GENE TRANSFER IN PHOTORECEPTOR CELLS

我们的长期目标是建立基因的分子基础 移植是防止或延缓退化的合理方法 这是遗传性疾病的特征 视网膜色素变性（Retinitis Pigmentosa，RP）最近 发展表明，治疗干预可能有一天 可能的，包括基因扩增或替换，旨在 防止视力丧失。这些疗法的最终成功将 依赖于感光细胞表达新的 引入基因。RP的基因治疗领域的进展已经 由于缺乏可感染成熟病毒的载体， 感光细胞然而，最近的发展，在设计一个 基于人类免疫缺陷病毒的逆转录病毒载体系统 (HIV)（称为慢病毒）已经显示出稳定的体内基因转移 最终分化成神经元。该提案代表了 一位分子生物学家和一位病毒学家合作， 构建基于HIV的载体，以携带在所述细胞中缺陷的基因， rd突变小鼠的感光细胞。视网膜细胞生物学家 将评估载体构建体在体外和体内的作用。 体内，以确定感染性的量和 转导的基因在感光细胞中发挥作用。这项建议 是基于我们的初步结果，显示改善的基因转移， 逆转录病毒连接到β-半乳糖苷酶报告基因进入视网膜 新生小鼠的外植体。慢病毒载体连接的构建 视蛋白启动子和报告基因的作用将在体外使用 分离的视网膜细胞和/或来自新生小鼠的视网膜外植体。 rd突变小鼠将被用来表明， 野生型基因纠正或改善异常表型。我们将 确定β-亚基的缺陷是否 rd小鼠的PR细胞中的磷酸二酯酶（β-PDE）可以是 通过病毒介导的β-PDE基因的递送来校正。后 成功完成体外研究，注射β- 将使用PDE慢病毒构建体进入rd的视网膜下腔 为了实现长期的光感受器退化的拯救， 变种人将通过以下方法评价β-PDE的治疗效果： 组织学检查和生物化学及分子生物学检查 方法慢病毒-β-PDE转导后。如果成功，这 该项目可能导致基因治疗方案的最终设计， 延缓光感受器退化或拯救光感受器细胞， RP患者

Our long range goal is to establish the molecular basis for gene transfer as a rational approach to prevent or retard the degeneration of photoreceptor cells that is characteristic of hereditary diseases of the outer retina such as Retinitis Pigmentosa (RP). Recent developments suggest that therapeutic intervention may some day be possible, including gene augmentation or replacement aimed at preventing loss of vision. The eventual success of these therapies will depend on the ability of photoreceptor cells to express newly introduced genes. Progress in the field of gene therapy for RP has been slowed by the lack of available vectors which infect mature photoreceptor cells. However, recent developments in the design of a retroviral vector system based on the human immunodeficiency virus (HIV) (termed a lentivirus) have shown stable in vivo gene transfer into terminally differentiated neurons. This proposal represents the collaboration of a molecular biologist and a virologist who will construct an HIV-based vector to carry the gene deficient in the photoreceptor cells of the rd mutant mouse. A retinal cell biologist will evaluate the action of the vector construct both in vitro and in vivo to determine the amount of infectivity and the ability of the transduced gene to function in the photoreceptor cells. This proposal is based on our preliminary results showing improved gene transfer of retrovirus linked to the beta-galactosidase reporter gene into retinal explants from neonatal mice. Construction of a lentiviral vector linked to an opsin promoter and reporter gene will be tested in vitro using dissociated retinal cells and/or retinal explants from neonatal mice. The rd mutant mouse will be used to show that the introduction of a wild-type gene corrects or ameliorates an abnormal phenotype. We will determine whether the deficiency in the beta-subunit of phosphodiesterase (beta-PDE) in PR cells of the rd mouse can be corrected by viral-mediated delivery of the beta-PDE gene. Upon successful completion-of the in vitro studies, injection of the beta- PDE lentiviral construct into the subretinal space of rd will be used to achieve long-term rescue of photoreceptor degeneration in this mutant. Therapeutic effects of beta-PDE will be evaluated by histological examination and by biochemical and molecular biological methods after lentiviral-beta-PDE transduction. When successful, this project may lead to the eventual design of gene therapy protocols to retard photoreceptor degeneration or rescue photoreceptor cells in patients with RP.