Aurintricarboxylic acid increases yield of HSV-1 vectors

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

The ICP27-deficient vectors d27-1, rHSV-rep2/cap2, and rHSV-EGFP strains were propagated in ICP27-complementing V27 cell line. The wtHSV-1 KOS strain and wtHSV-1 McIntyre strain were propagated in Vero or HEK-293 cell lines. ATA was applied to the media during the infection (ATA@step1) or dilution step (ATA@step2) (Figure 1a) . The HSV-1 infection at multiplicity of infection of 0.15 (typically 6 × 10 5 cells in a six-well plate) was performed in 40% (2/5 vol) of the total final media volume for 1-2 hours, and the remaining media (60% or 3/5 of the total final volume) was added during the dilution step. The cells were then incubated for 72 hours and supernatant harvested to perform assays to obtain titers in DRP/ml and PFU/ml. Infectious vector particles were harvested 72 hours postinfection by collecting the culture supernatant. The titers of HSV-1 stocks in DRP/ml were determined by Taqman assay. Viral genomes within crude culture medium were quantified via treatment in the presence of DNase I (Promega, Madison, WI) (50 U/ ml final concentration) at 37 °C for 60 minutes, followed by digestion with proteinase K (Invitrogen Life Technologies) (1 U/ml final concentration) at 50 °C for 60 minutes, and then denatured at 95 °C for 30 minutes. Linearized plasmid pZero 195 UL36, obtained from Applied Genetic Technologies Corporation, was used to generate standard curves. The primer-probe set was specific for the vector genome UL36 sequence (HSV-UL36 F: 5′-GTTGGTTATGGGGGAGTGTGG, HSV-UL36 R: 5′-TCCTTGTCTGGGGTGTCTTCG, and HSV-UL36 Probe: 5′-6FAM -CGACGAAGACTCCGACGCCACCTC-TAMRA). Amplification of the polymerase chain reaction (PCR) product was achieved with the following cycling parameters: 1 cycle at 50 °C for 2 minutes, 1 cycle at 95 °C for 10 minutes; 40 cycles at 95 °C for 15 seconds, and 40 cycles at 60 °C for 60 seconds. The results were expressed as a mean of rHSV DRP/ml titers ± SD and were statistically analyzed by Prism 5.0d GraphPad Software (GraphPad Software, La Jolla, CA). The titers of HSV-1 stocks in PFU/ml were also determined within crude culture medium. PFU/ ml were quantified by serial dilutions in DMEM 10% FBS, 1% penicillin/ streptomycin, and treatment to either V27 or HEK-293 cells. At 6 hours posttreatment, infectious media content was adjusted to DMEM/10% FBS/1% PenStrep/0.2% γ-globulins by spiking an appropriate 4% (w/v) γ-globulin in phosphate-buffered saline solution into DMEM/10% FBS/1% PenStrep. At 72 hours postinfection, media is removed, and plates dried and then fixed. Cell monolayers are treated with horse radish peroxidase-conjugated α-HSV antibody (Dako, Carpinteria, CA) followed by Vector VIP Peroxidase Substrate Kit (Vector Labs, Burlingame, CA) for enumeration and PFU titer calculation. The results were expressed as a mean of rHSV PFU/ml titers ± SD and were statistically analyzed by Prism 5.0d GraphPad Software. rAAV production HEK-293 cells (2.5 × 10 6 cells) were simultaneously coinfected with both rHSV-rep2/cap2 and rHSV-EGFP vectors as described by Kang et al. 11 At 2-4 hour postinfection, infectious medium was exchanged with DMEM + 10% FBS equivalent to double the preinfection culture volume. At the time of harvest, the cell pellet was frozen at −80 °C. DRP titers were quantified by real-time PCR in a 96-well block Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems Life Technologies, Grand Island, NY). Crude samples were subjected to three cycles of freezing and thawing, then incubat