Temperature-sensitive miR-483 is a conserved regulator of recombinant protein and viral vector production in mammalian cells.

Cell engineering and bioprocess optimizations such as low temperature cultivation represent powerful tools to improve cellular performance and product yields of mammalian production cells. Besides monoclonal antibodies (mABs), novel biotherapeutic formats such as viral vectors will gain increasing importance. Here, we demonstrate that similar to Chinese hamster ovary (CHO) cells, product yields of recombinant adeno-associated virus (rAAV) producing HeLa cells can be markedly increased by low temperature cultivation. MicroRNAs (miRNAs) are small non-coding RNAs that critically regulate cell phenotypes. We thus investigated differential miRNA expression in response to mild hypothermia in CHO and HeLa production cells. We discovered miR-483 to be substantially up-regulated upon temperature down-shift in both cell types. Functional validation experiments revealed that introduction of miR-483 mimics led to a significant increase in both rAAV and mAB production in HeLa and CHO cells, respectively. Furthermore, inhibition of miR-483 up-regulation during mild hypothermia significantly decreased product yields, suggesting that miR-483 is a key regulator of cellular productivity in mammalian cells. In addition, miRNA target gene identification indicated that miR-483 might regulate genes directly involved in cellular survival and protein expression. Our results highlight that miR-483 is a valuable tool for product-independent engineering of mammalian production cells.

Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing.