Preclinical development of gene-edited human hematopoietic stem cells

这个合作项目的总体目标是为 HIV感染的功能性治疗，患者的CD4+T细胞对 艾滋病毒感染(“防御”)，同时应用策略消除病毒库 (“销毁”)。这一策略的“防御”部分是以“柏林病人”为基础的 在接受无效的捐赠者的骨髓移植后治愈了艾滋病毒感染 CCR5基因突变，编码HIV感染所必需的辅助受体。虽然 从理论上讲，CCR5缺失的造血干细胞移植是一种有吸引力的方法 HIV的治疗策略，在实践中从CCR5-空的异基因骨髓移植 由于捐赠者有限，捐赠者不是治疗普通人群艾滋病毒的可行策略 可获得性和与该程序相关的风险，特别是移植物对宿主疾病。这里, 我们建议利用CRISPR-Cas9基因组编辑系统的强大功能来引入空 患者自身造血干细胞(HSCs)中CCR5的突变，从而允许 自体移植和HIV的功能性治愈，没有许多与 同种异体移植。提出的战略能否成功取决于最近的 发现的CRISPR-Cas9基因组编辑系统，它是一个高度可定制的系统，具有 在高百分比的目标中突变CCR5的两个拷贝所需的目标效率 细胞数量。作为全面翻译《保卫与毁灭》临床的第一步 战略，CRISPR治疗有限公司，这一申请的私营部门合作伙伴， 是否会在学术实验室制定中断HSC中CCR5的过渡策略(请参见 项目1)通过执行两个具体目标面向临床应用：1) 一种符合适用条件的人造血干细胞基因修饰方法的开发 以细胞为基础的医药产品管理条例；以及2)药理分析， 转基因肝星状细胞的生物分布、毒理学和非靶点效应。这个 这两个目标的实现将使这项技术为首次人体临床试验做好准备， 并将为艾滋病毒的长期功能性治愈提供基础。更广泛地说，这些研究 将建立一个基本的诊所就绪平台，总体战略的其他要素将在此基础上 如无毒调理方案(项目3)和加强HSC归巢或 可以增加对病毒库的破坏(项目1和2)。更广泛地说，这些方法 这里开发的用于在造血干细胞中进行符合GMP的基因编辑将得到广泛的应用 适用于各种遗传性血液疾病。

The overall goal of this collaborative project is to develop a “Defend and Destroy” strategy for the functional cure of HIV infection in which a patient's CD4+ T cells are rendered resistant to HIV infection (“Defend”) while simultaneously strategies are applied to eliminate viral reservoirs (“Destroy”). The “Defend” portion of this strategy is based upon the “Berlin Patient,” who was cured of HIV infection upon receipt of a bone marrow transplant from a donor harboring a null mutation in the CCR5 gene, which encodes a co-receptor essential for HIV infection. Although transplantation with CCR5 null hematopoietic stem cells (HSCs) is theoretically an attractive treatment strategy for HIV, in practice allogeneic bone marrow transplants from CCR5-null donors are not a viable strategy for treating HIV in the general population due to limited donor availability and the risks associated with the procedure, particularly graft vs. host disease. Here, we propose to harness the power of the CRISPR-Cas9 genome editing system to introduce null mutations in CCR5 in a patient's own hematopoietic stem cells (HSCs), thereby allowing for an autologous transplant and a functional cure of HIV without many of the risks associated with an allogeneic transplant. The success of the proposed strategy hinges upon the recently discovered CRISPR-Cas9 genome editing system, which is a highly customizable and has the on-target efficiency needed to mutate both copies of CCR5 in a high percentage of the target cell population. As the first step towards full clinical translation of the “Defend and Destroy” strategy, the company CRISPR Therapeutics Ltd, the private-sector partner in this application, will transition strategies for disrupting CCR5 in HSCs developed in the academic laboratory (see Project 1) towards clinical application through the execution of two specific aims: 1) Development of a process for genetically modifying human HSCs compliant with applicable regulations governing cell-based medicinal products; and 2) Analysis of pharmacology, biodistribution, toxicology and off-target effects of genetically modified HSCs. The accomplishment of these two aims will ready this technology for a first-in-humans clinical trial, and will provide the basis for a long-term functional cure of HIV. More broadly, these studies will establish a basic clinic-ready platform upon which other elements of the overall strategy such as non-toxic conditioning regimens (Project 3) and strategies to enhance HSC homing or destruction of viral reservoirs (Projects 1 and 2) can be added. More broadly, the methods developed here for conducting GMP-compliant gene editing in HSCs will have widespread applicability to a variety of genetic diseases of the blood.