Influence of sequence and size of DNA on packaging efficiency of parvovirus MVM-based vectors.
We have derived a vector from the autonomous parvovirus MVM(p), which expresses human IL-2 specifically in transformed cells (Russell et al., J. Virol 1992;66:2821-2828). Testing the therapeutic potential of these vectors in vivo requires high-titer stocks. Stocks with a titer of 10(9) can be obtained after concentration and purification (Avalosse et al., J. Virol. Methods 1996;62:179-183), but this method requires large culture volumes and cannot easily be scaled up. We wanted to increase the production of recombinant virus at the initial transfection step. Poor vector titers could be due to inadequate genome amplification or to inefficient packaging. Here we show that intracellular amplification of MVM vector genomes is not the limiting factor for vector production. Several vector genomes of different size and/or structure were amplified to an equal extent. Their amplification was also equivalent to that of a cotransfected wild-type genome. We did not observe any interference between vector and wild-type genomes at the level of DNA amplification. Despite equivalent genome amplification, vector titers varied greatly between the different genomes, presumably owing to differences in packaging efficiency. Genomes with a size close to 100% that of wild type were packaged most efficiently with loss of efficiency at lower and higher sizes. However, certain genomes of identical size showed different packaging efficiencies, illustrating the importance of the DNA sequence, and probably its structure.
Brandenburger
A
A
IRIBHN, Université Libre de Bruxelles, Brussels, Belgium. abranden@ulb.ac.be
Coessens
E
E
El Bakkouri
K
K
Velu
T
T
D010582:Bacteriophage lambda / Q000235:genetics; D002460:Cell Line; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D008915:Minute Virus of Mice / Q000235:genetics* / Q000502:physiology*; D010316:Particle Size; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D019065:Virus Assembly
D004279:DNA, Viral; D007376:Interleukin-2