Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing.

Recombinant adeno-associated viral (rAAV) vector-based gene therapy has been adapted for use in more than 100 clinical trials. This is mainly because of its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefit. However, the major challenge is to produce a high-titer, high-potency vector to achieve a better therapeutic effect. Even though the three plasmid-based transient transfection method is currently being used for AAV production in many clinical trials, there are complications associated with scalability and it is not cost-effective. Other methods require either large-scale production of two herpes simplex viruses, rHSV-RepCap and rHSV-GOI (gene of interest), with high titers, or a stable cell line with high titer wild-type adenovirus infection. Both of these options make the process even more complex. To address this issue, we have developed a stable cell line-based production with the use of only one rHSV-RepCap virus. Using this new methodology in small-scale production, we achieved ∼1-6 E + 04 vg/cell of AAV9 in the top producer clones. Large-scale production in 10-CS (10-Cell Stack) of one of the top producing clones resulted in ∼1-2 E + 13 vg/10-CS with 50% of full capsid ratio after purification. This method could potentially be adapted to suspension cells. The major advantage of this novel methodology is that by using the rHSV-RepCap virus, high titer AAV can be produced with any GOI containing a stable adherent or suspension producer cell line. The use of this AAV production platform could be beneficial for the treatment of many diseases.