Defining a therapeutic threshold in HSPC gene therapy for HIV-1 disease

描述（由申请人提供）：造血干祖细胞（HSPC）基因治疗HIV-1的成功部分取决于治疗性基因修饰达到足够水平的标记，以保护免疫系统免受HIV-1疾病的影响。我建议在这里测试的假设，需要一定的阈值水平的基因标记，以有效地取代艾滋病毒破坏的免疫系统与一个是耐HIV-1。低于这个阈值，免疫系统将得不到保护;高于这个阈值，免疫系统将得到保护，有效地预防艾滋病。所有过去和当前的HSPC临床试验-旨在实现尽可能高的基因标记水平，预计HIV-1将最终选择HIV保护的细胞-正在向前推进，而不了解这一要求。在此，我建议使用混合骨髓移植小鼠模型来检验这一假设。我将使用我开发的灵敏和高通量克隆定量测定和数学建模来研究在存在和不存在选择压力的情况下高于和低于该阈值的HSPC再增殖和T细胞动力学。加州大学洛杉矶分校艾滋病研究所所长、我的主要导师Irvin Chen博士和他的研究小组是第一个利用针对HIV-1辅助受体C-C趋化因子受体5型（CCR 5）的短发夹RNAi开发HIV-1基因治疗慢病毒载体的人，并且已经证明在非人灵长类动物和人源化小鼠中CCR 5的长期稳定减少。当我们准备将这种shRNA引入人类HSPC基因治疗时，我将使用CCR 5敲低作为受HIV-1感染保护的T细胞群体，并分析它们的再增殖动力学。从这些实验中获得的知识将帮助我们确定有效和安全选择工程化细胞的最佳条件，并创建可以指导工程化HSPC移植的进一步实验和临床前试验的数学模型。我在一个很好的位置，以追求这一重要的研究感谢陈博士和博士的上级指导生物数学肯尼斯兰格;我的研究和教育背景，在工程和基础生物学;和加州大学洛杉矶分校的丰富的研究环境。我的长期职业目标是成为干细胞基因治疗领域的独立和专业的翻译研究科学家。我目前的研究目标是帮助引入定量评估的实践，以更好地了解和改善治疗条件和诊断/预后评估在临床和临床前研究的艾滋病毒疾病。在K99/R 00奖的指导阶段，我计划扩大我在干细胞基因治疗和数学建模方面的研究专长。我将从这次培训机会中获得的研究经验，沿着独立阶段获得的财政支持，将大大帮助我追求我的长期职业和研究目标。 公共卫生相关性：基于HSPC的基因治疗是抗HIV药物治疗的创新替代方案，具有根除患者体内HIV并最终治愈艾滋病的潜力。使用高通量测试系统和动物模型，拟议的研究将调查在没有药物治疗的情况下有效保护身体免受艾滋病感染所需的HIV保护细胞的最低水平。从这项研究中获得的知识将有助于优化治疗条件和规划未来的临床试验。

DESCRIPTION (provided by applicant): The success of hematopoietic stem progenitor cell (HSPC) genetic therapy for HIV-1 is dependent in part upon achieving sufficient levels of marking with therapeutic gene modification to protect the immune system from HIV-1 disease. I propose here to test the hypothesis that a certain threshold level of gene marking is required to effectively replace the HIV-damaged immune system with one that is resistant to HIV-1. Below this threshold, the immune system would not be protected; above the threshold, the immune system would be protected and effectively prevent AIDS. All past and current HSPC clinical trials - which are designed to achieve as high a level of gene marking as is safely possible, anticipating that HIV-1 will ultimately select for the HIV-protected cells - are moving forward without an understanding of this requirement. Herein, I propose to test the hypothesis using mixed bone marrow transplant murine models. I will use a sensitive and high- throughput clonal quantification assay I developed and mathematical modeling to investigate HSPC repopulation and T-cell dynamics above and below this threshold in the presence and absence of selection pressure. Dr. Irvin Chen, Director of the UCLA AIDS Institute and my primary mentor, and his research group were the first to develop lentivirus vectors for HIV-1 gene therapy using short hairpin RNAi against the HIV-1 co-receptor, C-C chemokine receptor type 5 (CCR5), and have demonstrated long-term and stable reduction of CCR5 in both non-human primates and humanized mice. As we prepare to introduce this shRNA into HSPC gene therapy for humans, I will use CCR5 knockdown as the population of T-cells protected from HIV-1 infection and analyze their repopulation kinetics. Knowledge gained from these experiments will help us to determine optimal conditions for efficient and safe selection of engineered cells and to create a mathematical model that can guide further experiments and preclinical trials of engineered HSPC transplantation. I am in an excellent position to pursue this important research thanks to the superior mentoring of Dr. Chen and Dr. Kenneth Lange of Biomathematics; my research and educational backgrounds in both engineering and basic biology; and UCLA's rich research environment. My long-term career goal is to become an independent and specialized translational research scientist in the stem cell gene therapy field. My current research goal is t help introduce the practice of quantitative assessment to better understand and improve treatment conditions and diagnostic/prognostic evaluation in both clinical and preclinical studies of HIV diseases. During the mentored phase of the K99/R00 award, I plan to expand my research expertise in both stem cell gene therapy and mathematical modeling. The research experience I will gain from this training opportunity, along with the financial support granted in the independent phase, will significantly help me to pursue my long-term career and research goals. PUBLIC HEALTH RELEVANCE: HSPC-based gene therapy, an innovative alternative to anti-HIV drug therapy, has the potential to eradicate HIV in patients and ultimately to cure AIDS. Using a high-throughput testing system and animal models, the proposed study will investigate the minimum level of HIV-protected cells required to efficiently protect the body from AIDS without drug therapy. Knowledge gained from this study will be useful in optimizing treatment conditions and planning future clinical trials.