Hepatocyte-confined CRISPR/Cas9-based nanocleaver precisely eliminates viral DNA for efficient and safe treatment of hepatitis B virus infection

Abstract

Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9)-based therapies are attractive to achieve precise and targeted disruption of the hepatitis B virus (HBV) genome for HBV infection treatment. However, unspecific in vivo distribution of the CRISPR/Cas9 vector after systemic administration not only impedes CRISPR/Cas9 expression in the liver, but also causes undesired gene editing in off-site organs, decreasing the therapeutic efficacy and leading to potential side effects. Herein, we designed a hepatocyte-confined CRISPR/Cas9-based nanocleaver (HepCCCleaver) to realize precise and efficient cleavage of HBV DNA, specifically in hepatocytes, for safe and effective HBV treatment. This HepCCCleaver was formed by encapsulating the hepatocyte-specific CRISPR/Cas9 plasmid into a galactose-modified membrane-fusogenic carrier. Our galactose-modified membrane-fusogenic carrier allowed the HepCCCleaver to specifically accumulate in the liver and enter hepatocytes through membrane fusion, bypassing lysosome entrapment to avoid degradation for superior delivery efficiency. The hepatocyte-specific CRISPR/Cas9 plasmid released in the cytoplasm could switch on the gene expression only in hepatocytes, ensuring precise cleavage on viral DNA in HBV-infected but not other types of cells. By integrating the liver-targeting membrane-fusogenic delivery system with hepatocyte-specific CRISPR/Cas9 plasmid, this HepCCCleaver could precisely and efficiently confine over 95% events of viral DNA gene editing in hepatocytes. This HepCCCleaver is a universal gene editing platform enabling safe and efficient disease treatment, specifically in the cell type of interest.