In vivo gene editing of CCR5 in bone marrow using improved lentiviral vectors

项目摘要/摘要： 虽然开发高效抗逆转录病毒疗法的研究努力大大延长了患者的健康寿命 HIV-1感染者接受治疗，该病毒对发展构成独特的挑战 长期以来一直在寻求的治疗方法。病毒生命周期的一部分涉及将艾滋病毒基因组永久插入宿主 细胞的遗传物质，建立了一个长期的艾滋病毒感染细胞群体。预防的新战略 必须从这些细胞中扩散出来。与目前可用的抗病毒药物不同，抗艾滋病毒基因疗法 具有治疗甚至治愈HIV-1感染的潜力。基因治疗介导的基因敲除 HIV易感细胞中的HIV共受体CCR5在极大地降低HIV病毒载量方面显示出潜力 两种艾滋病毒疾病的小鼠模型和早期人类临床试验。令人惊讶的是，在罕见的试验参与者中， 即使停止抗逆转录病毒药物治疗，病毒载量的下降也会持续。然而，目前所有的抗艾滋病毒药物 基因治疗策略依赖于对艾滋病毒敏感细胞的分离和体外操作，其次是 将这些新的抗艾滋病毒细胞重新注入患者体内。此方法需要访问高级 细胞培养和骨髓移植设施，目前对大多数艾滋病毒来说昂贵得令人望而却步 居住在艾滋病毒流行继续恶化的地方的感染者。 为了应对这些技术、经济和生物挑战，廉价基因的新方法 必须开发治疗方法。CD34+造血干细胞和祖细胞(HSPC)是一个有吸引力的目标 对于基因治疗，鉴于它们是所有易受艾滋病毒感染的细胞的前身。然而，这些细胞具有 对当前可用的慢病毒载体的基因修改表现出抵抗力。在此介绍的新数据中 应用程序，我展示了在体外向CD34+HSPC传递以前未预见的水平的基因，通过以下方式实现 麻疹病毒血凝素和融合糖蛋白的假分型慢病毒载体。机械论 这项工作的见解指导了水泡性口炎病毒糖蛋白的突变，这种糖蛋白将用于 确定CD34+HSPC的限制因素。在这项应用中，我建议使用这些新型慢病毒载体来 使用人源化小鼠模型评估体内转导作为潜在的抗HIV策略的潜力 艾滋病毒感染的可能性。通过股内直接注射携带CRISPR/Cas9系统靶向的慢病毒载体 CCR5，我将评估这种方法是否能够实现足够高的基因敲除水平，以保护人们免受艾滋病毒感染 挑战。如果成功，这种方法有可能从根本上降低成本和技术难度 抗HIV基因治疗。

Project Summary/Abstract: While research efforts to develop highly-active antiretroviral therapy have greatly extended healthy lifespan for HIV-1 infected individuals receiving treatment, the virus poses unique challenges for the development of a long-sought after cure. Part of the viral lifecycle involves the permanent insertion of the HIV genome into a host cell's genetic material, establishing a long term population of HIV infected cells. New strategies to prevent spread from these cells must be developed. Unlike currently available antiviral drugs, anti-HIV gene therapy holds the potential for the treatment and even cure of HIV-1 infection. The gene therapy-mediated knockout of the HIV co-receptor CCR5 in HIV susceptible cells has shown potential in greatly reducing HIV viral loads in both mouse models of HIV disease and early phase human clinical trials. Surprisingly, in rare trial participants, this drop in viral load is sustained even when antiretroviral drug therapy is halted. However, all current anti-HIV gene therapy strategies rely on the separation and ex vivo manipulation of HIV susceptible cells, followed by re-infusion of these newly HIV-resistant cells back into patients. This approach requires access to advanced cell culture and bone marrow transplant facilities and is currently prohibitively expensive for the majority of HIV infected individuals living where the HIV epidemic continues to worsen. To address these technical, economic, and biological challenges, new approaches to inexpensive gene therapy must be developed. CD34+ hematopoietic stem and progenitor cells (HSPCs) are an attractive target for gene therapy, given their status as predecessors to all cells susceptible to HIV. However, these cells have shown resistance to genetic modification by currently available lentiviral vectors. In new data introduced in this application, I demonstrate previously unforeseen levels of gene delivery to CD34+ HSPCs in vitro, achieved by pseudotyping lentiviral vectors with the measles virus hemagglutinin and fusion glycoproteins. Mechanistic insights from this work has guided mutagenesis of vesicular-stomatitis virus glycoproteins which will be used to identify restriction factors in CD34+ HSPCs. In this application, I propose using these novel lentiviral vectors to evaluate the potential of in vivo transduction as a potential anti-HIV strategy, using a humanized mouse model of HIV infection. By direct intrafemoral injection of lentiviral vectors carrying the CRISPR/Cas9 system targeting CCR5, I will evaluate if this approach can achieve sufficiently high levels of gene knockout to protect from HIV challenge. If successful, this approach holds the potential to radically reduce the cost and technical difficulty of anti-HIV gene therapy.