Rescue of GUCY1*B Phenotype Using Somatic Gene Therapy

描述（由申请人提供）：本研究计划的长期目标是了解Leber先天性黑蒙-1（LCA 1）的细胞后果，并确定基于重组慢病毒载体的疗法是否可用于治疗这种导致新生儿失明或严重视力丧失的毁灭性常染色体隐性视网膜疾病。LCA 1是由编码光感受器鸟苷酸环化酶-1（GC 1）的基因突变引起的，其中大部分突变导致GC 1酶功能丧失。携带GC 1无效突变的GUCY 1 *B鸡是LCA 1的天然模型，具有视锥细胞显性视网膜，孵化时失明。该模型的分析，这作为一个焦点，为这项研究计划，提供了一个独特的机会，以提高我们的理解LCA 1和锥细胞对疾病的反应。该提议的目的1是检验GUCY 1 *B光感受器中正常GC 1的表达足以拯救光感受器功能并防止变性的假设。携带GC 1转基因的慢病毒将在胚胎发育期间给予GUCY 1 *B鸡，并在2-4周龄时通过检查（1）动物行为、（2）视网膜对光的反应性来评估治疗的有效性视网膜电图，（3）视网膜形态学，和（4）GC 1和鸟苷酸环化酶激活蛋白-1的表达。该提议的目的2是确定GUCY 1 *B视网膜中疾病进展与我们的基因替换策略拯救光感受器功能的能力之间的关系。由于GUCY 1 *B光感受器群体80%为视锥细胞，因此这些研究将与理解视锥细胞疾病和这些细胞对治疗性处理的反应特别相关。GC 1转基因在视网膜中的表达将延迟至孵化后10、21或60天，并且将通过将GC 1转基因置于四环素可调节启动子的控制下或通过改变GC 1慢病毒的施用时间来控制。将按照目标1所述评估延迟治疗的疗效。这些实验的结果将提高我们对LCA 1的理解和基因治疗治疗这种疾病的潜在用途。此外，他们将提供有关四环素调控表达系统控制视网膜中治疗基因表达的有效性的新信息。

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.