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（由申请人提供）：杆衍生的锥生活力因子（RDCVF）是一种新发现的生存因子，可延长锥形光感受器的健康和寿命。该因素引起了很大的兴趣，因为它可以潜在地维持各种视网膜变性疾病的患者的细胞健康，包括色素性视网膜炎（RP）和与年龄相关的黄斑变性（AMD）。如果该因子不断由锥形感光体（例如视网膜色素上皮（RPE）细胞）不断产生，则可能会发挥最大的治疗作用。实现此作用的一种方法是通过重组病毒载体（例如腺相关病毒（AAV））将编码RDCVF的cDNA编码为这些细胞。该建议旨在为测试AAV介导的RDCVF延迟视网膜变性的能力提供基础。在AIM 1中，我将使用靶向RPE和感光细胞的AAV矢量在体外提供RDCVF并验证适当的转基因表达。然后，我将使用一种新型的器官培养系统测试其促进锥生活力的能力，在该系统中，原发性RPE细胞感染了AAV.RDCVF，并将其与退化的全骨视网膜外植体共同培养。在AIM 2中，我将这些载体从锥形和杆变性中向下注入转基因小鼠，并寻找感光受体救援和作用保存。我还将进行研究，以比较小鼠与人视网膜中RDCVF的定位和表达水平，并将检查杆脱温的人视网膜，以减少RDCVF表达的降低，如小鼠所见。这些实验将在体内建立稳定的RDCVF输送系统，并在各种形式的视网膜变性中评估其生存力效应，并将提供有关将这些研究转化为人类应用至关重要的表达模式的信息。在AIM 3中，我将研究暴露在RDCVF短形式中的疏水片在介导其锥生活力效应中的作用；我还将探索其在介导RDCVF多聚化中的作用，这是基于初步数据假设的效果。我将使用缺失和定向诱变进行这些分析，并希望收集有关RDCVF对光感受器的保护作用的重要机械信息。总而言之，该项目将提供有关锥体保护神经营养因子RDCVF的生物学活性以及该蛋白在退化感光体上的病毒介导的视网膜表达的疗效。这项工作最终可以为人类临床试验铺平道路，以测试AAV介导的RDCVF递送防止遗传性或获得形式的视网膜变性形式的失明的能力。