A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography.
The extensive testing of adeno-associated virus (AAV) as a vector for human gene therapy has been hampered by low efficiency of the current packaging system, which is based on transient transfection with plasmid DNAs and infection with adenovirus in permissive cells. In an effort to resolve this problem, HeLa cell-based packaging cell lines were established. These packaging cells carry multiple copies of the AAV genome lacking the inverted terminal repeat (ITR) sequences. The AAV genes were silent in these cells but inducibly expressed by adenovirus infection. When the AAV vector plasmid containing the neoR gene flanked by the ITRs was also integrated into these cells, efficient production of the recombinant AAV particles occurred after adenovirus infection. AAV vector particles in cell lysates could be concentrated by sulfonated cellulose column chromatography. Using the packaging cells and the column chromatography technique, it is possible to prepare AAV vectors with the titer of higher than 10(8) cfu/ml or 5 x 10(10) particles/ml. This new strategy should be useful for testing AAV vectors in vivo.
Tamayose
K
K
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Hirai
Y
Y
Shimada
T
T
D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry; D002845:Chromatography; D004268:DNA-Binding Proteins / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D002482:Cellulose; C020254:cellulose sulfate