In vitro evaluation of an enhanced human serum amyloid a (SAA2) promoter-regulated soluble TNF receptor fusion protein for anti-inflammatory gene therapy

Tumour necrosis factor (TNF)-alpha contributes to the pathogenesis of many inflammatory diseases. Recombinant soluble TNF receptor fusion proteins (sT NFR:Ig) are potent TNF antagonists, both in vitro and in vivo. The concentr ation of serum amyloid A (SAA) increases by up to 1000-fold during inflamma tion, largely owing to cytokine-driven transcriptional upregulation. A repo rter plasmid, comprising the proximal 0.7 kb of the human SAA2 promoter fus ed to a luciferase gene, was used in transient transfection experiments in human HepG2 hepatoma cells to assess the quantitative and qualitative TNF a ntagonist properties of a construct in which sTNFR:Ig synthesis is under th e control of a chimera of the SAA2 promoter and a tat/HIV element. The SAA2 -tat/HIV-sTNFR:Ig construct retained the fine-tuned cytokine responsiveness of the SAA2 promoter, while exhibiting the quantitatively enhanced level o f protein expression conferred by the tat/HIV element. It produced a biolog ically significant TNF inhibition that was at least as strong as that achie ved using a CMV promoter-driven sTNFR:Ig construct. There was a dose- and t ime-dependent relationship between the pro-inflammatory cytokine used, and the generation of TNF antagonist activity by SAA2-tat/HIV-sTNFR:Ig. Althoug h sTNFR:Ig protein can be induced by either TNF-alpha or interleukin (IL)-1 beta, its antagonist activity is limited to the former cytokine. The SAA2- tat/HIV-sTNFR:Ig construct, and derivatives thereof, may therefore be ideal ly suited to gene therapy applications that require the local production of potent and specific immune modifiers only when there is active pathology. It may consequently be of particular use in the future treatment of disease s such as rheumatoid arthritis.