Journal Home > Volume 14 , Issue 2

COVID-19 is caused by severe acute respiratory SARS-CoV-2. Regardless of the availability of treatment strategies for COVID-19, effective therapy will remain essential. A promising approach to tackle the SARS-CoV-2 could be small interfering (si) RNAs. Here we designed the small hairpin RNA (named as shRNA688) for targeting the prepared 813 bp Est of the S protein genes (Delta). The conserved and mutated regions of the S protein genes from the genomes of the SARS-CoV-2 variants in the public database were analyzed. A 813 bp fragment encoding the most part of the RBD and partial downstream RBD of the S protein was cloned into the upstream red florescent protein gene (RFP) as a fusing gene in the pCMV-S-Protein RBD-Est-RFP plasmid for expressing a potential target for RNAi. The double stranded of the DNA encoding for shRNA688 was constructed in the downstream human H1 promoter of the plasmid in which CMV promoter drives enhanced green fluorescent protein (EGFP) marker gene expression. These two kinds of the constructed plasmids were co-transfected into HEK293T via Lipofectamine 2000. The degradation of the transcripts of the SARS-CoV-2 S protein fusing gene expressed in the transfected HEK293T treated by RNAi was analyzed by RT-qPCR with a specific probe of the targeted SARS-CoV-2 S protein gene transcripts. Our results showed that shRNA688 targeting the conserved region of the S protein genes could effectively reduce the transcripts of the S protein genes. This study provides a cell model and technical support for the research and development of the broad-spectrum small nucleic acid RNAi drugs against SARS-CoV-2 or the RNAi drugs for the other hazard viruses which cause human diseases.


menu
Abstract
Full text
Outline
About this article

RNAi Degrades the SARS-CoV-2 Spike Protein RNA for Developing Drugs to Treat COVID-19

Show Author's information Weiwei Zhang1,2,3Linjia Huang2Jumei Huang2Xin Jiang3,4Xiaohong Ren6Xiaojie Shi1Ling Ye2,3Shuhui Bian4Jianhe Sun2Yufeng Gao2Zehua Hu2Lintin Guo2Suyan Chen4Jiahao Xu2Jie Wu1,3Jiwen Zhang3,6( )Daxiang Cui5( )Fangping Dai1,2,3,5( )
Central Laboratory, Nantong Haimen People's Hospital, Nantong 226199, China
Genome-decoding Biomedical Technology Co., Ltd., Nantong 226126, China
Yangtze Delta Drug Advanced Research Institute, Nantong 226126, China
Precision-genes Bio-Technology Co., Ltd/Medical Laboratory, Nantong 226126, China
National Engineering Research Center for Nanotechnology, Shanghai Jiao Tong University, Shanghai 200241, China
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

Abstract

COVID-19 is caused by severe acute respiratory SARS-CoV-2. Regardless of the availability of treatment strategies for COVID-19, effective therapy will remain essential. A promising approach to tackle the SARS-CoV-2 could be small interfering (si) RNAs. Here we designed the small hairpin RNA (named as shRNA688) for targeting the prepared 813 bp Est of the S protein genes (Delta). The conserved and mutated regions of the S protein genes from the genomes of the SARS-CoV-2 variants in the public database were analyzed. A 813 bp fragment encoding the most part of the RBD and partial downstream RBD of the S protein was cloned into the upstream red florescent protein gene (RFP) as a fusing gene in the pCMV-S-Protein RBD-Est-RFP plasmid for expressing a potential target for RNAi. The double stranded of the DNA encoding for shRNA688 was constructed in the downstream human H1 promoter of the plasmid in which CMV promoter drives enhanced green fluorescent protein (EGFP) marker gene expression. These two kinds of the constructed plasmids were co-transfected into HEK293T via Lipofectamine 2000. The degradation of the transcripts of the SARS-CoV-2 S protein fusing gene expressed in the transfected HEK293T treated by RNAi was analyzed by RT-qPCR with a specific probe of the targeted SARS-CoV-2 S protein gene transcripts. Our results showed that shRNA688 targeting the conserved region of the S protein genes could effectively reduce the transcripts of the S protein genes. This study provides a cell model and technical support for the research and development of the broad-spectrum small nucleic acid RNAi drugs against SARS-CoV-2 or the RNAi drugs for the other hazard viruses which cause human diseases.

Keywords: COVID-19, Nanoparticles, RNAi, shRNA, Plasmids

References(37)

[1]

Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R. et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med, 2020, 382: 727-733.

[2]

Zhu, C., Lee, J.Y., Woo, J.Z., Xu, L., Nguyenla, X., Yamashiro, L.H., Ji, F., Biering, S.B., Van Dis, E., Gonzalez, F. et al. An intranasal ASO therapeutic targeting SARS-CoV-2. Nat Commun, 2022, 13: 4503.

[3]

Chang, Y.C., Yang, C.F., Chen, Y.F., Yang, C.C., Chou, Y.L., Chou, H.W., Chang, T.Y., Chao, T.L., Hsu, S.C., Ieong, S.M. et al. A siRNA targets and inhibits a broad range of SARS-CoV-2 infections including Delta variant. EMBO Mol Med, 2022, 14: e15298.

[4]

Wouters, O.J., Shadlen, K.C., Salcher-Konrad, M., Pollard, A.J., Larson, H.J., Teerawattananon, Y. and Jit, M. Challenges in ensuring global access to COVID-19 vaccines: production, affordability, allocation, and deployment. The Lancet, 2021, 397: 1023-1034.

[5]

Kuzmina, A., Khalaila, Y., Voloshin, O., Keren-Naus, A., Boehm-Cohen, L., Raviv, Y., Shemer-Avni, Y., Rosenberg, E. and Taube, R. SARS-CoV-2 spike variants exhibit differential infectivity and neutralization resistance to convalescent or post-vaccination sera. Cell Host Microbe, 2021, 29: 522-528, e522.

[6]

Cao, Y., Wang, J., Jian, F., Xiao, T., Song, W., Yisimayi, A., Huang, W., Li, Q., Wang, P., An, R. et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature, 2022, 602: 657-663.

[7]

McMillan, N.A.J., Morris, K.V. and Idris, A. (2022) RNAi to treat SARS-CoV-2-variant proofing the next generation of therapies. EMBO Mol Med, 2022, 14: e15811.

[8]

Traube, F.R., Stern, M., Tolke, A.J., Rudelius, M., Mejias-Perez, E., Raddaoui, N., Kummerer, B.M., Douat, C., Streshnev, F., Albanese, M. et al. Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs. Angew Chem Int Ed Engl, 2022: e202204556.

[9]

Merkel, O.M. Can pulmonary RNA delivery improve our pandemic preparedness? J Control Release, 2022, 345: 549-556.

[10]

Lin, P., Shen, G., Guo, K., Qin, S., Pu, Q., Wang, Z., Gao, P., Xia, Z., Khan, N., Jiang, J. et al. Type Ⅲ CRISPR-based RNA editing for programmable control of SARS-CoV-2 and human coronaviruses. Nucleic Acids Res, 2022, 50: e47.

[11]

Setten, R.L., Rossi, J.J. and Han, S.P. The current state and future directions of RNAi-based therapeutics. Nat Rev Drug Discov, 2019, 18: 421-446.

[12]

Hu, B., Zhong, L., Weng, Y., Peng, L., Huang, Y., Zhao, Y. and Liang, X.J. Therapeutic siRNA: state of the art. Signal Transduct Target Ther, 2020, 5: 101.

[13]

Carthew, R.W. and Sontheimer, E.J. Origins and Mechanisms of miRNAs and siRNAs. Cell, 2009, 136: 642-655.

[14]

Berber, B., Aydin, C., Kocabas, F., Guney-Esken, G., Yilancioglu, K., Karadag-Alpaslan, M., Caliseki, M., Yuce, M., Demir, S. and Tastan, C. Gene editing and RNAi approaches for COVID-19 diagnostics and therapeutics. Gene Ther, 2021, 28: 290-305.

[15]

Chowdhury, U.F., Sharif Shohan, M.U., Hoque, K.I., Beg, M.A., Sharif Siam, M.K. and Moni, M.A. A computational approach to design potential siRNA molecules as a prospective tool for silencing nucleocapsid phosphoprotein and surface glycoprotein gene of SARS-CoV-2. Genomics, 2021, 113: 331-343.

[16]

Donia, A. and Bokhari, H. RNA interference as a promising treatment against SARS-CoV-2. Int Microbiol, 2021, 24: 123-124.

[17]

Fu, Y. and Xiong, S. Tagged extracellular vesicles with the RBD of the viral spike protein for delivery of antiviral agents against SARS-COV-2 infection. J Control Release, 2021, 335: 584-595.

[18]

Abdullah Al Saba, M., Sajib Chakraborty, AHM Nurun Nabi Prediction of putative potential siRNAs for inhibiting SARS-CoV-2 strains, including variants of concern and interest. Future Microbiol, 2022, 17: 15.

[19]

Sohrab, S.S., El-Kafrawy, S.A. and Azhar, E.I. Effect of insilico predicted and designed potential siRNAs on inhibition of SARS-CoV-2 in HEK-293 cells. J King Saud Univ Sci, 2022, 34: 101965.

[20]

Khaitov, M., Nikonova, A., Shilovskiy, I., Kozhikhova, K., Kofiadi, I., Vishnyakova, L., Nikolskii, A., Gattinger, P., Kovchina, V., Barvinskaia, E. et al. Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation. Allergy, 2021, 76: 2840-2854.

[21]

Baldassi, D., Ambike, S., Feuerherd, M., Cheng, C.C., Peeler, D.J., Feldmann, D.P., Porras-Gonzalez, D.L., Wei, X., Keller, L.A., Kneidinger, N. et al. Inhibition of SARS-CoV-2 replication in the lung with siRNA/VIPER polyplexes. J Control Release, 2022, 345: 661-674.

[22]

Friedrich, M., Pfeifer, G., Binder, S., Aigner, A., Vollmer Barbosa, P., Makert, G.R., Fertey, J., Ulbert, S., Bodem, J., Konig, E.M. et al. Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2. Front Bioeng Biotechnol, 2022, 10: 801870.

[23]

Becker, J., Stanifer, M.L., Leist, S.R., Stolp, B., Maiakovska, O., West, A., Wiedtke, E., Borner, K., Ghanem, A., Ambiel, I. et al. Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors. Mol Ther, 2022, 30: 2005-2023.

[24]

Ambike, S., Cheng, C.C., Feuerherd, M., Velkov, S., Baldassi, D., Afridi, S.Q., Porras-Gonzalez, D., Wei, X., Hagen, P., Kneidinger, N. et al. Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread. Nucleic Acids Res, 2022, 50: 333-349.

[25]

Roden, C.A., Dai, Y., Giannetti, C.A., Seim, I., Lee, M., Sealfon, R., McLaughlin, G.A., Boerneke, M.A., Iserman, C., Wey, S.A. et al. Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures. Nucleic Acids Res, 2022, 50: 8168-8192.

[26]

Dutta, N.K., Mazumdar, K. and Gordy, J.T. The Nucleocapsid Protein of SARS-CoV-2: a Target for Vaccine Development. J Virol, 2020: 94.

[27]

Manfredonia, I., Nithin, C., Ponce-Salvatierra, A., Ghosh, P., Wirecki, T.K., Marinus, T., Ogando, N.S., Snijder, E.J., van Hemert, M.J., Bujnicki, J.M. et al. Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements. Nucleic Acids Res, 2020, 48: 12436-12452.

[28]

Vandelli, A., Monti, M., Milanetti, E., Armaos, A., Rupert, J., Zacco, E., Bechara, E., Delli Ponti, R. and Tartaglia, G.G. Structural analysis of SARS-CoV-2 genome and predictions of the human interactome. Nucleic Acids Res, 2020, 48: 11270-11283.

[29]

Hoffmann, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S., Schiergens, T.S., Herrler, G., Wu, N.H., Nitsche, A. et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 2020, 181: 271-280 e278.

[30]

Prandi, I.G., Mavian, C., Giombini, E., Gruber, C.E.M., Pietrucci, D., Borocci, S., Abid, N., Beccari, A.R., Talarico, C. and Chillemi, G. Structural Evolution of Delta (B. 1.617.2) and Omicron (BA. 1) Spike Glycoproteins. Int J Mol Sci, 2022: 23.

[31]

Scovino, A.M., Dahab, E.C., Vieira, G.F., Freire-de-Lima, L., Freire-de-Lima, C.G. and Morrot, A. SARS-CoV-2's Variants of Concern: A Brief Characterization. Front Immunol, 2022, 13: 834098.

[32]

Enjuanes, L., Sola, I., Zuniga, S., Honrubia, J.M., Bello-Perez, M., Sanz-Bravo, A., Gonzalez-Miranda, E., Hurtado-Tamayo, J., Requena-Platek, R., Wang, L. et al. Nature of viruses and pandemics: Coronaviruses. Curr Res Immunol, 2022, 3: 151-158.

[33]

Vatandaslar, H. A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase. Int J Mol Sci, 2022, 23.

[34]

Dai, F., Yusuf, F., Farjah, G.H. and Brand-Saberi, B. RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis. Dev Biol, 2005, 285: 80-90.

[35]

Zhang, Y., Almazi, J.G., Ong, H.X., Johansen, M.D., Ledger, S., Traini, D., Hansbro, P.M., Kelleher, A.D. and Ahlenstiel, C.L. Nanoparticle Delivery Platforms for RNAi Therapeutics Targeting COVID-19 Disease in the Respiratory Tract. Int J Mol Sci, 2022: 23.

[36]

Uludağ, H., Parent, K., Aliabadi, H.M. and Haddadi, A. Prospects for RNAi Therapy of COVID-19. Frontiers in Bioengineering and Biotechnology, 2020: 8.

[37]

Mehta, A., Michler, T. and Merkel, O.M. siRNA Therapeutics against Respiratory Viral Infections-What Have We Learned for Potential COVID-19 Therapies? Adv Healthc Mater, 2021, 10: e2001650.

Publication history
Copyright
Acknowledgements
Rights and permissions

Publication history

Received: 21 October 2022
Accepted: 07 November 2022
Published: 07 November 2022
Issue date: June 2022

Copyright

© Weiwei Zhang, Linjia Huang, Jumei Huang, Xin Jiang, Xiaohong Ren, Xiaojie Shi, Ling Ye, Shuhui Bian, Jianhe Sun, Yufeng Gao, Zehua Hu, Lintin Guo, Suyan Chen, Jiahao Xu, Jie Wu, Jiwen Zhang, Daxiang Cui, and Fangping Daii.

Acknowledgements

This research was supported by the Ministry of Science and Technology of China, Nantong Science and Technology Bureau (NT-HM-2020-09-24), the Shanghai Jingong Construction (Group) Health Fund (SHJG-2021-0402), and the Yangtze River Delta Drug Advanced Research Institute Foundation (NT-CGY202103).

Rights and permissions

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Return