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

项目摘要/摘要 这项提议的长期目标是实现高效的基因编辑和最大限度地植入 通过基因工程获得抗HIV的造血干/祖细胞(HSPC)，以实现HIV治愈。HSPC 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的基于基因治疗可以 提供针对艾滋病毒的长期保护，并有望实现治愈。但是，以前的 基于HSPC的基因治疗策略没有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的出现激发了人们对使用它来编辑 CCR5基因在HSPC基础上的抗HIV基因治疗策略。尽管如此，一种抗艾滋病毒药物的成功治愈 基于HSPC的基因治疗仍需克服两大障碍：1)CCR5基因修饰水平不足 2)基因修饰的HSPC植入率低。首先，最大化CCR5基因的水平 在没有残留遗传毒性的情况下对HSPC进行修饰，而不是与基于DNA的传递平台相关的遗传毒性 构建含CRISPR/Cas9基因的新型仙台病毒载体 人类HSPC的编辑。我们的SeV-Cas9以前所未有的效率转换和编辑这些HSPC(向上 到CCR5基因座的~80%)。其次，为了提高基因修饰的HSPC的植入率，我们开发了 一种新的体内化学选择策略，使用6-硫代鸟嘌呤(6TG)，这是一种临床上可用的前药， 需要次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HPRT)才能激活。HPRT的编辑允许预先 HPRT缺陷型HSPC在人源骨中的调理及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CCR5和HPRT基因在HSPC中的编辑及6TG Will体内选择 最大限度地植入抗艾滋病毒的HSPC。总而言之，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艾滋病毒- 具有抗药性的后代可能最终取代Hu BLT小鼠中潜伏感染艾滋病毒的细胞。为了实现我们的总体目标 目标，为了加快我们的基因治疗策略的临床转化，我们提出了以下具体目标： 目的1.构建高效表达CRISPR/Cas9基因的临床相关SeV载体 HSPC的改进型。 目标2.通过安全有效的预调节和选择策略最大限度地提高植入物的效率 对于CCR5和HPRT修饰的HSPC在Hu BLT小鼠体内的作用。 目的3.研究CCR5和HPRT修饰的HSPC在Hu BLT小鼠体内对HIV的抑制作用。

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.