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.

Autor: Srinivasan P; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Canova CT; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Sha S; Ultragenyx Pharmaceutical Inc., Novato, Cambridge, USA., Nguyen TNT; BioNTech SE, Cambridge, Massachusetts, USA., Joseph J; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Sangerman J; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Maloney AJ; Amgen, Cambridge, Massachusetts, USA., Katsikis G; Anthology, Cambridge, Massachusetts, USA., Ou RW; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Hong MS; School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea., Ng J; Stanford University School of Medicine, Stanford, California, USA., Yuan A; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Antov D; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Song S; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Chen W; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Neufeld C; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Wolfrum JM; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Barone PW; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Sinskey AJ; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Springs SL; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA., Braatz RD; Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2024 Aug 23. Date of Electronic Publication: 2024 Aug 23.
DOI: 10.1002/bit.28828
Abstrakt: 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.
(© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)
Databáze: MEDLINE
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