Construction Of CD3+ T Cell Specific And Chimeric Antigen Receptor Expressed Lentiviral Vector

OBJECTIVE:CAR-T cell therapy,as an effective method for tumor immunotherapy,can kill tumors by specifically recognize and bind tumor-associated antigens.CAR-T cell therapy has achieved great success in treating hematological malignancy,and its application in various solid tumors is also being explored.At present,there are more than 300 CAR-T related clinical trials in the world.In various clinical trials,the structure of CAR has been modified to improve the anti-tumor activity and safety of CAR-T cell.Currently,Two types of CD19 CAR-T cells have been approved by the Food and Drug Administration（FDA）for clinical use,while domestic CAR-T cell therapies are still in clinical trials.CAR-T cell therapy is promising in clinical practice,but the complex preparation process and high price of CAR-T cell limit the application of CAR-T cell therapy.In this study,CD3-specific lentivirus vectors were constructed to specifically infect CD3+T cells in vivo to express CAR and produce effector CAR-T cells,thus greatly simplifying the in-vitro production process of CAR-T cell.It makes CAR-T more applicable,and it is also a solution for universal CAR-T cells.In this study,a lentivirus vector was constructed,which can be used to produce lenti-virus that specifically bind cells carrying corresponding antigens,transfect and express target gene sequences,and was a flexible and stable gene editing tool.METHODS:In this study,we modified the pseudotyped envelope plasmid p-CMV-VSVG,which determines the LV infection ability in the second-generation LV three-plasmid packaging system.First,find the appropriate restriction site on the envelope plasmid p-CMV-VSVG,remove the VSVG fragment,and recover the remaining linear vector plasmid fragments p CMV-VSVG（SalⅠ,BglⅡ）.Then the modified measles virus hemagglutinin protein（H protein）DNA sequence was synthesized.This modified H protein of leprosy virus is suitable for LV pseudotyping and does not bind to its natural ligands CD46 and SLAM.Design appropriate primers,add a linker fragment（G₄S）₃of appropriate length after the DNA sequence,and obtain linear DNA fragment H-（G₄S）₃by PCR.Using a plasmid containing a CD3-specific single-chain variable fragment DNA fragment to design appropriate primers,and obtaining a linear CD3-specific single-chain antibody DNA sequence OKT3 by PCR.Each of the above linear DNA fragments has a homologous sequence of 20 bp before and after.The H-（G₄S）₃fragment was ligated with the OKT3 fragment using Overlap PCR to obtain a linear DNA fragment H-（G₄S）₃-OKT3.Finally,infusion was used to join the linear vector plasmid fragment p-CMV-VSVG（Sal I,Bgl II）with H-（G₄S）₃-OKT to obtain the plasmid p-CMV-Hm O.After the sequencing was confirmed to be correct,the second-generation LV triple plasmid packaging system was used to package and produce CD3-specific LV.CD3-specific LV infected 293T cells that did not express CD3 and Jurkat cells that stably expressed CD3,respectively,to detect their specific infectivity and expression.RESULTS:In this study,a new LV enveloped plasmid p-cmv-hmo was successfully constructed,and p-cmv-hmo was used to replace the original enveloped plasmid p-cmv-vsvg to produce a lentiviral vector that can specifically transfect CD3+cells and can stably express the target gene sequence in the vector plasmid.Conclusion:Using the envelope plasmid p-CMV-Hm O,we can produce CD3-specific LV.CD3 is highly expressed in almost all mature T cells in vivo.This CD3-specific LV can directly infect CD3+T cells in vivo and express CAR.Thus,CAR-T cells are produced in vivo and use CAR’s specific targeting of tumor-associated antigens to generate anti-tumor efficacy.This study also verified a lentiviral vector model.By modifying the enveloped plasmids of lentiviral vectors,LV can specifically target cells expressing corresponding antigens,transfect and express target gene sequences,which is a flexible and stable gene editing tool.