Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing

Background: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. Methods: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1-thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. Results: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. Conclusions: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.