Long-term anti-diabetogenic effects of GLP-1 gene therapy using double stranded Adeno-associated viral vector

Abstract

[한글]

[영문]Introduction: Diabetes is characterized by insulin resistance and a reduction of insulin secretion, which lead to progressive beta-cell failure and the loss of beta-cell mass. The central therapeutic issue is therefore how to restore the normal responsiveness of β-cells to glucose and how to counteract the defects in insulin secretion. Although glucagon-like peptide-1 (GLP-1) restores normal insulin secretion by making β-cells competent and diabetic β-cells more sensitive to glucose, native GLP-1 has the major drawback of being rapidly inactivated. In this study, we describe the construction and analysis of a GLP-1 plasmid and a double-stranded AAV expression vector that, when transduced, generate sufficient levels of circulating GLP-1 in vivo to overcome the rapid degradation of native GLP-1 and the limitations of gene therapy using ssAAV. Furthermore, we demonstrate the efficacy of our vectors in a type II diabetes mice model (db/db). Materials and methods: Double-stranded adeno-associated viral DNA was constructed by deleting the D-sequence of the 5′ ITR by MscI digestion. For stable gene expression, the CMV promoter was replaced with the CB promoter (CMV enhancer/chicken beta-actin promoter) and murine Ig κ-chain leader sequence linked to a hemagglutinin A (HA) epitope tag for expression. To create an active form of GLP-1, a furin recognition site-containing expression cassette was used. For in vivo GLP-1 expression, either dsAAV GFP or dsAAV rGLP-1 was injected intravenously into db/db mice. GLP-1 secretion and bioactivity were assayed using a GLP-1 RIA kit, and the bioactivity of the GLP-1 expressed from the viral vector was measured using cAMP induction and insulin secretion assays. Results: We successfully created a GLP-1 expression plasmid that can direct the secretion of active GLP-1 (7?37). Transfection experiments demonstrated that more efficient