Editing to Create and Correct Gene Variants

超过350个基因的突变被认为是导致原发性免疫缺陷(PID)的原因，但这些基因 这些突变导致了许多罕见的，但临床上严重的情况仍然未知，即使 整个外显子组测序。全基因组测序的使用有望揭示编码和非编码 T淋巴细胞缺乏症病例的突变不能通过整个外显子组测序解决。然而， 自信地从大量的良性变异中区分致病的PID突变 整个基因组是令人望而生畏的，因为每一种Pid的罕见发生率，对基因的不完全了解 T细胞发育所需的，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基因组编辑与我们的体外T细胞分化平台相结合，提供 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 从造血干细胞(HSPC)发展而来，最终达到了包括重写在内的新疗法 在患者的造血细胞中导致人类免疫疾病的突变。精确定位每一项的进展 患者的因果突变将开辟下一个前沿：对内源性致病因素的精确非病毒纠正 突变用于HSPC的自体基因治疗，避免使用不完全匹配的同种异体 供者移植，移植物排斥反应和移植物抗宿主病是潜在的破坏性并发症。 该项目将开发基于CRISPR的高效率、高通量的技术来识别 细胞发育、候选突变的快速功能测试和治疗性基因的必要基因T 纠正患者自己的HSPC。我们开发了CRISPR-CAS9作为一把分子手术刀来编辑特定的 人类原代细胞的基因组序列，最近改进了这项与治疗相关的技术 在hPSC中编辑。我们将进一步应用基于CRISPR的技术来高通量映射编码和 与SCID和其他形式的T细胞缺陷性PID相关的基因的非编码突变，我们将开发新的 原代人类HSPC的治疗性基因编辑技术。因此，该项目的三个核心目标 通过基因编辑解决根本性挑战：1)发现所有基因 可能导致T细胞缺陷的扰动，2)快速鉴定患有 未解决的遗传基础，以及3)技术的改进，以引入有效和特定的基因校正 作为个体化自体基因纠正以恢复免疫功能的先驱。

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.