Ra. Winegar et al., DETERMINATION OF TISSUE DISTRIBUTION OF AN INTRAMUSCULAR PLASMID VACCINE USING PCR AND IN-SITU DNA HYBRIDIZATION, Human gene therapy, 7(17), 1996, pp. 2185-2194
The increasing use of nucleic acid-based therapeutics has created a ne
ed for new methods of determining tissue distribution and levels. Radi
olabel methods may not always be appropriate because nucleic acids are
easily degraded. Quantitation using the polymerase chain reaction (PC
R) has the advantage that only continuous stretches of DNA will be amp
lified. In situ hybridization allows detection of specific sequences i
n histological preparations. We have used quantitative PCR and in situ
hybridization techniques to study the pharmacokinetics and distributi
on of PGagPol (a potential anti-HIV plasmid vaccine) in rabbits. Sampl
es were obtained 4 hr, 24 hr, 7 days, and 28 days after intramuscular
injection of 100 mu g or 400 mu g of plasmid. A simplified procedure f
or collecting and processing tissues for PCR that minimizes the risk o
f contamination was developed. Using PCR, plasmid was found principall
y in the skin and muscle of the injection site and in blood plasma. At
4 hr after dosing with 400 mu g, the plasmid was detected at the inje
ction site with mean copy numbers of 10(6) (in muscle) and 4 x 10(4) (
in skin) per microgram of tissue. Plasmid copy number declined rapidly
in muscle during the first 24 hr and was undetectable at 7 and 28 day
s after injection. The decline was slower in the skin, and the plasmid
was still detectable at 28 days. With in situ hybridization, plasmid
was detected in muscle, mainly in the perimysium and to a lesser degre
e in the endomysium and within the muscle fibers. These data indicate
that quantitative PCR and in situ hybridization are sensitive methods
for examining tissue distribution of DNA used for gene therapy.