Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination

Weiheng Su, Maria I. Patrício, Margaret R. Duffy, Jakub M. Krakowiak, Leonard W. Seymour () and Ryan Cawood
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Weiheng Su: University of Oxford
Maria I. Patrício: OXGENE Ltd
Margaret R. Duffy: University of Oxford
Jakub M. Krakowiak: OXGENE Ltd
Leonard W. Seymour: University of Oxford
Ryan Cawood: OXGENE Ltd

Nature Communications, 2022, vol. 13, issue 1, 1-14

Abstract: Abstract Recombinant adeno-associated virus (rAAV) shows great promise for gene therapy, however scalability, yield and quality remain significant issues. Here we describe an rAAV manufacturing strategy using a ‘helper’ adenovirus that self-inhibits its major late promoter (MLP) to truncate its own replication. Inserting a tetracycline repressor (TetR) binding site into the MLP and encoding the TetR under its transcriptional control allowed normal adenovirus replication in the presence of doxycycline but only genome amplification and early gene expression (the ‘helper’ functions) in its absence. Using this self-inhibiting adenovirus we demonstrate delivery of adenoviral helper functions, AAV rep and cap genes, and the rAAV genome to yield up to 30-fold more rAAV vectors compared to the helper-free plasmid approach and significant improvements in particle infectivity for a range of serotypes. This system allows significant improvements in the production of serotypes rAAV2, rAAV6, rAAV8 and rAAV9, and enables propagation of existing rAAV without transfection, a process that improves batch quality by depleting reverse packaged DNA contaminants. We propose this as a high-yielding, contaminant-free system suitable for scalable rAAV manufacture.

Date: 2022
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DOI: 10.1038/s41467-022-28738-2

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