Rod-dependent Cone Viability Factor: Towards a prevention of blindness

视杆细胞依赖性视锥细胞活力因子：预防失明

• 批准号: 7333034
• 负责人: Defne Audrey Amado
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333034
• 关键词: Age related macular degeneration; American; Animal Model; Area; Biological; Biological Preservation; Blindness; Cells; Clinical Trials; Coculture Techniques; Complementary DNA; Data; Degenerative Disorder; Dependovirus; Epithelial; Foundations; Future; Gel; Gene Delivery; Gene Transduction Agent; Genes; Genetic Transcription; Glean; Health; Human; Hybrids; Hydrophobic Surfaces; Immunohistochemistry; In Situ Hybridization; In Vitro; Individual; Inherited; Injection of therapeutic agent; Lead; Life; Mediating; Methods; Modeling; Mus; Names; Organ Culture Techniques; Patients; Pattern; Photoreceptors; Prevention; Protein Overexpression; Proteins; Recombinants; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinal Pigments; Retinitis Pigmentosa; Reverse Transcriptase Polymerase Chain Reaction; Role; Running; Site-Directed Mutagenesis; Structure of retinal pigment epithelium; Sum; System; Techniques; Testing; Therapeutic Effect; Time; Transgenic Mice; Translating; Translations; Vertebrate Photoreceptors; Viral; Viral Vector; Vision; Work; adeno-associated viral vector; base; cell type; cone-rod degeneration; day; disease-causing mutation; gene therapy; in vivo; insight; interest; neurotrophic factor; novel; prevent; protective effect; protein expression; research study; retinal rods; transgene expression; vector

DESCRIPTION (provided by applicant): Rod-Derived cone viability factor (RdCVF) is a newly discovered survival factor that prolongs the health and life of cone photoreceptors. This factor has attracted considerable interest because it could potentially maintain health of cells in patients with a diverse set of retinal degenerative diseases, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD). It is likely that this factor will exert maximal therapeutic effect if it is produced constantly by cells neighboring the cone photoreceptors, such as retinal pigment epithelial (RPE) cells. One approach to achieve this effect is to deliver the cDNA encoding RdCVF to these cells via a recombinant viral vector such as adeno-associated virus (AAV). This proposal aims to provide the foundation for testing the ability of AAV-mediated delivery of RdCVF to delay retinal degeneration in a diverse set of animal models. In Aim 1, I will use AAV vectors targeting RPE and photoreceptor cells to deliver RdCVF in vitro and verify appropriate transgene expression. I will then test its ability to promote cone viability, using a novel organ culture system in which primary RPE cells infected with AAV.RdCVF are co-cultured with degenerating all-cone retinal explants. In Aim 2, I will inject these vectors subretinally into transgenic mice with cone and rod degeneration, and look for photoreceptor rescue and funtional preservation. I will also perform studies comparing localization and expression levels of RdCVF in mouse versus human retinas, and will examine rod-degenerative human retinas for diminution of RdCVF expression as is seen in mice. These experiments will establish a stable RdCVF delivery system in vivo and assess its viability effects in a variety of forms of retinal degeneration, and will provide information about expression patterns critical for translation of these studies into human applications. In Aim 3, I will examine the role of the hydrophobic patch exposed on the short form of RdCVF in mediating its cone viability effects; I will also explore its role in mediating RdCVF multimerization, an effect hypothesized based on preliminary data. I will perform these analyses using deletion and site-directed mutagenesis, and hope to glean vital mechanistic information about the protective effects of RdCVF on photoreceptors. In sum, this project will provide important data about the biological activity of the cone-protective neurotrophic factor RdCVF and the efficacy of virally-mediated constitutive retinal expression of this protein on degenerating photoreceptors. This work could ultimately pave the way for a human clinical trial testing the ability of AAV-mediated delivery of RdCVF to prevent blindness in inherited or acquired forms of retinal degeneration.

描述(申请人提供)：视锥细胞存活因子(RdCVF)是新近发现的一种延长视锥细胞感光细胞健康和寿命的生存因子。这一因子已经引起了相当大的兴趣，因为它可以潜在地维持多种视网膜退行性疾病患者的细胞健康，包括视网膜色素变性(RP)和年龄相关性黄斑变性(AMD)。如果视锥感光细胞周围的细胞，如视网膜色素上皮(RPE)细胞不断产生该因子，可能会发挥最大的治疗效果。实现这一效果的一种方法是通过腺相关病毒(AAV)等重组病毒载体将编码RdCVF的cDNA传递给这些细胞。该建议旨在为在不同的动物模型中测试AAV介导的RdCVF延缓视网膜变性的能力提供基础。在目标1中，我将使用靶向RPE和光感受器细胞的AAV载体在体外传递RdCVF，并验证是否有合适的转基因表达。然后，我将使用一种新的器官培养系统来测试其促进视锥细胞存活的能力，在该系统中，感染AAV.RdCVF的原代RPE细胞与退化的全视锥视网膜外植体共培养。在目标2中，我将把这些载体注射到视锥和视杆变性的转基因小鼠视网膜下，寻找光感受器的挽救和功能保存。我还将进行研究，比较RdCVF在小鼠和人视网膜中的定位和表达水平，并将像在小鼠中看到的那样，检查杆状变性的人视网膜中RdCVF表达的减少。这些实验将在体内建立稳定的RdCVF递送系统，并评估其在各种形式的视网膜变性中的生存效果，并将提供有关表达模式的信息，这些信息对于将这些研究转化为人类应用至关重要。在目标3中，我将研究暴露在RdCVF短形上的疏水斑块在调节其锥体存活效应中的作用；我还将探讨它在介导RdCVF多聚化中的作用，这一效应是基于初步数据假设的。我将使用缺失和定点突变进行这些分析，并希望收集有关RdCVF对光感受器保护作用的重要机制信息。综上所述，该项目将提供有关锥体保护性神经营养因子RdCVF的生物学活性以及病毒介导的这种蛋白在退化的光感受器上的结构性视网膜表达的有效性的重要数据。这项工作最终可能为人类临床试验铺平道路，测试AAV介导的RdCVF传递预防遗传性或获得性视网膜变性失明的能力。