Chronic HBV Infection Using CRISPR/Cas9 Delivered by Novel pH-Sensitive Cationic Liposomes

Background: Current research focuses on reducing the level of HBV virus. We designed CRISPR/Cas9s that target the HBV genome. Methods: To prevent the emergence of escape mutant viruses, we mixed HBV-CRISPR/Cas9s (HBV-CRISPR/Cas9 complex). These HBV-CRISPR/Cas9s complexes were encapsulated in a nano-liposome complex, which is a hepatocyte-specific drug delivery system. CRISPR/Cas9 was used to assess delivery and knockout efficiencies of nano-liposome-gRNA-HBV complexes in the treatment of mice. The potency of the nano-liposome-gRNA-HBV complexes was evaluated in HepG2.2.15 cells and in HBV-Tg mice. Findings: Effective knockout of targets, efficient delivery of CRISPR/Cas9, and liver-specific delivery were observed with nano-liposome-gRNA-HBV complex treatment. Nano-liposome-gRNA-HBV complexes efficiently reduced HBsAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg when compared with nano-liposome-gRNA-HBV complex treatment. In addition, the suppressive effects of nano-liposome-gRNA-HBV complexes persisted for 21 days in vitro and in vivo. We indicated that treatment with nano-liposome-gRNA-HBV complexes controlled HBV infection more efficiently than ETV treatment did. Furthermore, the effect of a single dose of nano-liposome-gRNA-HBV complexes persisted for a long time. Interpretation: The results demonstrate that nano-liposome-gRNA-HBV complex may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors. Funding Statement: This study was supported by grants from the National Natural Science Foundation of China (Grant nos. 81602295 to Shuai Zhen). This study was supported by grants from Special Talent Training Program in Northwest Women and Children Hospital (2020ZD02) Declaration of Interests: The other authors declare that they have no competing interests. Ethics Approval Statement: All mouse experiments were conducted according to the National Institutes of Health Guidelines for Animal Care. All animal procedures were performed according to the guidelines developed by the China Council on Animal Care and the protocol was approved by the Xi’an jiaotong university.