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Mechanistic Model for Production of Recombinant Adeno-associated Virus via Triple Transfection of HEK293 Cells
Name
1-s2.0-S2329050121000723-main.pdf
Description
Published version
Size
1.76 MB
Format
Adobe PDF
Checksum (MD5)
158cca7895135050a2f57debe3cc0473
Author(s)
Nguyen, Tam NT
Sha, Sha
Hong, Moo Sun
Maloney, Andrew J
Barone, Paul W
Neufeld, Caleb
Wolfrum, Jacqueline
Springs, Stacy L
Sinskey, Anthony J
Braatz, Richard D
Date Issued
2021
Journal
Molecular Therapy — Methods & Clinical Development
Publisher
Elsevier BV
Version
Final published version
Abstract
Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
Persistent DSpace Link
DOI of Published Version
10.1016/j.omtm.2021.04.006
