Preparation Of Polymeric Gene Vectors And Research On Anti-tumor Gene Therapy And Application

Malignant tumors pose a grave threat to human health.With abundant exploration of and study on the mechanisms of occurrence and tumor progression,novel drugs and therapies emerge in endlessly.Known as one of the most important methods for treating tumors,gene therapy can cure tumors by correcting pathological defects at the gene level.Boasting high transfection efficiency,fine biocompatibility as well as low immunogenicity in vivo,high molecular genetic vectors can be mass-produced.Based on the merits of genetic vectors,this study conducted modification of cationic polymer(low molecular weight PEI 1.8k)by grafting and cross linking.Co-delivery of CpG and therapeutic gene with grafted products could be applied in anti-tumor immunotherapy in the scientific research.After stability test,cross linking products were developed as gene transfection kits.First,we utilized PEI1.8k to prepared a gene delivery system PEI-Lys(TOS)(KT)that had high transfection efficiency and biocompatibility,thus solving the problem of low transfection efficiency in the molecular weight gene vector system.Through systemically comparing the modified product KT with commercialized high molecular weight PEI25k and PEI1.8k,we proved it could be served as a good genetic vector.To promote in vivo application,natural hualuronic acid(HA)was introduced as a shielding carrier.A specific binding affinity between HA and high expression CD44 receptor on the tumor cell surface made the gene delivery system tumor-targeted,and the cationic gene delivery system’s poor stability in vivo circulation was overcome.The combination by charge attraction was adopted to enable co-delivery of negatively charged CpG and OX40L expression plasmid with KT gene carrier.CpG could induce increased expression of costimulatory molecule B7 on the surface of DC cell,and form in situ vaccines with OX40L expressed by tumor cells,thus activating T cells through costimulation.Through experiments of cell transfection in vitro,endocytosis and expression of antibody,we verified that when genetic vector KT carried OX40L plasmid and CpG,its transfection performance was not affected.In animal experiment,through determining the number of CD8+T、CD4+T、Treg、DC cells,the content of TNF-α IFN-γ、Granzyme B cytokines as well as the expression quantity of OX40 and OX40L in different treatment groups,we explored the regulatory effect of in situ vaccines on immunosuppression tumor microenvironment.To further verify that in situ vaccines could enhance sensibility to PD-1/PD-L1 blockade treatment,we used the tumor model of B16F10 cells with high PD-L1 expression.Dual immunity "engine" was used to change "cold" tumor into "hot"tumor,which made tumors more sensitive to PD-1/PD-L1 blockade treatment and effectively inhibit growth of tumors,thus further preventing recurrence and metastasis of tumors.In animal tests,through determining the content of PD-L1 on the surface of CD8+T、CD4+T tumor cells and that of TNF-α,IFN-γ and Granzyme B cytokines,we explored the level of antitumor immune response caused by the combined therapy and relative mechanisms of oncotherapy.By determination of memory T cells in spleens,we studied the memory effect induced by the therapy in fighting tumors and further explored the effect of the therapy on inhibiting recurrence and metastasis of tumors.Final results indicated the in situ vaccine strategy mediated by dual immunity"engine" could effectively improve the objective response rate of ICB therapy,thus inhibiting growth of multiple tumors.Finally,aiming at current international trade situation and heavy reliance on import of gene transfection reagents,through crosslinking modification of PEI 1.8k,we prepared a reduction sensitive genetic vector to be applied in development of gene transfection kits.By monitoring and tracing transfection performance,biocompatibility and products’ stability that clients paid particular attention to,we successfully developed commercialized,property-stable gene transfection reagents which can replace imported products.