Aurintricarboxylic acid increases yield of HSV-1 vectors

Pechan, Peter; Ardinger, Jeffery; Ketavarapu, Jyothi; Rubin, Hillard; Wadsworth, Samuel C; Scaria, Abraham

Effect of residual ATA in HSV stocks on the production of rAAV virions Because our ultimate goal is rAAV production, we investigated whether the presence of ATA in rHSV-1 stocks would influence rAAV yields. The rAAV-GFP vector was produced by coinfection of rHSV-rep2/cap2 and rHSV-EGFP vectors in HEK-293 cells using rHSV-1 stocks not containing ATA, or rHSV-1 stocks prepared under ATA@step1 protocol (see Materials and Methods). The effect of residual ATA in rHSV-rep2/cap2 stock (~3 µmol/l) resulted in a small 1.3-fold (statistically significant) increase in rAAV titer to 3.8 × 10 10 ± 2.4 × 10 9 DRP/ml (**P < 0.01) when compared with the rAAV DRP/ml titer made by using naive rHSV-1 stocks, 2.8 × 10 10 ± 2.8 × 10 9 DRP/ml (n = 4). As shown in Figure 4 , ATA also slightly increased rAAV yield (DRP/cell) when 10 µmol/l ATA was spiked directly into HEK-293 cell media during the rHSV-1 coinfection step. This effect was not observed when the ATA concentrations were higher than 15 µmol/l.