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

总体：项目总结/摘要 在我们的U19计划项目中要测试的假设是，基因工程细胞和细胞因子的联合治疗， （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”这一独特的病例中， CCR 5 Δ32供体细胞导致功能性治愈，没有剩余HIV-1的证据。然而，一个普遍的 基因工程造血干/祖细胞（HSPC）移植的未解决的局限性一直是 难以达到足以提供良好疗效的植入水平。我们在此建议， 基因工程细胞和体液免疫疗法。一种方法是CAR识别 HIV-1感染细胞。具有通过过继性T细胞疗法递送的肿瘤特异性汽车的T细胞免疫疗法具有以下优点： 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CAR-1的递送 T细胞可以增强所得工程化T细胞的数量和功能应答。以补充 T细胞介导的免疫的工程化，我们还提出工程化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HIV感染者病毒血症的前景。 动物模型和从幼年恒河猴和人类临床的血液和组织中清除SHIV 研究显示有效抑制。尽管如此，bNAb需要多次注射才能维持所需的水平。 抑制血浆中的病毒，而在慢性感染中没有清除病毒。我们将使用新型慢病毒载体 基因工程化以在体内靶向递送scFv-Fc bNA B b到B细胞中而无需离体操作。我们 假设该策略将促进bNAb产生、生物分布和基因的活化/分化， 修饰B细胞以清除HIV-1储库。最后，这些基因修饰疗法的成功取决于 获得足够的基因修饰细胞的全身水平。因此，另一个项目将具体处理 调节基因修饰细胞的水平以达到最大的治疗效果。此外，本发明还提供了一种方法， 如果观察到任何副作用，可以使用相同的试剂快速消除基因修饰的细胞， 提供了一个“安全开关”，从而为整个方法增加了一个安全元件。 Drs.厨房和陈（加州大学洛杉矶分校）将作为双PI。两人在一般干细胞方面都有丰富的经验， 生物学及其在HIV-1疾病中的应用。项目/核心领导人具有HIV-1生物学方面的专业知识， HIV-1疾病的基因治疗方法。专业知识的广度范围从载体和转基因 开发（Chen，An，Morizono，Kitchen，Symonds），HSPC生物学动物模型的开发和使用 （Kitchen，An，Morizono，Chen，Kiem），抗HIV-1免疫功能（Yang，Kitchen），了解HSPC 行为（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).