title: Determination of tissue distribution of an intramuscular plasmid vaccine using PCR and in situ DNA hybridization.
abstract: The increasing use of nucleic acid-based therapeutics has created a need for new methods of determining tissue distribution and levels. Radiolabel methods may not always be appropriate because nucleic acids are easily degraded. Quantitation using the polymerase chain reaction (PCR) has the advantage that only continuous stretches of DNA will be amplified. In situ hybridization allows detection of specific sequences in histological preparations. We have used quantitative PCR and in situ hybridization techniques to study the pharmacokinetics and distribution of PGagPol (a potential anti-HIV plasmid vaccine) in rabbits. Samples were obtained 4 hr, 24 hr, 7 days, and 28 days after intramuscular injection of 100 micrograms or 400 micrograms of plasmid. A simplified procedure for collecting and processing tissues for PCR that minimizes the risk of contamination was developed. Using PCR, plasmid was found principally in the skin and muscle of the injection site and in blood plasma. At 4 hr after dosing with 400 micrograms, the plasmid was detected at the injection 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 days 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 degree 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.
authors.affiliation: Toxicology Laboratory, SRI International, Menlo Park, CA 94025, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Winegar
authors.forename: J A
authors.initials: JA
authors.lastname: Monforte
authors.forename: K D
authors.initials: KD
authors.lastname: Suing
authors.forename: K G
authors.initials: KG
authors.lastname: O'Loughlin
authors.forename: C J
authors.initials: CJ
authors.lastname: Rudd
authors.forename: J T
authors.initials: JT
authors.lastname: Macgregor
mesh_terms: D000818:Animals; D001774:Blood Chemical Analysis; D015685:Fusion Proteins, gag-pol / Q000235:genetics* / Q000276:immunology* / Q000493:pharmacokinetics; D006678:HIV / Q000235:genetics* / Q000276:immunology*; D017403:In Situ Hybridization / Q000379:methods; D009132:Muscles / Q000737:chemistry; D010957:Plasmids / Q000276:immunology* / Q000493:pharmacokinetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D012680:Sensitivity and Specificity; D012867:Skin / Q000737:chemistry; D014018:Tissue Distribution / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics*
chemical_list: D015685:Fusion Proteins, gag-pol; D014614:Vaccines, Synthetic