Rescue of GUCY1*B Phenotype Using Somatic Gene Therapy

描述(申请人提供)：这项研究计划的长期目标是了解Leber先天性黑素瘤-1(LCA1)的细胞学后果，并确定基于重组慢病毒载体的疗法是否可以用于治疗这种导致新生儿失明或严重视力丧失的破坏性常染色体隐性视网膜疾病。LCA1是由编码光感受器鸟苷环化酶-1(Gc1)的基因突变引起的，其中大部分突变会导致Gc1酶功能的丧失。GUCY1*B鸡在GC1中携带零突变，是一种自然产生的LCA1模型，拥有视锥主导的视网膜，在孵化时是盲人的。对这一模型的分析是这项研究计划的焦点，它为我们提供了一个独特的机会来提高我们对LCA1和锥体对疾病的反应的理解。本研究的目的1是验证GUCY1*B光感受器中正常Gc1的表达足以挽救光感受器功能和防止退变的假设。携带GC1转基因的慢病毒将在胚胎发育期间注射给GUCY1*B鸡，治疗的有效性将在2-4周龄时通过检查(1)动物的行为，(2)视网膜电描记术对光的反应，(3)视网膜形态，以及(4)GC1和鸟苷环化酶激活蛋白-1的表达来评估。这项建议的目的2是确定GUCY1*B视网膜疾病进展与我们的基因替换策略挽救光感受器功能的能力之间的关系。由于GUCY1*B光感受器群体中80%是锥体细胞，这些研究将对了解锥体疾病和这些细胞对治疗的反应特别相关。Gc1转基因在视网膜中的表达将推迟到孵化后10、21或60天，并将通过将Gc1转基因置于四环素可调节启动子的控制下或通过改变Gc1慢病毒的给药时间来控制。延迟治疗的疗效将按照目标1的描述进行评估。这些实验的结果将提高我们对LCA1的理解，并提高基因疗法治疗这种疾病的潜在有效性。此外，他们还将提供有关四环素调控表达系统控制视网膜表达或治疗基因的有效性的新信息。

DESCRIPTION (provided by applicant): The long-range goals of this research program are to understand the cellular consequences of Leber congenital amaurosis - 1 (LCA1) and to determine if therapies based on recombinant lentiviral vectors can be used to treat this devastating autosomal recessive retinal disease that results in blindness or severe visual loss in newborn infants. LCA1 is caused by mutations in the gene encoding photoreceptor guanylate cyclase -1 (GC1), the majority of which lead to loss of function of the GC1 enzyme. The GUCY1*B chicken, which carries a null mutation in GC1, is a naturally occurring model of LCA1 that possesses a cone-dominant retina and is blind at hatching. Analyses of this model, which serves as a focal point for this research program, provide a unique opportunity to improve our understanding of LCA1 and of cone responses to disease. Aim 1 of this proposal is to test the hypothesis that the expression of normal GC1 in GUCY1*B photoreceptors is sufficient to rescue photoreceptor function and prevent degeneration. Lentivirus carrying a GC1 transgene will be administered to GUCY1*B chickens during embryonic development and the effectiveness of the treatment will be assessed at 2-4 weeks of age by examining (1) the behavior of the animal, (2) the responsiveness of the retina to light by electroretinography, (3) retinal morphology, and (4) the expression of GC1 and guanylate cyclase activating protein -1. Aim 2 of this proposal is to determine the relationship between disease progression in GUCY1*B retina and the ability of our gene replacement strategy to rescue photoreceptor function. Since the GUCY1*B photoreceptor population is 80% cone, these studies will be particularly relevant to understanding cone disease and the responses of these cells to therapeutic treatment. Expression of the GC1 transgene in retina will be delayed until 10, 21 or 60 days after hatching and will be controlled by either placing the GC1 transgene under the control of a tetracycline-regulatable promoter or by varying the time of administration of the GC1 lentivirus. The efficacy of the delayed treatments will be assessed as described for Aim 1. The results of these experiments will improve our understanding of LCA1 and the potential usefulness of gene therapy to treat this disease. In addition, they will provide new information about the effectiveness of tetracycline-regulated expression systems to control the expression or therapeutic genes in the retina.