Repression of GAD autoantigen expression in pancreas beta-cells by delivery of antisense plasmid/PEG-g-PLL complex

It was previously reported that silencing of the expression of glutamic aci d decarboxylase (GAD) in transgenic nonobese diabetic (NOD) mice completely protected islet beta -cells against development of diabetes. This suggests that the repression of GAD autoantigen by somatic gene delivery can preven t autoimmune destruction of pancreatic beta -cells. To repress GAD expressi on in islet beta -cells, we delivered an antisense GAD mRNA expression plas mid (pRIP-AS-GAD) using poly(ethylene glycol)-grafted poly-L-lysine (PEG-g- PLL) as a gene carrier. In a gel retardation assay, the pRIP-AS-GAD/PEG-g-P LL complex was completely retarded above a weight ratio of 1:1.5 (plasmid:P EG-g-PLL). PEG-g-PLL protected the plasmid DNA from DNase I for more than 6 0 minutes. In a reporter gene transfection assay, PEG-g-PLL showed the high est transfection efficiency at a weight ratio of 1:3. We also transfected p RIP-AS-GAD/PEG-g-PLL complex into a GAD-producing mouse insulinoma (MIN6) c ell line. The antisense mRNA was expressed specifically in p-cells and expr ession was dependent on glucose level. The repression of GAD after transfec tion of pRIP-AS-GAD was confirmed by immunoblot assay. In addition, in vivo expression of antisense RNA in pancreas was confirmed by RT-PCR after intr avenous injection of the complex into mice. Therefore, our study revealed t hat the pRIP-AS-GAD/PEG-g-PLL system is applicable for the repression of GA D autoantigen expression.