Plasmid DNA Malaria Vaccine: The Potential for Genomic Integration after Intramuscular Injection

Abstract

Plasmid-based (naked DNA) genetic vaccines are now entering clinical trials to test their safety and efficacy in healthy human volunteers. A safety concern unique to this new class of vaccines is the potential risk of deleterious integration into host cell genomic DNA following direct intramuscular injection. To address this issue experimentally, a preclinical safety study was conducted in mice to determine the structural nature of plasmid DNA sequences persisting in total muscle DNA at both 30 and 60 days following a single intramuscular injection of a plasmid expressing the Plasmodium falciparum circumsporozoite protein. In a protocol described for the first time, total DNA was extracted from muscle tissue and was subsequently linearized with a restriction endonuclease to enable agarose gel size fractionation of all extrachromosomal plasmid DNAs from high molecular weight mouse genomic DNA. Using PCR assays to quantitate plasmid-specific sequences, it was found that the amount of plasmid DNA persisting in muscle tissue varied but averaged about 10 fg per microgram of genomic DNA (in the range of 1500 copies per 150,000 genomes). In two of four separate experimental injections of mouse muscle, PCR assays of genomic DNA fractions indicated that agarose gel purification removed plasmid DNA down to a level of < or =3 copies per 150,000 mouse genomes. In the two other experimental samples, 3-30 copies of plasmid DNA remained associated with purified genomic DNA. The time following injection (i.e., 30 or 60 days) was not a factor in the number of copies of plasmid associating with genomic DNA and it was not possible to conclude if such sequences were covalently linked to genomic DNA or simply adventitiously associated with the genomic DNA. However, if an assumption is made that the highest level plasmid DNA found associated with genomic DNA (i.e., 30 copies) represented covalently integrated plasmid inserts and that each insert resulted in a mutational event, the calculated rate of mutation would be 3000 times less than the spontaneous mutation rate for mammalian genomes. This level of integration, if it should occur, was not considered to pose a significant safety concern.

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA

“…Unlike viruses, the plasmid has no potential for reactivation to a pathogenic state and some authors have recently demonstrated that, following intramuscular injection, integration of plasmid DNA into muscle genomic DNA, should it occur, is an extremely rare event (the calculated rate of integration would be 3000 times lower than the accepted rate of spontaneous mutation rate for mammalian genome). 21,43 In conclusion, our present study demonstrates the potential feasibility of the intramuscular gene transfer with naked plasmid DNA encoding apoE for the treatment of hyperlipidemic conditions. To date, however, several important issues remain to be addressed before this approach can be feasible for human gene therapy (ie higher levels of circulating proteins, increased efficiency of plasmid DNA uptake, increased half-life of the recombinant protein, systems for regulating recombinant gene expression, increased magnitude and temporal stability of transgene expression, 44 etc).…”

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).…”

Improved osteogenic vector for non-viral gene therapy

“…Advances in efficiency, specificity, duration of gene expression and safety led to a rising number of non-viral vector products entering clinical trials. However, there is still plenty of room for improvement of currently available non-viral vectors regarding expression rate and duration (Al-Dosari and Gao, 2009;Kay, 2011;Mingozzi and High, 2011;Wang et al, 2013). To promote further development of non-viral gene delivery approaches, non-viral expression systems were designed and tested for bone tissue engineering and regeneration.…”

“…However, compared to viral gene delivery methods, non-viral gene therapy shows generally low gene transfer efficiency, especially in vivo. Nevertheless, non-viral approaches do not trigger the host´s immune responses that much, due to lack of viral protein components and also have a very rare frequency of inserting into host genome (Martin et al, 1999). Furthermore, there is no size limitation concerning coding sequence and production is cost-efficient, safe and easy.…”

“…Therefore, instead of making viral gene transfer safer, currently a large number of on-going attempts are focussing on the enhancement of non-viral approaches (Bleiziffer et al, 2007;Glover et al, 2005). There is still plenty of room for improvement of the presently available non-viral vectors, regarding expression rate and duration (Al-Dosari and Gao, 2009;Kay, 2011;Mingozzi and High, 2011;Wang et al, 2013). Methods for improvement of bone morphogenetic protein 2 (BMP-2) expression systems are introduced in this work in order to promote further development of non-viral gene delivery approaches making safe non-viral systems even more applicable (Fig.…”