Insulin delivery with plasmid DNA

Success in controlling hyperglycemia in type I diabetics will require a res toration of basal insulin. To this end, three plasmid DNAs (pDNA) encoding preproinsulin were compared for constitutive expression and processing to i nsulin in nonendocrine cells in vitro. The pDNAs were designed to express r at proinsulin I (VR-3501), rat proinsulin I with the B10 aspartic acid poin t mutation (VR-3502), and a derivative of VR-3502 with a furin cleavage sit e added at the B-chain and C-peptide junction (VR-3503). Cells transfected with VR-3501 or VR-3502 were able to secrete only proinsulin, whereas trans fection with VR-3503 yielded 30-70% mature insulin, which could be increase d to > 99% by cotransfection with a furin expression plasmid (VR-3505). The insulin produced was biologically active. The bilateral injection of 100 m u g of VR-3502 plasmid into the tibialis anterior muscles of mice on two co nsecutive days yielded, on average, several hundred picograms of heterologo us proinsulin per milliliter of serum. In BALB/c mice, serum proinsulin pea ked 7-14 days postinjection and declined to preinjection levels by days 21- 28. In athymic nude mice, serum proinsulin was sustained for at least 6 wee ks. The therapeutic efficacy of delivering insulin via muscle injection of pDNA was evaluated in athymic nude mice made diabetic with the beta cell to xin streptozotocin (STZ). All animals given control DNA died within 1 week of receiving STZ while 40% of the mice coinjected with plasmids VR-3503 and VR-3505 lived through the duration of the 4-week experiment. Muscles of th e surviving animals contained 17-100 ng of immune-reactive insulin (IRI), 8 6-94% of which was mature insulin. The results suggest that heterologous in sulin made in muscle increased the survival rate. We propose that insulin p lasmid expression in skeletal muscle may be a valid approach to basal insul in delivery.