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  • Enhancing the production of recombinant adeno‐associated virus in synthetic cell lines through systematic characterization
Title: Enhancing the production of recombinant adeno‐associated virus in synthetic cell lines through systematic characterization
Abstract Recombinant adeno‐associated virus (rAAV) is among the most commonly used in vivo gene delivery vehicles and has seen a number of successes in clinical application. Current manufacturing processes of rAAV employ multiple plasmid transfection or rely on virus infection and face challenges in scale‐up. A synthetic biology approach was taken to generate stable cell lines with integrated genetic modules, which produced rAAV upon induction albeit at a low productivity. To identify potential factors that restrained the productivity, we systematically characterized virus production kinetics through targeted quantitative proteomics and various physical assays of viral components. We demonstrated that reducing the excessive expression of gene of interest by its conditional expression greatly increased the productivity of these synthetic cell lines. Further enhancement was gained by optimizing induction profiles and alleviating proteasomal degradation of viral capsid protein by the addition of proteasome inhibitors. Altogether, these enhancements brought the productivity close to traditional multiple plasmid transfection. The rAAV produced had comparable full particle contents as those produced by conventional transient plasmid transfection. The present work exemplified the versatility of our synthetic biology‐based viral vector production platform and its potential for plasmid‐ and virus‐free rAAV manufacturing.  more » « less
Award ID(s):
2011401
PAR ID:
10506868
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
Biotechnology and Bioengineering
Date Published:
Journal Name:
Biotechnology and Bioengineering
Volume:
121
Issue:
1
ISSN:
0006-3592
Page Range / eLocation ID:
341 to 354
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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