Rescue of GUCY1*B Phenotype Using Somatic Gene Therapy

描述（由申请人提供）：我们研究项目的目标是开发遗传性视网膜疾病的治疗方法，以支持长期感光功能和生存。三种方法主导了对这些疾病有效的开发：矫正基因，抗凋亡和神经营养疗法。从这些疗法中获得的益处相对短暂，许多减缓但不能防止感光细胞的退化。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配对的高度靶向组合治疗的治疗益处是否显著大于单独使用矫正基因治疗获得的治疗益处。我们将在LCA 1的GUCY 1 *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LCA 1的极好模型，而且还提供了检查联合治疗对视锥细胞的影响的独特机会。本项目分为两个研究目标：目的1 -开发双启动子、自失活、绝缘的慢病毒载体，能够将多种治疗性转基因递送到特定的视网膜细胞类型;目的2 -验证以下假设：将矫正基因治疗与靶向抗-凋亡或靶向神经营养疗法。对于目标1，将构建双启动子载体，其使用细胞特异性启动子将其治疗性货物的表达限制于（1）视锥细胞和视杆细胞，（2）单独的视杆细胞，和（3）M？ller细胞。将对具有所需细胞表达特征的载体进行修饰，以提供矫正（RetGC 1）、抗凋亡（X连锁凋亡抑制剂，XIAP）和神经营养（视杆细胞源性视锥细胞活力因子，RdCVF;胶质细胞源性神经营养因子，GDNF）疗法。每一种治疗性蛋白质或携带它们的载体都将被标记上一种独特的荧光蛋白，我们将用这种荧光蛋白来分析这些治疗对感光细胞存活的影响。将载体包装到慢病毒中，并使用荧光显微镜、免疫组织化学、RT-PCR和western blot分析其体内表达特性。对于目标2，将RetGC 1与XIAP、RdCVF或GDNF配对的组合治疗递送至GUCY 1 *B动物的视网膜，并且将使用视觉行为测定、视网膜电描记术、视网膜整体切片的荧光显微镜分析以及分子和蛋白质分析技术来评价这些治疗恢复光感受器的功能和促进光感受器存活的能力。将使用标准细胞计数方法以及最近邻和空间聚类分析技术分析经处理视网膜的整个安装的荧光照片。所有这些分析的结果将允许我们确定组合治疗是否有效地增加感光细胞的存活，以及是否存在增强感光细胞存活的递送治疗之间的协同作用的证据。 公共卫生相关性：影响视锥细胞的遗传性视网膜疾病是最使人衰弱的人类视网膜疾病之一。该提案中描述的研究旨在检查由矫正基因，抗凋亡和神经营养疗法组成的新组合治疗的能力，以支持和预防受遗传性视网膜疾病影响的视锥细胞和视杆细胞的变性。这项研究将使用具有丰富视锥细胞视网膜的遗传性视网膜疾病动物模型进行。

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.