A packaging-regulated adenoviral vector for rAAV production.

Recombinant adeno-associated virus (rAAV) holds immense potential for gene therapy, yet scalability, yield, and quality remain critical challenges. Herein, we describe a packaging-regulated adenovirus for rAAV production by integrating the protocatechuic acid repressor (PcaR) binding site into the major late promoter (MLP) and inserting PcaR into the E1 region. The packaging-regulated adenovirus allows normal replication and package in the presence of protocatechuic acid (PCA), but only permits early gene expression in its absence. Using this packaging-regulated adenoviral system to deliver the AAV Rep and Cap genes alongside the rAAV genome, we observed a 10-fold increase in genomic titer compared to the helper-free plasmid method, accompanied by a significant enhancement in particle infectivity. Notably, as a green tea-derived metabolite, PCA should exhibit a better safety profile compared to the use of antibiotics. This strategy enables high-yield, lower-contamination production of rAAV, thereby offering a promising solution to the challenges in rAAV manufacturing.