AAV-mediated gene correction in retina

描述（由申请人提供）：遗传性视网膜疾病是失明的主要原因，视网膜特异性和非特异性基因的许多突变已被确定导致光感受器变性。传统的基因治疗方法是基于基因添加策略，使用病毒载体，如腺病毒（Ad），腺相关病毒（AAV）和慢病毒。研究人员已经证明，在视网膜疾病的动物模型中，病毒介导的基因传递可以成功地延迟光感受器细胞的死亡；然而，特定的内源性基因突变没有得到纠正。基因靶向允许人们改变特定染色体位点的基因组序列，从而导致永久性的基因修饰。最近的报道表明，AAV可以在体外将靶向插入、缺失和替换引入同源染色体序列，其靶向率比传统转染或电穿孔方法高4-6倍。本提案中概述的实验将把AAV基因靶向策略应用于视网膜。病毒介导的修复效果将通过生化、组织学和功能分析进行评估。最初的研究将在转基因突变的β -半乳糖苷酶小鼠中进行，以评估视网膜发育和病毒载体设计是否影响aav介导的视网膜基因靶向的功效。实验随后将在-葡萄糖醛酸酶缺陷小鼠（gusmps/gusmps）中进行。这些动物将被用来评估aav介导的基因靶向治疗RPE疾病的潜力。从这些研究中收集的数据可能广泛应用于许多遗传性病理疾病，并可能最终导致永久性纠正导致这些疾病的基本遗传缺陷的治疗方法的发展。

DESCRIPTION (provided by applicant): Inherited retinal diseases are a major cause of blindness, and numerous mutations in both retina-specific and nonspecific genes have been identified that cause photoreceptor degeneration. Conventional gene therapy approaches are based on gene addition strategies using viral vectors such as adenovirus (Ad), adeno-associated virus (AAV) and lentivirus. Researchers have demonstrated that virus-mediated gene delivery may be used to successfully delay photoreceptor cell death in animal models of retinal disease; however, the specific endogenous gene mutations are not corrected. Gene targeting allows one to alter genomic sequences at specific chromosomal sites, resulting in permanent gene modifications. Recent reports indicate that AAV can be used to introduce targeted insertions, deletions and substitutions into homologous chromosomal sequences in vitro, with targeting rates that are 4-6 logs higher than those obtained by conventional transfection or electroporation methods. The experiments outlined in this proposal will apply AAV gene-targeting strategies to the retina. The efficacy of virus-mediated repair will be assessed through biochemical, histological and functional assays. Initially studies will be performed in transgenic mutant beta-galactosidase mice to assess if retinal development and viral vector design impact the efficacy of AAV-mediated gene targeting in the retina. Experiments will subsequently be performed in beta- glucuronidase- deficient mice (gusmps/gusmps). These animals will be used to evaluate the therapeutic potential of AAV-mediated gene targeting for RPE disease. The data gathered from these studies may have widespread applications for many inherited pathological conditions and may ultimately lead to the development of therapies that permanently correct fundamental genetic defects responsible for these conditions.