Engineering an HIV-resistant immune system for HIV cure

 描述(申请人提供)：候选人Hind Fadel，医学博士，梅奥临床医学院助理教授。她是医学系传染病科和梅奥诊所分子医学系的成员。法德尔博士是一名内科科学家，致力于基础和翻译HIV-1研究和基因治疗。她聚集了一支由导师、合作者和顾问组成的经验丰富的团队，并提出了一项全面的研究、课程工作和职业发展计划，这将使她成为一名成功的独立调查员。梅奥诊所提供的环境非常适合该奖项的研究和职业发展部分的成功。她还维护和确定了梅奥诊所以外的顾问和合作者。职业发展计划将使她能够磨练自己在基因组编辑方面的技能，通过基因工程定位核酸酶，洞察宿主因素在HIV-1生物学中的作用，并开发新的技能来操纵人类基因组，用于HIV-1治疗应用和基因治疗。该研究计划的重点是开发艾滋病毒治愈战略，该战略使用针对/编辑艾滋病毒-1依赖和限制因子基因的新型工程核酸酶，以创造艾滋病毒-1耐药性。这些方法将应用于原代CD34+造血干细胞(HSCs)，并在人源化小鼠身上进行研究。虽然抗逆转录病毒药物使艾滋病毒疾病的治疗成为可能，但它们在许多方面代表着一种“半途而废的技术”。它们使现有患者容易出现重大问题，包括耐药性、持续性免疫功能障碍和免疫衰老、复杂的代谢紊乱和加速衰老现象。第二个问题是，虽然药物治疗理论上可以防止进一步传播，但与终身供应药物组合和保持依从性相关的许多问题意味着疫情继续扩大。一个彻底的解决办法可以解决这两个问题。直到最近，CURE还被认为是未来主义的，但现在情况发生了变化。最近通过骨髓移植治愈了一名同时患有艾滋病毒和白血病的患者，该患者来自一名缺乏主要艾滋病毒细胞辅助因子(进入共同受体CCR5)的捐赠者，这给这一领域带来了活力。这种特殊的方法(异基因骨髓移植)对任何没有癌症的人来说都太有毒了。然而，针对HIV所需的基因(宿主辅因子)的价值已经明确。随着最近非常有前途的基因靶向技术的出现，以及我们对疾病过程本身的理解的进步，这位柏林患者帮助催化使治愈成为该领域的中心优先事项。修改患者自身的HSCs以在HIV/AIDS患者中再生抗HIV的免疫系统，再加上适当的温和调节，可能是一种有效的治疗策略。必须招募和发展年轻的研究人员，以便在这个肯定会是一个重要的、不断扩大的领域开展工作。法德尔博士建议针对HIV-1的两个弱点，她正在开发治愈的基因治疗方法：(I)消除HIV-1所需的宿主依赖因素，以及(Ii)编辑HIV-1进化为逃避的宿主限制因素，以恢复它们限制HV-1的能力。她的中心假设是，工程核酸酶将有效地靶向HIV-1依赖和限制因素，以产生对HIV-1感染的抵抗力，用于治疗应用，并加深我们对这些因素在HIV-1发病机制中的理解。主要目的是：1)在人的CD4+T细胞系中敲除HIV-1依赖因子，同时确定它们的病毒学作用，并确定它们作为治疗靶点的潜力；2)编辑人类限制因子，以恢复HIV-1的耐药性并确定对病毒复制的影响；3)在体外在CD34+HSC细胞中创建依赖因子敲除和限制因子敲入，并在人源化小鼠的临床前动物研究中鉴定它们的特征。这项拟议的研究有可能为多种宿主因素在HIV-1生物学中的作用提供洞察力，并导致治疗HIV-1疗法的翻译应用。开发HSC介导的治疗这种疾病的方法也可以促进其他感染性、自身免疫性和血液病的相关应用。

 DESCRIPTION (provided by applicant): The candidate, Hind Fadel, M.D., Ph.D., is an Assistant Professor at the Mayo Clinic College of Medicine. She is a member of the Division of Infectious Diseases in the Department of Medicine, and of the Department of Molecular Medicine at the Mayo Clinic. Dr. Fadel is a physician-scientist committed to a career in basic and translational HIV-1 research and gene therapy. She has gathered an experienced team of mentors, collaborators and advisors and is proposing a comprehensive research, course work, and career development plan that will position her to be a successful independent investigator. The environment provided by the Mayo Clinic is ideally suited for the success of both the research and career development components of the award. She has also maintained and identified advisors and collaborators outside Mayo Clinic. The career development plan will allow her to hone her skills in genome editing with engineered site-specific nucleases, to gain insights into host factors role in HIV-1 biology, and to develop new skills to manipulate human genome for HIV-1 cure applications and gene therapy. The research plan focuses on developing HIV cure strategies that use targeting/editing of HIV-1 dependency and restriction factor genes with novel engineered nucleases in order to create HIV-1 resistance. The methods will be applied to primary CD34+ hematopoietic stem cells (HSCs) and studied in humanized mice. While antiretroviral drugs have made treatment of HIV disease possible, they in many ways represent a "halfway technology." They leave existing patients vulnerable to major problems that include resistance, persistent immune dysfunction and immunosenescence, complex metabolic disturbances, and accelerated aging phenomena. The second problem is that while drug treatment can in theory prevent further transmission, the many problems associated with supplying the pill combinations life-long and with maintaining adherence have meant that the epidemic continues to expand. A definitive cure could solve both problems. Cure was until recently considered futuristic, but this has now changed. The recent cure of a patient with both HIV and leukemia -- the so-called "Berlin patient" -- by bone marrow transplantation from a donor lacking a main HIV cellular cofactor (the entry co-receptor CCR5) was energizing to the field. The specific approach (allogeneic BMT) is far too toxic for anyone without cancer. However, the value of targeting a gene that HIV needs (a host cofactor) has been made clear. Along with the very recent emergence of highly promising gene targeting technologies, and advances in our understanding of the disease process itself, the Berlin patient helped catalyze making cure a central priority in the field. Modification of a patient's own HSCs to regenerate an HIV- resistant immune system in subjects with HIV/AIDS, coupled with appropriately mild conditioning, could be an effective cure strategy. It is essential to recruit and develop young investigators to populate what is certain to be an important, expanding field. Dr. Fadel is proposing to target two vulnerabilities of HIV-1 as she develops curative gene therapy approaches: (i) eliminating host dependency factors HIV-1 requires, and (ii) editing host restriction factors that HIV-1 has evolved to evade in order to restore their ability to restrict HV-1. Her central hypothesis is that engineered nucleases will efficiently target HIV-1 dependency and restriction factors to generate resistance to HIV-1 infection for therapeutic application and t further our understanding of these factors in HIV-1 pathogenesis. The main aims are: 1) Knockout HIV-1 dependency factors in human CD4+ T-cell lines to simultaneously determine their virological roles and establish their potential for therapeutic targets for HIV cure applications; 2) Edit human restriction factors to restore HIV-1 resistance and determine effects on viral replication; 3) Create dependency factor knockouts and restriction factor knock-ins in CD34+ HSC cells ex-vivo and characterize them in pre-clinical animal studies in humanized mice. The proposed research has the potential to provide insights into the roles of multiple host factors in HIV-1 biology and to lead to translational applications for curative HIV-1 therapies. Developing HSC-mediated therapeutics in this disease can also advance related applications to other infectious, autoimmune and hematologic diseases.