title: Insulin delivery with plasmid DNA.
abstract: Success in controlling hyperglycemia in type I diabetics will require a restoration of basal insulin. To this end, three plasmid DNAs (pDNA) encoding preproinsulin were compared for constitutive expression and processing to insulin in nonendocrine cells in vitro. The pDNAs were designed to express rat proinsulin I (VR-3501), rat proinsulin I with the B10 aspartic acid point mutation (VR-3502), and a derivative of VR-3502 with a furin cleavage site 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 transfection with VR-3503 yielded 30-70% mature insulin, which could be increased to >99% by cotransfection with a furin expression plasmid (VR-3505). The insulin produced was biologically active. The bilateral injection of 100 microg of VR-3502 plasmid into the tibialis anterior muscles of mice on two consecutive days yielded, on average, several hundred picograms of heterologous proinsulin per milliliter of serum. In BALB/c mice, serum proinsulin peaked 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 weeks. The therapeutic efficacy of delivering insulin via muscle injection of pDNA was evaluated in athymic nude mice made diabetic with the beta cell toxin 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 the surviving animals contained 17-100 ng of immune-reactive insulin (IRI), 86-94% of which was mature insulin. The results suggest that heterologous insulin made in muscle increased the survival rate. We propose that insulin plasmid expression in skeletal muscle may be a valid approach to basal insulin delivery. The feasibility of plasmid DNA-based delivery of basal insulin was investigated. An expression system consisting of pDNAs encoding a selectively mutated rat preproinsulin and mouse furin was developed and characterized in vitro and in vivo. When injected with preproinsulin pDNA, the mouse tibialis anterior muscle expressed and released proinsulin into serum at levels comparable to normal basal insulin in rodents. These heterologous proinsulin levels were sustained for several weeks in immune-compromised nondiabetic mice. Mouse muscle coinjected with a pDNA encoding the endopeptidase furin and a pDNA encoding a pre-proinsulin modified to contain two furin cleavage sites produced fully processed insulin. This muscle-made insulin appears to have contributed to the survival of mice treated with a highly diabetogenic dose of streptozotocin, a beta cell toxin. The results demonstrate that skeletal muscle is able to express and deliver therapeutic insulin from plasmid DNA.
authors.affiliation: Department of Molecular Biology, Vical, Inc., San Diego, CA 92121, USA. aabai@vical.com
authors.forename: A M
authors.initials: AM
authors.lastname: Abai
authors.forename: P M
authors.initials: PM
authors.lastname: Hobart
authors.forename: K M
authors.initials: KM
authors.lastname: Barnhart
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy* / Q000378:metabolism; D016503:Drug Delivery Systems; D045683:Furin; D006801:Humans; D016867:Immunocompromised Host; D007273:Injections, Intramuscular; D007328:Insulin / Q000096:biosynthesis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011384:Proinsulin / Q000008:administration & dosage* / Q000235:genetics*; D011498:Protein Precursors / Q000008:administration & dosage* / Q000235:genetics*; D051381:Rats; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D007328:Insulin; D011498:Protein Precursors; C013737:preproinsulin; D004247:DNA; D011384:Proinsulin; D013381:Subtilisins; D045683:Furin