RECOMBINANT AAV6 GENE THERAPY VECTORS: PROCESS OPTIMIZATION AND SCALE UP INTO THE iCELLis NANO BIOREACTOR
BORGES-THESIS-2018.pdf (1.259Mb) (embargoed until: 2022-12-01)
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ABSTRACT Adeno-associated viral vector gene therapy is a treatment for genetic diseases that involves the introduction of a healthy copy of a mutated gene through the use of adeno-associated viral vectors. There has been a growing level of interest in gene therapy with as many as 2600 clinical trials reported as either completed, ongoing, or having approval as of 2018. With 56.8% of all trials in Phase I and 17.1% in Phase II, batch requirements that contain a good yield and are of high-purity are important. Industry has developed scalable processes for manufacture of large batches of highly-pure AAV product. These systems, while seemingly robust, have many challenges yet to overcome. One limitation to production are vector serotypes having lower yields compared to their counterparts. The AAV6 serotype, used to treat diseases such as cystic fibrosis and muscular dystrophy, have been observed to have a thirty-fold decrease in production compared to AAV9 serotype. To date, AAV6 production at Paragon has been confined to serum-free, suspension cell culture. By increasing the yield, we can affectively meet batch requirements for clinical trials while lowering the amount of resources and cost of goods. This study represents the first production of AAV6 at Paragon Bioservices using an adherent cell platform. Scale down studies were undertaken to increase production by optimizing transfection conditions such as the total DNA concentration during transfection, the transfection reagent-DNA ratio, the harvest days post transfection, seeding density, and plasmid ratio. The optimized conditions were tested in the iCELLis® Nano bioreactor and successful production of AAV6 was achieved at titers equivalent or better to those achieved in suspension culture.