CHARACTERIZATION OF PLASMID DNA TRANSFER INTO MOUSE SKELETAL-MUSCLE -EVALUATION OF UPTAKE MECHANISM, EXPRESSION AND SECRETION OF GENE-PRODUCTS INTO BLOOD
My. Levy et al., CHARACTERIZATION OF PLASMID DNA TRANSFER INTO MOUSE SKELETAL-MUSCLE -EVALUATION OF UPTAKE MECHANISM, EXPRESSION AND SECRETION OF GENE-PRODUCTS INTO BLOOD, Gene therapy, 3(3), 1996, pp. 201-211
The expression of naked plasmid DNA coding for firefly luciferase (pRS
Vluc) or a secreted protein, human-alpha-1-antitrypsin (pRcCMVhAAT) in
mouse skeletal muscle was characterized following administration by a
n improved intramuscular injection technique. Injection guided by inte
nse illumination along the longitudinal axis of the mouse quadriceps m
uscle and parallel to the myofibers yielded 200-fold higher levels of
luciferase expression than perpendicular injection. Luciferase express
ion was inhibition by an excess of non-coding DNA or dextran sulfate s
uggesting that muscle DNA uptake mechanism(s) can be saturated. Inject
ed plasmid DNA was rapidly eliminated from the muscle as evidenced by
tissue distribution studies of radiolabeled hAAT plasmid and Southern
analysis. However, PCR analysis demonstrated that hAAT cDNA persisted
in the muscle for at least 1 month after injection. Immunohistochemist
ry techniques indicated that the hAAT gene was expressed by the muscle
fibers. ELISA analysis of serum samples collected from intramuscularl
y injected mice demonstrated that secreted hAAT protein concentration
peaked in serum by day 7, started to decline by day 14 and was barely
detectable 21 days post-injection. RT-PCR analysis demonstrated that h
AAT transcript persisted at the site of injection for at least 1 month
indicating that the decline of serum hAAT concentration 21 days post-
injection was not due to the absence of hAAT transcript. However, the
decline of hAAT protein concentration in the serum was inversely corre
lated with accumulation of murine anti-hAAT antibodies in circulation.