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

总体：项目摘要/摘要 在我们的U19计划项目中要检验的假设是，基因工程细胞的联合疗法 (嵌合抗原受体(CAR))和体液(广谱中和抗体(BNAb))免疫试剂将 导致HIV-1疾病的治愈。在一个引人注目的“柏林病人”的案例中，异基因移植的 CCR5HIV-32供体细胞在没有残留Δ-1证据的情况下获得了功能性治愈。然而，一个万能的 基因工程造血干/祖细胞(HSPC)移植的局限性尚未解决 难以达到足以提供良好治疗效果的植入水平。我们建议在这里集中精力 关于建立基因工程细胞和体液免疫疗法。一种方法是汽车识别 HIV-1感染细胞。通过过继T细胞疗法提供的肿瘤特异性CARS的T细胞免疫疗法 在早期的人类研究中被证明对癌症有效。我们假设基于HSPC的汽车交付- T细胞可以增强所产生的工程化T细胞的数量和功能反应。来补充 在T细胞介导的免疫工程方面，我们还提出了工程B细胞来表达修饰为 单链变异体(scFv-Fc bNAb)。针对HIV-1的bNAb在抑制病毒血症方面显示出希望 幼年恒河猴和人类血液和组织中SIV的动物模型及清除 研究表明，抑制效果很好。尽管如此，bNAbs需要多次注射才能维持所需水平 抑制血浆中病毒并未清除慢性感染中的病毒。我们将使用新型慢病毒载体 基因工程的目的是将ScFv-Fc bNAb靶向体内的B细胞，而无需体外操作。我们 假设这一策略将促进bNAb的产生、生物分布和基因的激活/分化。 改良B细胞以清除HIV-1病毒储备库。最后，这些基因修饰疗法的成功取决于 达到足够的系统水平的转基因细胞。因此，另一个项目将专门处理手段 调节转基因细胞的水平，以达到最大的治疗效果。此外, 如果观察到任何不良反应，可以使用相同的试剂来快速消除转基因细胞， 提供了一个“终止开关”，从而为整个方法增加了一个安全元素。 Kitchen博士和Chen博士(加州大学洛杉矶分校)将担任双PI。两人在普通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/核心领导拥有艾滋病毒-1生物学和 HIV-1疾病的基因治疗方法。专业知识的范围从载体到转基因都有。 HSPC生物学动物模型的开发(Chen，An，Morizono，Kitchen，Symonds) (厨房，安，Morizono，Chen，Kiem)，抗HIV-1免疫功能(杨，厨房)，了解HSPC Behavior(Chen)到临床试验实施(Symonds)。

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).