In Vivo Introduction of an Extrachromosomal Plasmid Expressing Long-term Human Alpha-1-antitrypsin protects Islet Allografts (141.48)

Abstract

Alpha-1-antitrypsin (AAT) exerts anti-inflammatory and tolerogenic activities during islet allograft transplantation in diabetes mouse models. Although a serine protease inhibitor, evidence suggests that AAT possesses activities that are independent of protease inhibition. The previously reported plasmid, pEF-hAAT, contains the genomic sequence of human AAT (hAAT) and sustains circulating levels after single hydrodynamic tail-vein injection (HDI) in mice. To asses whether expression of hAAT by pEF-hAAT protects islets from acute rejection, pEF-hAAT (100 μg, n = 6) or PBS (n = 4, control) were hydrodynamically introduced to mice. Liver expression and circulating hAAT levels were determined. Supernatant of transfected Hepa1c cells exhibited anti-elastase activity. Sixteen days post HDI mice were injected streptozotocin (225 mg/kg) and grafted with 450 allogenic islets. Circulating hAAT levels (1-350 μg/ml) persisted for over 30 days. Normoglycemia was achieved and islets were accepted in 6 out of 6 recipients that expressed hAAT. All 4 control animals exhibited acute islet allograft rejection by day 12. We conclude that plasmid-derived long-term expression of as low as 1 μg/ml hAAT exhibits islet-allograft protection. Using site-directed mutagenesis of pEF-hAAT we will examine whether activities that are independent of protease inhibition partake in the protection of islet allografts.