Creation of a High-Yield AAV Vector Production Platform in Suspension Cells Using a Design-of-Experiment Approach.
Adeno-associated virus
affinity purification
design of experiment
production
rAAV
suspension cells
Journal
Molecular therapy. Methods & clinical development
ISSN: 2329-0501
Titre abrégé: Mol Ther Methods Clin Dev
Pays: United States
ID NLM: 101624857
Informations de publication
Date de publication:
11 Sep 2020
11 Sep 2020
Historique:
received:
10
02
2020
accepted:
29
05
2020
entrez:
17
7
2020
pubmed:
17
7
2020
medline:
17
7
2020
Statut:
epublish
Résumé
Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, but vector manufacturing remains a challenge. New methods are needed to increase rAAV yields and reduce costs. Past efforts to improve rAAV production have focused on optimizing a single variable at a time, but this approach does not account for the interactions of multiple factors that contribute to vector generation. Here, we utilized a design-of-experiment (DOE) methodology to optimize rAAV production in a HEK293T suspension cell system. We simultaneously varied the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density to systematically evaluate the impact of each variable across 52 conditions. The results revealed a unique set of parameters with a lower concentration of transgene plasmid, a higher concentration of packaging plasmid, and a higher cell density than previously described protocols. Using this DOE-optimized protocol, we achieved unpurified yields approaching 3 × 10
Identifiants
pubmed: 32671134
doi: 10.1016/j.omtm.2020.06.004
pii: S2329-0501(20)30126-1
pmc: PMC7334306
doi:
Types de publication
Journal Article
Langues
eng
Pagination
312-320Informations de copyright
© 2020 The Author(s).
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