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

使用改进的慢病毒载体对骨髓中的 CCR5 进行体内基因编辑

• 批准号: 10029620
• 负责人: Stosh Ozog
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-11 至 2021-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10029620
• 关键词: AIDS prevention; Address; Anti-Retroviral Agents; Antiviral Agents; Autoimmune Diseases; Back; Binding; Biochemical; Biological; Bone Marrow; Bone Marrow Transplantation; Bypass; CCR5 gene; CD34 gene; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Culture Techniques; Cell physiology; Cells; Chimeric Proteins; Clinical Trials; Communicable Diseases; Confocal Microscopy; Culture Techniques; Data; Defect; Development; Disease; Drops; Economics; Epidemic; Gene Delivery; Gene-Modified; Genes; Genetic; Genetic Materials; Glycoproteins; HIV; HIV Genome; HIV Infections; HIV resistance; HIV-1; Head; Hemagglutinin; Hematologic Neoplasms; Hematological Disease; Hematopoietic System; Hematopoietic stem cells; Highly Active Antiretroviral Therapy; Human; In Situ; In Vitro; Individual; Infection; Infusion procedures; Injections; Investigation; Knock-out; Lentivirus Vector; Longevity; Measles; Measles virus; Mediating; Membrane; Mendelian disorder; Methods; Modification; Mus; Mutagenesis; Participant; Patients; Pharmacotherapy; Phase; Phase I/II Clinical Trial; Phenotype; Population; Price; Proteins; Protocols documentation; Research; Resistance; Resources; Reverse Transcription; Site; Stem cell transplant; System; Techniques; Therapeutic; Translating; Vesicular stomatitis Indiana virus; Viral; Viral Load result; Viral Vector; Virus; Work; cell type; cellular transduction; combat; cost; experimental study; gene correction; gene therapy; genome editing; hematopoietic gene; humanized mouse; improved; in vivo; insight; knockout gene; mouse model; novel; novel strategies; prevent; reconstitution; small molecule; stem cells; success; tool; trafficking; vector

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.

项目摘要/摘要： 虽然开发高活动性抗逆转录病毒疗法的研究工作大大延长了健康的寿命 HIV-1感染了接受治疗的人，该病毒为发展带来了独特的挑战 长期治愈。病毒生命周期的一部分涉及将HIV基因组永久插入宿主 细胞的遗传物质，建立了长期的HIV感染细胞。防止的新策略 必须从这些细胞中扩散。与目前可用的抗病毒药物不同，抗HIV基因治疗 具有治疗甚至HIV-1感染的潜力。基因治疗介导的敲除 HIV易感细胞中的HIV共受体CCR5在大大降低HIV病毒载荷的潜力 HIV疾病的小鼠模型和早期人类临床试验。令人惊讶的是，在罕见的审判参与者中， 即使抗逆转录病毒药物治疗停止，病毒负荷下降也会持续。但是，所有当前的抗HIV 基因治疗策略依赖于HIV易感细胞的分离和离体操纵，其次是 将这些新的抗HIV细胞再浸入患者中。这种方法需要访问高级 细胞培养和骨髓移植设施，目前大多数HIV的昂贵 居住在艾滋病毒流行的地方继续恶化的感染者。 为了解决这些技术，经济和生物学挑战，新的方法廉价基因 必须开发治疗。 CD34+造血茎和祖细胞（HSPC）是一个有吸引力的靶标 对于基因治疗，鉴于它们作为所有容易受到艾滋病毒的细胞的前身。但是，这些细胞具有 显示了当前可用的慢病毒载体对遗传修饰的抗性。在此引入的新数据中 应用，我证明了先前无法预料的基因递送到CD34+ HSPC的体外，通过 与麻疹病毒血凝素和融合糖蛋白的假病毒载体的拟元分型载体。机理 这项工作的洞察力指导了囊泡炎性炎病毒糖蛋白的诱变，该诱变将用于 确定CD34+ HSPC中的限制因子。在此应用中，我建议使用这些新颖的慢病毒向量 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。通过直接携带CRISPR/CAS9系统的慢病毒向量的直接注射 CCR5，我将评估这种方法是否可以实现足够高的基因敲除以保护艾滋病毒 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。