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

Abstract

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.

Evaluation of life cycle defective adenovirus mutants for production of adeno‐associated virus vectors

“…Twenty‐four hours after seeding, cells were transfected with a total amount of 6 μg of DNA either comprising the All‐in‐One plasmid or co‐transfected using three plasmids encoding rep2, cap2 and AAV2 vector expressing a fusion protein of luciferase and GFP (LUC‐GFP) at 3:9:4 ratios. The plasmid constructs used for co‐transfection have been described elsewhere . For transfection Polyplus* PEIpro 1 mg/ml (Polyplus, Illkirch, France) was used at weight ratios of DNA to PEI of 1:1.…”

“…Infectivity of AAV2 vector was determined by GFP‐based flow cytometry analysis in a transduction assay adapted from a previous study and as described previously . HeLa‐t cells seeded the day before at a density of 2 × 10 4 cells per well in 96‐well plates were treated for 1 hour with 0.5 μg/ml mitomycin C (Applichem, Darmstadt, Germany) diluted in medium, at 37°C to force the cells into the S‐phase and to facilitate double‐strand DNA synthesis of the AAV2 vector genome after infection.…”

“…The plasmid constructs used for co-transfection have been described elsewhere. 33 For transfection Polyplus* PEIpro 1 mg/ml (Polyplus, Illkirch, France) was used at weight ratios of DNA to PEI of 1:1. Directly after transfection, A549 cells were transduced with helper virus at pMOI 500, if not stated otherwise.…”

“…Infectivity of AAV2 vector was determined by GFP-based flow cytometry analysis in a transduction assay adapted from a previous study 36 and as described previously. 33 HeLa-t cells seeded the day before at to 25% GFP-positive cells, each cell had been transduced by one AAV2 vector particle, thus leading to:…”

Advancements in the design and scalable production of viral gene transfer vectors

“…These libraries can then be screened via high-throughput methods for mutants with novel immune escape or tropism phenotypes. In the four-plasmid system, REP and CAP may be split onto separate plasmids to allow for optimization of viral protein ratios, which has shown considerable success in increasing yield (Emmerling et al, 2016). Two and four plasmid variations of this system also exist.…”