Investigation Of In Vivo Viral Tracking And T Cell Engineering Using Bioorthogonal Labeling Strategy

With the deterioration of living environment,the epidemic outbreak of major viruses and the increasing incidence of cancer have caused huge economic losses and social panic.Because virus infection and the occurrence of cancer are still not clear,the development of drugs for treating viruses and cancer are lagging behind.There is an urgent need to exploit an effective research strategy to clarify the mechanism of viral infection and break the bottleneck of cancer treatment.Recently,bioorthogonal chemistry based on metabolic engineering has been widely applied to living organisms labeling and tracking,and tumor-targeted therapy due to its non-destructive,high-efficiency,and specific targeting capabilities.The relevant applications in nano-biomedicine research are also continually expanding.Herein,a typical envelope virus（pseudotyped influenza virus,H5N1p）and non-enveloped virus（enterovirus 71,EV71）are studied for clarifying the key events during viral infection through metabolism engineering and bioorthogonal labeling,which is a universal virus labeling technique for tracking the dynamic of viral infection in vivo.Simultaneously,we also developed a safe and efficient viral transduction technology for T cell through glycometabolism engineering and bioorthogonal targeting,which provided new ideas for manufacture of clinical chimeric antigen receptor（CAR）-T cells.The main research contents include the following three aspects:1.Lipid metabolic modification of H5N1p with azide functional group and in situ fluorescent labeling of viruses in vivo.（1）Azide-Choline（AE-Cho）was incorporated into cell membrane by lipid metabolism,and azide（-N3）group was modified on viral envelope when the virion budding from host cell.A stable linkage was formed between viral particle and fluorescent probe by the bioorthogonal chemistry,thereby achieving the in situ fluorescent labeling for virus.The results showed that 92.5±4.1%of the virions could be successfully captured by the fluorescent probe,and the infectivity of in situ labeled virus also resulted in 1.5 folds of pre-labeled virus did.The results indicated that the in situ bioorthogonal labeling has a high-efficiency for viral labeling and can better preserve virus infectivity.（2）In the intranasal infection model,the bioorthogonal-targeting fluorescent probe rapidly captured influenza virus in the lungs.The in vivo imaging displayed an obvious dynamic process of virus fluorescence signal in mouse lung from 12-24 h.These data confirmed that in situ bioorthogonal labeling of virus was high efficiency and non-destruction,and can be successfully used for the viral tracking studies in vivo and in vitro.2.In situ bioorthogonal labeling for real-time tracking viral invasion and dynamic tissue tropism of EV71.（1）The dibenzocyclooctyne（DBCO）group was modified to viral surface using EDC/NHS cross-linking,and then fluorescent probe can conjugate to the DBCO-EV71 by in situ bioorthogonal chemistry for viral tracking in cells.（2）The neonatal ICR mice were intraperitoneal injected with DBCO-EV71,the paired fluorescent probe（N3-Cy5.5）effectively labeled the virion in vivo through bioorthogonal chemistry.The results showed that fluorescent probe successfully captured EV71 virus in vivo without damping viral infectivity,and viral copy number was positively correlated with its fluorescence intensity in different organs.（3）In situ bioorthogonal labeling strategy effectively captured EV71 in multiple organs without interfering viral spreading and tissue tropism,and accurately revealed dynamic dissemination and tissue tropism of EV71 with different pathogenicity.Especially,the significant neurological and respiratory tropism,a major clinical symptom caused by SC-EV71,were also confirmed,providing a reliable technique for visual studying EV71 infection mechanism in vivo.3.Development of PEI-DBCO/lentivirus transduction system for T cell using glycometabolism and bioorthogonal targeting.The PEI-DBCO polymer was successfully prepared by the conjugation between dibenzocyclooctyne and cationic polymer polyethyleneimine（PEI）,which further were used for the generation of PEI-DBCO/lentivirus nanocomplexes.Azide of Ac₄GlcNAz was incorporated into the T cell membrane through cell glycometabolism.Nanocomplexes effectively guided virus into T cell through the highly efficient and specific bioorthogonal reaction between DBCO group on nanocomplexes and N₃ on T cell.This process did not affect cell proliferation,activation and subpopulation,indicating a better safety of this strategy.Moreover,the prepared CAR-T cell can obviously increase the secretory of cytokines IL-2,TNF-?and INF-?,which further enhanced anti-tumor capability of CAR-T cells.Moreover,this system significantly enhanced the cytotoxicity of CAR-T cells for Raji cells in vivo,improving survival situation of mice.It solved problems of low efficiency,high toxicity of T cell transduction,and provided new ideas for further improving CAR-T cell for clinical therapy.In conclusion,this study demonstrated that the bioorthogonal chemistry based cell metabolism engineering can be used for viral capture,dynamic tracking in vivo,and the manufacture of safe and efficient CAR-T cells,which will provide solid theoretical foundation and reliable technical support for the development of antiviral drugs and clinical CAR-T or TCR-T cell therapy,respectively.