(Attack)2: Genetic engineering of cellular and humoral immunity to cure HIV

总体而言：项目摘要/摘要 我们的 U19 计划项目要测试的假设是，结合基因工程细胞疗法 （嵌合抗原受体（CAR））和体液（广泛中和抗体（bNAb））免疫试剂将 从而治愈 HIV-1 疾病。在“柏林病人”这一著名案例中，同种异体移植 CCR5Δ32 供体细胞实现了功能性治愈，且没有残留 HIV-1 的证据。然而，一个通用的 基因工程造血干细胞/祖细胞（HSPC）移植尚未解决的局限性是 难以达到足以提供良好治疗效果的植入水平。我们在这里建议重点 建立基因工程细胞和体液免疫疗法。一种方法是 CAR 识别 HIV-1感染的细胞。通过过继性 T 细胞疗法提供肿瘤特异性 CAR 的 T 细胞免疫疗法 早期人类研究证明它能有效对抗癌症。我们假设基于 HSPC 的 CAR- T 细胞可以增强所得工程化 T 细胞的数量和功能反应。来补充 T 细胞介导的免疫工程，我们还建议改造 B 细胞以表达修饰为 单链变体（scFv-Fc bNAb）。针对 HIV-1 的 BNAb 已显示出抑制病毒血症的前景 动物模型以及从幼年恒河猴的血液和组织中清除 SHIV 以及人类临床 研究显示有效的抑制。尽管如此，bNAb 需要多次注射才能维持所需的水平 能抑制血浆中的病毒，对慢性感染的病毒没有清除作用。我们将使用新型慢病毒载体 通过基因工程将 scFv-Fc bNAb 靶向递送至体内 B 细胞，无需离体操作。我们 假设该策略将促进 bNAb 的产生、生物分布和基因的激活/分化 改造 B 细胞以清除 HIV-1 储存库。最后，这些基因修饰疗法的成功取决于 达到足够的基因修饰细胞的全身水平。因此，另一个项目将专门解决方法 调节基因修饰细胞的水平，以达到最大的治疗效果。此外， 如果观察到任何不良影响，可以使用相同的试剂快速消除基因修饰细胞， 提供“终止开关”，从而为整体方法增加了安全元素。 博士。 Kitchen 和 Chen（加州大学洛杉矶分校）将担任双 PI。两人在一般干细胞领域都有丰富的经验 生物学及其在 HIV-1 疾病中的应用。项目/核心领导者拥有 HIV-1 生物学方面的专业知识和 HIV-1 疾病的基因治疗方法。专业知识的广度涵盖载体和转基因 HSPC 生物学动物模型的开发（Chen、An、Morizono、Kitchen、Symonds）、开发和使用 (Kitchen、An、Morizono、Chen、Kiem)、抗HIV-1免疫功能(Yang、Kitchen)、HSPC的理解 行为（陈）到临床试验实施（西蒙兹）。

Overall: Project Summary/Abstract The hypothesis to be tested in our U19 Program Project is that combining therapies of gene-engineered cellular (chimeric antigen receptor (CAR)) and humoral (broadly neutralizing antibodies (bNAb)) immune reagents will lead to a cure of HIV-1 disease. In the single remarkable case of the “Berlin patient”, allogeneic transplant of CCR5Δ32 donor cells resulted in a functional cure without evidence for remaining HIV-1. However, a universal unresolved limitation of transplant of gene-engineered hematopoietic stem/progenitor cells (HSPC) has been the difficulty of achieving engraftment levels sufficient to provide good therapeutic efficacy. We propose here to focus on building gene-engineered cellular and humoral immune therapeutics. One approach is a CAR recognizing HIV-1 infected cells. T cell immunotherapy with tumor specific CARs delivered by adoptive T cell therapy has proven to be effective against cancer in early human studies. We hypothesize that HSPC based delivery of CAR- T cells can enhance the number and functional responses of the resultant engineered T cells. To complement the engineering of T cell-mediated immunity, we also propose to engineer B cells to express bNAbs modified as single chain variants (scFv-Fc bNAb). BNAbs directed to HIV-1 have shown promise at suppressing viremia in animal models and clearing SHIV from the blood and tissues of infant rhesus macaques and human clinical studies show effective suppression. Nonetheless, bNAbs require multiple injections to maintain levels required to suppress virus in plasma and have not cleared virus in chronic infection. We will use novel lentiviral vectors gene-engineered to target delivery of scFv-Fc bNAb into B cells in vivo without ex vivo manipulation. We hypothesize that this strategy will promote bNAb production, biodistribution and activation/differentiation of gene- modified B cells to clear HIV-1 reservoirs. Finally, success of these gene-modifying therapeutics depends upon achieving sufficient systemic levels of gene-modified cells. Thus, another project will specifically address means to modulate up or down the levels of gene-modified cells to achieve maximum therapeutic efficacy. In addition, should any adverse effects be observed, the same reagent can be used to quickly eliminate gene-modified cells, providing a “kill-switch”, and thus an added safety element to the overall approach. Drs. Kitchen and Chen (UCLA) will serve as dual-PIs. Both have extensive experience in general stem cell biology and its applications to HIV-1 disease. The Project/Core Leaders have expertise in HIV-1 biology and gene therapeutic approaches to HIV-1 disease. The breadth of expertise ranges from vector and transgene development (Chen, An, Morizono, Kitchen, Symonds), development and use of animal models for HSPC biology (Kitchen, An, Morizono, Chen, Kiem), anti-HIV-1 immune function (Yang, Kitchen), understanding of HSPC behavior (Chen) to clinical trial implementation (Symonds).