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

Abstract

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 neo^R 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 10⁸ cfu/ml or 5 × 10¹⁰ particles/ml. This new strategy should be useful for testing AAV vectors in vivo. Adeno-associated virus (AAV) is a potential vector for human gene therapy. However, it has been difficult to prepare sufficient amounts of high-titer AAV vectors for in vivo application. In this work, we established HeLa cell lines containing multiple copies of the AAV genome and demonstrated that these cells can be used for packaging of recombinant AAV vectors. We also showed that Cellulofine sulfate column chromatography was useful for concentration of infectious AAV particles. Using these two techniques, large-scale preparation of high-titer AAV vectors became possible.