AA (secondary, reactive) amyloidosis is a disease caused by extracellular deposition of insoluble ?-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.
AA (reactive) amyloidosis is a progressive protein deposition disease that is associated with chronic inflammation and often leads to kidney failure. No specific therapy is available. Only nonspecific treatment to control inflammation and decrease levels of serum amyloid A (SAA), the amyloid precursor protein, has shown efficacy in slowing and/or decreasing progression of AA amyloidosis. We propose to inhibit liver production of SAA with an SAA-specific antisense oligonucleotide drug. By decreasing SAA production, there will be less of the amyloid precursor protein available to undergo amyloid fibril formation. In a separate aim we will investigate the role of in vivo modification of the amyloid precursor protein SAA in the initiation and perpetuation of AA amyloid fibril formation. Chemical modification of SAA may present a previously unrecognized risk factor that could be targeted by future therapeutic strategies.