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

描述(由申请人提供)：针对HIV-1的造血干细胞(HSPC)基因治疗的成功在一定程度上取决于通过治疗性基因修改达到足够的标记水平，以保护免疫系统免受HIV-1疾病的影响。我建议在这里测试这样一个假设，即需要某个阈值水平的基因标记才能有效地用对HIV-1具有抵抗力的免疫系统取代受艾滋病毒破坏的免疫系统。低于这一门槛，免疫系统将得不到保护；高于这一门槛，免疫系统将受到保护，并有效预防艾滋病。所有过去和目前的HSPC临床试验-旨在实现尽可能安全的最高水平的基因标记，预期HIV-1最终将选择受艾滋病毒保护的细胞-正在向前发展，而不了解这一要求。在这里，我建议使用混合骨髓移植小鼠模型来检验这一假设。我将使用我开发的一种灵敏和高通量的克隆定量分析和数学模型来研究在存在和不存在选择压力的情况下HSPC再繁殖和T细胞在这个阈值上下的动态。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首次开发出针对HIV-1共受体C-C趋化因子受体5(CCR5)的短发夹状RNAi用于HIV-1基因治疗的慢病毒载体，并在非人类灵长类动物和人源化小鼠中证明了CCR5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CCR5基因敲除作为保护T细胞免受HIV-1感染的群体，并分析它们的再群体动力学。从这些实验中获得的知识将帮助我们确定高效和安全地选择工程细胞的最佳条件，并创建一个数学模型来指导进一步的工程HSPC移植的实验和临床前试验。由于生物数学的陈博士和Kenneth Lange博士的卓越指导，以及我在工程学和基础生物学方面的研究和教育背景，以及加州大学洛杉矶分校丰富的研究环境，我能够从事这项重要研究，处于非常有利的地位。我的长期职业目标是成为干细胞基因治疗领域的一名独立而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艾滋病毒疾病的临床和临床前研究中的治疗条件和诊断/预后评估。在K99/R00奖项的指导阶段，我计划扩大我在干细胞基因疗法和数学建模方面的研究专长。我将从这次培训中获得的研究经验，以及在独立阶段给予的经济支持，将极大地帮助我追求我的长期职业和研究目标。

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.