Reduction of Amyloid Burden by Antisense APP

通过反义 APP 减少淀粉样蛋白负担

• 批准号: 6864811
• 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/6864811
• 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)：基本原理：淀粉样β-淀粉样前体蛋白(APP)导致形成淀粉样β-淀粉样蛋白(Abeta)是阿尔茨海默病(AD)发病机制中的一个关键事件。Abeta是由β位点APP裂解酶在残基671/673处裂解，然后由Gamma位点APP裂解酶在残基711/713处裂解而得到的。APP中的点突变，如瑞典的双重突变，已知会促进β-位点切割。因此，我们假设，在一个含有瑞典突变(KM670/671NL)的模型(TgAPPSWE或Tg2576)中，反义阻断突变的β-位点，而不是正常的伽马位点，将减少淀粉样变性APP-mRNA的翻译，但不会减少正常的APP-mRNA的翻译。我们在Tg2576(TgAPPSWE)转基因小鼠中用2‘-甲氧基-O-乙基(MOE)修饰的反义寡核苷酸(ODN)针对突变的β和正常伽马位点阻断APP-mRNA的翻译，从而验证了这一假设。数据显示，针对β-位点的2‘MoE修饰的反义寡核苷酸显著降低了大脑中可溶性Abeta40/42(sAbeta40/42)的水平，而总的可检测到的α-裂解可溶性APP(SAPPalpha)的水平略有增加[Siegel and Chauhan，SFN-2002,687.14]。反义封闭在伽马位点没有产生任何效果。这些结果表明，β位点阻断可能阻止了淀粉样变性基因的翻译，但不阻止小鼠APP的翻译，而伽马位点阻断是非特异性的。基于这些结果，目前的申请提出使用在APP中存在点突变的瑞典双突变模型(TgAPPSWE)(K670M/N671L)和过度表达正常野生型人类APP的模型(TgAPPWT)作为对照，以测试针对突变的APP的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有效性。本项目旨在测试针对β-位点的反义寡核苷酸作为治疗家族性AD的潜在用途。假设：TgAPPSWE和TgAPPWT中突变的β-位点的反义阻断将抑制淀粉样变性APP的翻译，而不会影响APP的正常处理。具体目的：1.通过对正常和突变的APP-mRNA的CRT-PCR定量和sAPPalpha的酶联免疫吸附试验，确定在突变的β-位点上的反义封闭是否会在不影响小鼠APP-mRNA的情况下降低可检测到的突变的APP-mRNA的水平，并在TgAPPSWE或TgAPPWT中评价正常的α-裂解APP的产生。2.用酶联免疫吸附试验和免疫细胞化学方法检测突变β位点的反义封闭是否会降低TgAPPSWE大鼠的脑淀粉样蛋白负荷。3.通过免疫细胞化学和突触标志物及反应性小胶质细胞和星形胶质细胞标志物的成像分析，确定在突变的β位点阻断反义封闭是否能预防和逆转Abeta诱导的突触毒性和小胶质细胞和星形胶质细胞的神经毒性反应。意义：通过反义合成的寡核苷酸靶向翻译淀粉样变性APP可能被证明是治疗家族性AD和其他遗传性疾病的一种有效和安全的基因治疗形式。