High-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling

High-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling

Abstract Triple transfection of HEK293 cells is the most widely used method for producing recombinant adeno-associated virus (rAAV), a leading gene delivery vector for human gene therapy. Despite its tremendous success, this approach generates several vector-related impurities that could potentially...

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Bibliographic Details
Main Authors: Hao Liu, Nan Liu, Chen Zhou, Ailing Du, Mayank Kapadia, Phillip W L Tai, Erik Barton, Guangping Gao, Dan Wang
Format: Article
Language:English
Published: Springer Nature 2025-05-01
Series:EMBO Molecular Medicine
Subjects:
AAV Manufacturing
Vector Impurities
Empty Capsid
Plasmid Backbone
AAVPureMfg
Online Access:https://doi.org/10.1038/s44321-025-00248-w
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Summary:Abstract Triple transfection of HEK293 cells is the most widely used method for producing recombinant adeno-associated virus (rAAV), a leading gene delivery vector for human gene therapy. Despite its tremendous success, this approach generates several vector-related impurities that could potentially compromise the safety and potency of rAAV. In this study, we introduce a method for high-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling (AAVPureMfg). Compared with traditional triple transfection, AAVPureMfg substantially improves vector purity by reducing prokaryotic DNA contaminants by 10- to 50-fold and increasing the full capsid ratio up to threefold. Mechanistically, Bxb1-mediated excision of the transgene cassette generates a minicircle cis construct devoid of bacterial sequences and ensures synchronized colocalization of trans and cis constructs in productive cells. Furthermore, we developed iterations that enhance vector genome homogeneity and streamline the production of rAAV with various transgenes, serotypes, and ITR configurations. Overall, our findings demonstrate that AAVPureMfg overcomes the inherent limitations associated with triple transfection, offering a broadly applicable and easy-to-implement method for producing high-purity rAAV with reduced plasmid costs.
ISSN:1757-4684

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