Pathogenesis and Treatment of AA Amyloidosis

AA(继发性，反应性)淀粉样变性是一种由不溶性β-褶皱在细胞外沉积引起的疾病 由淀粉样蛋白A(AA)蛋白组成的片状纤维，淀粉样蛋白A是急性时相蛋白血清的N末端片段 淀粉样蛋白A(SAA)。这些沉积物破坏了组织结构，最终损害了器官功能。普普通通 沉积部位包括肾、脾、肝和肠。人类肾小球蛋白尿 沉积通常是第一临床表现，随着沉积的增加，肾病综合征通常 进展为终末期肾病。AA淀粉样变性通常发展为慢性 炎症性疾病，如类风湿性关节炎、强直性脊柱炎、炎症性肠病，以及 遗传性自体炎症综合征(例如，家族性地中海热)。它也是以联想方式出现的 患有长期感染(骨髓炎、压疮、支气管扩张症)。截瘫退伍军人在 慢性压疮、尿路感染和骨髓炎导致AA淀粉样变性的风险。在……里面 此外，强直性脊柱炎在我们的男性退伍军人中很常见，这也增加了患AA淀粉样变性的风险。 目前，对AA淀粉样变性尚无特效治疗方法。只有控制促炎刺激才能减少 肝脏SAA合成已显示出对AA淀粉样蛋白进展的影响。在最近的一项第三阶段临床试验中， 潜在的新疗法[Eprodisate(Kiacta)]在延缓AA进展方面未能显示出显著意义。已提示 根据患者未得到满足的需求，我们的初步目标将是开始开发一种治疗AA淀粉样变性的方法。 以此为框架，我们对转甲状腺素(TTR)淀粉样变性进行了研究。这个 治疗策略将是给予人类SAA特异性反义寡核苷酸(ASO)， 促进SAA mRNA的降解，导致血液中SAA水平较低，可用于 淀粉样蛋白形成。携带人SAA1基因片段的转基因小鼠模型 启动子和调控元件将被生成并用于本研究。人类SAA1特异性ASO 将由Ionis制药公司提供，并筛选出下调人类SAA1表达的能力 细胞培养。有希望的化合物将与N-乙酰半乳糖胺(GalNAc)偶联；这种修饰 给予肝脏靶向递送，效力增加6-10倍，允许更低的剂量。在进一步 在正常小鼠中进行筛选以评估耐受性，将ASOS给予人SAA1转基因小鼠以 确定在不同剂量下达到的抑制水平。我们培育的人类SAA1转基因小鼠 将与不表达小鼠SAA1或SAA2的小鼠杂交；这些小鼠将由Dr. 弗雷德里克·德·比尔和南希·韦伯，肯塔基大学。这种杂交的后代，即小鼠 表达人SAA1而不是小鼠SAA1或SAA2的人将被诱导形成AA淀粉样变性 标准方案，也用精选的人类SAA特异性ASO和对照随机ASO治疗以 确定ASO治疗减少人类AA淀粉样蛋白沉积的程度。在第二个目标中，我们将 探讨SAA的翻译后修饰在淀粉样原纤维形成中的作用。我们的体外培养 研究表明，小鼠SAA1.1氨基末端残基的氨甲基化具有增强作用 对淀粉样蛋白形成的影响。SAA的这个区域对淀粉样蛋白的启动和繁殖是最关键的 纤维，这表明该区域的修饰可能在体内和体外都有重要的后果。 从人和小鼠的淀粉样沉积中提取的AA淀粉样蛋白，以及血清中的SAA，将是 用串联质谱仪进行分析，以确定修饰的残留物。将使用鼠标模型来测试 体内促进氨基甲基化的条件是否也促进AA淀粉样蛋白的发生。其他研究将 研究氨甲酰化对SAA与高密度脂蛋白结合的影响。如果发现修改会影响 体内淀粉样蛋白的形成，就像他们在体外所做的那样，靶向这样的修饰可能会提供新的治疗选择 治疗这种进行性的、致命的疾病。

AA (secondary, reactive) amyloidosis is a disease caused by extracellular deposition of insoluble β-pleated sheet fibrils composed of amyloid A (AA) protein, an N-terminal fragment of the acute phase protein serum amyloid A (SAA). The deposits disrupt tissue structure and eventually compromise organ function. Common sites of deposition include kidney, spleen, liver, and intestine. In humans proteinuria due to glomerular deposits is often the first clinical manifestation, and with increasing deposition, the nephrotic syndrome usually progresses to end-stage renal disease. AA amyloidosis usually develops as a complication of chronic inflammatory conditions such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, and the hereditary auto-inflammatory syndromes (e.g., familial Mediterranean fever). It also occurs in association with long-standing infections (osteomyelitis, decubitus ulcers, bronchiectasis). Veterans with paraplegia are at risk of developing AA amyloidosis due to chronic decubitus ulcers, urinary tract infections and osteomyelitis. In addition, ankylosing spondylitis is common in our male veterans which also increases risk for AA amyloidosis. At present, there is no specific therapy for AA amyloidosis. Only control of pro-inflammatory stimuli to reduce hepatic SAA synthesis has shown an effect on AA amyloid progression. In a recent Phase III clinical trial, a potential new therapy [eprodisate (Kiacta)] failed to show significance in retarding AA progression. Prompted by the unmet needs of patients, our initial aim will be to initiate development of a therapy for AA amyloidosis using as framework translational studies we have conducted on transthyretin (TTR) amyloidosis. The therapeutic strategy will be administration of human SAA-specific antisense oligonucleotides (ASO) that facilitate degradation of SAA mRNA resulting in lower SAA levels in blood and less substrate available for amyloid formation. A transgenic mouse model carrying a human SAA1 genomic segment encompassing SAA1 promoter and regulatory elements will be generated and employed for this study. Human SAA1-specific ASOs will be provided by Ionis Pharmaceuticals and screened for ability to downregulate SAA1 expression in human cell cultures. Promising compounds will be conjugated with N-acetyl galactosamine (GalNAc); this modification confers targeted delivery to the liver and increases potency 6-10-fold, allowing for lower dosing. After further screening in normal mice to evaluate tolerability, ASOs will be given to human SAA1 transgenic mice to determine the level of suppression achieved at various doses. The human SAA1 transgenic mice we generate will be crossed with mice that do not express mouse SAA1 or SAA2; these mice will be provided by Drs. Frederick de Beer and Nancy Webb, University of Kentucky. The offspring of this cross-breeding, i.e., mice expressing human SAA1 but not mouse SAA1 or SAA2, will be induced to develop AA amyloidosis using standard protocols and also treated with selected human SAA-specific ASOs and control random ASOs to determine the extent to which ASO therapy reduces deposition of human AA amyloid. In a second aim we will explore the role of post-translational modification of SAA in the genesis of amyloid fibril formation. Our in vitro studies show that carbamylation of residues in the amino-terminal portion of mouse SAA1.1 has a potentiating effect on amyloid formation. This region of SAA is most crucial to the initiation and propagation of amyloid fibrils, suggesting that modifications in this region may have important consequences in vivo as well as in vitro. AA amyloid protein extracted from amyloid deposits of humans and mice, as well as SAA in serum, will be analyzed by tandem mass spectrometry to identify modified residues. Mouse models will be used to test whether conditions that promote in carbamylation in vivo also promote AA amyloidogenesis. Other studies will investigate the effects of carbamylation on SAA association with HDL. If modifications are found to impact amyloid formation in vivo as they do in vitro, targeting such modifications may offer new therapeutic options to treat this progressive, fatal disease.