Rescue of GUCY1*B Phenotype Using Somatic Gene Therapy

描述（由申请人提供）：我们的研究项目的目标是开发支持长期光感受器功能和生存的遗传性视网膜疾病的治疗方法。三种方法主导了对这些疾病的有效治疗：纠正基因、抗凋亡和神经营养疗法。从这些疗法中获得的益处是相对短暂的，许多减缓但不能阻止光感受器细胞的退化。本提案中概述的研究旨在确定将矫正基因治疗与抗凋亡或神经营养治疗相结合的高靶向联合治疗的治疗益处是否明显大于单独使用矫正基因治疗获得的治疗益处。我们将在LCA1的GUCY1*B模型的富含视锥细胞的视网膜中进行这些实验，该模型系统不仅是LCA1的优秀模型，而且为检查联合治疗对视锥细胞的影响提供了独特的机会。该项目分为两个研究目标：AIM 1 -开发双启动子、自我灭活、绝缘的慢病毒载体，能够将多种治疗性转基因传递到特定的视网膜细胞类型；目的2：验证接受靶向矫正基因治疗的光感受器变性可以通过与靶向抗凋亡或靶向神经营养治疗相结合来预防的假设。对于Aim 1，将构建双启动子载体，将其治疗货物的表达限制为(1)锥细胞和杆状细胞，(2)单独的杆状细胞，以及(3)使用细胞特异性启动子的M¿ller细胞。具有所需细胞表达特征的载体将被修改以提供纠正（RetGC1），抗凋亡（X-linked inhibitor of apoptosis， XIAP）和神经营养（杆状锥体生存因子，RdCVF；胶质源性神经营养因子，GDNF）治疗。每一种治疗性蛋白，或携带它们的载体，都将被标记上一种独特的荧光蛋白，我们将用它来分析这些治疗对光感受器细胞存活的影响。载体将被包装成慢病毒，利用荧光显微镜、免疫组织化学、RT-PCR和western blot分析其在体内的表达特性。对于Aim 2，将RetGC1与XIAP、RdCVF或GDNF配对的联合治疗将被送到GUCY1*B动物的视网膜上，这些治疗恢复光感受器功能和启动子存活的能力将通过视觉行为分析、视网膜电图、视网膜全支架的荧光显微镜分析以及分子和蛋白质分析技术进行评估。采用标准细胞计数法、最近邻法和空间聚类分析技术，对处理后视网膜全片的荧光蒙太奇进行分析。所有这些分析的结果将使我们能够确定联合治疗是否有效地提高光感受器细胞的存活率，以及是否有证据表明在传递的治疗之间存在协同作用，以提高光感受器的存活率。

DESCRIPTION (provided by applicant): The goal of our research program is to develop therapies for inherited retinal diseases that support long-term photoreceptor function and survival. Three approaches have dominated efforts to develop effective for these diseases: corrective gene, anti-apoptotic, and neurotrophic therapies. The benefits obtained from these therapies have been relatively short-lived, many slowing but not preventing degeneration of the photoreceptor cells. The research outlined in this proposal is designed to determine if the therapeutic benefits of highly targeted combination treatments pairing corrective gene therapy with either anti-apoptotic or neurotrophic therapy are significantly greater than those obtained using corrective gene therapy alone. We will conduct these experiments in the cone-rich retinas of the GUCY1*B model of LCA1, a model system that is not only an excellent model for LCA1 but also provides a unique opportunity to examine the effects of combination treatments on cone cells. This project is divided into two research aims: AIM 1 - Develop dual-promoter, self inactivating, insulated lentiviral vectors capable of delivering multiple therapeutic transgenes to specific retinal cell types; AIM 2 -To test the hypothesis that degeneration of photoreceptors that have been treated with targeted corrective gene therapy can be prevented by combining corrective gene therapy with either targeted anti-apoptotic or targeted neurotrophic therapies. For Aim 1, dual-promoter vectors will be constructed that restrict expression of their therapeutic cargos to (1) cone and rod cells, (2) rod cells alone, and (3) M¿ller cells using cell-specific promoters. Vectors with the desired cellular expression characteristics will be modified to deliver corrective (RetGC1), anti-apoptotic (X-linked inhibitor of apoptosis, XIAP), and neurotrophic (rod derived cone viability factor, RdCVF; glial-derived neurotrophic factor, GDNF) therapies. Each of the therapeutic proteins, or the vectors carrying them, will be tagged with a unique fluorescent protein that we will use to analyze the effects of these treatments on photoreceptor cell survival. The vectors will be packaged into lentivirus and their expression characteristics will be analyzed in vivo using fluorescent microscopy, immuno-histochemistry, RT-PCR and western blot. For Aim 2, combination treatments pairing RetGC1 with either XIAP, RdCVF, or GDNF will be delivered to the retinas of GUCY1*B animals and the ability of these treatments to restore function to and promoter survival of photoreceptors will be evaluated using visual behavior assays, electroretinography, fluorescent microscopic analyses of retinal whole mounts, and molecular and protein analyses techniques. The fluorescent photomontages of whole mounts of the treated retinas will be analyzed using standard cell count methods and nearest neighbor and spatial clustering analyses techniques. The results of all of these analyses will permit us to determine if the combination treatments are effective in increasing survival of photoreceptor cells and if there is evidence of synergy between the delivered therapies that enhances photoreceptor survival. PUBLIC HEALTH RELEVANCE: Inherited retinal diseases that affect cone cells are among the most debilitating human retinal diseases. The research described in this proposal is designed to examine the ability of new combination treatments consisting of corrective gene, anti-apoptotic, and neurotrophic therapies to support and prevent degeneration of cone and rod cells affected by inherited retinal disease. This research will be carried out using an animal model of inherited retinal disease that possesses a cone-rich retina.