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 acid 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 prevent autoimmune destruction of pancreatic beta-cells. To repress GAD expression in islet beta-cells, we delivered an antisense GAD mRNA expression plasmid (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-PLL complex was completely retarded above a weight ratio of 1:1.5 (plasmid: PEG-g-PLL). PEG-g-PLL protected the plasmid DNA from DNase I for more than 60 minutes. In a reporter gene transfection assay, PEG-g-PLL showed the highest transfection efficiency at a weight ratio of 1:3. We also transfected pRIP-AS-GAD/PEG-g-PLL complex into a GAD-producing mouse insulinoma (MIN6) cell line. The antisense mRNA was expressed specifically in beta-cells and expression was dependent on glucose level. The repression of GAD after transfection 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 intravenous injection of the complex into mice. Therefore, our study revealed that the pRIP-AS-GAD/PEG-g-PLL system is applicable for the repression of GAD autoantigen expression.
Lee
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Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA. minhyung.lee@deans.pharm.utah.edu
Han
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Koh
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Park
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Yoon
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Kim
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D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D016373:DNA, Antisense / Q000008:administration & dosage / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use*; D004337:Drug Carriers / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005968:Glutamate Decarboxylase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007275:Injections, Intravenous; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D008297:Male; D051379:Mice; D009928:Organ Specificity; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics / Q000627:therapeutic use; D011092:Polyethylene Glycols / Q000378:metabolism*; D011107:Polylysine / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001324:Autoantigens; D016373:DNA, Antisense; D004337:Drug Carriers; D012333:RNA, Messenger; D011107:Polylysine; D011092:Polyethylene Glycols; D001616:beta-Galactosidase; D005968:Glutamate Decarboxylase