RNA interference (RNAi), which is mediated by small interfering RNAs (siRNAs) derived from viral genome or its replicative intermediates, is a natural antiviral defense in plants, fungi, and invertebrates. Whether RNAi naturally protects humans from viral invasion is still a matter of debate. Nevertheless, exogenous siRNAs are able to halt viral infection in mammals. The current review critically evaluates the production of antiviral siRNAs, delivery techniques to the infection sites, as well as provides an overview of antiviral siRNAs in clinical trials.

Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2′-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2′-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2′-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2′-F-cytidine and 2′-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2′-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2′-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2′ position, for further antiviral studies in vitro and in vivo .

Acyclovir is the drug of choice for the treatment of herpes simplex virus (HSV) infections. Acyclovir-resistant HSV strains may emerge, especially during long-term drug use, and subsequently cause difficult-to-treat exacerbations. Previously, we set up a novel treatment approach, based on enzymatically synthesized pools of siRNAs, or siRNA swarms. These swarms can cover kilobases-long target sequences, reducing the likelihood of resistance to treatment. Swarms targeting the UL29 essential gene of HSV-1 have demonstrated high efficacy against HSV-1 in vitro and in vivo. Here, we assessed the antiviral potential of a UL29 siRNA swarm against circulating strains of HSV-1, in comparison with acyclovir. All circulating strains were sensitive to both antivirals, with the half-maximal inhibitory concentrations (IC50) in the range of 350–1911 nM for acyclovir and 0.5–3 nM for the UL29 siRNA swarm. Additionally, we showed that an acyclovir-resistant HSV-1, devoid of thymidine kinase, is highly sensitive to UL29 siRNA treatment (IC50 1.0 nM; Imax 97%). Moreover, the detected minor variations in the RNAi target of the HSV strains had no effect on the potency or efficacy of UL29 siRNA swarm treatment. Our findings support the development of siRNA swarms for the treatment of HSV-1 infections, in order to circumvent any potential acyclovir resistance.

Steric exclusion chromatography (SXC) is a method for separation of large target solutes based on their association with a hydrophilic stationary phase through mutual steric exclusion of polyethylene glycol (PEG). Selectivity in SXC is determined by the size or shape (or both) of the solutes alongside the size and concentration of PEG molecules. Elution is achieved by decreasing the PEG concentration. In this study, SXC applicability for the separation and purification of single-stranded (ss) and double-stranded (ds) RNA molecules was evaluated for the first time. The retention of ssRNA and dsRNA molecules of different lengths on convective interaction media (CIM) monolithic columns was systematically studied under variable PEG-6000 and NaCl concentrations. We determined that over 90% of long ssRNAs (700-6374 nucleotides) and long dsRNAs (500-6374 base pairs) are retained on the stationary phase in 15% PEG-6000 and ≥0.4 M NaCl. dsDNA and dsRNA molecules of the same length were partially separated by SXC. Separation of RNA molecules below 100 nucleotides from longer RNA species is easily achieved by SXC. Furthermore, SXC has the potential to separate dsRNAs from ssRNAs of the same length. We also demonstrated that SXC is suitable for the enrichment of ssRNA (PRR1 bacteriophage) and dsRNA (Phi6 bacteriophage) viral genomes from contaminating cellular RNA species. In summary, SXC on CIM monolithic columns is an appropriate tool for rapid RNA separation and concentration.

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.