Optimized engineered meganucleases having specificity for a recognition sequence in the Hepatitis B virus genome
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 11-12×.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 consisting of 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, wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 12 or 13.
2. The engineered meganuclease of claim 1 , wherein said engineered meganuclease exhibits optimized characteristics selected from the group consisting of improved specificity, enhanced efficiency of cleavage, and enhanced efficiency of indel formation, when compared to the HBV 11-12×.26 meganuclease of SEQ ID NO: 14.
3. 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 said engineered meganuclease of claim 1 .
4. The engineered meganuclease of claim 1 , wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 12.
5. The engineered meganuclease of claim 1 , wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 13.
6. 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 therapeutically effective amount of said engineered meganuclease of claim 1 ; wherein said engineered meganuclease recognizes and cleaves said recognition sequence consisting of 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.