Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

Abstract

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 10¹⁴ vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 10¹⁵ VG in crude cell harvests and an average of 3.5 × 10¹⁴ total VG of purified rAAV9 material, resulting in greater than 1 × 10⁵ VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

Keywords: AAV vectors production; gene therapy; herpes simplex virus; suspension cell lines.

Figure 1.

Increased sodium chloride concentrations result in improved rAAV production after rHSV coinfection. (A and B) Transducing units measured in cell lysates 48 hr postinfection in 50-ml working volumes, with the medium supplemented with increasing percentages of the HSV matrix (5% FBS/5% glycerol/600 mM NaCl), or each component individually or in various combinations at a final concentration of 15% of total medium volume. (C–E) Increased rAAV9 capsid proteins, transducing units, and vector genomes measured in cell lysates 48 hr postinfection, in the presence of 0–90 mM salt supplementation. (F) rAAV9-GFP production in the absence or presence of salt 24, 48, and 72 hr postinfection. **p < 0.01, ***p < 0.001.

Figure 2.

Increased rAAV9 production after sodium chloride supplementation for other rAAV serotypes, genes of interest, and/or cell types. Total transducing units (A) and total vector genomes (B) were evaluated for rAAV2-GFP, rAAV5-GFP, and rAAV9-Des-GAA produced by rHSV coinfection in shaker flasks (50-ml working volume) in the presence of increased sodium chloride concentration (n = 2 for all). (C) Impact of increased sodium chloride on rAAV9-GFP production in BHK cells grown in DMEM with 5% FBS (BHK) or adapted to serum-free medium (BHK-SFX). (D) Total rAAV9-GFP vector genomes produced in BHK and BHK-SFX cells by rHSV coinfection in the absence or presence of optimal salt supplementation (30 and 60 mM, respectively). *p < 0.05, ***p < 0.001.

Figure 3.

Scalability of rAAV production in Expi293F cells. Transducing units for rAAV9-GFP (A) and total vector genomes for rAAV9-GFP and rAAV9-Des-GAA (B) were assessed in crude lysates from Expi293F cultures ranging from 50 to 3000 ml. (C) Total vector genome values are presented as vector genomes per cell at each scale.

Figure 4.

Characterization of rAAV vector made in Expi293F suspension system. (A and B) rAAV9-GFP was produced by rHSV infection in adherent HEK293 cells grown in 10-chamber CellSTACKs (CS10) under optimal conditions (approximately 3 × 10⁹ cells) either in DMEM containing 5% FBS alone (n = 4) or supplemented with 60 mM sodium chloride (n = 6), or in a 3-liter suspension culture containing 3 × 10⁹ Expi293F cells (n = 5). Total vector genome (A) and transducing unit (B) yields are shown. Total purified vector genomes are represented relative to units produced per cell (C) and per liter of medium (D). (E) The vector genome-to-transducing unit (VG/TU) ratio was calculated for each sample, and the averaged values for each group are shown. (F) Coomassie staining of rAAV9-GFP final stocks produced in HEK293 CS10 in the absence of salt supplementation (lane 1), or supplemented with sodium chloride (lane 2), or produced in Expi293F 3-liter cultures supplemented with sodium chloride (lanes 3–7). A total of 10¹¹ VG was loaded per well. *p < 0.05, **p < 0.01.

Figure 5.

Theoretical HSV system scalability. Extrapolation is based on current average rHSV yields for the constructs discussed in this work. HSV stocks are produced from adherent V27 cells grown in 10-layer flasks (CS10s). Number of production units (CS10s for adherent; liters for suspension), as well as cell type (V27, HEK293, or Expi293F), are indicated for each step and are rounded for clarity. HSV-GOI (gene of interest) and HSV-Rep2Cap9 seed stocks are produced from one and six CS10s, respectively, to support multiplicities of infection (MOIs) used in the adherent cell platform (2:12, respectively).