INTRODUCTION
Adeno-associated viruses (AAVs) are small, 25-nm-wide, icosahedral, non-enveloped viruses with a 4.7-kb-long single-stranded DNA genome belonging to the family Parvoviridae [1]. AAVs are non-pathogenic and replication-defective [2], depending on co-infection with helper viruses, such as Adenovirus or Herpes Simplex virus. First discovered from stocks of adenovirus in the 1960s [2, 3], AAVs have recently become a critical gene delivery vector for treating diseases. Glybera was the first AAV-based gene therapy drug approved in 2012 by the European Medicines Agency [4]. The US Food and Drug Administration (FDA) approved 2 AAV-based gene therapy drugs, Luxturna, in 2017 [5] and, Zolgesma, in 2019 [6].
To this date, based on phylogenetic analysis, 13 different serotypes and more than 100 variants of AAV have been identified [2, 3, 7-14]. Due to heterogeneity in capsid proteins, each serotype exhibits a distinct tropism and ability to transduce different cell types [15]. Serotype 6 has a wide range of target cells and has been shown to successfully transduce cells in the central nervous system [16], human prostate, breast, and liver cancer cells [17], melanocytes [18], skeletal muscle [19-21], heart [22-24], lung [25], and the eye [26]. Recently, AAV6 gained popularity due to its ability to transduce lymphocytes [27-29] and its use for generating Chimeric Antigen Receptor T cells [30-36].
The number of therapeutic AAV applications being investigated is steadily increasing, with more than 300 completed or ongoing clinical trials [37]. These applications require large amounts of AAV vectors to validate pre-clinical animal studies and clinical trials. Reported doses can reach up to 7.5 × 1015 VG for targeted delivery and up to 1.5 × 1017 VG for systemic delivery [38]. This large requirement in the number of viral vectors implies that an improvement in current production methods is necessary. This is especially true for AAV serotype 6, which requires up to 106 VG/cell during the transduction of T cells [27]. Recombinant AAV is produced by replacing the viral genes, Rep and Cap , with the gene of interest (GOI). Mammalian cells are transiently transfected with a GOI cassette flanked by the inverted terminal repeats, a plasmid carrying the Rep and Cap functions, and a third plasmid encoding the helper functions [39-42]. The transient transfection is generally done using the cost-effective cationic polymer, Polyethylenimine (PEI) [43]. However, adherent cell cultures are not deemed viable for AAV production at scales that exceed 1015 total VGs, making them unsuitable for late-phase clinical trials and commercial applications of these viral vectors [44]. The use of HEK293 cells in suspension for producing AAV vectors was first described in 2006 [45]. Despite many efforts in optimizing production, large-scale production of AAV vectors is considered a bottleneck in their implementation as a widespread type of viral vector for gene therapy and cell therapy [46], mainly because current HEK293-based production is done at low cell densities [45, 47-51]. Limiting the production of viral vectors to lower cell densities hinders the potential of this production process to be intensified [52]. On the other hand, the production of viral vectors at higher cell densities tends to be limited by the widely reported cell density effect (CDE), which results in diminished transfection and productivity [53-55]. In this manuscript, we demonstrate that the production of AAV serotype 6 via transient transfection is not limited to low-cell-density cultures.