Engineered Microvesicles For Delivery Of CRISPR/Cas9 System In Liver Cancer Treatment

Liver cancer is one of the most malignant cancers worldwide with high mortality,for which treatments are not much effective at the advanced stage of cancer till date.Sorafenib,a currently approved oral drug for liver cancer,still poses therapeutic challenges due to drug resistance of cancer cells.CRISPR/Cas9 is an attractive potential tool for treating drug resistance as it could target the underlying mechanism/pathway directly,whereas a safe,efficient and cancer cell specific delivery platform remains challenging.Microvesicles(MVs),the cell membrane budded nanovesicles involved in intercellular communication via transferring bio-active molecules,hold promising advantages to be used as delivery platform of gene editing components.Thus the study was designed to use MVs as a natural delivery platform of CRISPR/Cas9 in combination with conventional sorafenib treatment to achieve a better synergistic anticancer effect by targeting drug resistance.Main results achieved are summarized below:1.Different cell derived MVs have been investigated in which cancer-derived MVs were found to have a better cancer cell homing property,their cancer-related molecules transferring ability could induce sorafenib resistance and further promote the cancer progression.Simultaneously,HEK293-dervied MVs could act as the nano-vesicles for delivery of CRISPR system,mediating effective genome editing in target cells.2.Engineered MVs based CRISPR-Cas9 delivery system has been constructed and optimized.In this study,we report that normal HEK293 cell –derived MVs engineered to have Cas9 and donated as Cas9 MVs are capable of delivering sgRNA to cancer cell and editing relative gene.Further,those Cas9 MVs were engineered to have HN3(HN3LC9-293MVs),targets tumor cells efficiently.Our study results showed that HN3LC9-293 MVs were quickly absorbed by the recipient cancer cell in vitro.Anchoring HN3 to the membrane of the MVs using LAMP2,made HN3LC9-293 MVs to specifically enter the GPC3+ Hu H-7 cancer cells than the GPC3-LO2 cells in a coculture model which further supported the cancer cell homing ability of HN3LC9-293 MVs.3.Synergistic anti-cancer effect in liver cancer treatment has been achieved by utilizing engineered MVs to modulate sorafenib resistance in liver cancer cells as well as cancer stem cells(CSCs)stemness properties with sorafenib chemotherapy.Knocking out IQGAP1 which has been involved in activated PI3K/Akt signaling in sorafenib resistance could promote cancer cell response to drug administration.In the meantime,CSCs are a subpopulation of cancer cells with self-renewal ability and are considered to be one of the reasons for drug resistance.Thus,a dual sgRNA plasmid(sgIQ1.1/FOXM1.2)has been constructed and electroporated into above mentioned HN3LC9-293 MVs to target IQGAP1 and FOXM1(related with self-renewal ability of cancer stem cell which contribute to sorafenib resistance),resulting in the reduction of CD133+ liver CSCs.Furthermore,sgIQ1.1/sg FOXM1.2 loaded HN3LC9-293 MVs in combination with sorafenib showed more effective synergistic anti-proliferative and apoptotic effect.Taken together,by approaching sorafenib resistance with a combination therapy of engineered MVs loaded CRISPR/Cas9 and sorafenib which also could be used for targeting of CSCs,our study foreshadows better,safe and natural delivery systems for an accurate and successful anti-cancer therapy in the future.