Reduction of Amyloid Burden by Antisense APP

描述（由申请方提供）：依据：β-淀粉样前体蛋白（APP）的淀粉样蛋白生成加工导致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裂解酶在残基671/673处裂解，随后通过γ-位点APP裂解酶在残基711/713处裂解，依次衍生A β。 已知APP中的点突变（例如瑞典双突变）促进β位点切割。 因此，我们假设在瑞典突变（KM 670/671 NL）（TgAPPSWE或Tg 2576）模型中，反义阻断突变的β位点而不是正常的γ位点，将减少淀粉样蛋白生成APP-mRNA的翻译，但不减少正常APP-mRNA的翻译。 我们在Tg 2576（TgAPPSWE）转基因小鼠中通过用针对突变的β和正常的γ位点的2 '-甲氧基-O-乙基（莫伊）修饰的反义寡脱氧核苷酸（ODNs）阻断APP-mRNA的翻译来验证这一假设。 数据显示，针对β位点的2 'MOE修饰的反义ODN显著降低可溶性A β 40/42（sA β 40/42）的脑水平，而总的可检测的α切割的可溶性APP（sAPP α）的水平显示出小幅增加[Siegel和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蛋白的翻译，但不是小鼠APP，而γ-位点阻断是非特异性的。 基于这些结果，本申请提出使用在APP（K670 M/N671 L）中具有点突变的瑞典双突变体模型（TgAPPSWE）和过表达正常野生型人APP（TgAPPWT）的模型作为对照，以测试针对突变APP的β位点的反义ODN在降低淀粉样蛋白负荷和淀粉样蛋白相关毒性中的功效。 本研究旨在验证β-位点反义寡核苷酸在家族性AD治疗中的应用价值。 假设：在突变的β位点处的反义阻断将抑制淀粉样蛋白生成APP的翻译，而不影响TgAPPSWE和TgAPPWT中的正常APP加工。 具体目标：1。 通过正常和突变APP-mRNA的cRT-PCR定量和sAPP α的ELISA测量，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蛋白生成APP-mRNA的水平，而不影响小鼠APP-mRNA和TgAPPSWE中正常α切割APP或TgAPPWT中正常人和小鼠APP-mRNA的产生。 2. 通过ELISA和Abeta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降低TgAPPSWE中的脑淀粉样蛋白负荷。 3. 通过免疫细胞化学和突触标志物以及反应性小胶质细胞和星形胶质细胞标志物的成像分析，确定突变β位点的反义阻断是否会预防和逆转TgAPPSWE中小胶质细胞和星形胶质细胞的Abeta诱导的突触毒性和神经毒性反应。 重要性：反义寡核苷酸靶向淀粉样蛋白的翻译可能被证明是一种有效和安全的基因治疗形式，用于治疗家族性AD和其他遗传性疾病。

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.