Gene Therapy for Retinitis Pigmentosa

描述(申请人提供)：视网膜色素变性(RP)是美国最常见的遗传性失明原因之一，大约每3000人中就有1人受到影响。目前还没有治疗这种疾病的方法。这项研究的广泛和长期目标是开发一种治疗RP的方法。人类、狗和小鼠(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运送到RD视网膜，我们开发了一类新的更安全、毒性更低的腺病毒载体，称为微囊化腺病毒微型染色体-(EAMS或‘内脏’载体)。EAMS具有36Kb的克隆能力，允许插入用于受调控的转基因表达的大上游调控元件和/或包括多个治疗性转基因盒带。我们已经使用EAMS暂时挽救了Rd1小鼠大约10周的视网膜变性，否则将在出生后3周完成。我们现在的目标是将这一短暂的救援期延长到可能对人类更具治疗意义的救援期。EAM通过将类似天线的纤维蛋白附着在柯萨奇病毒B型腺病毒受体(CAR)上而与其靶细胞结合。我们已经确定CAR在小鼠和人的光感受器上没有显着表达，这可以解释EAMS和腺病毒载体低水平感染的原因。为了克服这个问题，我们建议修改EAMS的结构，使它们现在结合细胞表面唾液酸而不是CAR。唾液酸大量存在于杆状细胞膜上。我们还建议测试腺病毒纤维，最近被证明有显著增强的神经元的趋向性。我们将使用这些改进的EAMS将BPDE运送到RD10视网膜。我们将通过PDE分析、组织学、视网膜电信号和Western分析来评估对光感受器的治疗效果。在这项研究完成后，我们将构建并测试一种载体，该载体将有可能用于人类RP治疗的第一阶段临床试验。

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.