Oligonucleotide Repair of a Retinal Degenerarion Model

描述（由申请人提供）：编码杆状光感受器cGMP磷酸二酯酶(β - pde) β亚基的基因突变与色素性视网膜炎（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，因为这些表型是由编码β - pde的基因突变引起的。寡核苷酸定向基因校正是一种策略，其中治疗性寡核苷酸与其基因组靶序列之间的不匹配被内源性DNA修复机制识别，诱导它们将突变转化为野生型。与其他基因疗法相反，这种策略旨在永久修复染色体基因中的缺陷。本研究的主要目的是确定寡核苷酸定向基因校正是否可以用于修复rd1或rd10突变，防止光感受器变性，并保持视力。在Specific Aim 1中，寡核苷酸诱导的DNA修复蛋白的存在将通过免疫组织学和免疫印迹来确定。无细胞测定将用于确定是否寡核苷酸诱导修复测试DNA目标在视网膜和光感受器蛋白的存在。同样的方案将与单个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。在特异性目的2中，寡核苷酸将被注射并离子转运到新生小鼠的眼睛中。将通过免疫印迹和免疫组织化学检查视网膜的视紫红质和β - pde来评估光感受器的恢复。由于视紫红质是最丰富表达的光感受器标志物，监测其水平应该提供一个非常敏感和可靠的评估光感受器的救援。DNA修复将通过视网膜（-PDE）的免疫组织化学检查和视网膜提取物PDE6酶活性的测定来评估。也将采取无创的视觉功能测量（如ERG）。预防这些动物模型的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RP。

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.