Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies

Abstract

The ability of recombinant adeno-associated viral (rAAV) vectors to exhibit minimal immunogenicity and little to no toxicity or inflammation while eliciting robust, multiyear gene expression in vivo are only a few of the salient features that make them ideally suited for many gene therapy applications. A major hurdle for the use of rAAV in sizeable research and clinical applications is the lack of efficient and versatile large-scale production systems. Continued progression toward flexible, scalable production techniques is a prerequisite to support human clinical evaluation of these novel biotherapeutics. This review examines the current state of large-scale production methods that employ the herpes simplex virus type 1 (HSV) platform to produce rAAV vectors for gene delivery. Improvements have substantially advanced the HSV/AAV hybrid method for large-scale rAAV manufacture, facilitating the generation of highly potent, clinical-grade purity rAAV vector stocks. At least one human clinical trial employing rAAV generated via rHSV helper-assisted replication is poised to commence, highlighting the advances and relevance of this production method.

FIG. 1.

Schematic representations of adeno-associated virus (AAV) and herpes simplex virus (HSV) and vectors. Genomic organization of wild-type AAV-2 (left, top) and wild-type HSV-1 (right, top) viruses is shown with major genetic elements (not to scale); genomic organization of recombinant vectors thereof (left/right, middle); resulting viral vectors (left/right, bottom). ITR, inverted terminal repeat; Rep, AAV nonstructural protein open reading frame; Cap, AAV structural protein open reading frame; X, AAV serotype 1–10; GOI, gene of interest in the rAAV backbone; rHSV-GOI, rHSV carrying rAAV-GOI; IR, inverted repeat; L/S, long and short; U, unit; TK, thymidine kinase open reading frame; ICP, infection cell protein.

FIG. 2.

rAAV production methods by coinfection with rHSVs. (A) Adherent cell production of rAAV via rHSV coinfection. Cells are seeded and infected in 10-tray cell factories. (B) Suspension cell production of rAAV via rHSV coinfection. Cells are seeded and expanded in disposable bioreactors. (1) Generation of rHSV stocks; (2) rHSV vector recovery and concentration to high titer; (3) coinfection with rHSV vectors; (4) harvest and recovery of rAAV from cellular lysates. BHK, baby hamster kidney cells.