1 . 2024

Synthetic small interfering RNAs targeted to conservative region of the rhinovirus genome suppress the infection in vitro

Abstract

Introduction. Human rhinoviruses (HRVs) belong to the genus Enterovirus of the Picornaviridae family. HRV upper respiratory tract infection can be associated with various complications and exacerbation of chronic respiratory diseases such as bronchial asthma. Due to the large number of virus serotypes, vaccination against HRV isn’t possible. Despite advances in antiviral research no specific antiviral drug has been approved for the treatment of the disease caused by HRV. Antiviral agents based on the RNA-interference mechanism are developing fast. Such drugs block virus replication specifically in an infected cell.

Aim of this study – to develop siRNA molecules specifically inhibiting rhinovirus replication in human cells.

Material and methods. Bioinformatic analysis of rhinovirus genomes was carried out using the Vector NTI program. The design of siRNA molecules was carried out using the OligoWalk program. Antiviral activity experiments of the siRNAs were carried out in vitro in HeLa Ohio cells. The tested siRNAs were delivered into cells using commercial transfection reagent Lipofectamine 3000 or peptide-carrier KK-46. The antiviral activity was assessed by the ability to reduce the virus titer in cell supernatants and the number of viral RNA copies in cell lysates.

Results. The most conservative regions in the rhinovirus genome were identified and 125 siRNA complementary to them were designed. Based on bioinformatic analysis 7 variants were selected to study the antiviral activity in vitro. It was found that two variants siRV-5UTR-4-3 and siRV-5UTR-5-3 have the most evident antiviral effect. They both can significantly suppress HRV replication in mammalian cells. Based on these siRNA variants complexes with the peptide-carrier KK-46 were created. Other in vitro experiments have proven the antiviral activity of the siRV-5UTR-4-3/KK-46 and siRV-5UTR-5-3/KK-46 complexes against rhinovirus.

Conclusion. In this study siRNA molecules were designed against conservative regions of the rhinovirus genome and their antiviral effect was proved in vitro. The created siRNA molecules can be components of antiviral drugs.

Keywords:human rhinovirus; HRV; respiratory viruses; siRNA; RNA interference; antiviral drugs

For citation: Shilovskiy I.P., Timotievich E.D., Kovchina V.I., Nikolskii A.A., Yumashev K.V., Kaganova M.M., Rusak T.E., Tulubaev V.V., Smirnov V.V., Shatilov A.A., Shatilova A.V., Andreev S.M., Sergeev I.V., Maerle A.V., Kofiadi I.A., Kudlay D.A., Khaitov M.R. Synthetic small interfering RNAs targeted to conservative region of the rhinovirus genome suppress the infection in vitro. Immunologiya. 2024; 45 (1): 7–20. DOI: https://doi.org/10.33029/1816-2134-2024-45-1-7-20 (in Russian)

Funding. The study was carried according to state assignment and supported by the Federal Medical-Biological Agency. Open publication of the research results is allowed.

Conflict of interests. The authors declare no conflict of interests.

Authors’ contribution. Study conception and design – Shilovskiy I.P., Smirnov V.V., Khaitov M.R.; material collection and processing – Timotievich E.D., Nikolskii A.A., Kovchina V.I., Yumashev K.V., Kaganova M.M., Rusak T.E., Shatilov A.A., Shatilova A.V., Tulubaev V.V.; statistical processing – Andreev S.M., Kofiadi I.A.; manuscript preparation – Timotievich E.D., Shilovskiy I.P.; editing – Kudlay D.A., Sergeev I.V., Maerle A.V.

References

1. Jacobs S.E., Lamson D.M., Kirsten S., Walsh T.J. Human rhinoviruses. Clin Microbiol Rev. 2013; 26 (1): 135–62. DOI: https://doi.org/10.1128/CMR.00077-12

2. To K.W, Yip C.Y, Yuen K.Y. Rhinovirus – From bench to bedside. J Formos Med Assoc. 2017; 116 (7): 496–504. DOI: https://doi.org/10.1016/J.JFMA.2017.04.009

3. Rollinger J.M., Schmidtke M. The human rhinovirus: human-pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery. Med Res Rev. 2011; 31 (1): 42–92. DOI: https://doi.org/10.1002/MED.20176

4. Hayashi Y., Sada M., Shirai T., Okayama K., Kimura R., Kondo M., Okodo M., Tsugawa T., Ryo A., Kimura H. Rhinovirus Infection and Virus-Induced Asthma. Viruses. 2022; 14 (12): 2616. DOI: https://doi.org/10.3390/V14122616

5. Lee J.S., Kim S.R., Song J.H., Lee Y.P., Ko H.J. Anti-Human Rhinovirus 1B Activity of Dexamethasone viaGCR-Dependent Autophagy Activation. Osong Public Health Res Perspect. 2018; 9 (6): 334–9. DOI: https://doi.org/10.24171/J.PHRP.2018.9.6.07

6. Jackson D.J., Gern J.E. Rhinovirus Infections and Their Roles in Asthma: Etiology and Exacerbations. J Allergy Clin Immunol Pract. 2022; 10 (3): 673–81. DOI: https://doi.org/10.1016/J.JAIP.2022.01.006

7. Gunawardana N., Finney L., Johnston S.L., Mallia P. Experimental rhinovirus infection in COPD: implications for antiviral therapies. Antiviral Res. 2014; 102: 95–105. DOI: https://doi.org/10.1016/J.ANTIVIRAL.2013.12.006

8. Price A.S., Kennedy J.L. T-helper 2 mechanisms involved in human rhinovirus infections and asthma. Ann Allergy Asthma Immunol. 2022; 129 (6): 681–91. DOI: https://doi.org/10.1016/J.ANAI.2022.08.015

9. Casanova V., Sousa F.H., Stevens C., Barlow P.G. Antiviral therapeutic approaches for human rhinovirus infections. Future Virol. 2018; 13 (7): 505–18. DOI: https://doi.org/10.2217/FVL-2018-0016

10. Ruuskanen O., Waris M., Ramilo O. New aspects on human rhinovirus infections. Pediatr Infect Dis J. 2013; 32 (5): 553–5. DOI: https://doi.org/10.1097/INF.0B013E3182833C90

11. Coultas J.A., Cafferkey J., Mallia P., Johnston S.L. Experimental Antiviral Therapeutic Studies for Human Rhinovirus Infections. J Exp Pharmacol. 2021; 13: 645–59. DOI: https://doi.org/10.2147/JEP.S255211

12. Shilovskiy I.P., Yumashev K. V., Nikolsky A.A., Vishnyakova L.I., Khaitov M.R. Molecular and Cellular Mechanisms of Respiratory Syncytial Viral Infection: Using Murine Models to Understand Human Pathology. Biochemistry (Moscow). 2021; 86 (3): 290–306. DOI: https://doi.org/10.1134/S0006297921030068

13. Khaitov M.R., Litvin L.S., Shilovsky I.P., Bashkatova Y.N., Faizuloev E.B., Zverev V.V. RNA interference. New approaches to the development of antiviral agents. Immunologiya. 2010; 31: 69–76.

14. Whangbo J.S., Hunter C.P. Environmental RNA interference. Trends Genet. 2008; 24 (6): 297–305. DOI: https://doi.org/10.1016/J.TIG.2008.03.007

15. Khaitov M., Nikonova A., Shilovskiy I., Kozhikhova K., Kofiadi I., Vishnyakova L., Nikolsky A., Gattinger P., Kovchina V., Barvinskaya E., Yumashev K., Smirnov V., Maerle A., Kozlov I., Shatilov A., Timofeeva A., Andreev S., Koloskova O., Kuznetsova N., Vasina D., Nikiforova M., Rybalkin S., Sergeev I., Trofimov D., Martynov A., Berzin I., Gushchin V., Kovalchuk A., Borisevich S., Valenta R., Khaitov R., Skvortsova V. Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation. Allergy. 2021; 76 (9): 2840–54. DOI: https://doi.org/10.1111/all.14850

16. Khaitov M.R., Nikonova A.A., Shilovsky I.P., Kozhikhova K.V., Kofiadi I.A., Gudima G.O., Vishnyakova L.I., Nikolsky A.A., Kovchina V.I., Timotievich E.D., Yumashev K.V., Smirnov V.V., Maerle A.V., Kozlov I.B., Shatilov A.A., Shatilova A.V., Andreev S.M., Koloskova O.O., Kuznetsova N.A., Vasina D.V., Nikiforova M.A., Rybalkin S.P., Sergeev I.V., Trofimov D.Yu., Martynov A.I., Berzin I.A., Gushchin V.A., Kovalchuk A.V., Borisevich S.V., Skvortsova V.I. MIR 19^® is the world’s first specific antiviral drug for the treatment of COVID-19: development and preclinical studies. Immunologiya. 2023; 44 (3): 270–90. DOI: https://doi.org/10.33029/0206-4952-2023-44-3-270-290 (in Russian)

17. Khaitov M.R., Nikonova A.A., Kofiadi I.A., Shilovsky I.P., Smirnov V.V., Elisyutina O.G., Gudima G.O., Maerle A.V., Shatilov A.A., Shatilova A.V., Andreev S.M., Sergeev I.V., Trofimov D.Yu., Latysheva T.V., Ilyina N.I., Martynov A.I., Rabdano S.O., Ruzanova E.A., Savelyev N.S., Pletyukhina Yu.V. Safi A.S., Ratnikov V.A., Gorelov V.P., Kashchenko V.A., Kucherenko N.G., Umarova I.A., Moskaleva S.S., Fabrichnikov S.V., Zuev O .V., Pavlov N.B., Kryuchko D.S., Berzin I.A., Goryachev D.V., Merkulov V.A., Shipulin G.A., Yudin S.M., Trukhin V.P., Valenta R., Skvortsova V.I. Results of phase I and II clinical trials of the drug MIR 19^®. Immunologiya. 2023; 44 (3): 291–316. DOI: https://doi.org/10.33029/1816-2134-2023-44-3-291-316 (in Russian)

18. Harborth J., Elbashir S.M., Bechert K., Tuschl T., Weber K. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci. 2001; 114 (24): 4557–65. DOI: https://doi.org/10.1242/JCS.114.24.4557

19. Ui-Tei K., Naito Y., Takahashi F., Haraguchi T., Ohki-Hamazaki H., Juni A., Ueda R., Saigo K. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 2004; 3 2 (3): 936–48. DOI: https://doi.org/10.1093/nar/gkh247

20. Reynolds A., Leake D., Boese Q., Scaringe S., Marshall W.S., Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004; 22 (3): 326–30. DOI: https://doi.org/10.1038/nbt936

21. Gatta A.K., Hariharapura R.C., Udupa N., Reddy M.S., Josyula V.R. Strategies for improving the specificity of siRNAs for enhanced therapeutic potential. Expert Opinion on Drug Discovery. 2018; 13 (8): 709–25. DOI: https://doi.org/10.1080/17460441.2018.1480607

22. Koloskova O.O., Nosova A.S., Ilyukhina A.A., Shilovsky I.P., Sebyakin Yu.L., Khaitov M.R. Liposomal delivery vehicles for siRNA (review). Biopharmaceutical Journal. 2017; 9 (5). (in Russian)

23. Shilovsky I.P., Yumashev K.V., Kozhikhova K.V., Vishnyakova L.I., Smirnov V.V., Gudima G.O., Brylina V.E., Kaganova M.M., Nikolsky A. A.A., Timotievich E.D., Kovchina V.I., Rusak T.E., Shatilova A.V., Shatilov A.A., Andreev S.M., Kudlay D.A., Khaitov M.R. A synthetic peptide that mimics the antigenic site of the F protein suppresses respiratory syncytial virus infection in in vitro experiments. Immunologiya. 2023; 44 (2): 134–46. DOI: https://doi.org/10.33029/0206-4952-2023-44-2-134-146 (in Russian)

24. Shilovskiy I.P., Andreev S.M., Kozhikhova K.V., Nikolskii A.A., Khaitov MR. Prospects For the Use of Peptides against Respiratory Syncytial Virus. Mol Biol. 2019; 53 (4): 484–500. DOI: https://doi.org/10.1134/S0026893319040125/FIGURES/3

25. Vandini S., Biagi C., Fischer M., Lanari M. Impact of Rhinovirus Infections in Children. Viruses. 2019; 11 (6). DOI: https://doi.org/10.3390/V11060521

26. Casanova V., Sousa F.H., Stevens C., Barlow P.G. Antiviral therapeutic approaches for human rhinovirus infections. Future Virol. 2018; 13 (7): 505–18. DOI: https://doi.org/10.2217/FVL-2018-0016

27. Smee D.F., Evans W.J., Nicolaou K.C., Tarbet E.B., Day C.W. Susceptibilities of enterovirus D68, enterovirus 71, and rhinovirus 87 strains to various antiviral compounds. Antiviral Res. 2016; 131: 61–5. DOI: https://doi.org/10.1016/J.ANTIVIRAL.2016.04.003

28. Phipps K.M., Martinez A., Lu J., Heinz B.A., Zhao G. Small interfering RNA molecules as potential anti-human rhinovirus agents: In vitro potency, specificity, and mechanism. Antiviral Res. 2004; 61 (1): 49–55. DOI: https://doi.org/10.1016/j.antiviral.2003.08.005

29. Bochkov Y.A., Palmenberg A.C., Lee W.M., Rathe J.A., Amineva S.P., Sun X., Pasic T., Jarjour N., Liggett S., Gern J. Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C. Nature Medicine. 2011; 17 (5): 627–32. DOI: https://doi.org/10.1038/nm.2358

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