834 CRISPR-mediated in situ editing of liver resident macrophages for treating liver cancers

Background Liver cancer is one of the leading cause of cancer death worldwide with limited treatment options. The liver accommodates the largest population of tissue resident macrophages in the body, namely Kupffer cells. Immune deviation of hepatic immune responses from anti-tumor towards pro-tumor is crucial for cancer progression. This process is closely correlated with the functional polarization of these macrophages. In situ genome editing of liver resident macrophage with intention to shift macrophage function to stimulate anti-tumor immune responses is promising in treating liver cancers. Methods We have previously shown that Kupffer cells quickly capture and phagocytose circulating bacteria, making bacteria as a potential liver macrophage-specific deliver vector. Taking advantages of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, we have established a bacteria mediated genome editing methods for liver resident macrophages in vivo. Results We used a non-pathogenic Escherichia coli (E. coli) strain as a deliver vector for the CRISPR-Cas9 plasmids, essentially all liver resident macrophages but neither liver sinusoids endothelial cells nor hepatocytes were shown to taken up the bacteria, indicating the robustness and specificity of E. coli-mediated plasmid delivery. To test the genome editing efficiency, we chose VSIG4, Tim-4 and F4/80 that were highly expressed by Kupffer cells and validated the gene knockout/knockdown effects using intravital imaging. Expression of these receptors by Kupffer cells diminished by more than 90%. Simultaneously editing of multiple genes was also achieved with a slightly decreased efficiency when compared to single gene editing. The acute inflammatory responses and the hepatotoxity caused by bacteria were ameliorated by pre-immunization with the same E. coli strain, and can be further minimized by using a mutant E. coli strain that processed a modified LPS structure, which dramatically decreased the TLR-4 mediated inflammatory signaling and improved the safety of this method. Moreover, we have shown that not only embryonically-derived Kupffer cell but also monocyte-derived liver macrophages could be edited. The applications of this approach in treating primary liver cancers and liver metastasis are under investigation. Conclusions Taken together, we have established a rapid, efficient and convenient method to achieve in situ genome editing of liver resident macrophages in vivo. By targeting essential genes that instruct macrophage polarization, this method could be used as immunotherapy for liver diseases, including cancers.