Advanced generation infection-proof anti-HIV CAR-T with YY1 RNAi to block T cell exhaustion in NHP model

这一FastTrack第1/2阶段申请旨在创建一个新的IND就绪CAR-T平台， 在实验室开发并在非人类灵长类动物中测试的艾滋病毒治疗。分子 工程技术已被应用于产生表达的嵌合抗原受体（CAR 在T细胞中靶向HIV感染的细胞。基于CD 4的汽车被设计成实现免疫 通过识别感染细胞上的gp 120包膜蛋白根除HIV 1感染。 然而，先前的临床试验没有取得成功，我们建议通过该计划解决这一问题 of this research研究.预测影响CD 4 CAR效率的特征之一是T细胞耗竭， 其特征在于HIV特异性CD 8和CD 4 T细胞的高PD 1表达。本实验室 最近定义的转录因子YY 1是T细胞耗竭的主要调节因子，介导 检查点受体（CR）的上调和I型细胞因子的下调， 伴随细胞毒性失效。我们证实，在CD 4+细胞中，YY 1与PD 1平行增加， 慢性HIV感染中的T细胞YY 1的敲除恢复了临床前细胞因子IL 2的产生 检测和减少CR表达，包括耗竭标志物PD 1，其中PD 1被阻断， 恢复的IL 2与恢复的T细胞细胞毒性效力相关。现有的CD 4-T细胞的另一个缺点是， 基于CAR-T的一个新的特征是表达CD 4 CAR受体的CD 8 T细胞现在容易被感染， 被艾滋病毒消灭，这也可能阻碍先前人体试验的成功。最后，之前的测试 涉及信号传导潜力有限的第一代（gen）汽车，现在通过 添加共刺激，这可以进一步改善。我们的总体目标是创造一个更 有效的CAR-T用于控制HIV。我们的目标包括：（1）创造抗艾滋病毒 CAR-T细胞将通过掺入YY 1 shRNA抵抗T细胞耗竭，以持续抗- 艾滋病毒效力。此外，我们将（2）通过RNAi干预使CD 4 CAR-T抗感染， 阻断CAR-T中的感染和病毒复制。最后，我们将（3）进行补充 致力于产生新的第三代3-信号CD 4汽车（CAR 3）， 共刺激分子，以提高效力和再活化潜力。充分发挥两国 相当多的前期工作，我们将很快完成第一阶段的组成部分，以完成 在1-6个月的分子工程努力，直接进入体外2期，然后非 人类灵长类动物测试该计划是IT Bio，LLC（波士顿）和 史蒂文·布劳恩博士（杜兰大学）。杜兰国家灵长类动物研究中心（TNPRC）将进行 NHP实验在IT Bio，LLC的指导下通过收费服务合同进行。与 这三项努力-抑制T细胞耗竭，使CAR-T感染预防， 增加T细胞的效力和再活化潜力-我们希望获得一种新的，IND准备细胞 一种治疗剂，将提供持续的艾滋病毒控制，以平行最近的成功，在CAR- T治疗B细胞癌。

This FastTrack Phase 1/2 application aims to create a new IND-ready CAR-T platform for the treatment of HIV, developed in the laboratory and tested in non-human primates. Molecular engineering techniques have been applied to create chimeric antigen receptors (CAR) expressed in T cells to target HIV-infected cells. CD4-based CARs are designed to achieve immune eradication of HIV1 infections through recognizing gp120 envelope protein on infected cells. However, prior clinical trials did not meet with success, which we propose to address with the plan of this research. One of the features predicted to affect efficiency of CD4 CAR is T cell exhaustion, characterized by high PD1 expression of HIV-specific CD8 and CD4 T cells. Our laboratory recently defined transcription factor YY1 to be master regulator of T cell exhaustion, mediating upregulation of checkpoint receptors (CR) and downregulation of Type I cytokines with accompanying cytotoxic failure. We confirmed that YY1 is increased in parallel with PD1 in CD4 T cells in chronic HIV infection. Knockdown of YY1 restored cytokine IL2 production in preclinical testing and reduced CR expression, including exhaustion marker PD1, where blocked PD1 and restored IL2 correlated with recovered T cell cytotoxic potency. Another drawback of prior CD4- based CAR-T is that CD8 T cells expressing the CD4 CAR receptor are now readily infected and eliminated by HIV that could also have hampered success of prior human trials. Lastly, prior tests involved 1st generation (gen) CARs of limited signaling potential that are now improved with addition of costimulation that may be yet further improved. Our overall goal is to create a more effective CAR-T for the control of HIV. Our Aims for this proposal include (1) creating anti-HIV CAR-T cells that will resist T cell exhaustion with incorporation of YY1 shRNA for sustained anti- HIV potency. Further, we will (2) render the CD4 CAR-T infection-proof with RNAi intervention to block infection and virus replication in the CAR-T. Finally, we will (3) conduct a complementary effort to generate new 3rd gen 3-signal CD4 CARs (CAR3) that incorporate additional costimulatory molecules to improve potency and reactivation potential. Building on our considerable preliminary work, we will quickly finish the Phase 1 component to complete molecular engineering efforts in months 1-6, moving directly to Phase 2 in vitro and then non- human primate testing. This plan is a collaboration between IT Bio, LLC (Boston) and the lab of Dr Steven Braun (Tulane). The Tulane National Primate Research Center (TNPRC) will conduct the NHP experiments under the direction of IT Bio, LLC through a fee-for-service contract. With these three efforts – suppressing T cell exhaustion, rendering CAR-T infection-proof and increasing T cell potency and reactivation potential – we hope to obtain a novel, IND-ready cellular therapeutic agent that will provide a sustained control of HIV to parallel recent successes in CAR- T treatment of B cell cancers.