Inhibition of clinical pathogenic herpes simplex virus 1 strains with enzymatically created siRNA pools

Abstract

Herpes simplex virus (HSV) is a common human pathogen causing severe diseases such as encephalitis, keratitis, and neonatal herpes. There is no vaccine against HSV and the current antiviral chemotherapy fails to treat certain forms of the disease. Here, we evaluated the antiviral activity of enzymatically created small interfering (si)RNA pools against various pathogenic HSV strains as potential candidates for antiviral therapies. Pools of siRNA targeting 0.5-0.8 kbp of essential HSV genes UL54, UL29, or UL27 were enzymatically synthesized. Efficacy of inhibition of each siRNA pool was evaluated against multiple clinical isolates and laboratory wild type HSV-1 strains using three cell lines representing host tissues that support HSV-1 replication: epithelial, ocular, and cells that originated from the nervous system. The siRNA pools targeting UL54, UL29, and UL27, as well as their equimolar mixture, inhibited HSV replication, with the pool targeting UL29 having the most prominent antiviral effect. In contrast, the non-specific control siRNA pool did not have such an effect. Moreover, the UL29 pool elicited only a minimal innate immune response in the HSV-infected cells, thus evidencing the safety of its potential clinical use. These results are promising for the development of a topical RNA interference approach for clinical treatment of HSV infection. J. Med. Virol. 88:2196-2205, 2016. © 2016 Wiley Periodicals, Inc.

Novel activities of safe-in-human broad-spectrum antiviral agents

“…ZIKV titers were determined by plaque assay on VERO-E6 cells, as described earlier (Kuivanen et al, 2017). HSV-1 titers were determined by plaque titration in Vero cells in the presence of human immunoglobulin G (20 μg/mL), as described earlier (Paavilainen et al, 2016). CHIKV-2SG-NanoLuc and RRV-2SG-NanoLuc were titrated in BHK-21 cells using plaque assay, as described previously (Oo et al, 2018;Taylor et al, 2016).…”

Topical Treatment of Herpes Simplex virus Infection with Enzymatically Created siRNA Swarm

“…Real-time quantitative PCR was performed as previously described with Maxima SYBR green master mix and polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and was measured with Rotor-Gene Q real-time instrument (Qiagen) [11]. DNA samples were isolated with NucliSENS easyMag (bioMérieux, Marcy l'Etoile, France) following manufacturer's instructions.…”

“…We have developed RNA interference-based biological drugs effective against HSV infection [10,11]. Instead of using synthetic single site small interfering (si)RNA molecules, we utilize enzymatically produced siRNA swarms that cover hundreds of nucleotides (nt) of a targeted viral transcript.…”

“…Such Dicer-substrate siRNAs (DsiRNAs) can have enhanced potency and efficacy compared to canonical 21-nt siRNAs [12]. We have shown that the enzymatically created DsiRNA pools are effective against HSV in cell cultures, induce a favourable innate immunity profile, and also inhibit clinical HSV isolates [10,11,13]. Of several HSV targets tested, the UL29-DsiRNA swarm targeting gene UL29 had the best antiviral profile.…”

Gene Editing Approaches against Viral Infections and Strategy to Prevent Occurrence of Viral Escape

“…Another possibility is to combine CRISPR/Cas9 with RNAi to apply a double assault. RNAi approaches exist for a number of viruses, including HIV-1 [43, 79], HBV [80], HCV [81], HPV [82, 83], JCV [84], and HSV [85]. For example, RNAi against HIV-1 is already reaching the clinic [43, 79].…”