Reduction of Amyloid Burden by Antisense APP

通过反义 APP 减少淀粉样蛋白负担

• 批准号: 6780661
• 负责人: NEELIMA CHAUHAN
• 金额: $ 14.81万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6780661
• 关键词: amyloid proteins; antisense nucleic acid; enzyme linked immunosorbent assay; genetic translation; genetically modified animals; immunocytochemistry; laboratory mouse; messenger RNA; microglia; neurotoxicology; oligonucleotides; polymerase chain reaction; synapses

DESCRIPTION (provided by applicant): Rationale: Amyloidogenic processing of beta-amyloid precursor protein (APP) leading to the formation of beta amyloid (Abeta) is a key event in the pathogenesis of Alzheimer's disease (AD). Abeta is sequentially derived by a cleavage at the residues 671/673 by beta-site APP cleaving enzyme, followed by a cleavage at the residues 711/713 by gamma-site APP cleaving enzyme. Point mutations in APP such as in Swedish double mutations, are known to promote beta-site cleavage. Therefore, we hypothesize that antisense blocking of the mutated beta-site, and not of the normal gamma-site, will reduce translation of amyloidogenic APP-mRNA but not normal APP-mRNA, in a model harboring Swedish mutations (KM670/671NL) (TgAPPSWE or Tg2576). We tested this hypothesis in Tg2576 (TgAPPSWE) transgenic mice by blocking translation of APP-mRNA with 2'-Methoxy-O-Ethyl (MOE)-modified antisense oligodeoxynucleotides (ODNs) directed at the mutated beta-and normal gamma-sites. The data show that 2'MOE modified antisense ODNs directed at the beta-site significantly reduced cerebral levels of soluble Abeta40/42 (sAbeta40/42) while the levels of total detectable alpha-cleaved soluble APP (sAPPalpha) showed a small increase [Siegel and Chauhan, SFN-2002, 687.14]. Antisense blocking at the gamma-site did not produce any effect. These results indicate that beta-site blocking may have arrested the translation of amyloidogenic but not mouse APP, while gamma-site blocking is non-specific. Based on these results, current application proposes the use of a Swedish double mutant model (TgAPPSWE) harboring point mutations in APP (K670M/N671L), and a model overexpressing normal wild type human APP (TgAPPWT) as a control to test the efficacy of antisense ODNs directed at the beta-site of mutated APP in reducing amyloid burden and amyloid-associated toxicity. This project is to test the potential usefulness of antisense ODNs to the beta-site as therapy for familial AD. Hypothesis: Antisense blocking at the mutated beta-site will inhibit the translation of amyloidogenic APP without affecting normal APP processing in TgAPPSWE and TgAPPWT. Specific Aims: 1. Determine if antisense blocking at the mutated beta-site will reduce the levels of detectable mutated amyloidogenic APP-mRNA without affecting mouse APP-mRNA and production of normal alpha-cleaved APP in TgAPPSWE or normal human and mouse APP-mRNA in TgAPPWT as evaluated by cRT-PCR quantitation of normal and mutant APP-mRNA and ELISA measurement of sAPPalpha. 2. Determine if antisense blocking at the mutated beta-site will reduce cerebral amyloid load in TgAPPSWE, as evaluated by ELISA and immunocytochemistry of Abeta peptides. 3. Determine if antisense blocking at the mutated beta-site will prevent and reverse Abeta-induced synaptotoxicity and the neurotoxic response of microglia and astroglia in TgAPPSWE, as evaluated by immunocytochemistry and imaging analysis of synaptic markers and markers of reactive microglia and astroglia. Significance: Targeting the translation of amyloidogenic APP by antisense synthetic oligodeoxynucleotides may prove to be an effective and safe form of gene therapy for treating familial AD and other genetic diseases.

描述（由申请人提供）：理由：β -淀粉样蛋白前体蛋白（APP）的淀粉样变性加工导致β -淀粉样蛋白（Abeta）的形成是阿尔茨海默病（AD）发病机制中的关键事件。通过β位点APP切割酶在671/673残基上的切割，再通过γ位点APP切割酶在711/713残基上的切割，得到了β。APP中的点突变，如瑞典双突变，已知可促进β位点切割。因此，我们假设，在瑞典突变（KM670/671NL） （TgAPPSWE或Tg2576）模型中，突变β位点的反义阻断，而不是正常γ位点的反义阻断，会减少淀粉样蛋白源性APP-mRNA的翻译，而不是正常APP-mRNA的翻译。我们在Tg2576 （TgAPPSWE）转基因小鼠中验证了这一假设，方法是用2'-甲氧基- o -乙基（MOE）修饰的反义寡脱氧核苷酸（ODNs）阻断APP-mRNA对突变β和正常γ位点的翻译。数据显示，2'MOE修饰的针对β位点的反义odn显著降低了大脑中可溶性Abeta40/42 （sAbeta40/42）的水平，而总可检测的α -cleaved可溶性APP （sAPPalpha）的水平则小幅升高[Siegel and Chauhan， sfn - 2002,687 .14]。在γ位点的反义阻断没有产生任何效果。这些结果表明，β -位点阻断可能阻止了淀粉样变性的翻译，但对小鼠APP没有作用，而γ -位点阻断是非特异性的。基于这些结果，目前的应用建议使用在APP （K670M/N671L）中含有点突变的瑞典双突变模型（TgAPPSWE）和过表达正常野生型人类APP （TgAPPWT）模型作为对照，以测试针对突变APP β位点的反义odn在减少淀粉样蛋白负担和淀粉样蛋白相关毒性方面的效果。本项目旨在测试β位点的反义odn作为治疗家族性AD的潜在有用性。假设：突变β位点的反义阻断会抑制淀粉样变性APP的翻译，但不会影响TgAPPSWE和TgAPPWT的正常APP加工。具体目标：1；通过正常和突变的APP- mrna的rt - pcr定量和ELISA检测sAPPalpha，确定突变β位点的反义阻断是否会降低可检测的突变淀粉样变性APP- mrna的水平，而不影响TgAPPSWE中小鼠APP- mrna和正常α -裂解APP的产生，或TgAPPWT中正常人和小鼠APP- mrna的产生。2. 通过ELISA和β肽的免疫细胞化学评估，确定突变β位点的反义阻断是否会减少TgAPPSWE的脑淀粉样蛋白负荷。3. 通过免疫细胞化学和突触标记物以及反应性小胶质细胞和星形胶质细胞标记物的成像分析，确定突变β位点的反义阻断是否会预防和逆转β诱导的TgAPPSWE小胶质细胞和星形胶质细胞的突触毒性和神经毒性反应。意义：通过反义合成寡脱氧核苷酸靶向淀粉样蛋白APP的翻译可能是治疗家族性AD和其他遗传性疾病的一种有效且安全的基因治疗形式。