Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells

Abstract

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2’-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.

Single therapeutic dose of an antiviral UL29 siRNA swarm diminishes symptoms and viral load of mice infected intranasally with HSV‐1

“…Furthermore, the UL29 siRNA swarm exhibited proper antiviral efficacy in comparison to the nonspecific siRNA swarm ( p = 0.001), preventing over 99% of HSV‐1 replication (Figure 2B). These results are in line with our previous research 2,4–8 …”

“…These results are in line with our previous research. 2,[4][5][6][7][8] In mice, the anti-HSV UL29-targeted siRNA swarm treatment protected them from the onset of symptoms. The mice treated with the UL29 siRNA swarm developed symptoms as late as on day five, whereas 75% of placebotreated mice were already symptomatic at that time, with symptoms developing already from day one (Figure 3A).…”

“…We have previously shown that siRNA swarms targeting a 653 base pair sequence of the essential viral UL29 gene can inhibit active replication of circulating clinical HSV‐1 strains and acyclovir‐resistant strains in vitro, while being well tolerated by various cell types 2,4–8 . UL29 has previously proven to be the most suitable target for anti‐HSV siRNA swarms.…”

“…2 We have previously shown that siRNA swarms targeting a 653 base pair sequence of the essential viral UL29 gene can inhibit active replication of circulating clinical HSV-1 strains and acyclovir-resistant strains in vitro, while being well tolerated by various cell types. 2,[4][5][6][7][8] UL29 has previously proven to be the most suitable target for anti-HSV siRNA swarms. The gene encodes the essential protein ICP8, a single-stranded DNA-binding protein that is required throughout the replication cycle of HSV and is highly conserved.…”

Superior antiviral activity of IFNβ in genital HSV-1 infection

“…In our study we did not detect a significant increase in IFNγ expressing T or NK cells ( Figure 2C ; Supplementary Figures 3A, C, E ; Supplementary Figure 4A ), although we observed an induction of IFNγ in the vaginal lavages at 4 dpi which might be produced by other important immune cell subsets within vaginal tract like granulocytes, monocytes, or macrophages. Other important members of the type I IFN family (IFNϵ, IFNκ) were already characterized during viral infections like HSV-1/2, Zika virus or human papillomavirus (HPV) in the vaginal tract ( Reiser et al., 2011 ; Fung et al., 2013 ; Habiger et al., 2016 ; Woodby et al., 2018 ; Mungin et al., 2022 ) or in HSV-1-infected keratinocytes ( Li et al., 2020 ; Kalke et al., 2022 ). Mice deficient in IFNϵ, which is constitutively expressed by epithelial cells in the female reproductive tract, were highly susceptible to HSV-2 ( Fung et al., 2013 ).…”