Plasmid DNA Vaccines: Investigation of Integration into Host Cellular DNA following Intramuscular Injection in Mice

Abstract

The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/µg DNA (representing ∼150,000 diploid cells). Using this assay, we carried out integration studies of three different plasmid DNA vaccines, containing either the influenza hemagglutinin, influenza matrix or HIV gag gene. Six weeks after intramuscular injection, free plasmid was detected in treated muscle at levels ranging from approximately 1,000 to 4,000 copies/µg DNA. At 6 months, the plasmid levels ranged between 200 and 800 copies/µg DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was extrachromosomal. If integration had occurred, the frequency was ≤1–8 integrations per 150,000 diploid cells, which would be at least three orders of magnitude below the spontaneous mutation rate. Our results suggest that the risk of mutation due to integration of plasmid DNA vaccines following intramuscular injection is negligible.

Absolute Quantification of Plasmid DNA by Real-time PCR with Genomic DNA as External Standard and Its Application to a Biodistribution Study of an HIV DNA Vaccine

“…Frozen mice tissues were homogenized and divided into aliquots of about 20 mg, and total DNA was purified from an aliquot of the homogenate by boiling, 16 phenol-chloroform extraction 17 and DNA isolation kit. Briefly, for the boiling method, cell suspensions of different tissues were washed by PBS three times and suspended in 100 ml sterile ddH2O.…”

Plasmid-based gene therapy of diabetes mellitus

“…Animal studies involving plasmid DNA injections have shown that mutations from a potential integration event would be extremely infrequent, about 3000 times lower than the spontaneous mutation rate for mammalian genomes. [16][17][18] However, in the case of DNA vaccination against foreign antigens, it is of some concern that transfected muscle cells may be attacked and injured by the immune system, and this has been reported. 4,7,8 A related concern is the production of pathogenic anti-DNA antibodies, potentially induced by plasmid DNA and its immunostimulatory sequences (ISS), but the risk appears relatively small.…”

Illegitimate DNA integration in mammalian cells

“…This appears to happen at a low frequency. 16,17 However, we will see in this review that the resultant integrated structure can be surprisingly complex and unpredictable compared to the expected structure of targeted integration, making it difficult to evaluate the risks inherent in these events (see Figure 1).…”