Engineered meganucleases specific for recognition sequences in the hepatitis B virus genome
The present invention encompasses engineered meganucleases which recognize and cleave a recognition sequence within an open reading frame (ORF) of the genome of at least two genotypes of the Hepatitis B virus (HBV). The present invention also encompasses methods of using such engineered meganucleases in a pharmaceutical composition and in methods for treating or reducing the symptoms of a HBV infection, or treating hepatocellular carcinoma (HCC). 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 HCC.
1. An engineered meganuclease that binds and cleaves a recognition sequence consisting of SEQ ID NO: 16, 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 wherein said second subunit binds to a second recognition half-site of said recognition sequence and wherein said engineered meganuclease comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 33.
2. The engineered meganuclease of claim 1 , wherein said first subunit comprises an amino acid sequence having at least 96% sequence identity to residues 198-344 of SEQ ID NO: 33, and wherein said second subunit comprises an amino acid sequence having at least 96% sequence identity to residues 7-153 of SEQ ID NO: 33.
3. The engineered meganuclease of claim 1 , wherein said first subunit comprises residues 198-344 of SEQ ID NO: 33.
4. The engineered meganuclease of claim 1 , wherein said second subunit comprises residues 7-153 of SEQ ID NO: 33.
5. The engineered meganuclease of claim 1 , wherein said engineered meganuclease comprises the amino acid sequence of SEQ ID NO: 33.
6. A polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of claim 1 .
7. A recombinant DNA construct comprising a nucleic acid sequence encoding said engineered meganuclease of claim 1 .
8. A viral vector comprising a nucleic acid sequence encoding said engineered meganuclease of claim 1 .
9. A pharmaceutical composition for treatment of a subject having hepatitis B virus (HBV) or hepatocellular carcinoma caused by HBV, said pharmaceutical composition comprising a pharmaceutically acceptable carrier and:
(a) a nucleic acid encoding said engineered meganuclease of claim 1 ; or
(b) said engineered meganuclease of claim 1 .