94. A Scalable Recombinant HSV-Based Manufacturing System for Production of Highly Potent Adeno-Associated Type 9 Vectors

Abstract

Introduction: Adeno-associated virus vectors (AAV) have been utilized as a gene delivery tool to treat a multitude of diseases. As the potential uses for these vectors expand, the need for improved manufacturing methods becomes essential for large-scale implementation. Recombinant Herpes Simplex Virus (rHSV)-based method delivering AAV capsid and transgenes for recombinant AAV (rAAV) production has been shown to be a robust and scalable method for producing multiple rAAV serotypes for pre-clinical and clinical studies. Here we will present the first scalable production and purification method for rAAV serotype 9 utilizing the rHSV system. Methods: Recombinant rAAV9-GFP stocks were produced by transfection or rHSV co-infection on HEK 293. Vector genome titers were assessed by quantitative PCR, digital droplet PCR and dot blot. Infectivity was assessed by an infectious center assay, flow cytometry, and green cell transduction assay (GCA). Coomassie SDS-PAGE, or western blot analyses were performed to evaluate identity and purity of rAAV samples and electron microscopy to assess full versus empty ratio for each method. Results: rAAV vectors produced by the HSV method showed a 7 and 10 fold increase in total vector genomes and transducing units per CellStack®, respectively. Increased infectivity was observed in rHSV-made vectors as demonstrated by an increasing in transducing unit titers by GCA and infectious center assay. Flow cytometry of C12 cells infected with equal vector genome amounts of both transfection and rHSV made rAAV showed a 30% increase in GFP expressing cells. Coomassie staining and western blot analysis showed a 20-32% reduction in total capsid proteins in rAAV made by rHSV co-infection. Electron microscopy confirmed this reduction in total capsid protein was due to a decrease in empty particles in HSV-made rAAV samples. Overall, this method produced greater than 1×1014 rAAV9 vector genomes per liter equivalent of 293 cells of a final, purified product. Conclusions: Our data demonstrated the significant improvement in rAAV9 manufacturing by utilization of the rHSV based manufacturing methods. An improvement in both vector titer and infectivity was observed in rHSV made rAAV vectors compared to transfection-based production. Importantly, this scalable purification method is readily suitable for pre-clinical GLP studies and GMP manufacturing in our laboratory. Further improvements will be presented specifically evaluating rHSV production in a suspension-adapted HEK 293 cell system in view of a large scale manufacturing protocol of rAAV9 vectors for clinical studies.