Rhodopsin Gene Correction and Gene Knockout in Rod Cells

描述（由申请人提供）：将测试用于校正和敲除视杆细胞中视紫红质基因的策略，以确定如何操纵终末分化神经元中有害基因的结构和表达。我们试图了解允许这些细胞中靶向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细胞过程。在获得这种理解的过程中，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视网膜色素变性（ADRP）。ADRP的成功疗法将为显性眼病和其他神经退行性疾病的治疗提供范例。小鼠是这些研究的自然选择，因为它们的眼睛生理学与人类相似; ADRP疾病基因在小鼠中引起视网膜变性的速度比人类和大型动物更快，可以快速测试功效;小鼠基因组可以很容易地操纵。在此之前，我们融合了完整的人类视紫红质基因-治疗的最终目标-GFP基因，以产生一个可见的标记，视紫红质的表达，适当地定位到杆外节。在这里，我们建议使用我们现有的野生型rhodopsin-GTP小鼠，并产生四个新的小鼠品系携带突变的人rhodopsin-GFP基因，提供各种遗传操作的目标。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的基因。腺相关病毒载体将用于递送用于视紫红质校正和敲除的新型试剂。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，这些基因已被合理设计为在靶突变附近切割。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，以及内含子特异性ZFN，其被设计为刺激将毒害视紫红质基因表达的“杀手”外显子的掺入。用于视紫红质基因校正和敲除的测定将利用人视紫红质-GFP靶基因的荧光性质。

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.