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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

基本信息

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
项目类别：
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

项目摘要

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.

校正和敲除视杆光感受器细胞中视紫红质基因的策略将被测试