Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity

Longlong Si; Haiqing Bai; Crystal Yuri Oh; Amanda Jiang; Fan Hong; Tian Zhang; Yongxin Ye; Tristan X. Jordan; James Logue; Marisa McGrath; Chaitra Belgur; Karina Calderon; Atiq Nurani; Wuji Cao; Kenneth E. Carlson; Rachelle Prantil-Baun; Steven P. Gygi; Dong Yang; Colleen B. Jonsson; Benjamin R. tenOever; Matthew Frieman; Donald E. Ingber

doi:10.1016/j.omtn.2022.08.031

Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity

Longlong Si, Haiqing Bai, Crystal Yuri Oh, Amanda Jiang, Fan Hong, Tian Zhang, Yongxin Ye, Tristan X. Jordan, James Logue, Marisa McGrath, Chaitra Belgur, Karina Calderon, Atiq Nurani, Wuji Cao, Kenneth E. Carlson, Rachelle Prantil-Baun, Steven P. Gygi, Dong Yang, Colleen B. Jonsson, Benjamin R. tenOeverMatthew Frieman, Donald E. Ingber

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5′-C; antisense strand, 3′-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.

Original language	English
Pages (from-to)	923-940
Number of pages	18
Journal	Molecular Therapy - Nucleic Acids
Volume	29
DOIs	https://doi.org/10.1016/j.omtn.2022.08.031
State	Published - 13 Sep 2022

Keywords

Hoogsteen G-G base pairing
MT: Oligonucleotides
RIG-I
SARS-CoV-2
Therapies and Applications
antiviral therapeutic
immunostimulatory RNA
influenza
interferon
organ on a chip

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10.1016/j.omtn.2022.08.031

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Si, L., Bai, H., Oh, C. Y., Jiang, A., Hong, F., Zhang, T., Ye, Y., Jordan, T. X., Logue, J., McGrath, M., Belgur, C., Calderon, K., Nurani, A., Cao, W., Carlson, K. E., Prantil-Baun, R., Gygi, S. P., Yang, D., Jonsson, C. B., ... Ingber, D. E. (2022). Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity. Molecular Therapy - Nucleic Acids, 29, 923-940. https://doi.org/10.1016/j.omtn.2022.08.031

@article{c04ecbeaba6f4f9f85c0d0bd690f3ce9,

title = "Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity",

abstract = "The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5′-C; antisense strand, 3′-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.",

keywords = "Hoogsteen G-G base pairing, MT: Oligonucleotides, RIG-I, SARS-CoV-2, Therapies and Applications, antiviral therapeutic, immunostimulatory RNA, influenza, interferon, organ on a chip",

author = "Longlong Si and Haiqing Bai and Oh, {Crystal Yuri} and Amanda Jiang and Fan Hong and Tian Zhang and Yongxin Ye and Jordan, {Tristan X.} and James Logue and Marisa McGrath and Chaitra Belgur and Karina Calderon and Atiq Nurani and Wuji Cao and Carlson, {Kenneth E.} and Rachelle Prantil-Baun and Gygi, {Steven P.} and Dong Yang and Jonsson, {Colleen B.} and tenOever, {Benjamin R.} and Matthew Frieman and Ingber, {Donald E.}",

note = "Funding Information: We thank IDT Inc. for synthesizing the RNA oligonucleotides, X. Song and H. Queen at Creative-Biolabs Inc. for carrying SPR experiments, and Genewiz Inc. for carrying out the RNA-seq work. We thank the animal care staff at the University of Tennessee Health Science Center, Regional Biocontainment Laboratory for their support of these studies. This work was supported by the NIH (NCATS UH3-HL-141797 to D.E.I.) and the Defense Advanced Research Projects Agency under Cooperative Agreements (HR00111920008 and HR0011-20-2-0040 to D.E.I.). L.S. H.B. and D.E.I. conceived this study. L.S. and H.B. conducted in vitro experiments and analyzed data with assistance from C.O. A.J. C.B. W.C. K.C. and R.P. T.Z. and S.P.G. performed TMT mass spectrometry and data analysis. F.H. performed the native gel electrophoresis experiments. Y.Y. performed the analysis of CCC sequence distribution in human mRNAs and lncRNAs. T.J. J.L. M.M. M.F. and B.R.t. performed the experiments on SARS-CoV-2, SARS-CoV, and MERS-CoV viruses. A.N. performed western blotting experiments. R.P.-B. assisted in the propagation and characterization of HCoV-NL63 virus. K.E.C. and C.B.J. designed and coordinated experiments to test RNA-1 activities in K18-ACE2 mouse models, which were carried out by D.Y. The manuscript was written by L.S. H.B. and D.E.I. with input from other authors. D.E.I. is a founder, board member, SAB chair, and equity holder in Emulate Inc. D.E.I. L.S. H.B. C.O. and R.P. are inventors on relevant patent applications held by Harvard University. Funding Information: We thank IDT Inc. for synthesizing the RNA oligonucleotides, X. Song and H. Queen at Creative-Biolabs Inc. for carrying SPR experiments, and Genewiz Inc. for carrying out the RNA-seq work. We thank the animal care staff at the University of Tennessee Health Science Center, Regional Biocontainment Laboratory for their support of these studies. This work was supported by the NIH (NCATS UH3-HL-141797 to D.E.I.) and the Defense Advanced Research Projects Agency under Cooperative Agreements ( HR00111920008 and HR0011-20-2-0040 to D.E.I.). Publisher Copyright: {\textcopyright} 2022 The Author(s)",

year = "2022",

month = sep,

day = "13",

doi = "10.1016/j.omtn.2022.08.031",

language = "English",

volume = "29",

pages = "923--940",

journal = "Molecular Therapy - Nucleic Acids",

issn = "2162-2531",

publisher = "Cell Press",

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Si, L, Bai, H, Oh, CY, Jiang, A, Hong, F, Zhang, T, Ye, Y, Jordan, TX, Logue, J, McGrath, M, Belgur, C, Calderon, K, Nurani, A, Cao, W, Carlson, KE, Prantil-Baun, R, Gygi, SP, Yang, D, Jonsson, CB, tenOever, BR, Frieman, M & Ingber, DE 2022, 'Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity', Molecular Therapy - Nucleic Acids, vol. 29, pp. 923-940. https://doi.org/10.1016/j.omtn.2022.08.031

TY - JOUR

T1 - Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity

AU - Si, Longlong

AU - Bai, Haiqing

AU - Oh, Crystal Yuri

AU - Jiang, Amanda

AU - Hong, Fan

AU - Zhang, Tian

AU - Ye, Yongxin

AU - Jordan, Tristan X.

AU - Logue, James

AU - McGrath, Marisa

AU - Belgur, Chaitra

AU - Calderon, Karina

AU - Nurani, Atiq

AU - Cao, Wuji

AU - Carlson, Kenneth E.

AU - Prantil-Baun, Rachelle

AU - Gygi, Steven P.

AU - Yang, Dong

AU - Jonsson, Colleen B.

AU - tenOever, Benjamin R.

AU - Frieman, Matthew

AU - Ingber, Donald E.

N1 - Funding Information: We thank IDT Inc. for synthesizing the RNA oligonucleotides, X. Song and H. Queen at Creative-Biolabs Inc. for carrying SPR experiments, and Genewiz Inc. for carrying out the RNA-seq work. We thank the animal care staff at the University of Tennessee Health Science Center, Regional Biocontainment Laboratory for their support of these studies. This work was supported by the NIH (NCATS UH3-HL-141797 to D.E.I.) and the Defense Advanced Research Projects Agency under Cooperative Agreements (HR00111920008 and HR0011-20-2-0040 to D.E.I.). L.S. H.B. and D.E.I. conceived this study. L.S. and H.B. conducted in vitro experiments and analyzed data with assistance from C.O. A.J. C.B. W.C. K.C. and R.P. T.Z. and S.P.G. performed TMT mass spectrometry and data analysis. F.H. performed the native gel electrophoresis experiments. Y.Y. performed the analysis of CCC sequence distribution in human mRNAs and lncRNAs. T.J. J.L. M.M. M.F. and B.R.t. performed the experiments on SARS-CoV-2, SARS-CoV, and MERS-CoV viruses. A.N. performed western blotting experiments. R.P.-B. assisted in the propagation and characterization of HCoV-NL63 virus. K.E.C. and C.B.J. designed and coordinated experiments to test RNA-1 activities in K18-ACE2 mouse models, which were carried out by D.Y. The manuscript was written by L.S. H.B. and D.E.I. with input from other authors. D.E.I. is a founder, board member, SAB chair, and equity holder in Emulate Inc. D.E.I. L.S. H.B. C.O. and R.P. are inventors on relevant patent applications held by Harvard University. Funding Information: We thank IDT Inc. for synthesizing the RNA oligonucleotides, X. Song and H. Queen at Creative-Biolabs Inc. for carrying SPR experiments, and Genewiz Inc. for carrying out the RNA-seq work. We thank the animal care staff at the University of Tennessee Health Science Center, Regional Biocontainment Laboratory for their support of these studies. This work was supported by the NIH (NCATS UH3-HL-141797 to D.E.I.) and the Defense Advanced Research Projects Agency under Cooperative Agreements ( HR00111920008 and HR0011-20-2-0040 to D.E.I.). Publisher Copyright: © 2022 The Author(s)

PY - 2022/9/13

Y1 - 2022/9/13

N2 - The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5′-C; antisense strand, 3′-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.

AB - The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5′-C; antisense strand, 3′-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.

KW - Hoogsteen G-G base pairing

KW - MT: Oligonucleotides

KW - RIG-I

KW - SARS-CoV-2

KW - Therapies and Applications

KW - antiviral therapeutic

KW - immunostimulatory RNA

KW - influenza

KW - interferon

KW - organ on a chip

UR - http://www.scopus.com/inward/record.url?scp=85137409393&partnerID=8YFLogxK

U2 - 10.1016/j.omtn.2022.08.031

DO - 10.1016/j.omtn.2022.08.031

M3 - Article

AN - SCOPUS:85137409393

SN - 2162-2531

VL - 29

SP - 923

EP - 940

JO - Molecular Therapy - Nucleic Acids

JF - Molecular Therapy - Nucleic Acids

ER -

Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity

Abstract

Keywords

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