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: 7686532
• 负责人: JOHN H WILSON
• 金额: $ 4.94万
• 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/7686532
• 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; 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

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¿tothe 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 designedto 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 humanrhodopsin-GFPtarget gene.

将测试Rod感光细胞中视紫红质基因校正和敲除策略 确定操纵终端中有害基因的结构和表达所需的内容 分化的神经元。我们试图了解允许靶向基因的基本细胞过程 这些细胞中的修复和突变以及可以控制有毒蛋白质表达的细胞。在此过程中 为了获得这种理解，我们旨在开发有效的方法来治疗常染色体显性视网膜炎 由视紫红质基因缺陷引起的色素（ADRP）。成功的ADRP疗法将提供 用于治疗主要眼病和其他神经退行性疾病的范例。老鼠是自然的 这些研究的选择是因为它们的眼睛生理类似于人类； ADRP疾病基因引起 小鼠的视网膜变性比人类和大型动物更快，可以快速测试 效率;和小鼠基因组可以轻易操纵。以前，我们融合了完整的人 视紫红质基因€tothe GFP基因生成视紫红质的可见标记 表达适当地定位于杆外段。在这里，我们建议使用现有的野生型 Rhodopsin-GTP小鼠并生成四种新的小鼠系，载有突变的人类Rhopopsin-GFP基因 为各种遗传操作提供目标。这些改良的人类视蛋白GFP基因将 包括三个定义的ADRP突变€p23h，Q64Ter和Q344ter¿以及一个带有内部的基因 复制。腺相关病毒载体将用于提供新型的视紫红质校正试剂 和淘汰赛。对于基因校正，我们将在存在和不存在的情况下测试视紫红质DNA的片段 锌指核病（ZFN）的基因经过合理设计，可在目标突变附近清除。 对于基因敲除，我们将测试外显子特异性ZFN，直接突变Rhopopsin-GFP基因，并内含子 - 特定的ZFN旨在刺激“杀手”外显子的感染，该外显子会毒害表达 视紫红质基因。视紫红质基因校正和敲除测定法将利用 人hopopsin-gfptarget基因。