Aurintricarboxylic acid increases yield of HSV-1 vectors

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

Recombinant adenovirus-associated virus (rAAV) vectors have been successfully introduced in several human gene therapy clinical trials because of their nonpathogenic nature, low toxicity, minimal immunogenicity, and long-term persistence. Production of large quantities of clinical grade rAAV vectors for gene therapy has been challenging due to limitations in scalability of the commonly used co-transfection protocol. 1 AAVs are not able to replicate by themselves and were first found to propagate only when adenoviruses or herpes viruses coinfected the same cells. 2, 3 The first scalable rAAV protocol was based on adenovirus infection of rAAV/Rep-Cap cell lines. 4 Besides adenoviruses, also herpesviruses have been shown to provide complete helper virus functions for the production of AAV virions. 5, 6 The minimal set of herpes simplex virus type-1 (HSV-1) genes required for AAV replication and packaging has been identified as the HSV-1 early genes UL5, UL8, UL52, and UL29. 7 These genes encode components of the HSV-1 core replication machinery-the helicase, primase, and primase accessory proteins (U L 5, U L 8, and U L 52) and the single-stranded DNA-binding protein (U L 29). A protocol for production of rAAV serotype 2 (rAAV2) vectors using HSV-1 amplicons expressing AAV2 Rep and Cap in combination with rHSV-1 helper vectors has been described, 8 and this protocol was modified and further optimized by several groups using coinfection of two rHSV-1 vectors, both replication-deficient infected-cell protein ICP27-mutants, one carrying rAAV provirus and a second bearing AAV2 Rep-cap genes, [9] [10] [11] The use of rHSV-1 vectors has been historically limited by their relatively low titers and the rAAV vector yields in the rHSV-based manufacturing, thus would be affected by the titers of rHSV-1 helper. 12, 13 Therefore, several methods to improve rHSV-1 yield have been studied, i.e., changing rHSV-1 propagation conditions [12] [13] [14] or using anti-inflammatory compounds known to inhibit the host defense mechanism, like dexamethasone or valproic acid. 15, 16 In our search for compounds that would increase HSV-1 yield, we investigated another anti-inflammatory compound, aurintricarboxylic acid (ATA). ATA is a heterogeneous mixture of polymers accredited with a continuously growing number of biological activities. [17] [18] [19] [20] ATA is mostly known as an antiviral agent to several viruses like HIV, herpesvirus HHV-7, SARS-CoV, and others. [20] [21] [22] [23] However, ATA did not block the replication of adenovirus type 5 24 and has been reported to increase adenovirus type 5 titer in human embryonic kidney (HEK)-293 cells. 25 Here, we investigate the effects of ATA on HSV-1 vector production yield in V27, Vero, and HEK-293 cells. We further tested the rHSV-1 stocks produced in the presence of ATA in the HSV-1-based rAAV manufacturing protocol.