Rhodopsin Gene Correction and Gene Knockout in Rod Cells

矫正和敲除视杆细胞视紫红质基因的策略将被测试， 确定需要什么来操纵有害基因的结构和表达， 分化的神经元我们试图了解基本的细胞过程，使靶基因 这些细胞中的修复和突变，以及那些可以控制有毒蛋白质表达的细胞。过程中 为了获得这种理解，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， 色素沉着症（ADRP）由视紫红质基因缺陷引起。ADRP的成功疗法将提供 用于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 这些研究的选择，因为他们的眼睛生理学类似于人类;ADRP疾病基因导致 小鼠的视网膜变性比人类和大型动物更快，可以快速检测 功效;并且小鼠基因组可以容易地操纵。之前，我们融合了完整的人类 视紫红质基因作为治疗的最终靶点，与绿色荧光蛋白基因结合，产生视紫红质的可见标记物 表达，适当地定位到杆外段。在这里，我们建议使用我们现有的野生型 视紫红质-GTP小鼠并产生四种携带突变的人视紫红质-GFP基因的新小鼠系， 为各种基因操作提供了目标。这些修饰的人类视紫红质-GFP基因将 包括三个确定的ADRP突变<$P23 H，Q64 ter和Q344 ter <$和一个携带内部 重复。腺相关病毒载体将被用于递送用于视紫红质校正的新型试剂 和击倒。对于基因校正，我们将在存在和不存在以下物质的情况下测试视紫红质DNA片段： 锌指核酸酶（ZFN）的基因已经被合理地设计成在靶突变附近切割。 对于基因敲除，我们将测试外显子特异性ZFN以直接突变视紫红质-GFP基因，并测试内含子-GFP基因。 特定的ZFN旨在刺激“杀手”外显子的掺入，从而毒害细胞的表达 视紫红质基因视紫红质基因校正和敲除的测定将利用 人视紫红质-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.