Rhodopsin Gene Correction and Gene Knockout in Rod Cells

视杆细胞中的视紫红质基因校正和基因敲除

• 批准号: 7186678
• 负责人: JOHN H WILSON
• 金额: $ 36.41万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7186678
• 关键词: Animals; Biological Assay; Cell physiology; Cells; Cleaved cell; Count; DNA; DNA Damage; Defect; Dependovirus; Detection; Disease; Double Strand Break Repair; Effectiveness; Event; Exons; Eye diseases; Fluorescence; Foundations; Frequencies; Gene Expression; Gene Targeting; Genes; Genome; Genomics; Goals; Grant; Green Fluorescent Proteins; Human; Individual; Introns; Knock-in Mouse; Knock-out; Lead; Localized; Measures; Messenger RNA; Metabolism; Methods; Mus; Mutate; Mutation; Neurodegenerative Disorders; Neurons; Nonsense-Mediated Decay; Ocular Physiology; Photoreceptors; Poison; Procedures; Process; Property; Proteins; Range; Reagent; Research Personnel; Retina; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Rod Outer Segments; Site; Structure; System; Testing; Therapeutic; Zinc Fingers; adeno-associated viral vector; base; design; fusion gene; gene correction; gene repair; genetic manipulation; homologous recombination; knockout gene; mouse genome; mutant; novel; nuclease; programs; repaired; research study; retinal rods; subretinal injection; therapy design

DESCRIPTION (provided by applicant): Strategies for correction and knockout of the rhodopsin gene in rod photoreceptor cells will be tested to determine what it takes to manipulate the structure and expression of deleterious genes in terminally differentiated neurons. We seek to understand the fundamental cellular processes that allow targeted gene repair and mutation in these cells, as well as those that can control expression of toxic proteins. In the process of gaining this understanding, we aim to develop effective methods for treating autosomal dominant retinitis pigmentosa (ADRP) caused by defects in the rhodopsin gene. Successful therapies for ADRP will provide a paradigm for treatment of dominant eye diseases and other neurodegenerative disorders. Mice are a natural choice for these studies because their eye physiology resembles that of humans; ADRP disease genes cause retinal degeneration in mice more quickly than in humans and large animals, allowing rapid testing of efficacy; and mouse genomes can be readily manipulated. Previously, we fused the complete human rhodopsin gene-the ultimate target for therapy-to the GFP gene to generate a visible marker for rhodopsin expression that localizes properly to rod outer segments. Here, we propose to use our existing wild type rhodopsin-GTP mice and generate four new mouse lines carrying mutant human rhodopsin-GFP genes to provide targets for various kinds of genetic manipulation. These modified human rhodopsin-GFP genes will include three defined ADRP mutations-P23H, Q64ter, and Q344ter-and a gene that carries an internal duplication. Adeno-associated virus vectors will be used to deliver novel reagents for rhodopsin correction and knockout. For gene correction, we will test segments of rhodopsin DNA in the presence and absence of genes for zinc-finger nucleases (ZFNs) that have been rationally designed to cleave near the target mutations. For gene knockout, we will test exon-specific ZFNs to mutate the rhodopsin-GFP gene directly, and intron- specific ZFNs designed to stimulate incorporation of a 'killer' exon that will poison expression of the rhodopsin gene. Assays for rhodopsin gene correction and knockout will utilize the fluorescent properties of the human rhodopsin-GFP target gene.

描述（由申请人提供）：将测试杆状光感受器细胞中校正和敲除视紫红质基因的策略，以确定如何操纵终末分化神经元中有害基因的结构和表达。我们试图了解这些细胞中允许靶向基因修复和突变的基本细胞过程，以及那些可以控制有毒蛋白表达的细胞过程。在获得这一认识的过程中，我们的目标是开发有效的方法来治疗由视紫红质基因缺陷引起的常染色体显性视网膜色素变性（ADRP）。ADRP的成功治疗将为主要眼病和其他神经退行性疾病的治疗提供一个范例。老鼠是这些研究的自然选择，因为它们的眼睛生理与人类相似；ADRP疾病基因在小鼠中比在人类和大型动物中更快地引起视网膜变性，从而可以快速测试其功效；而且老鼠的基因组很容易被操纵。在此之前，我们将完整的人类视紫红质基因（治疗的最终目标）与GFP基因融合，以产生一个可见的视紫红质表达标记，该标记适当地定位于杆外段。本研究拟利用现有野生型视紫红质- gtp小鼠，培育4个携带人类视紫红质- gfp突变基因的小鼠新品系，为各种基因操作提供靶点。这些修饰的人类视紫红质- gfp基因将包括三个定义的ADRP突变- p23h， Q64ter和q344ter -和一个携带内部复制的基因。腺相关病毒载体将用于提供新的试剂，用于视紫红质校正和敲除。对于基因校正，我们将测试紫红质DNA片段在存在和不存在锌指核酸酶（ZFNs）基因的情况下，锌指核酸酶（ZFNs）被合理地设计为在目标突变附近切割。对于基因敲除，我们将测试外显子特异性ZFNs，直接使视紫红质- gfp基因突变，以及内含子特异性ZFNs，旨在刺激“杀手”外显子的结合，从而毒害视紫红质基因的表达。检测视紫红质基因的校正和敲除将利用人视紫红质- gfp靶基因的荧光特性。