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

• 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
• 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

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）：本项目的重点是阐明未折叠蛋白反应（UPR）在常染色体显性视网膜色素变性（ADRP）发病机制中的作用，以及基于UPR信号标志物调节的基因治疗的开发。视网膜色素变性（RP）是最常见的遗传性失明形式，影响全世界所有种族群体中每4000人中约有1人。RP可以作为常染色体显性（ADRP），常染色体隐性（ARRP）或X连锁性状传播。超过100个视紫红质突变约占ADRP病例的30%，具有不同程度的视力损害。错误折叠的视蛋白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）的信号转导级联反应。如果不加抑制，该途径可能通过细胞凋亡触发感光细胞死亡。虽然在某些情况下补充维生素A可能是有益的，但目前还没有有效的药物治疗ADRP。因此，本提案的主要目的是确定基于异常视紫红质引起的ER应激反应的重编程的基因治疗是否是可行的治疗，不受视紫红质突变（P23 H和T17 M）的不同定位的限制。 在两种ADRP小鼠模型中，我们计划通过病毒递送分子伴侣GRP 78/BiP和递送靶向caspase-7、caspase-12和促凋亡CHOP/GADD 153 mRNA的小干扰siRNA来重新编程ER应激信号传导，以降低ADRP光感受器中的凋亡水平。对于每种ADRP模型，我们计划：（1）通过BiP蛋白的过表达调节UPR以有利于促存活途径的激活;（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量来监测光感受器的存活，并使用特异性抗体和RT-PCR来测量ER应激和细胞凋亡的激活。我们还将使用验光法测量视力的改善，验光法是一种可以评估小鼠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，但使用伴侣抑制ER应激和细胞凋亡是新颖的。这种方法可以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。 公共卫生相关性：我们的目标是设计基因治疗致盲性疾病称为常染色体显性视网膜色素变性（ADRP）。我们计划研究光感受器死亡的细胞机制，并开发基于ADRP光感受器凋亡阻断的基因治疗。使用无害的病毒，我们计划提供分子伴侣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。