Expression of Viral DNA Polymerase in Synthetic Recombinant Adeno‐Associated Virus Producer Cell Line Enhances Full Particle Productivity

Recombinant adeno‐associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high‐quality products is required. We have developed a synthetic biology strategy to generate HEK293‐based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction. One such cell line, GX6B, produced up to 106 capsids per cell, but only a much lower level of rAAV genomes. The low AAV genome titer limited its rAAV productivity and increased empty viral particle content. To boost AAV genome amplification, the coding sequence of the DNA polymerase complex (UL30/UL42) from helper Herpes Simplex Virus type 1 (HSV‐1) was placed under an inducible promoter control and integrated into GX6B genome at a relatively low level. The resulting clones produced significantly higher titer of viral genomes, while their capsid level was unaffected. As a result, the encapsidated rAAV2 titer and the full particle content were significantly increased. We further demonstrated that this strategy of expressing HSV‐1 DNA polymerase to increase full particle productivity could be implemented in a synthetic cell line producing another serotype rAAV8.