Gene delivery using anti-inflammatory HMGB-1 box A peptide

Abstract

High mobility group box-1 (HMGB-1) is an abundant nuclear and cytoplasmic protein. HMGB-1 is composed of three domains which have box A and box B of two positively charged domains and negatively charged carboxyl terminus (acidic tail). HMGB-1 containing cytokine activity induces inflammatory response via the receptor for advanced glycation end products (RAGE). On the other hand, recombinant HMGB-1 box A (rHMGB-1A) has antagonist function of this cytokine activity. Boxes A and B are DNA binding domains and have high contents of basic amino acids. In this study, rHMGB-1A was produced by recombinant DNA technology and evaluated as a siRNA delivery carrier with anti-inflammatory effect. The HMGB-1A cDNA was amplified and cloned into the pET-21a, constructing pET-21a-HMGB-1A. rHMGB-1A was overexpressed and purified by Nickel affinity and cationic exchange chromatography. Polymyxin B column was used to eliminate endotoxin. The purified rHMGB-1A was analyzed in SDS-PAGE. Anti-inflammatory effect of rHMGB-1A was evaluated using activated macrophage, Raw 264.7 cells. The Raw 264.7 cells were preincubated with purified rHMGB-1A for 1 hr. Raw 264.7 cells were activated by wild type HMGB-1 (wtHMGB-1) or lipopolysaccharides (LPS). The results showed that rHMGB-1A reduced the secretion of TNF-α effectively in the cells activated with wtHMGB-1 or LPS. Luciferase siRNA was complexed with rHMGB-1A. Gel retardation assay showed that siRNA was completely retarded at a 1:3 siRNA:rHMGB-1A weight ratio. Heparin competition assay suggests that rHMGB-1A releases siRNA more easily than high molecular weight polyethylenimine (PEI 25K). Transfection assay showed that rHMGB-1A had higher siRNA delivery efficiency than PEI 25K. rHMGB-1A almost have not toxicity in HEK 293 and Raw 264.7 cells. The results suggest that rHMGB-1A may be useful for siRNA therapy for inflammatory diseases.