Aptabinding of tumor necrosis factor- (TNF) inhibits its proinflammatory effects and alleviates islet inflammation

Pancreatic islet cell transplantation (ICT) has emerged as an effective therapy for diabetic patients lacking endogenous insulin production. However, the islet graft function is compromised by a nonspecific inflammatory and thrombotic reaction known as the instant blood-meditated inflammatory reaction (IBMIR). Here, we report the characterization of four single-stranded DNA aptamers that bind specifically to TNF alpha - a pivotal cytokine that causes proinflammatory signaling during the IBMIR process - using single molecule binding analysis and functional assays as a means to assess the aptamers' ability to block TNF alpha activity and inhibiting the downstream proinflammatory gene expression in the islets. Our single-molecule fluorescence analyses of mono- and multivalent aptamers showed that they were able to bind effectively to TNF alpha with monoApt2 exhibiting the strongest binding (K-d similar to 0.02 +/- 0.01 nM), which is similar to 3 orders of magnitude smaller than the K-d of the other aptamers. Furthermore, the in vitro cell viability analysis demonstrated an optimal and safe dosage of 100 mu M for monoApt2 compared to 50 mu M for monoApt1 and significant protection from proinflammatory cytokine-mediated cell death. More interestingly, monoApt2 reversed the upregulation of IBMIR mediating genes induced by TNF alpha in the human islets, and this was comparable to established TNF alpha antagonists. Both monoaptamers showed high specificity and selectivity for TNF alpha. Collectively, these findings suggest the potential use of aptamers as anti-inflammatory and localized immune-modulating agents for cellular transplant therapy.