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.

项目摘要/摘要该提案的长期目标是实现高效的基因编辑并最大程度地植入基因工程的抗HIV造血干/祖细胞（HSPC）以实现HIV治疗。 HSPC 基于基因疗法导致植入患者衍生的血液干细胞的抗HIV抗性后代可以提供对艾滋病毒的长期保护，并有可能实现治愈的可能性。但是，以前基于HSPC的基因治疗策略没有提供明显的治疗作用。最近，轻松和多功能性 CRISPR/CAS9介导的基因编辑技术对使用它的编辑引起了极大的兴趣基于HSPC的抗HIV基因治疗策略中的CCR5基因。尽管如此，反HIV成功治愈基于HSPC的基因疗法仍然必须克服2个主要障碍：1）CCR5基因修饰水平不足在HSPC和2）基因修饰的HSPC的植入不良。首先，要最大化CCR5基因的水平在没有与基于DNA的递送平台相关的残留遗传毒性的HSPC中进行的修改，我们开发了一种基于RNA的新型仙台病毒（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以前所未有的效率转导和编辑这些HSPC（提高在CCR5基因座时约80％。其次，为了改善基因修饰的HSPC的植入，我们已经开发了一种新型的体内化学选择策略，采用6-硫代氨酸（6TG），这是一种临床上的前药，需要低黄嘌呤 - 瓜氨酸磷酸糖基转移酶（HPRT）才能进行活性。 HPRT的编辑允许人性化骨中HPRT缺乏HSPC的体内化学选择和移植后的体内化学选择骨髓，肝脏和胸腺移植（HU BLT）小鼠模型。我们假设提供了有效的SEV CRISPR/CAS9在HSPC中介导的CCR5和HPRT基因的编辑，然后使用6TG进行体内选择最大化抗HIV的HSPC的植入。在TOTO中，我们的策略在功能上最大化了Ex Vivo HPSC 基因修饰和体内植入效率，这将不断提供足够数量的HIV- 抗性后代最终可能替代HU BLT小鼠中受感染的细胞的艾滋病毒。为了实现我们的整体目标，并加快基因治疗策略的临床翻译，我们提出以下特定目的：目标1。开发高效CRISPR/CAS9介导的基因的临床相关SEV载体 HSPC的修改。目标2。通过安全有效的预处理和选择策略最大化植入的效率对于CCR5和HPRT修改了HU BLT小鼠的HSPC。 AIM 3。研究HU BLT小鼠中CCR5和HPRT修饰的HSPC的植入HIV抑制。