Rhodopsin Gene Correction and Gene Knockout in Rod Cells

纠正和敲除视紫红质基因的策略将在视杆感光细胞中进行测试 确定需要什么才能最终操纵有害基因的结构和表达 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，以及那些可以控制有毒蛋白表达的细胞。在这个过程中 为了了解这一点，我们的目标是开发治疗常染色体显性视网膜炎的有效方法。 由视紫红质基因缺陷引起的色素变性(ADRP)。ADRP的成功治疗将提供一种 治疗显性眼病和其他神经退行性疾病的范例。老鼠是天生的 选择这些研究是因为他们的眼睛生理与人类相似；adrp疾病基因导致 小鼠的视网膜退化比人类和大型动物更快，这使得快速检测 药效；小鼠基因组很容易被操纵。之前，我们融合了完整的人类 视紫红质基因是治疗视紫红质的终极靶点，而GFP基因是视紫红质的可见标志物 正确定位到杆状外部线段的表达式。在这里，我们建议使用我们现有的野生型 视紫红质-GTP小鼠，并产生四个新的携带突变的人视紫质-GFP基因的小鼠株系 为各种基因操作提供靶标。这些经过修饰的人类视紫红质-GFP基因将 包括三个已定义ADRP突变P23H、Q64ter和Q344ter以及一个携带内部 复制。腺相关病毒载体将用于运送视紫红质矫正的新型试剂 和淘汰赛。对于基因校正，我们将测试视紫红质DNA片段在存在和不存在的情况下 锌指核酸酶(ZFN)的基因被合理地设计为在目标突变附近切割。 对于基因敲除，我们将检测外显子特异的ZFN直接突变视紫红质-GFP基因，而内含子- 特定的ZFN被设计成刺激结合将毒化 视紫红质基因。视紫红质基因校正和敲除的检测将利用 人视紫质-GFP靶基因。

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.