Rhodopsin Gene Correction and Gene Knockout in Rod Cells

描述(申请人提供)：将测试纠正和敲除视紫红质基因的策略，以确定在终末分化的神经元中操纵有害基因的结构和表达所需的条件。我们试图了解在这些细胞中允许有针对性的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质表达的过程。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遗传性视网膜色素变性(ADRP)。ADRP的成功治疗将为治疗显性眼病和其他神经退行性疾病提供一个范例。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疾病基因在老鼠身上导致视网膜退化的速度比人类和大型动物更快，从而能够快速测试疗效；而且老鼠的基因组很容易被操纵。此前，我们将完整的人视紫质基因--治疗的最终目标--与GFP基因融合，生成视紫红质表达的可见标记，该标记正确地定位于视杆外部节段。在这里，我们计划利用我们现有的野生型视紫红质-GTP小鼠，产生四个新的携带突变的人视紫质-GFP基因的小鼠系，为各种遗传操作提供靶点。这些修改后的人类视紫红质-GFP基因将包括三个明确的ADRP突变-P23H、Q64ter和Q344ter-以及一个携带内部复制的基因。腺相关病毒载体将被用来运送用于矫正和敲除视紫红质的新型试剂。对于基因校正，我们将在存在和不存在锌指核酸酶(ZFN)基因的情况下测试视紫红质DNA片段，这些基因被合理地设计为在目标突变附近切割。对于基因敲除，我们将测试外显子特定的ZFN，以直接突变视紫红质-GFP基因，以及内含子特定的ZFN，旨在刺激将毒害视紫红质基因表达的‘杀手’外显子的加入。检测视紫红质基因的校正和敲除将利用人类视紫红质-GFP靶基因的荧光特性。

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.