Exploring Template-free CRISPR/Cas9 Genome Editing as a Novel Strategy for Personalized Gene Therapy of Monogenic Blood Disorders

很大一部分遗传性单基因疾病是由散布在受影响基因的整个基因座上的患者特有的突变引起的。通过将健康的基因拷贝引入患病细胞的基因组中来纠正这些突变，在几个临床试验中被证明是成功的。在大多数这些试验中，健康基因被逆转录病毒载体随机插入整个基因组，从而剥夺了它们的内源控制元件。结果，并发症出现了，包括基因沉默和致癌基因的激活。为了避免这些问题，我们建议开发一种基于CRISPR/Cas9的无模板基因组编辑方法，用于原位纠正导致慢性肉芽肿(X-CGD)或严重免疫缺陷疾病(X-SCID)的点突变，这两种疾病都是遗传性单基因血液疾病。为此，我们将建立表达携带患者特定突变的单拷贝CYBB或IL2R-γ基因的人造血细胞系，并将这些细胞系用于初步筛选，以校正基于CRISPR/Cas9的整合酶缺陷慢病毒载体(IDLV)的基因。在本屏幕中发现的高度可纠正的突变将通过单胚胎注射引入小鼠生殖系，以获得X-CGD和X-SCID的小鼠模型。然后，这些模型将用于自体骨髓移植实验，旨在通过体外将CRISPR/Cas9 IDLV导入造血干细胞来治疗这种疾病。总体而言，本项目旨在为个性化基因治疗提供概念证明。

A significant fraction of inherited monogenic disorders are caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical trials. In most of these trials the healthy genes were inserted randomly throughout the genome by retroviral vectors and were thus deprived of their endogenous control elements. As a result complications arose including gene silencing and activation of cancer causing genes. To circumvent these problems, we propose to develop a template-free CRISPR/Cas9 based genome editing approach for the in situ correction of point mutations causing chronic granulomatous (X-CGD) - or severe immunodeficiency disease (X-SCID), which are both inherited monogenic blood disorders. For this, we will generate human hematopoietic cell lines expressing single copy CYBB or IL2R-gamma genes harboring patient specific mutations and use these cell lines in a primary screen for gene correcting CRISPR/Cas9 based, integrase deficient lentiviral vectors (IDLVs). Highly correctable mutations identified in this screen will be introduced into the mouse germline by single embryo injections to obtain mouse models of X-CGD and X-SCID. These models will then be used in autologous bone marrow transplantation experiments aimed at curing the disease by ex vivo CRISPR/Cas9 IDLV delivery into hematopoietic stem cells. Overall, this project seeks to provide proof of concept for personalized gene therapy.