Cellular contaminants of adeno-associated virus vector stocks can enhance transduction.
Transduction efficiency of different types of recombinant (r)AAV-2 based vectors preparations markedly differed, with apparently no correlation with the replicative titers. Using HeLa cells as target for transduction, 105 and 30 infectious units were necessary to observe one transductant using respectively cesium-chloride-purified rAAV and crude lysates of producer cells obtained by sonication. The purified vectors were however able to transduce HEK-193 cells efficiently, but transgene expression was detected with some delay compared with crude lysates. The unexpected high transduction efficiency of sonicated crude lysates was due to virally mediated gene transfer, since similar sonicated crude lysates, but with no AAV rep and cap genes, did not lead to detection of transgene products after incubation with HeLa cells. Furthermore, sonicated cellular extracts of 293 or 293/T cells given in trans stimulate transduction of HeLa cells by purified rAAV. In contrast, neither extracts from the adenovirus E1-transformed 911 cell line, nor from other cell lines not harboring any adenovirus gene, had enhancing effect on rAAV-mediated transduction. These data suggest that 293 sonicated extracts contain factors which stimulate rAAV-mediated transduction of cells that are normally poorly transduced and offer a system to identify such factors and to characterize further the steps limiting the transfer of gene by AAV vectors.
Tenenbaum
L
L
IRIBHN, Campus Erasme, Université Libre de Bruxelles, Germany.
Hamdane
M
M
Pouzet
M
M
Avalosse
B
B
Stathopoulos
A
A
Jurysta
F
F
Rosenbaum
C
C
Hanemann
C O
CO
Levivier
M
M
Velu
T
T
D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors / Q000592:standards*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D014162:Transfection; D014779:Virus Replication / Q000235:genetics
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins