GENE TRANSFER IN PHOTORECEPTOR CELLS

我们的长期目标是建立基因的分子基础 转移是预防或延缓退变的合理途径 是遗传性疾病的特征的感光细胞 外视网膜，如视网膜色素变性(RP)。近期 事态发展表明，有一天治疗性干预可能会 可能，包括针对以下目标的基因增强或替换 防止失明。这些疗法的最终成功将 依赖于光感受器细胞新表达的能力 引入了基因。RP基因治疗领域的研究进展 由于缺乏感染成熟病毒的可用媒介而变得缓慢 感光细胞。然而，最近在设计一种 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)在体内显示出稳定的基因转移。 分化成终末分化的神经元。这项提案代表了 分子生物学家和病毒学家的合作 构建携带人类免疫缺陷病毒缺陷基因的HIV载体 RD突变小鼠光感受器细胞。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 活体以确定传染性的数量和能力 转导基因在光感受器细胞中发挥作用。这项建议 是基于我们的初步结果显示改善了基因转移 与β-半乳糖苷酶报告基因连锁的逆转录病毒进入视网膜 取自新生小鼠的外植体。慢病毒连锁载体的构建 到视蛋白启动子和报告基因的体外测试将使用 分离的视网膜细胞和/或新生小鼠的视网膜外植体。 RD突变小鼠将被用来证明引入一个 野生型基因可以纠正或改善异常的表型。我们会 确定脑脊液β亚基是否存在缺陷 RD小鼠PR细胞中的磷酸二酯酶(β-PDE) 通过病毒介导的β-PDE基因的传递而纠正。vt.在.的基础上 成功地完成了体外研究，注射了β- 将使用pDe慢病毒构建到RD的视网膜下空间 为了实现对光感受器变性的长期抢救 变种人。将通过以下方法评估β-PDE的治疗效果 组织学检查及生化和分子生物学 方法慢病毒-β-PDE转导后。当成功时，这是 该项目可能导致最终设计基因治疗方案，以 延缓感光细胞退化或挽救感光细胞 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.