Glucagon-like Peptide-1 Plasmid Construction and Delivery for the Treatment of Type 2 Diabetes

Glucagon-like peptide-1 (GLP-1) is a 30-amino-acid hormone produced by intestinal L cells. It has been proposed that GLP-1 can be used as a new treatment for type 2 diabetes mellitus because it acts to augment insulin secretion and its effectiveness is maintained in type 2 diabetic patients. Despite its many remarkable advantages as a therapeutic agent for diabetes, GLP-1 is not immediately clinically applicable because of its extremely short half-life. One way to overcome this drawback is GLP1 gene delivery, which enables GLP-1 production in the body. In this study, the effect of GLP1 gene delivery was evaluated both in vitro and in vivo using a new plasmid constructed with a GLP1 (7–37) cDNA. The expression of the GLP1 gene was driven by a SV40 promoter/enhancer. To increase the expression level of GLP-1, nuclear factor B binding sites were introduced. The in vitro results showed expression of GLP-1 and in vitro activity of GLP-1, which is a glucose-dependent insulinotropic action. A single systemic administration of polyethyleneimine/pSIGLP1NF B complex into DIO mice resulted in increasing insulin secretion and decreasing blood glucose levels for a duration longer than 2 weeks.
Introduction
GLP-1 is a peptide hormone secreted from the intestinal L cells in response to nutrient ingestion . GLP-1 administration has been shown to reduce hyperglycemia with type 2 diabetic patients. GLP-1 is a potent insulinotropic hormone, and its insulinotropic effect is glucose-dependent. It stimulates not only insulin gene transcription, but also all steps of insulin biosynthesis. GLP-1 has been shown to be capable of inducing new cells in subjects with an insufficient number of these cells. Recent results on the actions of GLP-1 show that it stimulates cell differentiation and proliferation and also inhibits apoptosis in cells. These actions would increase the cell mass and function over a long-term perspective. In addition to its effect on the cells, GLP-1 reduces glucagon secretion. Also, it inhibits gastrointestinal motility, especially gastric emptying. All these effects render GLP-1 extremely desirable as a therapeutic agent for type 2 diabetes.
For the treatment of type 2 diabetes, sulfonylurea has been most commonly used because it is potent, effective, and inexpensive. However, sulfonylurea stimulation of insulin secretion is not glucose-dependent, and hence hypoglycemia is an adverse effect of the treatment. Other current therapies also have significant side effects and have limited efficacy. By using GLP-1, it is possible to prevent the risk of hypoglycemia by potentiating insulin secretion in a glucose-dependent manner.
Despite numerous advantages as a diabetes treatment, there is a problem limiting the usefulness of GLP-1. GLP-1 is degraded rapidly due to the presence of a ubiquitously expressed enzyme, DPP-IV. The conversion of intact, biologically active GLP-1 to its inactive metabolites occurs within 2 min. Therefore, continuous infusion or multiple injections are required for clinical application of GLP-1.
To delay the degradation of GLP-1, the properties of the injectable form have been modified with protamine or zinc. However, the results from using various methods have shown that the half-life is still too short. A number of new approaches are currently under investigation for using GLP-1 as a therapeutic agent. Since the extremely short half-life is due to DPP-IV activity, DPP-IV-resistant analogs and DPP-IV inhibitors have been developed as alternatives. Even though they have shown considerable promise, they still require at least everyday injection to get the therapeutic effect.
If GLP-1 plasmid can be delivered and the genetic information can be effectively processed, it would allow enough GLP-1 to stay in the body for treatment of type 2 diabetes. Nonviral gene delivery is more desirable than viral gene delivery because of its excellent safety profile and ability to carry large amounts of DNA. However, the efficiency of nonviral gene delivery is not sufficient for therapeutic application. In this study, a GLP-1 plasmid was designed to achieve enough expression of GLP-1. The GLP-1 plasmid was constructed by using optimal transcriptional regulatory elements. Also, the GLP-1 plasmid was further modified with nuclear factor B (NF B) binding sites to enhance nuclear import. One way to enhance gene expression efficiency is to increase nuclear transport of plasmids into cells. Nuclear factor B is a family of transcription factors present in every cell type. In one study, under nonstimulatory conditions, transfection was enhanced by 2.6- to 5.8-fold by incorporating NF B binding sites into plasmids. In addition, it has been suggested that the activation of NF B occurs in chronic disorders, including diabetes.