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

Weiheng Su,Ryan Cawood,Jakub M Krakowiak,Margaret R Duffy,Maria I Patrício,Leonard W Seymour

doi:10.1038/s41467-022-28738-2

Weiheng Su, Ryan Cawood + Show 4 more

Open Access

https://doi.org/10.1038/s41467-022-28738-2

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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.

Highlights

Recombinant adeno-associated virus shows great promise for gene therapy, scalability, yield and quality remain significant issues
By cotransfecting with plasmid pCMV-tetracycline repressor (TetR) in HEK293 cells and treating with doxycycline we found that EGFP expression could be regulated using a TetR binding element downstream of the major late promoter (MLP) TATA box (Supplementary Fig. 1d)
We achieved this by inserting a tetracycline TetO site downstream of the adenovirus MLP TATA box and encoding TetR under its transcriptional control, creating the Tetracycline-Enabled Self-Silencing Adenovirus’ (TESSA) adenoviral vector in which the MLP is capable of self-repression

Summary

Introduction

Recombinant adeno-associated virus (rAAV) shows great promise for gene therapy, scalability, yield and quality remain significant issues. 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 show that TESSA vectors encoding both AAV rep and cap genes allow the direct propagation of rAAV particles, and that this approach can reduce the level of DNA contaminants within an existing rAAV preparation

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