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

此FastTrack阶段1/2应用程序旨在为以下应用程序创建新的Ind-Ready CAR-T平台 艾滋病毒的治疗，在实验室开发，并在非人类灵长类动物身上进行测试。分子 工程技术已被应用于创造嵌合抗原受体(CAR)表达 在T细胞中以HIV感染细胞为靶点。基于CD4的汽车旨在实现免疫 通过识别感染细胞上的gp120膜蛋白根除HIV1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来解决这一问题 这项研究的最新进展。据预测，影响CD4CAR效率的特征之一是T细胞耗尽， 以高表达HIV特异性CD8和CD4T细胞的PD1为特征。我们的实验室 新近确定的转录因子YY1是T细胞耗竭的主要调节因子，介导 上调检查点受体(CR)和下调I型细胞因子 伴随着细胞毒性衰竭。我们证实，在CD4中，YY1与PD1平行增加 慢性HIV感染中的T细胞。在临床前抑制YY1恢复细胞因子IL2的产生 检测和减少CR表达，包括耗竭标记PD1，其中阻止PD1和 恢复的IL2与恢复的T细胞细胞毒力相关。以前的CD4的另一个缺点是- 基于CAR-T的是表达CD4CAR的CD8T细胞现在很容易感染和 被艾滋病毒消除，这也可能阻碍之前的人体试验的成功。最后，之前的测试 涉及信号潜力有限的第一代(Gen)汽车，现在已通过 增加了共刺激作用，可能还会进一步改进。我们的总体目标是创造一个更 有效控制艾滋病毒的CAR-T。我们这项提议的目标包括：(1)创造抗艾滋病毒 Car-T细胞通过掺入YY1 shRNA来抵抗T细胞耗竭，以持续抗- 艾滋病病毒的效力。此外，我们将(2)通过RNAi干预使CD4CAR-T免受感染，以 阻断CAR-T的感染和病毒复制。最后，我们将(3)进行补充 努力生产新的第三代3信号CD4汽车(CAR3)，其中包括更多 共刺激分子，以提高效力和再激活潜力。建立在我们的 前期工作相当多，我们将很快完成第一阶段的组件工作 分子工程在1-6个月内的努力，直接进入体外第二阶段，然后非 人类灵长类测试。该计划是IT Bio，LLC(波士顿)和 史蒂文·布劳恩博士(杜兰)。杜兰国家灵长类研究中心将进行 NHP在IT Bio，LLC的指导下，通过收费服务合同进行试验。使用 这三项努力-抑制T细胞耗竭，使CAR-T免受感染和 提高T细胞的效力和再激活潜能--我们希望获得一种新型的、可供IND使用的细胞 一种可持续控制艾滋病毒的治疗剂，可与最近在治疗方面取得的成功相媲美 B细胞癌的T治疗。

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.