Oligonucleotide Repair of a Retinal Degenerarion Model

描述(申请人提供)：编码杆状光感受器cGMP磷酸二酯酶(β-PDE)的β亚基的基因突变与视网膜色素变性(RP)有关，RP是一种导致人类失明的疾病家族。小鼠Rd1(Pde6b Rd1)和Rd10(Pde6b Rd10)视网膜变性表型是研究人类RP的模型，因为这些表型是编码β-PDE基因突变的结果。寡核苷酸导向的基因校正是一种通过内源性DNA修复机制识别治疗性寡核苷酸与其基因组靶序列之间的不匹配，诱导它们将突变转化为野生型的策略。与其他基因疗法不同，这种策略旨在永久修复染色体基因的缺陷。这项拟议的研究的广泛目标是确定是否可以使用寡核苷酸指导的基因校正来修复RD1或RD10突变，防止光感受器退化，并保护视力。在特定的目标1中，将通过免疫组织学和免疫印迹来确定是否存在寡核苷酸诱导的DNA修复蛋白。无细胞实验将被用来确定在视网膜和光感受器蛋白存在的情况下，寡核苷酸是否会诱导测试DNA目标的修复。同样的方案将与个别修复蛋白的抑制剂结合，以测试它们在修复反应中的重要性。在特定目标2中，将寡核苷酸注射到新生小鼠的眼睛中并进行离子转移。光感受器的挽救将通过免疫印迹和视网膜视紫红质和β-PDE的免疫组织化学检查来评估。由于视紫红质是最丰富表达的光感受器标记物，监测它的水平应该能为光感受器的挽救提供一个非常敏感和可靠的评估。DNA修复将通过视网膜的免疫组织化学检查(-PDE)和检测视网膜提取物的PDE6酶活性来评估。还将采取非侵入性的视觉功能测量(例如ERG)。在这些动物模型中预防视力丧失应该指导我们对遗传性视网膜疾病的基因治疗，如人类常染色体隐性遗传性视网膜病变。

DESCRIPTION (provided by applicant): Mutations in the gene encoding the beta subunit of rod photoreceptor cGMP phosphodiesterase (beta-PDE) are associated with retinitis pigmentosa (RP), a disease family that leads to blindness in humans. The mouse rd1 (Pde6b rd1) and rd10 (Pde6b rd10) retinal degeneration phenotypes are models for the study of human RP because these phenotypes result from mutations in the gene encoding beta-PDE. Oligonucleotide-directed gene correction is a strategy in which a mismatch between a therapeutic oligonucleotide and its genomic target sequence is recognized by endogenous DNA repair mechanisms, inducing them to convert the mutation to wild type. As opposed to other gene therapies, this strategy is intended to permanently repair the defect in the chromosomal gene. The broad objective of this proposed research is to determine whether oligonucleotide-directed gene correction can be used to repair the rd1 or rd10 mutations, prevent photoreceptor degeneration, and preserve vision. In Specific Aim 1, the existence of oligonucleotide-inducible DNA repair proteins will be determined by immunohistology and immunoblotting. A cell-free assay will be used to determine whether oligonucleotides induce repair of test DNA targets in the presence of retina and photoreceptor proteins. The same protocol will be coupled with inhibitors of individual repair proteins to test for their importance in the repair reaction. In Specific Aim 2, oligonucleotides will be injected and iontophoresed into eyes of neonatal mice. Photoreceptor rescue will be assessed by immunoblotting and immunohistochemical examination of retinas for rhodopsin and beta-PDE. Since rhodopsin is the most abundantly expressed photoreceptor marker, monitoring its levels should provide an exquisitely sensitive and robust assessment of photoreceptor rescue. DNA repair will be assessed by immunohistochemical examination of retinas for (-PDE and by assaying retinal extracts for PDE6 enzymatic activity. Noninvasive measures of visual function (e.g., ERG) will be taken also. Preventing vision loss in these animal models should direct our efforts for gene therapy of inherited retinal diseases such as autosomal recessive RP in humans.