Exploiting Adenovirus Biology for Advancing Adeno-Associated Viral Vector Manufacturing

Recombinant adeno-associated virus (AAV) vectors hold significant promise for gene therapy; however, challenges persist in achieving efficient and scalable production. To solve this AAV manufacturing challenge, we developed a novel helper adenovirus genetically engineered to deliver AAV genes, while self-inhibiting its own replication cycle to prevent the production of adenoviral structural protein upon infection in HEK293 cells. Here, we strategically inserted tetracycline repressor binding sites into the adenovirus Major Late Promoter and encoded the tetracycline repressor under its transcriptional control to allow for normal adenovirus replication in the presence of doxycycline, but only enhanced genome amplification and early gene expression (important for the ‘helper’ functions) in its absence.

This platform entitled 'Tetracycline-Enabled Self-Silencing Adenovirus' (TESSA®) enables robust and versatile approaches for producing AAV, offering flexibility through two distinct approaches. The process variation between the two models differs by the approach in which the gene of interest (GOI) is introduced into the HEK293 cells. 

A TESSA® vector encoding Rep and Cap can be combined with either a TESSA® vector (TESSA® Duo model) or an AAV (TESSA® Pro model) to deliver the GOI. This flexibility enables the production of cost-effective AAV vectors using TESSA® in a rapid and scalable manner, yielding up to 20-fold more rAAV vectors with productivities exceeding >1E+6 vector genome copies (GC) per cell.

Importantly, AAV manufacture using the TESSA® platforms can be scaled up efficiently and economically. In 50L and 200L bioreactors, the TESSA® Duo produces yields exceeding 7E+11 and 3E+11 GC/mL of AAV6 and AAV2, respectively.