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疾病。 血液和免疫系统的遗传性疾病。使用靶向核酸酶，随后递送DNA 使用腺相关病毒（AAV）载体的同源定向修复（HDR）的供体模板，我们和 其他人已经在多个治疗相关的遗传基因座上实现了高效的基因校正。人类 HSPCs可以进行高效的校正，并在体内重建血液和免疫系统， 移植到免疫受损的NSG小鼠中。然而，通过AAV载体的转导损害了细胞的功能。 存活、增殖和植入效率。这项提案旨在了解先天免疫细胞 在HSPC中在大量群体和单细胞水平上对AAV载体的应答，以获得高表达。 有效的基因纠正，而不损害HSPC的生存或自我更新能力。 尽管HSPC可以经历高效HDR，但是这些细胞对于HDR的表达是显著受损的（4-10倍）。 与未处理的对照相比，移植物的存活率降低，我们已经确定这是由于AAV载体。剂量 反应实验中，高拷贝数转导表现出降低的细胞活力和增殖 以及通过集落形成单位测定测量的祖细胞存活减少， 与通过数字液滴PCR测量的AAV基因组的增加的核积累相关。我们 因此，我们建议研究两种抗病毒途径，NF-κ B（Aim 1）和cGAS（Aim 2），在一系列的抗病毒药物中， AAV拷贝数，以通过测量这些途径的激活来确定其对HSPC存活的影响， 以及使用GFP表达载体的转导和CFU测定的抑制和敲除实验 对于祖细胞存活来说。在对祖细胞存活的初步研究之后，我们将确定是否存在短暂的 NF-kB和cGAS抑制通过植入改善长期造血干细胞的自我更新 NSG小鼠。我们将同时在目标3中使用无偏假设生成方法来确定 通过单细胞RNA-seq实验， 在目标1和2实验中，使用整个群体或大的亚群体可能会遗漏。 我的科学环境和培训计划是成功完成这项研究的理想选择 提议斯坦福大学干细胞研究所拥有许多才华横溢的干细胞研究人员， 正如我们与Irv Weissman博士的RNA-seq合作所证明的那样。科学的财富 资源，包括我们专门的FACS核心和职业资源，通过博士后办公室提供 事务，以及科学和职业培训，从我的导师博士波特乌斯将使我实现我的 科学和职业目标。成功完成此建议书将加深我们对HSPC的了解 生物学和自我更新的背景下，抗病毒反应，以及改善发展的基因， 在HSPC中的基于编辑的疗法。

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.