T. Martin et al., Plasmid DNA malaria vaccine: The potential for genomic integration after intramuscular injection, HUM GENE TH, 10(5), 1999, pp. 759-768
Plasmid-based (naked DNA) genetic vaccines are now entering clinical trials
to test their safety and efficacy in healthy human volunteers. A safety co
ncern unique to this new class of vaccines is the potential risk of deleter
ious 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 se
quences persisting in total muscle DNA at both 30 and 60 days following a s
ingle intramuscular injection of a plasmid expressing the Plasmodium falcip
arum 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 fou
nd that the amount of plasmid DNA persisting in muscle tissue varied but av
eraged about 10 fg per microgram of genomic DNA (in the range of 1500 copie
s per 150,000 genomes). In two of four separate experimental injections of
mouse muscle, PCR assays of genomic DNA fractions indicated that agarose ge
l purification removed plasmid DNA down to a level of less than or equal to
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 pos
sible to conclude if such sequences were covalently linked to genomic DNA o
r simply adventitiously associated with the genomic DNA. However, if an ass
umption is made that the highest level plasmid DNA found associated with ge
nomic DNA (i.e., 30 copies) represented covalently integrated plasmid inser
ts and that each insert resulted in a mutational event, the calculated rate
of mutation would be 3000 times less than the spontaneous mutation rate fo
r mammalian genomes. This level of integration, if it should occur, was not
considered to pose a significant safety concern.