Developing Non-viral Targeted Integration Of Chimeric Antigen T Receptor By CRISPR/Cas9

In recent years,the rapid development of cancer immunotherapy has brought hope to many cancer patients.Among them,the chimeric antigen receptor(CAR)T-cell therapy has shown a good effect in the clinical treatment and has attracted much attention.Especially it has great potential and advantages in the treatment of blood tumors.In children and adults with acute lymphocytic leukemia,the complete remission rate of anti-CD19 CAR-T therapy can be as high as 90%.Most of CAR-T cells use the lentivirus infection to overexpress CAR receptor on T cells,which has the disadvantages of high production cost and tedious process.Moreover,the use of virus as delivery vector has the hazard of random insertion.CRISPR/Cas9 technology,as the third generation of gene editing tool,has the advantages of high efficient targeted modification and simple operation.Therefore,we use it to prepare CAR-T cells which are targeted integration with non-virus,in order to solve the problems in the traditional lentivirus preparation.Our study mainly carried out two aspects.On the one hand,we explore the methods to improve the efficiency of CAR targeted integration.On the other hand,we test the functions of the non-viral targeted integration CAR-T cells.First,we optimized the conditions of gene editing to obtain CAR-T cells with high efficient of integration and viability.Comparing with delivering plasmids and linear double-stranded DNA,we determined that linear double-stranded DNA as template has higher viability.Then we compared the recombination rate of different templates including homology-directed repair(HDR),microhomology-mediated end joining(MMEJ),and homology-independent targeted integration(HITI),and found that HDR template has higher recombination rate.Comparing the length of homologous arms on recombination and viability,we finally determined that the length of homologous arms is 800 bp.In order to obtain more CAR positive cells,we use small molecule compounds that inhibit cell cycle or change the signal pathway of DNA damage repair.Through the exploration of concentration and processing time,we successfully found that small molecule B-27 can effectively improve the integration efficiency of CAR elements.On the basis of increasing the recombination rate of CAR,we selected AAVS1 safety site to insert CAR sequence targeted CD19(AAVS1-19bbz),and tested the functions of the CAR-T cells.Comparing to viability,expansion,surface markers,cytokine release and tumor killing ability,we have identified that AAVS1-19 bbz prepared by gene editing is significantly different from the control group,which shows that CAR element inserted can effectively perform the functions.At the same time,we compared with the CAR-T cells(LV-19bbz)prepared by the traditional lentivirus.We found that surface markers and cytokine release were difference between them,but they had similar characteristics in most functions,and can effectively kill tumor cells.On the other hand,we also verified the small molecular B-27 increased the recombination rate of CAR,but had little effect on its functions.In conclusion,we have successfully constructed CAR-T cells with non-viral targeted integration by CRISPR/Cas9,and proved that this CAR-T cells can play an effective role in vitro.Our new technology not only overcomes the disadvantages of using virus system,but also provides the basic for the diversified transformation and specific regulation of CAR-T cells,which helps to improve the safety and effectiveness of CAR-T cells to promote the further development of CAR-T technology.