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

摘要 尽管造血干细胞和祖细胞(HSPC)移植现在是两个艾滋病毒临床病例的基础- 1缓解/功能治愈，将这种方法应用于更广泛的患者的手段尚未被 已确认身份。在这个项目中，我们将解决基于HSPC的抗HIV策略的一个关键限制：嫁接 和基因编辑的HSPC及其后代的效力。尽管我们之前的发现证明了基因 编辑的HSPC在体内长期植入，只有有限的比例随着时间的推移而存留，并且不能 支持无抗逆转录病毒疗法(ART)的病毒缓解。为了解决这一问题，我们已经i)采用了更多 编辑我们感兴趣的轨迹的高级战略，CCR5，II)确定了一种方法，不仅可以扰乱CCR5 基因，但同时插入治疗性抗艾滋病毒转基因，以及iii)设计实验来评估这一点 在我们抑制HIV感染的非人类灵长类动物(NHP)模型中的策略。我们将理性地将两个目标 针对经基因编辑的CCR5基因座设计的高效抗HIV转基因：病毒特异性嵌合抗原 受体CD4CAR和广谱中和抗体样分子eCD4-Ig。我们的初步数据 展示我们有能力在初级CCR5等位基因中插入高达50%的已定义遗传序列 NHP HSPC。在这里，我们将优化我们的方法来注入CD4CAR或eCD4-Ig，并安全地植入 含有CD4CAR∆CCR5和eCD4-Ig∆CCR5 HSPC的自体HSPC产品。 由于我们正在引入两个治疗性转基因并同时破坏CCR5辅助受体，我们参考 这是一种“三合一”的做法。除了产生有效且高效地修改的HSPC产品之外， 我们将与我们U19联合体中的每个项目密切合作。我们将与Project 3/Cannon协调以确定 在我们各自的动物模型进行活体研究之前，修改HSPC是最有效的手段。使用项目 1/斯卡登，我们将评估一种旨在增强骨髓冷冻剂(BMC)分化的支架 来源于HSPC的T细胞，即CD4CAR∆CCR5T细胞。最后，我们将调查SAFER的影响， 以Project 2/Magenta为特征的非遗传毒性调节(NGC)方案治疗猴/人感染 免疫缺陷病毒(SIV)和ART的抑制。我们相信，安全有效的植入术 基因修饰的、病毒特异性的HSPC及其后代将实现对新城疫病毒的强大保护 挑战，并对受感染、受抑制的动物的病毒库产生重大影响。

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.