Editing to Create and Correct Gene Variants

超过350个基因的突变被认为是原发性免疫缺陷（PID）的驱动因素，但这些基因 突变导致许多这些罕见的，但临床上严重的条件仍然未知，即使 全外显子组测序全基因组测序有望揭示编码和非编码 对于T淋巴细胞缺乏的情况，突变不能通过全外显子组测序解决。然而，在这方面， 有信心地区分致病性PID突变与大量的良性变异， 由于每种PID的发病率很低，对基因的了解不完整，整个基因组令人畏惧 我们缺乏基于序列的规则来预测哪些非编码变体可能 是致病的。CRISPR-Cas9基因组编辑结合我们的体外T细胞分化平台， 直接测试人类基因序列如何控制免疫细胞发育的前所未有的机会 从造血干祖细胞（HSPC），并最终达到新的疗法，包括重写 在患者的造血细胞中引起人类免疫疾病的突变。在查明 患者的因果突变将打开下一个前沿：精确的内源性致病的非病毒校正 突变用于HSPC中的自体基因治疗，避免了使用不完全匹配的同种异体 供者移植，其中移植物排斥和移植物抗宿主病是潜在的破坏性并发症。 该项目将开发高效、高通量的基于CRISPR的技术来识别 用于细胞发育的必需基因T，候选突变的快速功能测试，以及治疗性遗传学 纠正患者自身的HSPC。我们已经开发了CRISPR-Cas9作为分子手术刀来编辑特定的 在原代人类细胞中的基因组序列，最近改进了这项技术， 在HPSC中编辑。我们将进一步应用基于CRISPR的技术进行编码的高通量映射， 与SCID和其他形式的T细胞缺陷PID相关的基因中的非编码突变，我们将开发新的 在原代人类HSPC中进行治疗性基因编辑的技术。因此，该项目的三个核心目标 解决通过基因编辑治愈PID的根本挑战：1）发现所有基因 可能导致T细胞缺陷的干扰，2）快速鉴定PID病例的因果突变， 未解决的遗传基础，以及3）改进技术以引入有效和特定的基因校正 作为个体化自体基因校正的先导，以恢复免疫功能。

Mutations in over 350 genes have been implicated as drivers of primary immunodeficiency (PID), but the genes that are mutated to cause many of these rare, but clinically serious conditions remain unknown, even despite whole exome sequencing. The use of whole genome sequencing promises to reveal coding and non-coding mutations for cases of T lymphocyte deficiency that cannot be solved by whole exome sequencing. However, confidently distinguishing pathogenic PID mutations from the exceedingly large number of benign variants across the entire genome is daunting, due to the rare incidence of each PID, incomplete knowledge of the genes required for T cell development, and our lack sequence-based rules to predict which non-coding variants may be pathogenic. CRISPR-Cas9 genome editing combined with our in vitro T cell differentiation platform offers unprecedented opportunities to test directly how human genetic sequences control immune cell development from hematopoietic stem progenitor cells (HSPCs) and ultimately to arrive at new therapies consisting of rewriting mutations that cause human immune diseases in patient blood-forming cells. Progress in pinpointing each patient’s causal mutation will open the next frontier: precise non-viral correction of endogenous disease-causing mutations for autologous gene therapy in HSPCs, avoiding the necessity to use imperfectly matched allogeneic donor transplants, for which graft rejection and graft vs. host disease are potentially devastating complications. This project will develop high-efficiency, high-throughput CRISPR-based technologies for identification of essential genes T for cell development, rapid functional testing of candidate mutations, and therapeutic genetic correction of a patient’s own HSPCs. We have developed CRISPR-Cas9 as a molecular scalpel to edit specific genome sequences in primary human cells and recently improved this technology for therapeutically-relevant editing in HPSCs. We will further apply CRISPR-based technologies for high-throughput mapping of coding and non-coding mutations in genes related to SCID and other forms of T-cell deficient PID, and we will develop new technologies for therapeutic gene editing in primary human HSPCs. Thus this project’s three central aims address fundamental challenges to achieving cures for PID through gene editing: 1) Discovery of all gene perturbations that could result in T cell deficiency, 2) Rapid identification of causal mutations for PID cases with unsolved genetic basis, and 3) Improvement in technology to introduce efficient and specific gene corrections into primary HPSCs as a forerunner to personalized autologous gene correction to restore immune function.