Systematic comparison of rAAV vectors manufactured using large-scale suspension cultures of Sf9 and HEK293 cells.
AAV packaging
HEK293
Sf9
adeno-associated virus
gene therapy
Journal
Molecular therapy : the journal of the American Society of Gene Therapy
ISSN: 1525-0024
Titre abrégé: Mol Ther
Pays: United States
ID NLM: 100890581
Informations de publication
Date de publication:
20 Nov 2023
20 Nov 2023
Historique:
received:
13
09
2023
revised:
11
10
2023
accepted:
17
11
2023
pubmed:
22
11
2023
medline:
22
11
2023
entrez:
22
11
2023
Statut:
aheadofprint
Résumé
Recombinant adeno-associated virus (rAAV) vectors could be manufactured by plasmid transfection into human embryonic kidney 293 (HEK293) cells or baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. However, systematic comparisons between these systems using large-scale, high-quality AAV vectors are lacking. rAAV from Sf9 cells (Sf9-rAAV) at 2-50 L and HEK293 cells (HEK-rAAV) at 2-200 L scales were characterized. HEK-rAAV had ∼40-fold lower yields but ∼10-fold more host cell DNA measured by droplet digital PCR and next-generation sequencing, respectively. The electron microscope observed a lower full/empty capsid ratio in HEK-rAAV (70.8%) than Sf9-rAAV (93.2%), while dynamic light scattering and high-performance liquid chromatography analysis showed that HEK-rAAV had more aggregation. Liquid chromatography tandem mass spectrometry identified different post-translational modification profiles between Sf9-rAAV and HEK-rAAV. Furthermore, Sf9-rAAV had a higher tissue culture infectious dose/viral genome than HEK-rAAV, indicating better infectivity. Additionally, Sf9-rAAV achieved higher in vitro transgene expression, as measured by ELISA. Finally, after intravitreal dosing into a mouse laser choroidal neovascularization model, Sf9-rAAV and HEK-rAAV achieved similar efficacy. Overall, this study detected notable differences in the physiochemical characteristics of HEK-rAAV and Sf9-rAAV. However, the in vitro and in vivo biological functions of the rAAV from these systems were highly comparable. Sf9-rAAV may be preferred over HEK293-rAAV for advantages in yields, full/empty ratio, scalability, and cost.
Identifiants
pubmed: 37990495
pii: S1525-0016(23)00623-8
doi: 10.1016/j.ymthe.2023.11.022
pii:
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
Copyright © 2023. Published by Elsevier Inc.
Déclaration de conflit d'intérêts
Declaration of interests J.L., S.P., N.L., A.C., M.D., Y.L., K.L., D.H., S. Li, L.O., and S. Liu. are employees of Avirmax Biopharma Inc. Other authors declare no conflict of interest.