Polymer mediated gene delivery for type 2 diabetes gene therapy

Abstract

Glucagon-like peptide 1 (GLP-1) is a insulinotropic protein, which increases the insulin secretion from islet β-cells at a high glucose concentration. It was reported that the continuous infusion of GLP-1 normalized the blood glucose level in type 2 diabetes animal model1. However, the extremely short half-life of GLP-1 has limited its application2 and prompted the gene therapy with the GLP-1 expression plasmid3. pβ-GLP-1, a GLP-1 expression plasmid, was transfected to cells using polyethylenimine (PEI) as a gene carrier. The in vitro results showed a dose dependent expression of GLP-1. Co-culture assay of the GLP-1 plasmid transfected cells with isolated rat islet cells demonstrated that GLP-1 increased insulin secretion, compared to controls during a hyperglycemic challenge. A single injection of PEI/pβ-GLP-1 complex into ZDF rats resulted in increasing insulin secretion and decreasing blood glucose level that was maintained for 2 weeks. However, the glucose level was not completely normalized, suggesting that the expression level of GLP-1 was not enough to normalize the blood glucose level. More efficient GLP-1 expression systems were developed using two step transcription amplification (TSTA) or nuclear factor-κb (NF-κb) mediated nuclear transport. To apply the TSTA system to the GLP-1 expression plasmid, pβ-Gal4-p65 and pUAS-GLP-1 were constructed and co-transfected to 293 cells using PEI as a gene carrier. The pβ-Gal4-p65/pUAS-GLP-1 showed higher mRNA expression than pβ-GLP-1 in RT-PCR. In addition, the GLP-1 protein level by pβ-Gal4-p65/pUAS-GLP-1 was 4 times higher than pβ-GLP-1. The highest transgene expression was obtained at a 2:1 pUAS-GLP-1:pβ-Gal4-p65 ratio. The ratio between the two plasmids had significant effect of the trasngene expression. In the second strategy, NF-κb binding sites were introduced into the GLP-1 expression plasmid. The in vitro results showed higher expression of GLP-1 compared to the control plasmid. A single systemic administration of PEI/pSi-GLP-1-NF-κb complex into DIO mice resulted in increasing insulin secretion and decresing blood glucose levels, compared to the control GLP-1 plasmid without NF-κb binding sites. This GLP-1 gene delivery system may provide an effective treatment modality for type 2 diabetes.