Efficient Sendai virus mediated CRISPR/Cas9 gene editing to protect hematopoietic stem cells from HIV

高效仙台病毒介导的 CRISPR/Cas9 基因编辑保护造血干细胞免受 HIV 感染

基本信息

批准号：
10402835
负责人：
Dong Sung An
金额：
$ 61.66万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-06-11 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10402835
关键词：
6-Mercaptopurine Alleles BLT mice Berlin Biological Bone Marrow Purging Bone Marrow Transplantation CCR5 gene CD34 gene CRISPR/Cas technology Cells Chemicals Chimeric Proteins Clinical Complex DNA Defect Disease Engraftment Fetal Liver Frequencies Gene Transduction Agent Gene-Modified Generations Genes Genetic Engineering Goals HIV HIV resistance Hematopoiesis Hematopoietic Hematopoietic stem cells Human Hypoxanthine Phosphoribosyltransferase Immune system Individual Knock-out Long-Term Effects Mediating Modification Mus National Institute of Allergy and Infectious Disease Patients Pharmaceutical Preparations Prodrugs Protocols documentation RNA Research Residual state Safety Sendai virus Speed T-Lymphocyte Technology Temperature Therapeutic Effect Thioguanine Transfection Transplantation United States National Institutes of Health base clinical translation clinically relevant falls gene therapy genome editing genotoxicity improved in vitro Assay in vivo in vivo engraftment insertion/deletion mutation interest mouse model novel peripheral blood plasmid DNA post-transplant preconditioning procedure safety programs protein complex reconstitution stem cells success vector

项目摘要

Project summary/abstract The long-term objective of this proposal is to achieve highly efficient gene editing and maximize engraftment of genetically engineered HIV-resistant hematopoietic stem/progenitor cells (HSPCs) to achieve HIV cure. HSPC based gene therapy that results in HIV-resistant progenies from engrafted patient derived blood stem cells can provide long-term protection against HIV with the promising possibility of achieving a cure. However, previous HSPC based gene therapy strategies did not provide clear therapeutic effects. Recently, the ease and versatility of the CRISPR/Cas9-mediated gene editing technology has spurred an immense interest in using it to edit the CCR5 gene in HSPC based anti-HIV gene therapy strategies. Nonetheless, a successful cure by an anti-HIV HSPC based gene therapy still has to overcome 2 major barriers: 1) insufficient levels of CCR5 gene modification in HSPC and 2) poor engraftment of gene modified HSPCs. Firstly, to maximize the levels of CCR5 gene modification in HSPCs without the residual genotoxicity associated with DNA based delivery platforms, we developed a novel RNA-based Sendai virus (SeV) vector for highly efficient CRISPR/Cas9-mediated gene editing of human HSPC. Our SeV-Cas9 transduces and edits these HSPCs with unprecedented efficiency (up to ~80% at the CCR5 locus). Secondly, to improve the engraftment of gene modified HSPCs, we have developed a novel in vivo chemoselection strategy that employs 6-thioguanine (6TG), a clinically available prodrug that requires hypoxanthine-guanine phosphoribosyl-transferase (HPRT) for activity. Editing of HPRT allows for pre- conditioning and post-transplant in vivo chemoselection of HPRT-deficient HSPC in the humanized bone marrow, liver and thymus transplanted (hu BLT) mouse model. We hypothesize that efficient SeV delivered CRISPR/Cas9 mediated editing of CCR5 and HPRT genes in HSPC followed by in vivo selection with 6TG will maximize the engraftment of HIV-resistant HSPC. In toto, our strategy functionally maximizes ex vivo HPSC gene modification and in vivo engraftment efficiency, which will continuously provide sufficient numbers of HIV- resistant progenies that might ultimately replace HIV latently infected cells in hu BLT mice. To achieve our overall goal, and to speed the clinical translation of our gene therapy strategy, we propose the following Specific Aims: AIM 1. Develop a clinically relevant SeV vector for highly efficient CRISPR/Cas9 mediated gene modification of HSPC. AIM 2. Maximize the efficiency of engraftment by a safe and effective pre-conditioning and selection strategy for CCR5 and HPRT modified HSPC in hu BLT mice. AIM 3. Investigate HIV inhibition by engraftment of CCR5 and HPRT modified HSPC in hu BLT mice.

项目概要/摘要该提案的长期目标是实现高效的基因编辑并最大限度地植入基因工程抗艾滋病毒造血干/祖细胞（HSPC）以实现艾滋病毒治愈。 HSPC 基于基因疗法，从移植的患者来源的血液干细胞中产生抗艾滋病毒的后代，可以提供针对艾滋病毒的长期保护，并有望实现治愈。然而，之前的基于 HSPC 的基因治疗策略并未提供明确的治疗效果。最近，易用性和多功能性 CRISPR/Cas9 介导的基因编辑技术激起了人们对使用它来编辑基因的巨大兴趣。基于 HSPC 的抗 HIV 基因治疗策略中的 CCR5 基因。尽管如此，抗艾滋病毒的成功治愈基于HSPC的基因治疗仍需克服2大障碍：1）CCR5基因修饰水平不足 HSPC 中的缺陷和 2) 基因修饰 HSPC 的植入不良。首先，最大化CCR5基因水平对 HSPC 进行修饰，而不会产生与基于 DNA 的递送平台相关的残留遗传毒性，我们开发了一种新型基于 RNA 的仙台病毒 (SeV) 载体，用于高效 CRISPR/Cas9 介导的基因人类 HSPC 的编辑。我们的 SeV-Cas9 以前所未有的效率（高达 CCR5 位点达到约 80%）。其次，为了提高基因修饰 HSPC 的植入，我们开发了一种新颖的体内化学选择策略，采用 6-硫鸟嘌呤 (6TG)，一种临床可用的前药，需要次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HPRT) 才能发挥活性。 HPRT 的编辑允许预人源化骨中 HPRT 缺陷 HSPC 的调理和移植后体内化学选择骨髓、肝脏和胸腺移植（hu BLT）小鼠模型。我们假设高效的 SeV 交付 CRISPR/Cas9 介导的 HSPC 中 CCR5 和 HPRT 基因的编辑，然后使用 6TG 进行体内选择最大限度地提高抗 HIV HSPC 的植入。总的来说，我们的策略在功能上最大化了离体 HPSC 基因修饰和体内植入效率，将持续提供足够数量的HIV- 抗性后代可能最终取代 hu BLT 小鼠体内 HIV 潜伏感染的细胞。为了实现我们的整体目标，并加速我们的基因治疗策略的临床转化，我们提出以下具体目标：目的 1. 开发临床相关的 SeV 载体，用于高效 CRISPR/Cas9 介导的基因 HSPC 的修饰。目标 2. 通过安全有效的预处理和选择策略最大限度地提高植入效率对于 hu BLT 小鼠中的 CCR5 和 HPRT 修饰的 HSPC。目标 3. 通过在 hu BLT 小鼠中植入 CCR5 和 HPRT 修饰的 HSPC 来研究 HIV 抑制作用。