Unfolded Protein Response as a Therapeutic Target for ADRP Animal Models

未折叠蛋白反应作为 ADRP 动物模型的治疗靶点

基本信息

批准号：
7948809
负责人：
Marina Gorbatyuk
金额：
$ 21.75万
依托单位：
UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7948809
关键词：
11 cis Retinal Accounting Affect Animal Model Antibodies Apoptosis Apoptotic Biological Preservation Blindness Caspase Cell Death Cessation of life Comparative Study Contrast Sensitivity Data Deterioration Development Disease Electroretinography Endoplasmic Reticulum Ethnic group Evaluation GADD45 Gene Transfer Genes Genetic Variation Goals Golgi Apparatus Inherited Investigational Drugs Knockout Mice Lead Location Measures Mediating Messenger RNA Methods Modeling Molecular Molecular Chaperones Monitor Mus Mutation Nucleosomes Opsin Optometry Pathogenesis Pathway interactions Patients Photoreceptors Proteins Rattus Resistance Retina Retinal Retinal Degeneration Retinitis Pigmentosa Reverse Transcriptase Polymerase Chain Reaction Rhodopsin Role Serotyping Severities Signal Transduction Small Interfering RNA Small RNA Structure Supplementation Techniques Testing Therapeutic Transgenic Mice Viral Virus Vision Visual impairment Vitamin A X inherited trait base biological adaptation to stress caspase 12 caspase-7 defined contribution design dosage drug discovery endoplasmic reticulum stress gene therapy improved functioning molecular chaperone GRP78 morphometry mouse model mutant novel programs promoter protein misfolding protein transport response success therapeutic target trafficking transcription factor CHOP vector

项目摘要

DESCRIPTION (provided by applicant): This project is focused on the elucidation of the role of the Unfolded Protein Response (UPR) in autosomal dominant retinitis pigmentosa (ADRP) pathogenesis and development of the gene therapy based on modulation of the UPR signaling markers. Retinitis pigmentosa (RP) is the most common inherited form of blindness, affecting about 1 in every 4000 people in all ethnic groups worldwide. RP can be transmitted either as an autosomal dominant (ADRP), autosomal recessive (ARRP), or X-linked trait. More than 100 mutations in rhodopsin account for approximately 30% of ADRP cases with varying severity of visual impairment. Misfolded opsin interferes with the trafficking of wild-type rhodopsin, accumulates in the endoplasmic reticulum (ER) and stimulates a signal transduction cascade known as the Unfolded Protein Response (UPR). If unchecked, this pathway triggers photoreceptor death, presumably through apoptosis. Although supplementation with vitamin A may be beneficial in some cases, currently, there is no effective pharmacological therapy for ADRP. Therefore, the major objective of this proposal is to determine whether the gene therapy based on the re-programming of the ER stress response caused by aberrant rhodopsin is a viable treatment, unlimited by different localizations of rhodopsin mutations (P23H and T17M). In two mouse models of ADRP, we plan to reprogram the ER stress signaling by viral delivery of the molecular chaperone GRP78/BiP and delivery of small interfering siRNAs targeting caspase-7, caspase-12 and pro-apoptotic CHOP/GADD153 mRNAs to diminish the level of apoptosis in ADRP photoreceptors. For each ADRP model, we plan to: (1) modulate the UPR in favor of activation of pro-survival pathway by over-expression of BiP protein; (2) suppress apoptosis by diminishing levels of activated caspase-7 and caspase-12 and (3) inhibit the CHOP-associated apoptosis by targeting CHOP mRNA. We will monitor survival of photoreceptors using electroretinography and morphometry and will measure the activation of the ER stress and apoptosis using specific antibodies and RT-PCR. We will also measure improvement in vision using Optometry, a technique that can evaluate both acuity and contrast sensitivity in mice. We anticipate that the success of this approach will also require the appropriate combination of AAV serotype, vector dosage, photoreceptor specific promoter and optimized expression for the chaperone BiP. While AAV mediated gene transfer is being developed for treatment of RP, the suppression of ER stress and of apoptosis using chaperones is novel. This approach may overcome the genetic diversity of this disease and reveal the pathways of cell death that lead from mutation to retinal degeneration. PUBLIC HEALTH RELEVANCE: Our goal is to design gene therapy for blinding disorder known as Autosomal Dominant Retinitis Pigmentosa (ADRP). We plan to study cellular mechanisms involved in photoreceptor death and develop a gene therapy based on the blockage of apoptosis in ADRP photoreceptors. Using a harmless virus, we plan to deliver molecular chaperon BiP and small RNA molecules to improve function and structure of photoreceptors and also intend to study the mechanism by which these therapeutic molecules provide therapy.

描述（由申请人提供）：该项目的重点是阐明未折叠蛋白反应（UPR）在常染色体显性色素性视网膜炎（ADRP）发病机制中的作用，并开发基于 UPR 信号标记调节的基因治疗。色素性视网膜炎 (RP) 是最常见的遗传性失明，全世界各族裔中每 4000 人中约有 1 人受影响。 RP 可以通过常染色体显性 (ADRP)、常染色体隐性 (ARRP) 或 X 连锁性状传播。超过 100 种视紫质突变导致 ADRP 病例中约 30% 具有不同严重程度的视力障碍。错误折叠的视蛋白会干扰野生型视紫红质的运输，在内质网 (ER) 中积累，并刺激称为未折叠蛋白反应 (UPR) 的信号转导级联。如果不加以控制，该途径可能会通过细胞凋亡触发光感受器死亡。尽管补充维生素 A 在某些情况下可能有益，但目前尚无针对 ADRP 的有效药物治疗方法。因此，该提案的主要目的是确定基于异常视紫红质引起的内质网应激反应重新编程的基因治疗是否是一种可行的治疗方法，不受视紫红质突变（P23H和T17M）不同定位的限制。在两个 ADRP 小鼠模型中，我们计划通过病毒递送分子伴侣 GRP78/BiP 和递送靶向 caspase-7、caspase-12 和促凋亡 CHOP/GADD153 mRNA 的小干扰 siRNA 来重新编程 ER 应激信号，以减少ADRP 光感受器细胞凋亡水平。对于每个 ADRP 模型，我们计划：（1）通过 BiP 蛋白的过度表达来调节 UPR，以有利于促生存途径的激活； (2) 通过降低活化的 caspase-7 和 caspase-12 的水平来抑制细胞凋亡，(3) 通过靶向 CHOP mRNA 来抑制 CHOP 相关的细胞凋亡。我们将使用视网膜电图和形态测定法监测光感受器的存活，并使用特异性抗体和 RT-PCR 测量 ER 应激和细胞凋亡的激活。我们还将使用验光来测量视力的改善情况，这是一种可以评估小鼠的敏锐度和对比敏感度的技术。我们预计这种方法的成功还需要 AAV 血清型、载体剂量、光感受器特异性启动子和伴侣 BiP 的优化表达的适当组合。虽然 AAV 介导的基因转移正在开发用于治疗 RP，但使用分子伴侣抑制 ER 应激和细胞凋亡是新颖的。这种方法可能会克服这种疾病的遗传多样性，并揭示从突变到视网膜变性的细胞死亡途径。公共健康相关性：我们的目标是设计针对常染色体显性视网膜色素变性（ADRP）的致盲性疾病的基因疗法。我们计划研究光感受器死亡的细胞机制，并开发一种基于阻断 ADRP 光感受器细胞凋亡的基因疗法。我们计划使用无害的病毒来传递分子伴侣 BiP 和小 RNA 分子，以改善光感受器的功能和结构，并打算研究这些治疗分子提供治疗的机制。