The application of 'gene repair' to hematopoietic genetic disorders requires intra-disciplinary expertisederived from recent advances in: 1) genome engineering methods; 2) methods for gene delivery to HSC; and3) 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 ofdouble 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) candeliver a specific LAGLIDADG homing endonuclease (LHE; for induction of site specific double strandbreaks)Nand a donor DMA repair template into murine HSC at levels appropriate to induce gene repair; and2) 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 relativeselective advantage for gene corrected cells. First, we will generate a novel murine model of X-linked severecombined immunodeficiency (XSCID) that will permit us evaluate gene repair under near ideal conditionsusing 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 generepair. Following establishment of successful gene repair, we will utilize the identical LHE-XSCID animalmodel and delivery systems to evaluate the functional activity of an engineered l-Anil enzyme (developed inthe 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 theX-linked immunodeficiency (XID) locus. NIL vectors, carrying the XID-specific LHE and a repair template withor without a cis-linked selection marker, will be used to determine if drug selection can enrich gene repairedstem 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 usingengineered LHEs and NIL delivery systems.
