Gene Therapy for Retinitis Pigmentosa

描述（由申请人提供）：色素性视网膜炎（RP）是美国遗传性失明的最常见原因之一，每3000人中约有1人患病。目前没有治疗这种疾病的方法。本研究的广泛、长期目标是开发RP的治疗方法。人、犬和小鼠（rd）中的某些形式的RP是由cGMP磷酸二酯酶（bPDE）的β亚基突变引起的。为了将编码bPDE的正常cDNA递送至rd视网膜，我们已经开发了一类新的更安全且毒性更低的腺病毒载体，称为包膜腺病毒微型染色体（EAM或“挖空”载体）。EAM具有36 Kb克隆能力，允许插入用于调节转基因表达的大的上游调节元件和/或包含多个治疗性转基因盒。我们已经使用EAM暂时挽救了rd 1小鼠约10周的视网膜变性，否则将在出生后3周完成。我们的目标现在集中在延长这一短暂的救援时间，以一个可能更有治疗意义的人类。EAM通过将触角样纤维蛋白附着到柯萨奇病毒B-腺病毒受体（CAR）上而与其靶细胞结合。我们已经确定CAR在鼠和人光感受器上不显著表达，这可以解释EAM和腺病毒载体的低水平感染。为了克服这个问题，我们建议修改EAM的结构，使得它们现在结合细胞表面唾液酸而不是CAR。唾液酸大量存在于视杆细胞膜上。我们还建议测试腺病毒纤维最近显示有显着增强的神经元向性。我们将使用这些改进的EAM将bPDE递送到rd 10视网膜。我们将通过PDE测定、组织学、视网膜电图和Western分析来测量对光感受器的治疗效果。本研究完成后，我们将构建并测试一种载体，该载体将在治疗人类RP的I期临床试验中具有潜在用途。

DESCRIPTION (provided by applicant): Retinitis Pigmentosa (RP) is one of the most frequent causes of hereditary blindness in the United States, affecting approximately 1 in every 3000 individuals. There is currently no treatment available for this disease. The broad, long term objectives of this study are to develop a therapy for RP. Some forms of RP in humans, dogs and mice (rd) are caused by mutations in the beta subunit of cGMP phosphodiesterase (bPDE). In order to deliver a normal cDNA encoding bPDE to the rd retina, we have developed a novel class of safer and less-toxic adenovirus vectors termed Encapsidated Adenovirus Minichromosomes - (EAMs or 'gutted' vectors). EAMs have a 36Kb cloning capacity, allowing insertion of large upstream regulatory elements for regulated transgene expression and/or inclusion of multiple therapeutic transgene cassettes. We have used EAMs to temporarily rescue retinal degeneration in rd1 mice by approximately 10 weeks, which would otherwise be complete by 3 weeks postnatal. Our objectives are now focused on extending this short period of rescue to one that might be more therapeutically relevant to humans. EAMs bind to their target cells by attachment of the antenna-like fiber protein to the Coxsackievirus B-adenovirus Receptor (CAR). We have determined that CAR is not significantly expressed on murine and human photoreceptors which could explain the low levels of infection by EAMs and adenovirus vectors. In order to overcome this problem, we propose to modify the structure of EAMs such that they now bind cell surface sialic acid instead of CAR. Sialic acid is abundantly present on the rod cell membrane. We also propose to test adenovirus fibers recently shown to have significantly enhanced tropism for neurons. We will use these improved EAMs to deliver bPDE to the rd10 retina. We will measure therapeutic effects on photoreceptors by PDE assays, histology, electroretinograms and western analysis. Upon completion of this study, we will have constructed and tested a vector which will have potential use in a Phase 1 clinical trial for treatment of RP in humans.