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: 8575057
• 负责人: Marina Gorbatyuk
• 金额: $ 24.13万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8575057
• 关键词: Unfolded; Protein; Response; Therapeutic; Target

Unfolded Protein Response as a Therapeutic Target for ADRP Animal Models 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.

未折叠蛋白反应作为 ADRP 动物模型的治疗靶点 该项目的重点是阐明未折叠蛋白反应 (UPR) 在常染色体中的作用 显性视网膜色素变性（ADRP）发病机制及基因治疗的进展 UPR 信号标记的调节。色素性视网膜炎 (RP) 是最常见的遗传形式 失明，影响全世界各族群中每 4000 人中约 1 人。 RP 可以传输 作为常染色体显性 (ADRP)、常染色体隐性 (ARRP) 或 X 连锁性状。超过100个突变 视紫红质约占 ADRP 病例的 30%，具有不同严重程度的视力障碍。错误折叠 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。如果未选中，则此 途径可能通过细胞凋亡触发光感受器死亡。虽然补充维生素A 在某些情况下可能是有益的，目前，ADRP 尚无有效的药物治疗方法。所以， 该提案的主要目标是确定基于重编程的基因治疗是否 由异常视紫质引起的内质网应激反应是一种可行的治疗方法，不受不同部位的限制 视紫红质突变（P23H 和 T17M）。 在两个 ADRP 小鼠模型中，我们计划通过病毒传递来重新编程 ER 应激信号 分子伴侣 GRP78/BiP 和针对 caspase-7、caspase-12 的小干扰 siRNA 的递送 和促凋亡 CHOP/GADD153 mRNA 可降低 ADRP 光感受器的凋亡水平。为了 对于每个 ADRP 模型，我们计划：（1）通过过度调节 UPR 以有利于促生存途径的激活 BiP蛋白的表达； (2) 通过降低活化的 caspase-7 和 caspase-12 水平来抑制细胞凋亡 (3)通过靶向CHOP mRNA抑制CHOP相关的细胞凋亡。我们将监测生存情况 使用视网膜电图和形态测量来测量光感受器，并测量内质网应激的激活 使用特异性抗体和 RT-PCR 进行细胞凋亡。我们还将使用以下方法来衡量视力的改善情况： 验光，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。而AAV介导的基因 正在开发转移治疗 RP、抑制 ER 应激和细胞凋亡 伴侣很新颖。这种方法可以克服这种疾病的遗传多样性，并揭示 导致从突变到视网膜变性的细胞死亡途径。