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.