Selection and Validation of siRNAs Preventing Uptake and Replication of SARS-CoV-2

In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by...

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Vydáno v:	Frontiers in bioengineering and biotechnology Ročník 10; s. 801870
Hlavní autoři:	Friedrich, Maik, Pfeifer, Gabriele, Binder, Stefanie, Aigner, Achim, Vollmer Barbosa, Philippe, Makert, Gustavo R., Fertey, Jasmin, Ulbert, Sebastian, Bodem, Jochen, König, Eva-Maria, Geiger, Nina, Schambach, Axel, Schilling, Erik, Buschmann, Tilo, Hauschildt, Sunna, Koehl, Ulrike, Sewald, Katherina
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	Switzerland Frontiers Media SA 02.03.2022 Frontiers Media S.A
Témata:	ACE2 Angiotensin-converting enzyme 2 Bioengineering and Biotechnology Cancer Cells Conserved sequence coronavirus Coronaviruses COVID-19 COVID-19 vaccines Cytoplasmic membranes Disease transmission Drug development Gene silencing Genomes Immunization mRNA Nsp1 Pandemics Penicillin Protein sorting signals Proteins Replication Respiratory tract RNA-mediated interference SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 siRNA Structural proteins therapeutic siRNA Viral infections United States > US COVID-19 ACE2 RNAi SARS-CoV-2 therapeutic siRNA Nsp1 coronavirus
ISSN:	2296-4185, 2296-4185
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Abstract	In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. ‘host shutoff factor’) as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.
AbstractList	In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. 'host shutoff factor') as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention. In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. 'host shutoff factor') as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.In 2019, the novel highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak rapidly led to a global pandemic with more than 346 million confirmed cases worldwide, resulting in 5.5 million associated deaths (January 2022). Entry of all SARS-CoV-2 variants is mediated by the cellular angisin-converting enzyme 2 (ACE2). The virus abundantly replicates in the epithelia of the upper respiratory tract. Beyond vaccines for immunization, there is an imminent need for novel treatment options in COVID-19 patients. So far, only a few drugs have found their way into the clinics, often with modest success. Specific gene silencing based on small interfering RNA (siRNA) has emerged as a promising strategy for therapeutic intervention, preventing/limiting SARS-CoV-2 entry into host cells or interfering with viral replication. Here, we pursued both strategies. We designed and screened nine siRNAs (siA1-9) targeting the viral entry receptor ACE2. SiA1, (siRNA against exon1 of ACE2 mRNA) was most efficient, with up to 90% knockdown of the ACE2 mRNA and protein for at least six days. In vitro, siA1 application was found to protect Vero E6 and Huh-7 cells from infection with SARS-CoV-2 with an up to ∼92% reduction of the viral burden indicating that the treatment targets both the endosomal and the viral entry at the cytoplasmic membrane. Since the RNA-encoded genome makes SARS-CoV-2 vulnerable to RNA interference (RNAi), we designed and analysed eight siRNAs (siV1-8) directly targeting the Orf1a/b region of the SARS-CoV-2 RNA genome, encoding for non-structural proteins (nsp). As a significant hallmark of this study, we identified siV1 (siRNA against leader protein of SARS-CoV-2), which targets the nsp1-encoding sequence (a.k.a. 'host shutoff factor') as particularly efficient. SiV1 inhibited SARS-CoV-2 replication in Vero E6 or Huh-7 cells by more than 99% or 97%, respectively. It neither led to toxic effects nor induced type I or III interferon production. Of note, sequence analyses revealed the target sequence of siV1 to be highly conserved in SARS-CoV-2 variants. Thus, our results identify the direct targeting of the viral RNA genome (ORF1a/b) by siRNAs as highly efficient and introduce siV1 as a particularly promising drug candidate for therapeutic intervention.
Author	Binder, Stefanie Sewald, Katherina Pfeifer, Gabriele Buschmann, Tilo König, Eva-Maria Koehl, Ulrike Schambach, Axel Friedrich, Maik Makert, Gustavo R. Ulbert, Sebastian Geiger, Nina Fertey, Jasmin Bodem, Jochen Schilling, Erik Hauschildt, Sunna Aigner, Achim Vollmer Barbosa, Philippe
AuthorAffiliation	1 Institute of Clinical Immunology , Faculty of Leipzig University of Leipzig , Max-Bürger-Forschungszentrum (MBFZ) , Leipzig , Germany 3 Rudolf Boehm Institute for Pharmacology and Toxicology , Clinical Pharmacology , Leipzig University , Faculty of Medicine , Leipzig , Germany 7 Boston Children’s Hospital , Harvard Medical School , Boston , MA , United States 8 Leipzig University , Institute of Biology , Leipzig , Germany 6 REBIRTH Research Center for Translational Regenerative Medicine , Hannover Medical School , Hannover , Germany 5 Institute of Virology , Julius-Maximilians-Universität Würzburg , Würzburg , Germany 9 Institute for Cellular Therapeutics , Hannover Medical School , Hannover , Germany 2 Department of Vaccines and Infection Models , Fraunhofer Institute for Cell Therapy and Immunology IZI , Leipzig , Germany 4 Institute of Experimental Hematology , Hannover Medical School , Hannover , Germany 10 Fraunhofer Institute of Toxicology and Experimental Medicine , Biomedical Research
AuthorAffiliation_xml	– name: 4 Institute of Experimental Hematology , Hannover Medical School , Hannover , Germany – name: 9 Institute for Cellular Therapeutics , Hannover Medical School , Hannover , Germany – name: 3 Rudolf Boehm Institute for Pharmacology and Toxicology , Clinical Pharmacology , Leipzig University , Faculty of Medicine , Leipzig , Germany – name: 1 Institute of Clinical Immunology , Faculty of Leipzig University of Leipzig , Max-Bürger-Forschungszentrum (MBFZ) , Leipzig , Germany – name: 10 Fraunhofer Institute of Toxicology and Experimental Medicine , Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH) of the German Center for Lung Research (DZL) , Hannover , Germany – name: 2 Department of Vaccines and Infection Models , Fraunhofer Institute for Cell Therapy and Immunology IZI , Leipzig , Germany – name: 5 Institute of Virology , Julius-Maximilians-Universität Würzburg , Würzburg , Germany – name: 6 REBIRTH Research Center for Translational Regenerative Medicine , Hannover Medical School , Hannover , Germany – name: 7 Boston Children’s Hospital , Harvard Medical School , Boston , MA , United States – name: 8 Leipzig University , Institute of Biology , Leipzig , Germany
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Copyright	Copyright © 2022 Friedrich, Pfeifer, Binder, Aigner, Vollmer Barbosa, Makert, Fertey, Ulbert, Bodem, König, Geiger, Schambach, Schilling, Buschmann, Hauschildt, Koehl and Sewald. 2022. This work is licensed under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. Copyright © 2022 Friedrich, Pfeifer, Binder, Aigner, Vollmer Barbosa, Makert, Fertey, Ulbert, Bodem, König, Geiger, Schambach, Schilling, Buschmann, Hauschildt, Koehl and Sewald. 2022 Friedrich, Pfeifer, Binder, Aigner, Vollmer Barbosa, Makert, Fertey, Ulbert, Bodem, König, Geiger, Schambach, Schilling, Buschmann, Hauschildt, Koehl and Sewald
Copyright_xml	– notice: Copyright © 2022 Friedrich, Pfeifer, Binder, Aigner, Vollmer Barbosa, Makert, Fertey, Ulbert, Bodem, König, Geiger, Schambach, Schilling, Buschmann, Hauschildt, Koehl and Sewald. – notice: 2022. This work is licensed under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. – notice: Copyright © 2022 Friedrich, Pfeifer, Binder, Aigner, Vollmer Barbosa, Makert, Fertey, Ulbert, Bodem, König, Geiger, Schambach, Schilling, Buschmann, Hauschildt, Koehl and Sewald. 2022 Friedrich, Pfeifer, Binder, Aigner, Vollmer Barbosa, Makert, Fertey, Ulbert, Bodem, König, Geiger, Schambach, Schilling, Buschmann, Hauschildt, Koehl and Sewald
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Notes	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Reviewed by: Wei-Hua Chen, Huazhong University of Science and Technology, China Edited by: Weipeng Wang, Soochow University, China Martina Anton, Klinikum rechts der Isar der TUM, Germany This article was submitted to Preclinical Cell and Gene Therapy, a section of the journal Frontiers in Bioengineering and Biotechnology Muhammad Nawaz, University of Gothenburg, Sweden
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SubjectTerms	ACE2 Angiotensin-converting enzyme 2 Bioengineering and Biotechnology Cancer Cells Conserved sequence coronavirus Coronaviruses COVID-19 COVID-19 vaccines Cytoplasmic membranes Disease transmission Drug development Gene silencing Genomes Immunization mRNA Nsp1 Pandemics Penicillin Protein sorting signals Proteins Replication Respiratory tract RNA-mediated interference SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 siRNA Structural proteins therapeutic siRNA Viral infections
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