Potential DNA Vaccine Integration into Host Cell Genome

Abstract

Studies have been designed to examine the potential integration of DNA vaccines into the host cell genome. This is of concern because of the possibility of insertional mutagenesis resulting in the inactivation of tumor suppressor genes or the activation of oncogenes. The requirements for adequate testing were determined to be (1) a method to purify host cell genomic DNA from nonintegrated free plasmid, (2) a sensitive method to detect integrated plasmid in the purified genomic DNA, and (3) stringent methods to avoid contamination. These requirements were fulfilled by agarose-gel electrophoresis, the polymerase chain reaction, and separation of each activity with stringent handling procedures, respectively. An exploratory experiment was carried out in which mice were injected with 100 micrograms of vaccine plasmid DNA in each quadriceps. Examination of quadriceps and 12 other tissues at several time points failed to reveal any evidence of integration at a sensitivity level that could detect 1 to 7.5 integrations in 150,000 nuclei. A worst-case scenario determined that this would be at least 3 orders of magnitude below the spontaneous mutation frequency.

Cellular immunity induced by a novel HPV18 DNA vaccine encoding an E6/E7 fusion consensus protein in mice and rhesus macaques

“…DNA vaccines appear to be very well tolerated in humans. Preclinical safety studies indicate that there was little evidence of plasmid integration (13,14). DNA vaccines can also be used for repeat administration as the efficacy of plasmid vectors are not influenced by preexisting neutralizing antibodies (15).…”

Perspectivas para nuevas vacunas antituberculosas en la era posgenómica.

“…La actividad adyuvante de los CpG se debe a que éstos se unen a los receptores tipo Toll 9 (TLR9) e inducen la producción de IL-12 (que estimula la producción de IFNγ por las células NK, y dirigen la respuesta Th1), IFNα, TNFα e IL-6 (52). Es interesante que, después de muchos meses, el patrón de metilación del ADN de los plásmidos inyectados continúa siendo de tipo bacteriano (adenosinas metiladas) lo cual indica que éstos no se replican en las células eucarióticas y que, por lo tanto, su capacidad de hacer recombinaciones homólogas, así como su potencial mutagénico y carcinogénico es muy bajo (53).…”

Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo

“…1 Mouse skeletal muscle injected with plasmid DNA alone results in long-term protein expression. 2 Plasmid DNA is neither replicated nor integrated into the host cell genome, but remains in its episomal form 3 and is expressed in both dividing and nondividing cells. The injection of DNA does not result in the production of anti-DNA antibodies, 4,5 which allows for multiple treatments.…”

“…DNA dose dependence was investigated in order to maximize tumor reporter protein expression. Quantities ranging from 0.1 to 2 g per mm 3 tumor volume were examined ( Figure 3). Luciferase expression showed a DNA dose dependence, with the highest tested activity at 2 g per mm 3 tumor volume.…”