BACKGROUND: The ability of tissues to take up naked plasmid DNA in vivo suggests an approach for reconstituting systemic metabolic deficiencies without the disadvantages of viral vectors and lipid-DNA complexes. Plasmid-mediated gene transfer into skeletal muscle was investigated as a means of providing a therapeutic source of insulin. METHODS: Four plasmid constructs, each bearing a mouse furin cDNA transgene and rat proinsulin cDNA (modified for processing by furin) driven by four different promoters were injected into the calf muscles of male Balb/c mice. Insulin and C-peptide concentrations were measured by radio-immunoassays having minimal crossreactivity for proinsulin and partially processed proinsulin. RESULTS: Intramuscular insulin concentrations increased by up to 3.6-fold over controls seven days after single injections of CMV, beta-actin, hsp70 and myoglobin promoter constructs. The optimal dose for most constructs was 100 micrograms plasmid DNA. Intramuscular plasmid injection into streptozotocin-induced diabetic Balb/c mice raised plasma insulin and C-peptide concentrations, and reduced hyperglycaemia. Two injections (100 micrograms plasmid DNA each) caused higher plasma insulin concentrations and significantly reduced hyperglycemia in diabetic mice than a single injection. Best results were obtained when plasmid injections preceded induction of diabetes by 14 days. CONCLUSIONS: Skeletal muscle is a potentially useful platform for ectopic secretion of insulin using naked plasmid as a gene transfer vector. Injection at two sites 14 days before the onset of severe hyperglycemia is optimal. This approach could protect Type I diabetics from fatal ketoacidosis and enhance the action of agents that sensitize tissues to insulin in type II diabetes.
BACKGROUND: The ability of tissues to take up naked plasmid DNA in vivo suggests an approach for reconstituting systemic metabolic deficiencies without the disadvantages of viral vectors and lipid-DNA complexes. Plasmid-mediated gene transfer into skeletal muscle was investigated as a means of providing a therapeutic source of insulin. METHODS: Four plasmid constructs, each bearing a mousefurin cDNA transgene and ratproinsulin cDNA (modified for processing by furin) driven by four different promoters were injected into the calf muscles of male Balb/c mice. Insulin and C-peptide concentrations were measured by radio-immunoassays having minimal crossreactivity for proinsulin and partially processed proinsulin. RESULTS: Intramuscular insulin concentrations increased by up to 3.6-fold over controls seven days after single injections of CMV, beta-actin, hsp70 and myoglobin promoter constructs. The optimal dose for most constructs was 100 micrograms plasmid DNA. Intramuscular plasmid injection into streptozotocin-induced diabetic Balb/c mice raised plasma insulin and C-peptide concentrations, and reduced hyperglycaemia. Two injections (100 micrograms plasmid DNA each) caused higher plasma insulin concentrations and significantly reduced hyperglycemia in diabeticmice than a single injection. Best results were obtained when plasmid injections preceded induction of diabetes by 14 days. CONCLUSIONS: Skeletal muscle is a potentially useful platform for ectopic secretion of insulin using naked plasmid as a gene transfer vector. Injection at two sites 14 days before the onset of severe hyperglycemia is optimal. This approach could protect Type I diabetics from fatal ketoacidosis and enhance the action of agents that sensitize tissues to insulin in type II diabetes.