ONLY A SMALL FRACTION OF CELLS PRODUCE ASSEMBLED CAPSIDS DURING
TRANSFECTION-BASED MANUFACTURING OF ADENO-ASSOCIATED VIRUS VECTORS
Abstract
Plasmid transfection of mammalian cells is the dominant platform used to
produce adeno-associated virus (AAV) vectors for clinical and research
applications. Low yields from this platform currently make it difficult
to supply these activities with adequate material. In an effort to
better understand the current limitations of transfection-based
manufacturing, this study examines what proportion of cells in a model
transfection produce appreciable amounts of assembled AAV capsid. Using
conformation-specific antibody staining and flow cytometry we report the
surprising result that despite obtaining high transfection efficiencies
and nominal vector yields in our model system, only 5-10% of cells
appear to produce measurable levels of assembled AAV capsids. This
finding implies that considerable increases in vector titer could be
realized through increasing the proportion of productive cells.
Furthermore, we suggest that the flow cytometry assay used here to
quantify productive cells may be a useful metric for future optimization
of transfection-based AAV vector manufacturing platforms.