Development of Splice Modulating Oligonucleotides Targeting RAGE as a Treatment for Alzheimer's Disease

项目摘要/摘要 阿尔茨海默病(AD)是一种以进行性为特征的严重神经退行性疾病 记忆力丧失、语言缺陷、人格改变和痴呆症，最常见于65岁以上的专利。当前 治疗只对认知功能下降有轻微和暂时的影响，突显了创新的必要性 可以阻止AD进展的治疗方法。 而常见的阿尔茨海默病是由淀粉样前体蛋白常染色体显性突变和 早老素，大多数AD病例被认为是散发性的，既有遗传的，也有 环境风险因素。然而，淀粉样β蛋白(Aβ)斑块，过度磷酸化的tau神经纤维 缠结(NTS)和严重的胶质增生都是所有形式的AD的共同神经退行性特征。感受器 晚期糖基化终产物(RAGE)在AD患者的大脑中上调，并直接相互作用 在神经细胞、免疫细胞和血管细胞中有Aβ。RAGE信号也可以诱导tau的过度磷酸化 并与散发性AD的共病危险因素(如心血管疾病)的发病机制有关 和糖尿病)。因此，RAGE是家族性和散发性AD治疗发展的主要靶点。 RAGE前信使核糖核酸产生相互排斥的选择性剪接变体，要么是膜- 结合并能够发出信号(MBRAGE)，或截断并内源性分泌以清除RAGE配体 没有信令(EsRAGE)。在AD小鼠中，MBRAGE的过表达加速了病理、认知、 和AD的行为特征，以及输入合成esRAGE降低了Aβ和NT的病理以及 改善学习/记忆和突触功能。因此，开发了一种减少RAGE信令的新策略 一种称为剪接调控寡核苷酸(SMO)的化合物，能够控制Pre-mRNA的替代 剪接降低mbRAGE的表达，同时增加esRAGE的表达。SMO是一种 一类合成的基于RNA的化合物，它以空间位阻或减弱分子间的相互作用 剪接机和前信使核糖核酸。SMO以非凡的效力、特异性、长时间与其目标绑定 行动的持续时间和可以忽略的偏离目标的效果。以前的靶向配体的药理学方法- 狂怒的相互作用被偏离目标的效果或短暂的半衰期所阻碍，没有人有双重的影响。 提出了增加天然esRAGE和降低mBRAGE异构体表达的机制。 靶向RAGE的SMO将在硅胶中设计，并针对效力和特异性进行提炼，首先是体外细胞 培养(目标1)选择主要的SMO候选药物。然后，每个SMO候选人将被特色化并 进一步优化RAGE转基因小鼠体内的安全性和效力(目标2)。最后，SMO将成为 评价链脲佐菌素(STZ)诱导的AD模型预防/逆转AD的治疗作用 认知缺陷和tau蛋白过度磷酸化(目标3)。最终目标是开发一个铅愤怒SMO作为 治疗阿尔茨海默病的潜在治疗方法。

Project Summary/Abstract Alzheimer`s Disease (AD), is a severe neurodegenerative disorder characterized by progressive memory loss, language deficits, personality changes, and dementia, most prevalent in patents over 65. Current treatments only have modest and temporary effects on cognitive decline, highlighting the need for novel treatments which can halt the progression of AD. While familiar AD is caused by autosomal dominant mutations in amyloid precursor protein and presenilin, the majority of AD cases are considered sporadic with a combination of both genetic and environmental risk factors. However, amyloid-beta (Aβ) plaques, hyper-phosphorylated tau neurofibrillary tangles (NTs), and severe gliosis are all neurodegenerative hallmarks common to all forms of AD. The receptor for advanced glycation end-products (RAGE) is upregulated in the brains of AD patients and interacts directly with Aβ in neuronal, immune and vascular cells. RAGE signaling can also induce hyper-phosphorylation of tau and is implicated in the pathogenesis of co-morbid risk factors for sporadic AD (e.g. cardiovascular disease and diabetes). Thus, RAGE is a prime target for therapeutic development in both familial and sporadic AD. The RAGE pre-mRNA yields mutually exclusive alternative splice variants that are either, membrane- bound and capable of signaling (mbRAGE), or truncated and endogenously secreted to clear RAGE ligands without signaling (esRAGE). In AD mice, overexpression of mbRAGE accelerated the pathologic, cognitive, and behavioral hallmarks of AD, and infusion of synthetic esRAGE decreased Aβ and NT pathology as well as improved learning/memory and synaptic function. Thus, a novel strategy to reduce RAGE signaling is develop compounds called splice modulating oligonucleotides (SMOs) capable of controlling pre-mRNA alternative splicing to decrease expression of mbRAGE and simultaneously increase expression of esRAGE. SMOs are a class of synthetic RNA based compounds that sterically block or weaken interactions between elements of the splicing machinery and the pre-mRNA. SMOs bind to their targets with exceptional potency, specificity, long duration of action and negligible off-target effects. Previous pharmacological approaches to target ligand- RAGE interactions have been hampered by off-target effects or a short half-life, and none have the dual mechanism of both increasing natural esRAGE and decreasing mbRAGE isoform expression proposed here. SMOs targeting RAGE will be designed in silico and refined for potency and specificity, first in vitro cell culture (Aim 1) to select lead SMO drug candidates. Each SMO candidate will then be characterized and further optimized for safety and potency in vivo in RAGE transgenic mice (Aim 2). Finally, SMOs will be evaluated as therapeutics in an acute streptozotocin (STZ) induced model of AD for prevention/reversal of cognitive deficits and tau hyper-phosphorylation (Aim 3). The ultimate goal is to develop a lead RAGE SMO as potential therapeutic for the treatment of AD.