A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination

Recent work highlights the potential usefulness of MVM-based vectors as sel ective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790-796). To implement this strategy however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-tra nsfecting permissive cell lines with a plasmid carrying the vector genome a nd a helper plasmid expressing the capsid genes (replaced with a foreign ge ne in the vector genome). The resulting stocks, however, are always heavily contaminated with replication-competent viruses (RCV), which precludes the ir use in vivo and particularly in gene therapy. In the present work we hav e developed a second-generation MVMp-based vector system specifically desig ned to reduce the probability of RCV generation by homologous recombination . We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code t o further decrease vector-helper homology. In this system, the left homolog ous region was almost completely eliminated and the right sequence overlap was reduced to 74 nt with only 61% homology. We were thus able to substanti ally reduce (similar to 200 x), but not completely eliminate, generation of contaminating viruses in medium-scale rMVMp preparations. Since the remain ing sequence homology between the new vector and helper genomes is weak, ou r results suggest that contaminating viruses in this system are generated b y nonhomologous recombination. It is important to note, unlike the autonomo usly replicating helper viruses produced from the first-generation vector/h elper genomes, the contaminating viruses arising from the new packaging sys tem cannot initiate secondary infection rounds (so they are not 'replicatio n-competent viruses'). Our findings have important implications for the des ign of new MVMp-based vectors and for the construction of trans-complementi ng packaging cell lines.