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

小鼠造血干细胞中的基因修复（11 部分中的 6 部分）

• 批准号: 7503424
• 负责人: David J Rawlings
• 金额: $ 45.5万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7503424
• 关键词: Address; Adoptive Transfer; Affect; Agammaglobulinemia; Animal Model; Antigens; B-Cell Development; B-Lymphocytes; Benchmarking; Bone Marrow; Bone Marrow Cells; Cells; Cellular Structures; Cleaved cell; Condition; DNA Repair; Defect; Development; Disease; Engineering; Engraftment; Enzymes; Evaluation; Exons; Gene Delivery; Genes; Genome engineering; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Hereditary Disease; Homing; Homologous Gene; Human; Immune; Immune system; Immunologic Deficiency Syndromes; Immunologics; Interleukin 2 Receptor Gamma; Knock-in Mouse; Lentivirus Vector; Link; Lymphoid; Marrow; Methods; Modeling; Mus; O6-benzylguanine/Temozolomide; Patients; Pharmaceutical Preparations; Phenotype; Protocols documentation; Range; Rate; Reagent; Receptor Signaling; Relative (related person); Site; Stem cells; System; TEC Protein Tyrosine Kinase; Target Populations; Terminator Codon; Testing; Toxic effect; Variant; X-Linked Severe Combined Immunodeficiency; base; design; embryonic stem cell; endonuclease; gene correction; gene repair; immunodeficient mouse model; in vivo; male; mutant; novel; progenitor; reconstitution; repaired; research study; vector

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.

将“基因修复”应用于造血遗传疾病需要学科内的专业知识