RESCUE OF THE RD PHENOTYPE USING SOMATIC GENE THERAPY

描述(改编自申请者摘要)：长期目标 这项研究计划是为了了解生物化学过程中 光感受器细胞如果被破坏，会导致细胞功能障碍和 退化。本程序中研究的模型系统是视网膜 变性(RD)鸡--唯一的遗传性视网膜动物模型 一种视网膜视锥占优势的疾病。在之前的资助中 期间，研究人员确定RD基因编码 光感受器鸟苷环化酶1(Gc1)，一个对视锥细胞和 棒状光转导。他们的分析结果表明，RD 鸡是Leber先天性黑色素症(LCA1)的模型，LCA1是一种遗传性 视网膜色素性视网膜病家系视网膜疾病 导致新生儿失明的退化。他们提议 RD鸡Gc1零突变导致异常低 视锥细胞和视杆细胞中cGMP的水平，光转导的丧失，以及 最终导致光感受器退化。这些研究的目标是 在这份提案中概述的是挽救视网膜变性 应用体细胞基因治疗的RD鸡的表型。他们将测试 RD光感受器细胞中正常Gc1表达的假说 足以恢复光感受器功能并防止 感光细胞死亡。这项建议的目的是：(1)确定 能够指导表达的GCAP1启动子片段 报告基因在体外对光感受器的作用(鸡胚胎视网膜 细胞培养)和体内(鸡胚胎)；(2)检测能力 选择GCAP1启动子驱动光感受器中Gc1的表达 RD/RD视网膜培养中的细胞；以及(3)挽救视网膜 慢病毒感染RD雏鸡的退变表型 将GCAP1/GC1转基因到视网膜祖细胞或有丝分裂后的视网膜细胞。 他们将确定GCAP1的转录起点并检查 GCAP1启动子片段的特异性和活性水平 鸡视网膜细胞培养及病毒瞬时转染法的研究 9-11期鸡胚的转导。GCAP1的能力 启动子驱动瞬时转基因RD/RD中Gc1的表达 视网膜培养将通过测量Gc1活性进行检查。评估 Gc1基因治疗的有效性，他们将检查(1) RD/RD视网膜的视网膜电信号反应，(2)视网膜 形态，(3)Gc1表达，和(4)表达谱 昼夜节律调控基因的正常时间表达模式为 RD/RD视网膜受损。不同处理对RD/RD鸡的影响比较 胚胎发育与孵化的对比将使人们能够确定 如果把这种疗法用在病人身上，疗效就会提高。 正常基因在体内最早的表达。

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.