Gene Repair in Murine Hematopoietic Stem Cells (Component 6 of 11)

将“基因修复”应用于造血遗传疾病需要学科内的专业知识 源自以下方面的最新进展：1）基因组工程方法; 2）用于将基因递送至HSC的方法;以及 3)操作HSC用于移植。西北基因组工程联盟（Northwest Genome Engineering Consortium，NGEC） 组件，我们将通过设计和评估系统来解决其中两个关键问题， 双链断裂诱导试剂和修复模板进入鼠造血干细胞（HSC）; 和优化修复的HSC的植入潜力。 我们将检验以下假设：1）自身失活的非整合慢病毒载体（NIL载体）可以 递送特异性LAGLIDADG归巢核酸内切酶（LHE;用于诱导位点特异性双链 将供体DNA修复模板以适于诱导基因修复的水平插入鼠HSC中;和 2)NIL载体修复的HSC能有效地再植入并重建淋巴造血系统。 我们的研究将利用两个独立的免疫缺陷小鼠模型，其在相对免疫缺陷方面不同。 基因校正细胞的选择优势。首先，我们将建立一个新的X连锁严重的小鼠模型， 联合免疫缺陷（XSCID），使我们能够在接近理想的条件下评估基因修复 使用充分表征的LHE，l-Scel.该模型将允许我们评估一系列NIL载体设计， 转导方案和HSC靶群体，以确定体内基因转导的最佳方法。 修复.在建立成功的基因修复后，我们将使用相同的LHE-XSCID动物 模型和递送系统，以评估工程化的l-Anil酶（开发于 组件2-5的LHE设计周期）。 在随后的目标中，我们将评估第二个工程化LHE，其设计为在Btk基因内切割， X连锁免疫缺陷（XID）基因座。携带XID特异性LHE和修复模板的NIL载体， 或没有顺式连锁的选择标记，将用于确定药物选择是否可以富集修复的基因 干细胞，从而提高该动物模型中的免疫重建速率。 我们的综合研究将提供一个关键的基准，以判断基因修复方法， 工程化LHE和NIL递送系统。

The application of "gene repair" to hematopoietic genetic disorders requires intra-disciplinary expertise derived from recent advances in: 1) genome engineering methods; 2) methods for gene delivery to HSC; and 3) manipulation of HSC for transplantation. In this Northwest Genome Engineering Consortium (NGEC) Component, we will address two of these key issues by designing and evaluating systems for delivery of double strand break-inducing reagents and repair templates into murine hematopoietic stem cells (HSC); and optimization of the engraftment potential of the repaired HSC. We will test the hypotheses that: 1) Self inactivated, non-integrating lentiviral vectors (NIL vectors) can deliver a specific LAGLIDADG homing endonuclease (LHE; for induction of site specific double strand breaks)Nand a donor DMA repair template into murine HSC at levels appropriate to induce gene repair; and 2) NIL vector-repaired HSC can effectively re-engraft and reconstitute the lympho-hematopoietic system. Our studies will utilize two independent immunodeficient mouse models that differ in regard to the relative selective advantage for gene corrected cells. First, we will generate a novel murine model of X-linked severe combined immunodeficiency (XSCID) that will permit us evaluate gene repair under near ideal conditions using the well characterized LHE, l-Scel. This model will allow us to evaluate a range of NIL vector designs, transduction protocols, and HSC target populations in order to identify optimal methods for in vivo gene repair. Following establishment of successful gene repair, we will utilize the identical LHE-XSCID animal model and delivery systems to evaluate the functional activity of an engineered l-Anil enzyme (developed in the LHE design cycle by Components 2-5). In subsequent Aims, we will evaluate a second engineered LHE, designed to cut within the Btk gene at the X-linked immunodeficiency (XID) locus. NIL vectors, carrying the XID-specific LHE and a repair template with or without a cis-linked selection marker, will be used to determine if drug selection can enrich gene repaired stem cells and, thereby, enhance the rate of immune reconstitution in this animal model. Our combined studies will provide a key benchmark against which to judge gene repair approaches using engineered LHEs and NIL delivery systems.