Investigation Of PD-1 And IL-6 Genes Silencing Efficiency Using Lentiviral Vector-Mediated ShRNA Technology In Effector Cells

Over the last few years,with rapid development and advance knowledge about human immunology anticipated the feasibility and breakthrough a thriving cellular immunotherapeutic modality termed chimeric antigen receptor T cell therapy(CART).This novel transfusion medicine involving the infusion of engineered lymphocytes to mediate antitumoral effect without any impairment on normal tissues.Despite encouraging outcomes has observed with this cell therapy,CAR-T restrained with negative regulators such as programmed cell death 1(PD-1)receptor following immune activation that limit the antitumor responses of cell therapy.Mainly,PD-1 inhibits T cells activity by engaging with its cognate ligands on tumor cells and promote T cell dysfunction and exhaustion.Besides,following in vivo expansion of CAR-T cells,cytokine release syndrome(CRS)recognized as a life-threatening factor due to elevated IL-6 levels which is termed as hallmarks of CRS.Simultaneously,antibodies targeting these molecules could block the inhibitory axis to substantially boost the efficacy,but ambiguous activation of T cells retard the immunity with severe adverse events.Intriguingly,RNA interference(RNAi)may characterize as a promising combinatorial tool to disrupt cell intrinsic pathway of inhibitory receptor PD-1 and CRS responsive IL-6 receptor as RNAi can specifically intrude the “undruggable” genes.Therefore,this study intended to investigate the downregulation of PD-1 and IL-6 gene of T cells with RNAi effector shRNA and to impose release the “brakes” of natural immune system.The goal of this project is to analyze the knockdown efficiency PD-1 and IL-6 gene through infecting the T cells(Jurkat cells)with lentivirus containing PD-1 and IL-6 shRNA,respectively.The inhibition of PD-1 and IL-6 expression on effector T cells following shRNA techniques can improve the anticancer effects and achieved substantial sustained efficacy.To implement this project,lentivirus(LV)encoding PD-1 and IL-6 fusion construct was produced and transduced into jurkat effector cells to generate corresponding stable cell pool that overexpress PD-1 and IL-6,respectively.Fluorescence microscopy and qRT-PCR results confirmed the successful stable expression of PD-1 and IL-6 in jurkat cell.Afterwards,recombinant LV(rLV)containing eight kinds of shRNA constructs were designed and generated against PD-1 and IL-6 gene,separately.rLV containing shRNA oligonucleotides were transduced to corresponding jurkat overexpressing cells to evaluate the knockdown efficiency and to select the effective shRNA.By assessment of mRNA level with qPCR of each shRNA transduced cells resulted the effective shRNA reduce around 50% of PD-1 protein and 40 % reduction of IL-6 expression.Moreover,the result suggested that rLV can be successfully used for efficient gene delivery and gene knockdown studies of effector cells.This investigation sets the platform for using rLV technology to investigate the cellular intrinsic disruption of PD-1 and IL-6 gene in effector cells.Further in-vivo work is needed to validate and improve LV containing shRNA mediated gene silencing in effector cells.