Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno‐associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids
Name
Biotech Bioengineering - 2024 - Srinivasan - Multidose transient transfection of human embryonic kidney 293 cells.pdf
Description
Published version
Size
2.96 MB
Format
Adobe PDF
Checksum (MD5)
539822e1f9951a66c171948bee09ac03
Author(s)
Srinivasan, Prasanna
Canova, Christopher T
Sha, Sha
Nguyen, Tam NT
Joseph, John
Sangerman, Jose
Maloney, Andrew J
Katsikis, Georgios
Ou, Rui Wen
Hong, Moo Sun
Date Issued
2024
Journal
Biotechnology and Bioengineering
Publisher
Wiley
Citation
Srinivasan, P., Canova, C. T., Sha, S., Nguyen, T. N. T., Joseph, J., Sangerman, J., Maloney, A. J., Katsikis, G., Ou, R. W., Hong, M. S., Ng, J., Yuan, A., Antov, D., Song, S., Chen, W., Neufeld, C., Wolfrum, J. M., Barone, P. W., Sinskey, A. J., … Braatz, R. D. (2024). Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno-associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids. Biotechnology and Bioengineering, 121, 3694–3714.
Version
Final published version
Abstract
Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1%–30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid-filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.
MIT Department
Massachusetts Institute of Technology. Center for Biomedical Innovation
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1002/bit.28828
