Study On The Inhibitory Effect Of RAAV Expressing MDA-7/IL-24 And Oncolytic Adenovirus Carrying α-Tim3 On CRC

Colorectal cancer（CRC）is the third most common cancer worldwide,and its occurrence and development are mainly attributed to key gene mutations and microsatellite instability.Surgery and chemotherapy often lead to serious adverse reactions and a high recurrence rate.In recent years,the development of viral therapy and immunotherapy has become a hot topic in cancer treatment research.However,Insufficient ability to kill cancer cells solely using viral vectors,and due to acquired drug resistance,many patients do not respond to single immunotherapy.Therefore,there is an urgent need to develop new cancer treatment strategies.Leucine rich repeat G protein coupled receptor 5（LGR5）is overexpressed in colon cancer cells and is one of the markers of intestinal cancer cell population.In this study,a recombinant adeno-associated virus（rAAV）and oncolytic adenovirus vector regulated by the LGR5promoter were designed,and a strategy of combining targeted gene viral therapy with immunotherapy was used for the treatment of colon cancer.The aim is to provide some reference for the treatment and medication of colon cancer.In this study,we successfully screened the core sequence of the LGR5 promoter and constructed a recombinant adeno-associated virus AAV-LGR5-IL-24-WPRE（AAV-lgr5-IL-24）that regulates MDA-7/IL-24 expression using the LGR5 promoter using genetic engineering techniques,as well as a recombinant oncolytic adenovirus Ad-lgr5-α-Tim3（AdLP-α-Tim3）that regulates the expression of immune checkpoint molecule TIM3 antibody.In the study of rAAV,the first step is to establish a batch production system for rAAV using suspended HEK293 cells;At the in vitro level,WPRE elements were identified by flow cytometry to significantly increase the expression level of exogenous genes;MTT detection showed that AAV-lgr5-IL-24infection showed a trend of inhibiting colon cancer cells proliferation;Exploring the mechanism of rAAV inhibiting colon cancer cell growth through Western Blot and q PCR analysis,the results showed that AAV-lgr5-IL-24 can induce apoptosis in colon cancer cells and prevent tumor cell migration and tumor angiogenesis;In vivo studies,AAV-lgr5-IL-24 treatment can significantly inhibit tumor growth and prolong the survival period of tumor bearing mice.In the study of oncolytic adenoviruses,we first utilized offspring replication and MTT to investigate whether the LGR5 promoter can increase the targeted killing ability of adenoviruses.Subsequently,AdLP-α-Tim3 was discovered through MTT,crystal violet staining,and flow cytometry experiments has a significant induced apoptosis and inhibitory effect on colon cancer cells.Then,q PCR was used to detect changes in immune related factors,and the results showed that AdLP-α-Tim3 promotes the activation of the immune system to a certain extent and participates in anti-tumor immune activation.Finally,by co-culturing Jurkat ^CD3 cells with tumor cells,it was found that AdLP-α-Tim3 treatment can significantly inhibit the proliferation of colon cancer cells through Jurkat^CD3.In summary,the results of this study confirm that the recombinant adeno-associated virus AAV-lgr5-IL-24 can inhibit the proliferation and development of colon cancer cells,significantly prolonging the survival period of transplanted tumor mice;Recombinant oncolytic adenovirus AdLP-α-Tim3 has a significant ability to kill colon cancer cells,demonstrating good anti-tumor ability.Therefore,the above treatment strategies provide certain reference value for the treatment of malignant tumors in clinical practice and provide new ideas for the development of anti-tumor drugs.