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(申请人提供)：本项目致力于阐明未折叠蛋白反应(UPR)在常染色体显性遗传性视网膜色素变性(ADRP)发病机制中的作用，以及基于UPR信号标志物调控的基因治疗的发展。视网膜色素变性(RP)是最常见的遗传性失明形式，全世界所有种族中每4000人中就有1人受到影响。RP可作为常染色体显性遗传(ADRP)、常染色体隐性遗传(ARRP)或X连锁遗传。超过100个视紫红质突变约占ADRP病例的30%，其视力损害的严重程度各不相同。错误折叠的视蛋白干扰野生型视紫红质的运输，聚集在内质网(ER)，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，这一途径可能会通过细胞凋亡来触发感光器死亡。虽然补充维生素A在某些情况下可能是有益的，但目前还没有有效的药物治疗ADRP。因此，这项建议的主要目的是确定基于视紫红质异常引起的ER应激反应的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。在两种adrp小鼠模型中，我们计划通过病毒传递分子伴侣GRP78/Bip和针对caspase-7、caspase-12和促凋亡的CHOP/GADD153 mRNAs的小干扰siRNA来重新编程ER应激信号，以减少adrp光感受器的凋亡水平。对于每种ADRP模型，我们计划：(1)通过过度表达Bip蛋白来调节UPR，有利于激活促生存途径；(2)通过降低激活的caspase-7和caspase-12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描记术和形态计量学来监测光感受器的存活，并将使用特异性抗体和RT-PCR来测量内质网应激和细胞凋亡的激活情况。我们还将使用验光技术来测量视力的改善，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蛋白Bip的优化表达的适当组合。虽然AAV介导的基因转移正在被开发用于治疗RP，但使用伴侣蛋白抑制内质网应激和细胞凋亡是新的。这种方法可能会克服这种疾病的遗传多样性，并揭示导致从突变到视网膜退化的细胞死亡途径。 公共卫生相关性：我们的目标是设计针对常染色体显性遗传性视网膜色素变性(ADRP)的失明疾病的基因疗法。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光感受器凋亡的基因疗法。利用一种无害的病毒，我们计划运送分子伴侣Bip和小RNA分子来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。