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

非基因毒性调理可增强非人灵长类动物的干细胞工程和病毒特异性免疫力

• 批准号: 10163912
• 负责人: HANS-PETER KIEM
• 金额: $ 59.28万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10163912
• 关键词: Address; Alleles; Allogenic; Animal Model; Animals; Antiviral Agents; Autologous; Autopsy; B-Lymphocytes; Bar Codes; Berlin; Bone Marrow; Boston; Brain; CCR5 gene; Cell Transplantation; Cells; Cellular Immunity; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Repair Pathway; Data; Development; Disease remission; Dose; Engraftment; Flow Cytometry; Gene-Modified; Genes; Genetic; Genetic Engineering; Goals; HIV; HIV Infections; Hematopoietic stem cells; Humoral Immunities; Immune; Immune response; Immune system; Immunity; Immunohistochemistry; Impairment; Infection; Infusion procedures; Kinetics; Letters; Life; London; Measures; Methodology; Methods; Mississippi; Modeling; Mutate; Nonhomologous DNA End Joining; Oligonucleotides; Patients; Pharmaceutical Preparations; Phenotype; Population; Property; Protocols documentation; Regimen; Resistance; Rosaniline Dyes; Sentinel; Site; Source; Stem cell transplant; T-Lymphocyte; Therapeutic; Time; Tissues; Transgenes; Viral reservoir; Viremia; Virus; Virus Activation; Virus Diseases; Virus Latency; Whole-Body Irradiation; Work; antiretroviral therapy; base; cell type; chimeric antigen receptor; conditioning; cryogel; deep sequencing; design; engineered stem cells; experience; experimental study; gene therapy; hematopoietic differentiation; improved; in vivo; inhibitor/antagonist; interest; lymph nodes; neutralizing antibody; news; nonhuman primate; novel; peripheral blood; preference; repaired; resistance gene; response; scaffold; simian human immunodeficiency virus; stem cell expansion; stem cells; therapeutic transgene; trafficking

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.

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，我们将优化我们的方法来插入CD 4 CAR或eCD 4-IG，并安全地植入一个 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。