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 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 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。