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.

项目概要/摘要： 虽然开发高效抗逆转录病毒疗法的研究工作大大延长了老年人的健康寿命， HIV-1感染者接受治疗，该病毒对开发一种 长期寻求治愈。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，建立一个长期的人口艾滋病毒感染的细胞。预防的新战略 从这些细胞中扩散出来的病毒必须被开发出来。与目前可用的抗病毒药物不同， 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CCR 5显示出极大降低HIV病毒载量的潜力， 艾滋病小鼠模型和早期人类临床试验。令人惊讶的是，在少数试验参与者中， 这种病毒载量的下降即使在抗逆转录病毒药物治疗停止时也会持续。然而，目前所有的抗艾滋病毒药物 基因治疗策略依赖于HIV易感细胞的分离和离体操作， 将这些新的抗艾滋病毒细胞重新输回患者体内。这种方法需要访问先进的 细胞培养和骨髓移植设施，目前对大多数艾滋病毒感染者来说， 生活在艾滋病毒流行继续恶化的地方的受感染者。 为了应对这些技术、经济和生物学挑战， 治疗必须发展。CD 34+造血干细胞和祖细胞（HSPC）是一个有吸引力的目标 因为它们是所有易感染艾滋病毒的细胞的前身。然而，这些细胞具有 显示出对目前可用的慢病毒载体的遗传修饰的抗性。在新的数据中， 应用中，我证明了以前无法预见的水平的基因递送到CD 34 + HSPC体外，实现了 用麻疹病毒血凝素和融合糖蛋白假型化慢病毒载体。机械论 从这项工作中获得的见解指导了水泡性口炎病毒糖蛋白的诱变， 鉴定CD 34 + HSPC中的限制性因子。在本申请中，我建议使用这些新的慢病毒载体， 使用人源化小鼠模型评估体内转导作为潜在抗HIV策略的潜力 艾滋病毒感染。通过直接股内注射携带CRISPR/Cas9系统靶向的慢病毒载体， CCR 5，我将评估这种方法是否可以达到足够高的基因敲除水平，以保护免受艾滋病毒感染 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