Plasmid DNA vaccines: Investigation of integration into host cellular DNA following intramuscular injection in mice

The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purif ication of high-molecular-weight genomic DNA away from free plasmid using g el electrophoresis, such that the genomic DNA can then be assayed for integ rated plasmid using a sensitive PCR method. The assay sensitivity was appro ximately 1 plasmid copy/mug DNA (representing similar to 150,000 diploid ce lls). Using this assay, we carried out integration studies of three differe nt plasmid DNA vaccines, containing either the influenza hemagglutinin, inf luenza matrix or HIV gag gene. Six weeks after intramuscular injection, fre e plasmid was detected in treated muscle at levels ranging from approximate ly 1,000 to 4,000 copies/mug DNA. At 6 months, the plasmid levels ranged be tween 200 and 800 copies/mug DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was ex trachromosomal. If integration had occurred, the frequency was less than or equal to1-8 integrations per 150,000 diploid cells, which would be at leas t three orders of magnitude below the spontaneous mutation rate. Our result s suggest that the risk of mutation due to integration of plasmid DNA vacci nes following intramuscular injection is negligible. Copyright (C) 2001 S. Karger AG, Basel.