在前期研究中，我们研发了以HIV-1广谱中和抗体为胞外结构的VC-CAR-T细胞可以特异性杀伤HIV-1感染细胞。进一步研究发现，经过长期扩增的CAR-T细胞，杀伤活性明显减弱。通过表达谱分析发现PD-1、Tim-3、Lag-3等抑制性受体表达明显上升。因此本项目拟在VC-CAR的模块上共表达几种shRNA特异性敲除以上受体，从而增强CAR-T细胞的功能。进一步实验显示共表达shRNA的VC-CAR-T细胞可以长期维持记忆性表型，并且更有效地杀伤靶细胞。本项目拟采用分子病毒学技术、免疫学技术和人源化小鼠等方法，旨在证明共表达shPD-1/Tim-3/Lag-3可以使CAR-T细胞自我更新增强、功能耗竭减缓、体内半衰期延长，长期且持续地发挥抗病毒作用，从而在HIV-1感染患者样本中更有效地清除重激活的病毒储存库，并优化出更有效的CAR-T疗法，为功能性治愈HIV-1提供更好的候选策略。

Our previous studies have shown that CAR-T cells guided by the single-chain Fv of a broadly neutralizing antibody specifically eradicate the HIV-1 infected cells. However, the efficiency of CAR-T cell is compromised after excessive expansion. Our group identified several checkpiont receptors, such as PD-1, Tim-3 and Lag-3, are significantly up-regulated after long-term expansion through analyzing the transcriptional changes. Therefore, we co-expressed CAR-T moiety with shRNA targeted these inhibitory molecules to augment the potency of CAR-T cells. We demonstrated that the modified CAR-T cells maintained memory-like phenotype and robust proliferation during long-term culture, and induced specific cytolysis of cells expressing HIV-1 Env proteins. In this project, we will utilize in-vitro HIV-1 latency model and humanized mice to examine if the shRNA-CAR-T-expressing cells increase the self-renewal capability, prevent exhausion, and persistently suppress HIV-1 rebound. Furthermore, we will verify whether shRNA-CAR-T cells exert more potency to induce the cytolysis of latency reversing agent (LRA)-reactivated HIV-1-infected CD4+ T lymphocytes isolated from infected individuals receiving suppressive cART. Based on these works, we will optimize a CAR-T cell-based immunotherapy strategy for therapeutic application in efforts to reach a functional HIV cure.