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

应用“基因修复”治疗血液系统遗传病需要专业知识。 源自以下方面的最新进展：1)基因组工程方法；2)向HSC传递基因的方法；以及 3)造血干细胞移植的操作。在这个西北基因组工程联盟(NGEC) 组件，我们将通过设计和评估交付的系统来解决其中两个关键问题 双链断裂诱导剂和修复模板转化为小鼠造血干细胞； 并对修复后的HSC植入能力进行了优化。 我们将检验以下假设：1)自灭活的、非整合的慢病毒载体(nil载体)可以 递送特异性LAGLIDG归巢内切酶(LHE；用于诱导位点特异性双链 将DNA修复模板和供体DNA修复模板以适当的水平插入小鼠HSC，以诱导基因修复；以及 (2)NIL载体修复的HSC能有效地再移植和重建淋巴-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，它们在亲缘关系上是不同的 基因修饰细胞的选择优势。首先，我们将建立一种新的X连锁重症小鼠模型 联合免疫缺陷(XSCID)将使我们能够在接近理想的条件下评估基因修复 利用特征化的LHE，L-SCEL。该模型将允许我们评估一系列零向量设计， 转导方案和HSC靶群，以确定体内基因的最佳方法 修理。在成功建立基因修复后，我们将利用相同的LHE-XSCID动物 用于评价工程L-苯丙胺酶功能活性的模型和给药系统 按组件2-5划分的LHE设计周期)。 在随后的AIMS中，我们将评估第二个工程LHE，其设计目的是在BTK基因的 X连锁免疫缺陷(XID)基因座。空载体，携带XID特定的LHE和修复模板 或者没有顺式连锁选择标记，将被用来确定药物选择是否可以丰富修复的基因 干细胞，从而提高这种动物模型的免疫重建率。 我们的联合研究将提供一个关键的基准，根据它来判断基因修复方法 设计了LHEs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.