sequence encoding one or more of said engineered meganucleases of any one of  claims 1 - 16 , wherein said promoter drives expression of said polycistronic nucleic acid sequence to generate a polycistronic mRNA in a target cell. 
     
     
         35 . The recombinant DNA construct of  claim 34 , wherein said polycistronic mRNA is said polycistronic mRNA of any one of  claims 28 - 30 . 
     
     
         36 . The recombinant DNA construct of any one of  claims 31 - 35 , wherein said recombinant DNA construct encodes a viral vector comprising said nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 25 . 
     
     
         37 . The recombinant DNA construct of  claim 36 , wherein said viral vector is a recombinant adeno-associated viral (AAV) vector. 
     
     
         38 . A viral vector comprising a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 25 . 
     
     
         39 . The viral vector of  claim 38 , wherein said viral vector is a recombinant AAV vector. 
     
     
         40 . The viral vector of  claim 38  or  claim 39 , wherein said viral vector comprises a cassette comprising a promoter and a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 25 . 
     
     
         41 . The viral vector of any one of  claims 38 - 40 , wherein said viral vector comprises at least a first cassette and a second cassette, wherein said first cassette comprises a promoter and a nucleic acid sequence encoding said engineered meganuclease of any one of  claims 1 - 25 , and wherein said second cassette comprises a promoter and a nucleic acid sequence encoding a second engineered meganuclease which recognizes and cleaves a second recognition sequence which is present in a Hepatitis B virus genome but differs from SEQ ID NO: 10. 
     
     
         42 . The viral vector of any one of  claims 38 - 40 , wherein said viral vector comprises a cassette comprising a promoter and a polycistronic nucleic acid sequence encoding one or more of said engineered meganucleases of any one of  claims 1 - 25 , wherein said promoter drives expression of said polycistronic nucleic acid sequence to generate a polycistronic mRNA in a target cell. 
     
     
         43 . The viral vector of  claim 42 , wherein said polycistronic mRNA is said polycistronic mRNA of any one of  claims 28 - 30 . 
     
     
         44 . A pharmaceutical composition for treatment of a subject having Hepatitis B virus or hepatocellular carcinoma caused by Hepatitis B virus, said pharmaceutical composition comprising a pharmaceutically acceptable carrier and:
 (a) a nucleic acid encoding said engineered meganuclease of any one of  claims 1 - 25 ; or   (b) said engineered meganuclease of any one of  claims 1 - 25 .   
     
     
         45 . The pharmaceutical composition of  claim 44 , wherein said nucleic acid encoding said engineered meganuclease is said mRNA of any one of  claims 27 - 30 . 
     
     
         46 . The pharmaceutical composition of  claim 45 , wherein said pharmaceutical composition comprises said recombinant DNA construct of any one of  claims 31 - 37 . 
     
     
         47 . The pharmaceutical composition of  claim 45 , wherein said pharmaceutical composition comprises said viral vector of any one of  claims 38 - 43 . 
     
     
         48 . The pharmaceutical composition of  claim 45 , wherein said pharmaceutical composition comprises said engineered meganuclease of any one of  claims 1 - 25 . 
     
     
         49 . The pharmaceutical composition of  claim 45 , wherein said pharmaceutical composition comprises an engineered meganuclease of any one of  claims 1 - 25 , and a second engineered meganuclease which recognizes and cleaves a second recognition sequence which is present in a Hepatitis B virus genome but differs from SEQ ID NO: 10. 
     
     
         50 . The pharmaceutical composition of  claim 45 , wherein said pharmaceutical composition comprises a nucleic acid encoding said engineered meganuclease of any one of  claims 1 - 25 , and a nucleic acid encoding a second engineered meganuclease which recognizes and cleaves a second recognition sequence which is present in a Hepatitis B virus genome but differs from SEQ ID NO: 10. 
     
     
         51 . The pharmaceutical composition of  claim 45 , wherein said pharmaceutical composition comprises one or more of said mRNAs of any one of  claims 27 - 30  encapsulated within lipid nanoparticles. 
     
     
         52 . A lipid nanoparticle or lipid nanoparticle formulation comprising one or more of said mRNAs of any one of  claims 27 - 30 . 
     
     
         53 . A method for treating a subject having Hepatitis B virus or hepatocellular carcinoma caused by Hepatitis B virus, said method comprising delivering to a target cell in said subject:
 (a) a therapeutically effective amount of a nucleic acid encoding said engineered meganuclease of any one of  claims 1 - 25 , wherein said engineered meganuclease is expressed in said target cell in vivo; or   (b) a therapeutically effective amount of said engineered meganuclease of any one of  claims 1 - 16 ;   wherein said engineered meganuclease recognizes and cleaves said recognition sequence comprising SEQ ID NO: 10 within the Hepatitis B virus genome, and wherein the infection and/or proliferation of said Hepatitis B virus in said subject is reduced or eliminated.   
     
     
         54 . The method of  claim 53 , wherein said method comprises administering to said subject said pharmaceutical composition of any one of  claims 44 - 51 . 
     
     
         55 . The method of  claim 53  or  claim 54 , wherein said engineered meganuclease, or said nucleic acid encoding said engineered meganuclease, is delivered to a target hepatocyte cell. 
     
     
         56 . The engineered meganuclease of any one of  claims 1 - 25  or the polynucleotide of any one of  claims 26 - 30  for use in medicine. 
     
     
         57 . The engineered meganuclease of any one of  claims 1 - 25  or the polynucleotide of any one of  claims 26 - 30  for use in the treatment of Hepatitis B virus invention or hepatocellular carcinoma caused by Hepatitis B virus.