Nongenotoxic conditioning to enhance stem cell engineering and virus-specific immunity in nonhuman primates

摘要 虽然造血干细胞和祖细胞（HSPC）移植现在是两个HIV临床病例的基础， 1缓解/功能性治愈，将这种方法应用于更广泛的患者的手段尚未被 鉴定在本项目中，我们将解决基于HSPC的抗HIV策略的关键限制： 和基因编辑的HSPC及其后代的效力。尽管我们之前的研究结果表明， 编辑的HSPC在体内长期移植，只有有限的比例随着时间的推移而持续，并且不能移植。 支持无抗逆转录病毒疗法（ART）的病毒缓解。为了解决这个问题，我们i）采用了一种更 编辑我们感兴趣的基因座CCR 5的先进策略，ii）确定了一种不仅可以破坏CCR 5的方法， 基因，但同时插入治疗性抗HIV转基因，和iii）设计实验来评估这一点 策略在我们强大的非人灵长类动物（NHP）模型抑制HIV感染。我们将理性地瞄准两个 针对基因编辑的CCR 5基因座设计的高效抗HIV转基因：病毒特异性嵌合抗原 受体CD 4 CAR和广泛中和抗体样分子eCD 4-IG。我们的初步数据 证明我们有能力在原发灶中高达50%的靶向CCR 5等位基因处插入定义的遗传序列， NHP HSPC。在这里，我们将优化插入CD 4 CAR或eCD 4-IG的方法，并安全地植入 将含有CD 4 CAR-β CCR 5和eCD 4-IG β CCR 5 HSPC的自体HSPC产物植入同一动物。 当我们引入两种治疗性转基因并同时破坏CCR 5辅助受体时，我们参考 这是一个“三为一”的方法。除了产生有效和有效修饰的HSPC产物之外， 我们将与U19联盟的每个项目密切合作。我们将与Project 3/Cannon协调， 在我们各自的动物模型中进行体内研究之前，最有效的方法来修饰HSPC。与项目 1/Scadden，我们将评估一种骨髓冷冻凝胶（BMC）支架，该支架旨在增强骨髓基质细胞的分化， HSPC衍生的T细胞，即CD 4CAR + CCR 5 T细胞。最后，我们将研究更安全， 以项目2/洋红为特征的非遗传毒性预处理（NGC）方案用于猴/人感染 免疫缺陷病毒（SHIV）和ART抑制。我们认为， 基因修饰的病毒特异性HSPC及其后代将能够对新生SHIV产生强大的保护作用 挑战，并显着影响感染，抑制动物的病毒水库。

ABSTRACT Although hematopoietic stem and progenitor cell (HSPC) transplantation now underlies two clinical cases of HIV- 1 remission/functional cure, a means to apply this approach to a wider array of patients has not yet been identified. In this project, we will address a key limitation for HSPC-based anti-HIV strategies: the engraftment and potency of gene-edited HSPC and their progeny. Although our previous findings demonstrate that gene edited HSPCs engraft long-term in vivo, only a limited proportion persist over time, and are incapable of supporting antiretroviral therapy (ART)-free virus remission. To address this, we have i) adapted a more advanced strategy to edit our locus of interest, CCR5, ii) identified an approach to not only disrupt the CCR5 gene, but simultaneously insert therapeutic anti-HIV transgenes, and iii) designed experiments to evaluate this strategy in our robust nonhuman primate (NHP) model of suppressed HIV infection. We will target two rationally designed, highly potent anti-HIV transgenes to the gene-edited CCR5 locus: the virus-specific chimeric antigen receptor CD4CAR, and the broadly neutralizing antibody-like molecule eCD4-Ig. Our preliminary data demonstrate our ability to insert defined genetic sequences at up to 50% of targeted CCR5 alleles in primary NHP HSPCs. Here, we will optimize our approach to insert CD4CAR or eCD4-Ig, and safely engraft an autologous HSPC product containing both CD4CAR∆CCR5 and eCD4-Ig∆CCR5 HSPCs into the same animal. As we are introducing two therapeutic transgenes and simultaneously disrupting the CCR5 coreceptor, we refer to this as a “three for one” approach. In addition to generating a potent and efficiently modified HSPC product, we will work closely with each project in our U19 consortium. We will coordinate with Project 3/Cannon to identify the most efficient means to modify HSPCs, prior to in vivo studies in our respective animal models. With Project 1/Scadden, we will evaluate a bone marrow cryogel (BMC) scaffold designed to enhance the differentiation of HSPC-derived T-cells, namely CD4CAR∆CCR5 T-cells. Finally, we will investigate the impact of safer, nongenotoxic conditioning (NGC) regimens characterized by Project 2/Magenta on infection with simian/human immunodeficiency virus (SHIV) and suppression by ART. We believe that safe and efficacious engraftment of gene-modified, virus-specific HSPCs and their progeny will enable robust protection against de novo SHIV challenge, and significantly impact viral reservoirs in infected, suppressed animals.