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

项目概要/摘要 该提案的长期目标是实现高效的基因编辑，并最大限度地增加基因的植入。 基因工程抗HIV造血干/祖细胞（HSPC），以实现HIV治愈。HSPC 基于基因治疗的从移植的患者来源的造血干细胞产生抗HIV后代的方法， 提供长期的艾滋病毒防护，并有可能治愈。但此前的 基于HSPC的基因治疗策略未提供明确的治疗效果。最近， CRISPR/Cas9介导的基因编辑技术引起了人们对使用它来编辑 CCR 5基因在基于HSPC的抗HIV基因治疗策略中的应用尽管如此，一种抗艾滋病毒药物的成功治愈 基于HSPC的基因治疗仍然必须克服2个主要障碍：1）CCR 5基因修饰水平不足 2）基因修饰的HSPC移植不良。首先，最大化CCR 5基因的水平 在HSPC中进行修饰，而没有与基于DNA的递送平台相关的残留遗传毒性，我们 开发了一种新型的基于RNA的仙台病毒（SeV）载体，用于高效CRISPR/Cas9介导的基因 编辑人类HSPC。我们的SeV-Cas9以前所未有的效率转导和编辑这些HSPC（高达2000倍）。 在CCR 5基因座处至约80%）。其次，为了改善基因修饰的HSPCs的植入，我们开发了 一种新的体内化学选择策略，其采用6-硫代鸟嘌呤（6 TG），6-硫代鸟嘌呤是一种临床可用的前药， 需要次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）的活性。HPRT的编辑允许预- 人源化骨中HPRT缺陷型HSPC的调节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提供了 CRISPR/Cas9介导的HSPC中CCR 5和HPRT基因的编辑，随后用6 TG进行体内选择， 最大化HIV抗性HSPC的植入。总的来说，我们的策略在功能上最大化了体外HPSC 基因修饰和体内植入效率，这将持续提供足够数量的HIV- 最终可能取代hu BLT小鼠中HIV潜伏感染细胞的抗性后代。为了实现我们的整体 为了实现这一目标，并加快我们基因治疗策略的临床转化，我们提出了以下具体目标： AIM 1.开发用于高效CRISPR/Cas9介导基因的临床相关SeV载体 HSPC的改进。 AIM 2.通过安全有效的预处理和选择策略最大限度地提高植活效率 对于在hu BLT小鼠中CCR 5和HPRT修饰的HSPC。 AIM 3.通过在hu BLT小鼠中植入CCR 5和HPRT修饰的HSPC研究HIV抑制。

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.