Association between adeno‐associated virus genomic titers and intracellular plasmid levels

Abstract

Abstract The recombinant adeno‐associated viral (rAAV) vector is one of the most effective viral vectors in gene therapy because of its low immunogenicity, high transduction efficiency, broad tissue specificity, and long‐term transgene expression ability. HEK293T cells are widely used to produce rAAV vectors, and acquisition of high rAAV‐producing cells is one of the essential processes for higher rAAV vector production. To acquire such cells, a method to identify cells with high expression of transgene protein was developed; however, the relationship between high expression of transgene protein and increased production of rAAV has not been sufficiently studied to date. We used the fluorescent protein ZsGreen1 as a transgene, subdivided the adherent cells according to the intensity of transgene expression protein, and reconfirmed the relationship between rAAV vector production and the amount of intracellular plasmid. We found that the amount of rAAV produced was not correlated with the intensity of expression of the transgene protein but may be correlated with the amount of intracellular plasmid. We also found that cells with high expression of transgene protein might not necessarily produce large amounts of rAAV vector. Based on these results, it was suggested that intracellular plasmid copy number could be used as a new marker for cells producing high levels of rAAV vector.

Keywords

• adeno‐associated virus
• cell sorting
• fractionation
• genome titer
• intracellular plasmid copies
• transfection