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分散在受影响基因的整个位点上的患者特异性突变引起的。通过将健康基因拷贝引入患病细胞的基因组来纠正这些突变在几项临床试验中证明是成功的。在大多数试验中，健康基因通过逆转录病毒载体随机插入整个基因组中，因此失去了它们的内源性控制元件。结果出现了并发症，包括基因沉默和致癌基因的激活。为了解决这些问题，我们提出开发一种基于无模板CRISPR/Cas9的基因组编辑方法，用于原位校正引起慢性肉芽肿性（X-CGD）或严重免疫缺陷病（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，我们将产生表达携带患者特异性突变的单拷贝CYBB或IL 2 R-γ基因的人造血细胞系，并将这些细胞系用于基于基因校正CRISPR/Cas9的整合酶缺陷型慢病毒载体（IDLV）的初步筛选。将通过单胚胎注射将在该筛选中鉴定的高度可校正的突变引入小鼠种系中以获得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，旨在通过将CRISPR/Cas9 IDLV离体递送到造血干细胞中来治愈疾病。总的来说，该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.