Antiviral modified siRNA swarms for treatment of herpes simplex virus infection

Herpes simplex virus type 1 (HSV-1) is a common virus of humans carried by half of the global population. After the primary infection, HSV has the ability to establish life-long latency, wherefrom it can reactivate. The latent state cannot be eliminated with modern pharmaceuticals, nor is there a vaccine available, despite massive efforts. Instead, the treatment focuses on diminishing viral replication. The current treatment, however, is insufficient, as it relies almost solely on acyclovir (ACV), and its derivatives, which share their mechanism of action, making ACV-resistant infections almost untreatable. Unfortunately, such infections are rather common, as severe HSV infections require long-term prophylactic treatment to prevent recurrences, which selects for ACV-resistant variants. The lack of treatment diversity against HSV-1 infections encourages for research on novel therapies. Previously, enzymatically synthetized swarms of small interfering (si)RNA have been established as feasible means to treat HSV infection in vitro and in vivo. They differ from regular siRNA by their enzymatic synthesis and by their substantially longer target sequence. Thus, the emergence of resistance, even during long-term prophylactic treatment, is unlikely. However, as all RNA therapy, siRNA swarms face challenges with RNA stability. Therefore, in this study, the goal was to improve the siRNA swarms by synthesizing novel anti-HSV siRNA swarms with chemical 2′-fluoro-modifications to increase RNA efficacy and stability. The modified siRNA swarms, representing modifications of each nucleotide, were first validated in vitro in cells of the nervous system. The research was continued in a highly translational cell line representing the human cornea, which we first validated for use in antiviral RNAi studies. In both cell types, the modified siRNA swarm(s) proved well tolerated and potent beyond the unmodified counterparts, with only modest effects on the host innate responses, even in the presence of viral challenge. Furthermore, all studied HSV-1 strains, including various clinical isolates, were highly sensitive to both modified and unmodified siRNA swarms, whereas their ACV sensitivity varied, proving the potential of siRNA swarms for future therapeutic use. This study shows that incorporation of modified nucleotides to the anti-HSV siRNA swarms is advantageous, and should therefore be preferred in future studies

Treatment of herpes simplex virus infection using antiviral siRNA swarms with 2’-fluoro-modifications

Herpes simplex virus 1 (HSV-1) is a very common pathogen. Besides mostly harmless oral lesions, HSV-1 causes severe diseases such as neonatal herpes, herpes encephalitis and herpes keratitis, the primary cause of infectious blindness worldwide. The available anti-herpes chemotherapy is efficient but depends on a functional viral thymidine kinase. Long-term treatment, required especially in severe diseases, promotes emergence of thymidine kinase mutant strains. These strains are multi-drug resistant, and may lead to dangerous untreatable exacerbations, demonstrating an evident unmet medical need. Small interfering RNA (siRNA) swarms are a novel antiviral approach with extensive tolerance for pathogen mutations. In contrast to regular siRNAs targeting around twenty nucleotides, swarms can target thousands, and thus overcome major challenges of regular antiviral-siRNAs, such as emergence of resistant mutant strains. The most extensively studied siRNA swarm target is the essential UL29 gene of HSV-1. The UL29 targeting siRNA swarm has proven antiviral efficacy against multiple patient-derived strains in vitro and significant inhibition of virus replication in vivo. Here, the swarm is improved by 2’-fluoro-modifications to achieve advanced stability and potency. In this Master’s thesis, effects of incorporated 2’-fluoro-nucleotides on cellular tolerability, host responses and antiviral efficacy are studied in vitro. According to the results, the modified siRNA swarms are well tolerated and demonstrate high antiviral efficacy in prophylactic and therapeutic settings in vitro. The modified siRNA swarms were better than or equal to the nonmodified siRNA swarms in every studied aspect. Overall, the results encourage for subsequent in vivo experiments utilizing the modified siRNA swarms.Herpes simplex virus tyyppi 1 (HSV-1) on yleinen taudinaiheuttaja, joka tunnetaan parhaiten aiheuttamistaan epämiellyttävistä ja toistuvista yskänrokoista. HSV-1 voi kuitenkin aiheuttaa myös vakavampia tautitiloja, kuten sarveiskalvontulehdusta. HSV-1:n aiheuttama sarveiskalvontulehdus on maailman yleisin sokeuteen johtava infektioperäinen sairaus, johon nykyinen lääkehoito on riittämätön. Herpesinfektioiden nykyinen lääkehoito on tehokasta, mutta edellyttää viruksen oman tymidiinikinaasigeenin toimintaa. Erityisesti vakavammissa sairauksissa vaadittava pitkäaikainen ja ennaltaehkäisevä lääkehoito voi johtaa tymidiinikinaasimutatoituneiden viruskantojen ilmaantumiseen. Nämä lääkeresistentit viruskannat ovat selkeä puute nykyisessä lääkehoidossa, ja siten tärkeä lääkekehityskohde. Pienet häiritsevät RNA:t eli siRNA:t (engl. small interfering RNA) johtavat geenien hiljentymiseen. Niitä voidaan käyttää estämään virusten lisääntymistä kohdistamalla siRNA:t kohdeviruksen välttämättömään geeniin. Tavallisesti siRNA:t on kohdistettu verrattain lyhyeen sekvenssijaksoon. Pieni kohdealue altistaa tehon menetykseen joko lähisukuisten virusten perimän monimuotoisuuden vuoksi tai mutaation kautta. SiRNA-parvet tuotetaan entsymaattisesti pitkästä kohdealueesta, jolloin niiden teho kattaa viruskantojen monimuotoisuuden ja mahdolliset mutaatiot kohdesekvenssissä. SiRNA-parvia on tutkittu lääkkeenä herpesinfektioon sekä in vitro että in vivo erittäin lupaavin tuloksin. SiRNA:t kuitenkin hajoavat nopeasti elimistössä, mikä vähentää hoitomuodon potentiaalia, annostelutavasta riippuen. SiRNA:n kestävyyttä pystytään kuitenkin parantamaan kemiallisilla modifikaatioilla. Tässä Pro Gradu -tutkielmassa selvitettiin siRNA-parviin sisällytettyjen 2’-fluoro-nukleotidien vaikutusta parvien tehoon herpestä vastaan in vitro. Lisäksi selvitettiin solujen luonnollisen immuniteetin vaste muunnelluille siRNA-parville. Tutkimuksissa käytetyt solulinjat edustivat hermostoa ja sarveiskalvoa, jotka ovat olennaisia herpesinfektion kohdekudoksia. Muunnellut siRNA-parvet ovat tulosten perusteella vähintään yhtä turvallisia ja jopa tehokkaampia kuin perinteiset siRNA-parvet. Molempien parvityyppien havaittiin lisäksi estävän virusinfektiota ainakin viiden vuorokauden ajan kerta-annostelun jälkeen. Tulokset valottavat perinteisten ja erityisesti muunneltujen siRNA-parvien potentiaalia terapeuttisena ja ennaltaehkäisevänä lääkehoitomuotona ja kannustavat niiden jatkotutkimuksiin in vivo

Native RNA purification method for small RNA molecules based on asymmetrical flow field-flow fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules.Peer reviewe

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

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

Peer reviewe

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

The In Vitro Replication, Spread, and Oncolytic Potential of Finnish Circulating Strains of Herpes Simplex Virus Type 1

Herpes simplex virus type 1 (HSV-1) is the only FDA- and EMA- approved oncolytic virus, and accordingly, many potential oncolytic HSVs (oHSV) are in clinical development. The utilized oHSV parental strains are, however, mostly based on laboratory reference strains, which may possess a compromised cytolytic capacity in contrast to circulating strains of HSV-1. Here, we assess the phenotype of thirty-six circulating HSV-1 strains from Finland to uncover their potential as oHSV backbones. First, we determined their capacity for cell-to-cell versus extracellular spread, to find strains with replication profiles favorable for each application. Second, to unfold the differences, we studied the genetic diversity of two relevant viral glycoproteins (gB/UL27, gI/US7). Third, we examined the oncolytic potential of the strains in cells representing glioma, lymphoma, and colorectal adenocarcinoma. Our results suggest that the phenotype of a circulating isolate, including the oncolytic potential, is highly related to the host cell type. Nevertheless, we identified isolates with increased oncolytic potential in comparison with the reference viruses across many or all of the studied cancer cell types. Our research emphasizes the need for careful selection of the backbone virus in early vector design, and it highlights the potential of clinical isolates as backbones in oHSV development

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity

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

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity

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

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity

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