RESCUE OF THE RD PHENOTYPE USING SOMATIC GENE THERAPY

描述（改编自申请人摘要）：长期目标 这项研究计划是为了了解生物化学过程， 感光细胞，如果破坏，导致细胞功能障碍， 退化在这个程序中研究的模型系统是视网膜 鸡视网膜变性（RD）是遗传性视网膜病变的唯一动物模型， 一种视锥细胞占优势的视网膜疾病。在此前的融资中， 期间，研究人员确定rd基因编码 光感受器鸟苷酸环化酶1（GC 1），一个对视锥细胞和 视杆光转导他们的分析结果表明， 鸡是Leber先天性黑蒙（LCA 1）的模型， 视网膜色素变性视网膜疾病 导致新生儿失明的变性。他们提出 rd鸡的GC 1无效突变导致了异常低的 视锥细胞和视杆细胞中的cGMP水平，光转导损失，以及 最终导致感光细胞退化。研究的目标是 在这项建议中概述的是拯救视网膜变性 体细胞基因治疗对rd鸡表型的影响他们将测试 rd光感受器细胞中正常GC 1表达假说 足以恢复光感受器功能并防止 感光细胞死亡。本提案的目的是：（1）确定 能够指导表达的GCAP 1启动子的片段 报告基因的光感受器在体外（胚胎鸡视网膜 细胞培养）和体内（鸡胚）;（2）检查 选择的GCAP 1启动子驱动GC 1在感光细胞中的表达 RD/RD视网膜培养物中的细胞;和（3）拯救视网膜 使用慢病毒在RD鸡中递送 GCAP 1/GC 1转基因至视网膜祖细胞或有丝分裂后视网膜细胞。 他们将确定GCAP 1的转录起始点， GCAP 1启动子片段的特异性和活性水平， 鸡视网膜细胞培养物的瞬时转染和通过病毒 第9-11阶段鸡胚胎的转导。GCAP 1的能力 在瞬时转染的rd/rd中驱动GC 1表达的启动子 通过测量GC 1活性来检查视网膜培养物。评估 GC 1基因治疗的有效性，他们将检查（1） RD/RD视网膜的视网膜电图反应，（2）视网膜电图反应 形态学，（3）GC 1表达，和（4） 昼夜节律调节基因，其正常的时间表达模式是 在RD/RD视网膜中破坏。期间处理的RD/RD鸡的比较 胚胎发育与孵化时的对比将使人们能够确定 如果将其施用于患者， 正常基因在体内的最早表达。

DESCRIPTION (Adapted from applicant's abstract): The long-term goal of this research program is to understand the biochemical processes in photoreceptor cells that if disrupted, lead to cell dysfunction and degeneration. The model system studied in this program is the retinal degeneration (rd) chicken, the only animal model for inherited retinal disease that possesses a cone-dominant retina. In the previous funding period, the investigators determined that the rd gene encoded photoreceptor guanylate cyclase 1 (GC1), a gene critical for cone and rod phototransduction. The results of their analyses show that the rd chicken is a model for Leber's congenital amaurosis (LCA1), an inherited retinal disease of the retinitis pigmentosa family of retinal degenerations that causes blindness in newborn infants. They propose that the GC1 null mutation in the rd chicken leads to abnormally low levels of cGMP in cone and rod cells, loss of phototransduction, and eventually to photoreceptor degeneration. The goal of the studies outlined in this proposal is to rescue the retinal degeneration phenotype of the rd chicken using somatic gene therapy. They will test the hypothesis that expression of normal GC1 in rd photoreceptor cells is sufficient to restore photoreceptor function and prevent photoreceptor cell death. The aims of this proposal are: (1) to identify fragments of the GCAP1 promoter that are capable of directing expression of reporter genes to photoreceptors in vitro (embryonic chicken retina cell culture) and in vivo (chicken embryos); (2) to examine the ability of selected GCAP1 promoters to drive expression of GC1 in photoreceptor cells in rd/rd retinal cultures; and (3) to rescue the retinal degeneration phenotype in the rd chicken using lentivirus to deliver a GCAP1/GC1 transgene to retinal progenitor or post-mitotic retinal cells. They will determine the transcription start point of GCAP1 and examine the specificity and activity levels of GCAP1 promoter fragments by transient transfection of chicken retinal cell cultures and by viral transduction of stage 9-11 chicken embryos. The ability of the GCAP1 promoter to drive GC1 expression in transiently-transfected rd/rd retinal cultures will be examined by measuring GC1 activity. To assess the effectiveness of the GC1 gene therapy, they will examine (1) the electroretinographic responses of the rd/rd retina, (2) retinal morphology, (3) GC1 expression, and (4) expression profiles of circadian-regulated genes whose normal temporal expression pattern is disrupted in rd/rd retina. Comparisons of rd/rd chickens treated during embryonic development versus at hatching will allow one to determine if the efficacy of the therapy is enhanced if it is administered to the earliest expression of the normal gene in vivo.