Recombinant adeno-associated virus (rAAV) is the vector of choice for in vivo gene therapy; however, scalability, yield and quality remain significant issues for rAAV manufacture. We recently described a new helper adenovirus system entitled ‘Tetracycline-Enabled Self-Silencing Adenovirus’ (TESSA) wherein the adenovirus Major Late Promoter (MLP) was modified in situ to enable selfrepression of promoter activity and truncate the adenovirus replication cycle for efficient and contaminant-free manufacture of rAAV. We previously reported two approaches of using TESSA for rAAV manufacture, both yielding significantly more rAAV with improved infectivity compared to the helper-free rAAV production system (Su et al. 2022) but still requiring two viral vector species as input materials. To further simplify the rAAV manufacture process, here we used the phiC31 system to integrate the rAAV genome into a cGMP compliant suspension serum-free HEK293 cell line, and the engineered producer cells are capable of producing rAAV at high titres by simple infection with a single TESSA vector expressing the AAV Capsid of interest.
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