Gene therapy of streptozotocin-induced diabetes by intramuscular delivery of modified preproinsulin genes.

BACKGROUND: Despite improvements in insulin preparation and delivery, physiological normoglycemia is not easily achieved in diabetics. Therefore, there has been considerable interest in developing gene therapy approaches to supply insulin. We studied a nonviral muscle-based method of gene therapy and demonstrated that it could prevent hyperglycemia in murine streptozotocin (STZ)-induced diabetes. METHODS: A plasmid encoding mouse furin-cleavable preproinsulin II cDNA (FI), or its B10-analogue (B10FI), and a plasmid encoding furin were coinjected into muscle of CD-1 mice, who were treated a day later with STZ to induce diabetes. Electroporation was applied to increase gene transfer. Blood glucose was measured in fed and fasting mice, and fasting plasma insulin was measured by radioimmunoassay. The form of insulin produced and the presence of C-peptide were analyzed by gel filtration chromatography. RESULTS: A B10FI plasmid codelivered with a furin plasmid reduced fed and fasting blood glucose levels in STZ-treated diabetic mice. The (pro)insulin levels in plasma were increased by up to 70-fold versus blank plasmid-treated diabetic mice. The administration of FI with furin was less effective. (Pro)insulin levels were greatly increased by using two plasmids carrying different promoter elements (CMV and SV40). Insulin was identified in muscle cells by immunohistochemistry. In plasma, 40-70% of the (pro)insulin was processed to the mature form and free C-peptide was identified. Insulin gene-treated mice had improved growth rates and appeared healthier. A single injection of B10FI with SV40Furin DNA increased plasma (pro)insulin for at least 8 weeks and reduced fed blood glucose levels for 5 weeks and fasting levels for 8 weeks. CONCLUSIONS: This is the first report that electroporation-enhanced intramuscular gene therapy with B10FI can prevent hyperglycemia in murine STZ-induced diabetes. Gene therapy using various routes and methods of furin-cleavable insulin gene delivery has been previously explored but, in muscle, results comparable to ours have not been reported.

Regulatory cytokine production stimulated by DNA vaccination against an altered form of glutamic acid decarboxylase 65 in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop a T-cell dependent autoimmune form of diabetes, in which glutamic acid decarboxylase 65 (GAD65) is an important islet target antigen. Intramuscular DNA vaccination with a plasmid encoding native GAD65 (a cytosolic antigen) did not significantly alter the incidence of diabetes, but vaccination against an altered form of GAD65 with a signal peptide (spGAD), which is secreted in vitro, was protective. The preventive effect was further enhanced by repeated injections of the spGAD plasmid. Following DNA injection into muscle GAD65 was expressed for several months, and this was not accompanied by an inflammatory response. Immunization against GAD65 was not associated with substantial alterations in cytokine production by splenic lymphocytes stimulated with immunogenic GAD65 peptides. In contrast, spGAD induced increased secretion of both interleukin 10 and interferon gamma and a striking decrease in the interferon gamma/interleukin 10 ratio in culture supernatants. Similarly, spGAD-immunized mice had higher serum interleukin 10 levels and lower serum interferon gamma levels than other groups, suggesting a systemic effect. In nondiabetic mice there was increased basal production of transforming growth factor beta(1), which was enhanced by antigenic stimulation. These alterations in regulatory cytokine production were apparent both early and late after the treatment was initiated. These findings suggest that DNA vaccination against spGAD protects NOD mice by increasing regulatory cytokine production.