Exosome-mediated delivery of Cas9 ribonucleoprotein complexes for tissue-specific gene therapy of liver diseases.

Abstract

CRISPR-Cas9 gene editing has emerged as a powerful therapeutic technology, but the lack of safe and efficient in vivo delivery systems, especially for tissue-specific vectors, limits its broad clinical applications. Delivery of Cas9 ribonucleoprotein (RNP) owns competitive advantages over other options; however, the large size of RNPs exceeds the loading capacity of currently available delivery vectors. Here, we report a previously unidentified genome editing delivery system, named exosome^RNP, in which Cas9 RNPs were loaded into purified exosomes isolated from hepatic stellate cells through electroporation. Exosome^RNP facilitated effective cytosolic delivery of RNP in vitro while specifically accumulated in the liver tissue in vivo. Exosome^RNP showed vigorous therapeutic potential in acute liver injury, chronic liver fibrosis, and hepatocellular carcinoma mouse models via targeting p53 up-regulated modulator of apoptosis (<i>PUMA</i>), cyclin E1 (<i>CcnE1</i>), and K (lysine) acetyltransferase 5 (<i>KAT5</i>), respectively. The developed exosome^RNP provides a feasible platform for precise and tissue-specific gene therapies of liver diseases.