Rescue of GUCY1*B Phenotype Using Somatic Gene Therapy

使用体细胞基因疗法拯救 GUCY1*B 表型

• 批准号: 7995194
• 负责人: SUSAN Lynn SEMPLE-ROWLAND
• 金额: $ 33.95万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7995194
• 关键词: Affect; Aftercare; Animal Model; Animals; Antibodies; Apoptotic; Behavior; Biological Assay; Biological Models; Birds; Blindness; CD44 gene; Cell Count; Cell Survival; Cells; Cessation of life; Characteristics; Chickens; Clinic; Clinical Trials; Cluster Analysis; Combined Modality Therapy; Complement; Control Groups; Data; Development; Disease; Effectiveness; Electroretinography; Exhibits; Eye; GDNF gene; GRK1 gene; Gene Delivery; Genes; Glial Fibrillary Acidic Protein; Goals; Histocytochemistry; Human; Immunohistochemistry; Inherited; Intervention; Investigation; Knockout Mice; Lead; Leber' s amaurosis; Lentivirus Vector; Life; Light; Mediating; Methods; Microscopic; Microscopy; Modeling; Molecular; Monitor; Morphology; Mus; Neuroglia; Performance; Peripheral; Phenotype; Photoreceptors; Protein Isoforms; Proteins; RNA Interference; Rattus; Relative (related person); Reporter; Research; Research Personnel; Retina; Retinal; Retinal Cone; Retinal Diseases; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Series; Solutions; Somatic Gene Therapy; Structure; Subfamily lentivirinae; Techniques; Testing; Therapeutic; Therapeutic Intervention; Time; Transgenes; Vertebrate Photoreceptors; Vision; Visual; Visual Fields; Western Blotting; Yang; behavior test; blind; caspase-3; catalyst; cell type; cost; design; disease phenotype; effective therapy; follow-up; functional restoration; gene therapy; guanylate cyclase 1; hatching; human BIRC4 protein; improved; in vivo; light microscopy; loss of function; meetings; neurotrophic factor; novel; photoreceptor degeneration; prevent; programs; promoter; protective effect; public health relevance; research study; response; retinal rods; therapeutic gene; therapeutic protein; therapeutic transgene; therapy design; therapy development; treatment effect; treatment strategy; vector; vector control

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.

描述(由申请人提供)：我们研究计划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。有三种方法主导了开发有效治疗这些疾病的努力：纠正性基因疗法、抗凋亡疗法和神经营养疗法。从这些疗法中获得的益处是相对短暂的，许多疗法减缓了光感受器细胞的退化，但并不能阻止退化。这项提案中概述的研究旨在确定将纠正性基因疗法与抗凋亡或神经营养疗法搭配使用的高度靶向性联合治疗的治疗益处是否显著大于仅使用纠正性基因疗法获得的疗效。我们将在LCA1的GUCY1*B模型的富含视锥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，而且提供了一个独特的机会来检验联合治疗对视锥细胞的影响。该项目分为两个研究目标：目的1-开发双启动子、自我灭活、隔离的慢病毒载体，能够向特定类型的视网膜细胞输送多个治疗性转基因；目的2-验证靶向纠正性基因治疗可以通过结合靶向抗凋亡或靶向神经营养治疗来防止光感受器退化的假设。对于目标1，将构建双启动子载体，将其治疗货物的表达限制在(1)视锥和视杆细胞，(2)视杆细胞，和(3)使用细胞特异性启动子的M？ler细胞。具有所需细胞表达特性的载体将被改造，以提供纠正性(RetGC1)、抗凋亡(X连锁细胞凋亡抑制物，XIAP)和神经营养(杆状衍生锥体生存因子，RdCVF；胶质衍生神经营养因子，GDNF)疗法。每一种治疗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，我们将使用该蛋白来分析这些治疗对光感受器细胞存活的影响。将载体包装成慢病毒，利用荧光显微镜、免疫组织化学、RT-PCR和免疫印迹等方法对其体内表达特性进行分析。对于目标2，将把RetGC1与XIAP、RdCVF或GDNF配对注射到GUCY1*B动物的视网膜，这些治疗方法恢复光感受器功能和促进光感受器存活的能力将通过视觉行为分析、视网膜电图、视网膜整体的荧光显微镜分析以及分子和蛋白质分析技术来评估。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整个处理过的视网膜的荧光照片。所有这些分析的结果将使我们能够确定联合治疗在提高光感受器细胞存活率方面是否有效，以及是否有证据表明所提供的治疗方法之间存在协同作用，以提高光感受器细胞的存活率。 公共卫生相关性：影响视锥细胞的遗传性视网膜疾病是人类视网膜最虚弱的疾病之一。这项建议中描述的研究旨在检验由纠正性基因疗法、抗凋亡疗法和神经营养疗法组成的新的联合疗法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退化的能力。这项研究将使用遗传性视网膜疾病的动物模型进行，该动物模型拥有富含视锥的视网膜。