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.