High-cell-density production of adeno-associated viral vector serotype 6 by triple transfection in suspension HEK293 cell cultures

Abstract

The use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has risen considerably in recent years. Consequently, the amount of AAV vectors required during the validation and clinical trials has also increased. AAV serotype 6 (AAV6) is well-documented for its efficiency in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG). Overall, this means that large-scale production of AAV6 is needed. Suspension cell-based platforms are currently limited to low-cell-density productions, hindering the potential of this production process to increase yields. Here, we investigate the improvement of the production of AAV6 at higher cell densities. The production was performed by transient transfection of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the production can be carried out at medium cell density without effects on cell-specific titer or particle functionality, resulting in titers above 1010 VG/mL. Medium supplementation alleviated the cell density effect, in terms of VG/cell, at high-cell-density productions. On the other hand, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations. The medium-cell-density production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.

Tuning capsid formation dynamics in recombinant adeno‐associated virus producing synthetic cell lines to enhance full particle productivity

“…[47,48] Enhancing cellular metabolism through process optimization may elevate rAAV productivity. [49,50] Our synthetic cell lines included only a minimal set of AAV and helper genes. While productivity achieved has been good, it is possible that additional helper viral genes, such as E4orf6/7 and others, which modulate host cell functions like DNA replication, cell cycle progression, and DNA repair, could aid in virus genome replication.…”

“…[ 47,48 ] Enhancing cellular metabolism through process optimization may elevate rAAV productivity. [ 49,50 ]…”

Model-based characterization of metabolism of the recombinant adeno-associated virus (rAAV) production via Human Embryonic Kidney (HEK293) cells

“…In this study, we utilized a previously published dataset [19] to developed a metabolic model framework based on flux kinetics related to AAV production in the HEK293 cell line platform.…”

“…However, since such information is only available for five of the species, adjustments to the initial concentration of the remaining species, as well as the model parameters, were made during the optimization iterations. Further details about this real-world example can be found in another source [19].…”

Enhancing the production of adeno‐associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design‐of‐experiment optimization of triple plasmid ratio

“…Adeno-associated virus (AAV) vectors are increasingly being used in gene therapy because of their safety, diverse tropism, sustained expression, and ability to effectively infect both dividing and nondividing cells. [1][2][3][4][5] To date, six AAV-based gene therapies have been approved for clinical use by regulatory agencies and more than 255 clinical trials have been conducted or are ongoing. [6] To date, more than 100 AAV serotypes, including synthetic serotypes, have been identified that exhibit various tropisms and characteristics in human tissues.…”