Innate cellular responses against Adeno-associated virus in hematopoietic stem and progentitor cells influence cell survival and repopulation capacity

造血干细胞和祖细胞(HSPC)的基因编辑有可能治愈许多 血液和免疫系统的遗传性疾病。使用靶向核酸酶，然后递送DNA 使用腺相关病毒载体进行同源定向修复(HDR)的供体模板，我们和 其他人则在多个治疗相关的遗传位点上实现了高效的基因校正。人类 HSPC在体内可以进行高效的纠正和重建血液和免疫系统 移植到免疫受损的NSG小鼠体内。然而，AAV载体的转导会损害细胞 存活、增殖和植入效率。这项提议旨在了解先天免疫细胞 在HSPC的群体和单细胞水平上对AAV载体的应答，以获得高 在不损害HSPC存活或自我更新能力的情况下，有效地进行基因纠正。 尽管HSPC可以经历高效率的HDR，但这些细胞在 与未经处理的对照相比，我们已经确定这是由于AAV载体所致。In剂量 反应实验，高拷贝数转导显示细胞存活率和增殖能力下降。 以及通过集落形成单位实验测量的祖细胞存活率下降，毒性 与数字滴状聚合酶链式反应检测到的AAV基因组核积累增加有关。我们 因此，建议研究两种抗病毒途径，核因子-kB(目标1)和cGAS(目标2)，跨越一系列 AAV拷贝数通过测量这些通路的激活来确定它们对HSPC存活的影响 以及GFP表达载体转导和CFU检测的抑制和基因敲除实验 为了祖细胞的存活。在对祖细胞存活进行初步研究后，我们将确定是否存在暂时性 抑制核因子-kB和cGAS通过植入促进长期造血干细胞的自我更新 在NSG小鼠中。我们将同时使用目标3中的无偏假设生成方法来确定 通过单细胞RNA-SEQ实验在HSPC亚群中发生对AAV的细胞反应 在使用整个种群或大的亚种群的目标1和2实验中可能会遗漏。 我的科学环境和培训计划是成功完成这项研究的理想条件 求婚。斯坦福干细胞研究所有许多才华横溢的干细胞研究人员，他们非常 协作，我们与Irv Weissman博士的RNA-SEQ合作证明了这一点。科学的财富 资源包括我们专门的FACS核心和通过博士后办公室提供的职业资源 事务，以及来自我的导师Porteus博士的科学和职业培训将使我实现我的 科学和职业目标。这项建议的成功完成将加深我们对HSPC的了解 生物学和自我更新在抗病毒反应的背景下，以及改善基因的发展- 在HSPC中编辑基于治疗学。

Gene editing of hematopoietic stem and progenitor cells (HSPCs) has the potential to cure many genetic diseases of the blood and immune system. Using targeted nucleases followed by delivery of a DNA donor template for homology directed repair (HDR) using an adeno-associated viral (AAV) vector, we and others have achieved high efficiency gene correction at multiple therapeutically relevant genetic loci. Human HSPCs can undergo high efficiency correction and reconstitute the blood and immune system in vivo after engraftment into immune-compromised NSG mice. However, transduction by the AAV vector impairs cell survival, proliferation, and engraftment efficiency. This proposal aims to understand the innate immune cellular responses to the AAV vector at the bulk population and single-cell level in HSPCs in order to obtain high efficiency gene correction without impairment of HSPC survival or self-renewal capacity. Although HSPCs can undergo high efficiency HDR, these cells are drastically (4-10 fold) impaired for engraftment compared to untreated controls, which we have determined is due to the AAV vector. In dose response experiments, high copy number transduction demonstrated decreased cell viability and proliferation as well as decreased progenitor cell survival as measured by Colony Forming Unit assay, toxicity which correlates with increased nuclear accumulation of AAV genomes as measured by digital droplet PCR. We therefore propose to investigate two antiviral pathways, NF-kB (Aim 1) and cGAS (Aim 2), across a range of AAV copy numbers to determine their effect on HSPC survival by measuring activation of these pathways as well as inhibition and knock-out experiments using transduction of GFP expressing vectors and CFU assays for progenitor cell survival. After initial studies on progenitor cell survival we will determine whether transient NF-kB and cGAS inhibition improves self-renewal of long-term hematopoietic stem cells through engraftment in NSG mice. We will simultaneously use an unbiased hypothesis generating approach in Aim 3 to determine cellular responses to AAV occurring in HSPC subpopulations through single-cell RNA-seq experiments which may be missed in Aim 1 and 2 experiments using the whole population or large subpopulations. My scientific environment and training plan are ideal for successful completion of this research proposal. The Stanford Stem Cell Institute has many talented stem cell researchers which are highly collaborative as demonstrated by our RNA-seq collaboration with Dr. Irv Weissman. The wealth of scientific resources including our dedicated FACS core and career resources offered through the Office of Post-doctoral Affairs, as well as scientific and career training from my mentor Dr. Porteus will allow me to achieve my scientific and career goals. Successful completion of this proposal will both further our understanding of HSPC biology and self-renewal in the context of antiviral responses, as well as improve the development of gene- editing based therapeutics in HSPCs.