Unfolded Protein Response as a Therapeutic Target for ADRP Animal Models

未折叠蛋白反应作为adrp动物模型治疗靶点的研究 本项目致力于阐明常染色体上未折叠蛋白反应(UPR)的作用。 显性视网膜色素变性(ADRP)的发病机制及基因治疗研究进展 UPR信令标记的调制。视网膜色素变性(RP)是最常见的遗传性视网膜色素变性 失明，全世界所有民族中每4000人中就有1人失明。RP可以通过以下方式传输 常染色体显性(ADRP)、常染色体隐性(ARRP)或X连锁性状。人类基因组中有100多个突变 视紫红质约占ADRP病例的30%，并伴有不同程度的视力损害。折叠错误 视蛋白干扰野生型视紫红质的运输，聚集在内质网(ER)和 刺激一个称为未折叠蛋白反应(UPR)的信号转导级联反应。如果未选中，则此 通路可能通过细胞凋亡来触发感光细胞的死亡。虽然补充维生素A 在某些情况下可能是有益的，目前，还没有有效的药物治疗ADRP。因此， 这项提议的主要目标是确定基于重新编程的基因治疗是否 视紫红质异常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 视紫红质基因突变(P23H和T17M)。 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及其靶向caspase-7、caspase-12小干扰siRNA的传递 和促凋亡的CHOP/GADD153mRNAs，以降低ADRP光感受器的凋亡水平。为 在每种ADRP模型中，我们计划：(1)通过过度调节UPR，使其有利于促进生存通路的激活。 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。我们将监测儿童的存活情况 光感受器使用视网膜电图术和形态计量学，并将测量内质网应激的激活 用特异性抗体和RT-PCR检测细胞凋亡。我们还将使用以下工具测量视力的改善 验光，这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、 伴侣蛋白Bip的光感受器特异性启动子及其优化表达。而AAV介导的基因 转运蛋白正在被开发用于治疗RP，抑制ER应激和细胞凋亡 监护人是一种新奇的东西。这种方法可能克服这种疾病的遗传多样性，并揭示 从突变到视网膜退化的细胞死亡途径。

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.