Increased recombinant adeno‐associated virus production by HEK293 cells using small molecule chemical additives

Abstract

Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here, we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by human embryonic kidney (HEK) cells up to three-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume and reduced viable cell density relative to untreated rAAV producing cells at harvest. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures. Further investigation showed nocodazole addition to cultures to be time critical. Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a three-fold increase in genome titre compared to untreated cells.

Critical challenges and advances in recombinant adeno‐associated virus (rAAV) biomanufacturing

“…In addition to comparing producer/non‐producer states, different cell lines, clones from the same cell line but with varying productivities can be further investigated using transcriptomics to understand the cellular features for high productivity. Based upon transcriptomic results, media supplementation strategies (Scarrott et al, 2022) and cell engineering targets (Abaandou et al, 2021; Formas‐Oliveira et al, 2020) can be proposed and then validated to achieve the desired productivity or quality attributes. According to Scarrott et al's (2022) research, adding small molecule chemical additives during production as a media supplementation strategy can enhance rAAV productivity by up to three times in HEK293 cells.…”

“…Based upon transcriptomic results, media supplementation strategies (Scarrott et al, 2022) and cell engineering targets (Abaandou et al, 2021; Formas‐Oliveira et al, 2020) can be proposed and then validated to achieve the desired productivity or quality attributes. According to Scarrott et al's (2022) research, adding small molecule chemical additives during production as a media supplementation strategy can enhance rAAV productivity by up to three times in HEK293 cells. In addition, several published resources will be very helpful for the transcriptomics study, which includes human transcriptome databases (Futschik et al, 2018) and transcriptome data for HEK293 cells (Lin et al, 2014) and HeLa cells (Adey et al, 2013; Landry et al, 2013).…”

Current avenues of gene therapy in Pompe disease

“…New technology has emerged to scale transient transfections, including packed bed reactors [86–88], conversion to suspension HEK293 lines [89], AAV process intensification [90 ▪ ,91 ▪ ] and the use of novel chemical additives as transfection reagents [84,89,92–94,95 ▪ ,96 ▪ ]. Nonplasmid DNA sources such as doggybone DNA and novel transfection reagents could facilitate manufacturing significantly, along with the use of certain chemical additives to boost rAAV production [95 ▪ ,97–99].…”

Multi‐omics kinetic analysis of recombinant adeno‐associated virus production by plasmid transfection of HEK293 cells

“…Different approaches have been taken to increase rAAV productivity of plasmid transfection, including optimization of plasmid design, 12 transfection process, 13 and treatment with small molecule chemicals. 14 A number of alternative production systems use viral vectors based on Baculovirus or HSV. 15 Each system employs two recombinant viral vectors, one carries rAAV genome, the other encodes Rep and Cap proteins.…”

“…Additionally, HEK293 cells harbor another Ad helper gene E1. Different approaches have been taken to increase rAAV productivity of plasmid transfection, including optimization of plasmid design, 12 transfection process, 13 and treatment with small molecule chemicals 14 . A number of alternative production systems use viral vectors based on Baculovirus or HSV 15 .…”