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WO2022023734A1 - Signaux d'emballage coronaviraux - Google Patents

Signaux d'emballage coronaviraux Download PDF

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Publication number
WO2022023734A1
WO2022023734A1 PCT/GB2021/051934 GB2021051934W WO2022023734A1 WO 2022023734 A1 WO2022023734 A1 WO 2022023734A1 GB 2021051934 W GB2021051934 W GB 2021051934W WO 2022023734 A1 WO2022023734 A1 WO 2022023734A1
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vlp
urgu
urru
motif
urau
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Sam CLARK
Richard John BINGHAM
Reidun TWAROCK
Peter George Stockley
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University of Leeds
University of Leeds Innovations Ltd
University of York
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University of Leeds
University of Leeds Innovations Ltd
University of York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to virus like particles, vaccines and delivery vectors utilising coronavirus packaging signals, and therapeutic agents targeting such packaging signals.
  • Background of the Invention Since the turn of the century, we have experienced outbreaks of coronaviral infections every eight to ten years with increasingly severe impacts on global health and economy. After severe acute respiratory syndrome coronavirus (SARS-CoV-1) in 2002, and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 1 , we are currently facing the COVID- 19 (SARS-CoV-2) pandemic. In future, additional pathogens including more deadly variants may evolve 2 .
  • Coronavirus disease 2019 (Covid-19) is caused by severe, acute respiratory syndrome coronavirus (SARS-CoV-2), a recently emerging lineage B betacoronavirus (Sarbecovirus).
  • the viral particle ( ⁇ 100 nm dia. 4 ) is bounded by a membrane envelope studded with multiple copies of three types of glycoproteins: the envelope (E), membrane (M), and spike (S) proteins.
  • E envelope
  • M membrane
  • S spike
  • the latter binds the ACE-2 receptor on human cells 5 , a vital step during cell entry. It is therefore the principal antigen in vaccine design 6,7 , and is under investigation as a potential target for anti-viral therapy 8 .
  • its sequence is known to be mutating within the pandemic strains 9 , making the production of a broadly neutralising vaccine more problematic.
  • PSs packaging signals
  • Lineage B Lineage B coronavirus
  • Lineage C Vector C
  • PSs Lineage B coronavirus
  • PSs Lineage C coronavirus
  • These PSs include new motifs that differ from PS motifs previously identified for coronaviruses.
  • the new motifs are conserved across coronaviruses and the inventors have also identified new motifs and PSs in other coronaviruses, including multiple alphacoronaviruses and betacoronaviruses.
  • VLP virus like particles
  • the identification of these packaging signals is significant, because it allows the production of virus like particles (VLP) with nucleic acid sequence comprising the PSs.
  • the presence of the PSs will cause the VLPs to assemble more efficiently, to be more stable, and to present antigens in a native formation. Therefore, the VLPs will be particularly useful in vaccine compositions and as gene delivery vectors.
  • the packaging signals present novel drug targets that are less likely to elicit escape mutants.
  • Targeting PSs is an established modality for treating viral infections and inhibiting replication of viruses (WO2019/241631). Therefore, the new PSs identified by the inventors provide new methods and agents for treating coronavirus infections, and new methods for identifying agents for treating coronavirus infections.
  • the invention provides a virus like particle (VLP) comprising a nucleic acid sequence that includes a packaging signal, wherein the VLP is a coronavirus VLP and the packaging signal comprises the motif: a) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), b) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), c) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3), or d) 5'-URAU-(N) 8 -UGAU-3' (SEQ ID NO: 4), or wherein the VLP is a Lineage B (Sarbecovirus) coronavirus or Lineage C (Merbecovirus) coronavirus VLP and the packaging signal comprises the motif 5'-URRU- (N) 8 -URRU-3' (SEQ ID NO: 5), wherein R represents a purine, and wherein the VLP is replication-incompetent
  • VLP virus like particle
  • the invention also provides a virus like particle (VLP) comprising a nucleic acid sequence that includes a packaging signal, wherein the VLP is a coronavirus VLP and the packaging signal comprises the motif a) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), b) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), c) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3), or d) 5'-URAU-(N) 8 -UGAU-3' (SEQ ID NO: 4), or wherein the VLP is a Lineage B (Sarbecovirus) betacoronavirus or Lineage C (Merbecovirus) betacoronavirus VLP and the packaging signal comprises the motif 5'- URRU-(N
  • the packaging signal comprises two copies of said motif separated by 8-11 nucleotides, as identified in the Examples. Such packaging signals are likely to be dominant, of higher affinity, and more effective for improving packaging efficiency and stability.
  • the nucleic acid sequence comprises multiple of said packaging signals, such as 2, 3, 4, 5, 10, 20, or 30. The Examples demonstrate that multiple packaging signals are present in coronaviruses and involved in packaging, so multiple packaging signals may further improve packaging efficiency and stability.
  • the VLP is a Lineage B (Sarbecovirus) coronavirus or Lineage C (Merbecovirus) coronavirus VLP
  • the packaging signal comprises the motif: a) 5'-URAU-(N) 8 -URAU-3' (SEQ ID NO: 6), b) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), c) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), or d) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3).
  • the VLP is a Lineage B (Sarbecovirus) coronavirus or Lineage C (Merbecovirus) coronavirus VLP
  • the packaging signal comprises the motif: a) 5'-UAAU-(N) 8 -UAAU-3' (SEQ ID NO: 7), b) 5'-UGAU-(N) 8 -UAAU-3' (SEQ ID NO: 8), c) 5'-UAAU-(N) 8 -UGAU-3' (SEQ ID NO: 9), d) 5'-UGAU-(N) 8 -UGAU-3' (SEQ ID NO: 10), e) 5'-UAGU-(N) 8 -UAAU-3' (SEQ ID NO: 11), f) 5'-UGGU-(N) 8 -UAAU-3' (SEQ ID NO: 12), g) 5'-UAGU-(N) 8 -UGAU-3' (SEQ ID NO: 13), h) 5'-UGGU-(N) 8 -
  • the VLP is a coronavirus VLP
  • the packaging signal comprises the motif: a) 5'-UAGU-(N) 8 -UAAU-3' (SEQ ID NO: 11), b) 5'-UGGU-(N) 8 -UAAU-3' (SEQ ID NO: 12), c) 5'-UAGU-(N) 8 -UGAU-3' (SEQ ID NO: 13), d) 5'-UGGU-(N) 8 -UGAU-3' (SEQ ID NO: 14), e) 5'-UAAU-(N) 8 -UAGU-3' (SEQ ID NO: 15), f) 5'-UGAU-(N) 8 -UAGU-3' (SEQ ID NO: 16), g) 5'-UAAU-(N) 8 -UGGU-3' (SEQ ID NO: 17), h) 5'-UGAU-(N) 8 -UGGU-3' (SEQ ID NO: 18), i) 5'-UAGU-(N) 8
  • the packaging signal, or the nucleic acid forms a secondary structure shown in Figure 4.
  • the coronavirus VLP is of a coronavirus that infects humans.
  • the coronavirus VLP is a betacoronavirus VLP, such as of HCoV- OC43 or HCoV-HKU1.
  • the coronavirus VLP is a alphacoronavirus VLP, such as of HCoV-NL63 or HCoV-229E.
  • the Lineage B (Sarbecovirus) betacoronavirus VLP is a SARS-CoV VLP, such as a SARS-CoV-1 or SARS-CoV-2 VLP.
  • the Lineage C (Merbecovirus) betacoronavirus VLP is a MERS-CoV VLP.
  • the packaging signal comprises a sequence selected from Tables 1 and 3-8, with 0, 1, 2, 3, or 4 nucleotide substitutions, additions or deletions in the sequence outside the URRU (SEQ ID NO: 27) submotif.
  • the packaging signal comprises a sequence selected from those in Table 1 marked with an asterisk, with 0, 1, 2, 3 or 4 nucleotide substitutions, additions or deletions in the sequence outside the URRU (SEQ ID NO: 27) submotif.
  • the packaging signal comprises a sequence marked as a nested or double motif, with 0, 1, 2, 3 or 4 nucleotide substitutions, additions or deletions in the sequence outside the URRU (SEQ ID NO: 27) submotif.
  • the Examples demonstrate that variation in the sequence between the submotifs is permitted. Double and nested motifs may be dominant, of higher affinity, and more effective for improving packaging efficiency and stability.
  • the nucleic acid sequence comprises a sequence selected from the sequences in Figures 6-8, or a sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity to a sequence in Figures 6-8.
  • the nucleic acid consists of a sequence selected from the sequences in Figures 6-8, or a sequence with at least 80%, 85%, 90%, 95% or 99% sequence identity to a sequence in Figures 6-8.
  • the sequences adopt the structures displayed in Figures 9-11.
  • the nucleic acid sequence is a non-replicating nucleic acid. Such VLPs are useful in vaccine and gene therapy applications.
  • the nucleic acid sequence does not comprise a complete spike (S) gene, does not comprise a complete envelope (E) gene or does not comprise a complete membrane (M) gene, or does not comprise any of a complete envelope (E) gene, a complete spike (S) gene or a complete membrane (M) gene.
  • VLPs are useful in vaccine and gene therapy applications.
  • the nucleic acid sequence encodes a nucleocaspid gene.
  • the VLP comprises nucleocapsid protein bound to the nucleic acid sequence.
  • the VLP comprises multiple nucleocapsid proteins bound to the nucleic acid sequence. The Examples demonstrate that the nucleocapsid protein may aid in packaging.
  • the nucleic acid sequence does not encode any polypeptide sequence. In certain embodiments, at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the nucleic acid sequence consists of the packaging signal motif. In certain embodiments, the nucleic acid sequence does not comprise any coronavirus sequence except for the packaging signal, or except for the packaging signal motif. In certain embodiments, the nucleic acid sequence comprises one, two, three or all of the spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes.
  • the VLP comprises or consists of membrane (M) and nucleocapsid (N) proteins; membrane (M) and envelope (E) proteins; membrane (M), nucleocapsid (N) and envelope (E) proteins; membrane (M), envelope (E) and spike (S) proteins, or membrane (M), nucleocapsid (N), envelope (E) and spike (S) proteins.
  • the nucleic acid sequence also comprises sequence encoding a non-viral therapeutic polypeptide, antisense oligonucleotide or siRNA. The VLPs of the invention will be useful as delivery vectors for such sequences.
  • the packaging signal comprises a sequence selected from those in Table 9 with a positive RR1 & RR2 average figure in the final column (highlighted in bold), or such a sequence comprising 0, 1, 2, 3, or 4 nucleotide substitutions, additions or deletions in the sequence outside the URRU (SEQ ID NO: 27) submotif.
  • the Examples demonstrate that these sites are more protected in the particle.
  • the VLP is a variant selected from Table 10. In certain embodiments, at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the nucleic acid sequence is not derived from a coronavirus.
  • the nucleic acid sequence comprises multiple of said packaging signals, such as 2, 3, 4, 5, 10, 20, or 30, separated by sequence that is not derived from a coronavirus.
  • the packaging signal or each packaging signal is less than 200 nucleotides in length, such as less than 150, 100, 80, 60, 50 or 45 nucleotides in length, such as 43, 42, 41, 40 or 16 nucleotides in length.
  • the nucleic acid sequence comprises multiple of said packaging signals, optionally separated by sequence that is not derived from a coronavirus.
  • the invention provides a vaccine composition comprising any VLP described above.
  • the vaccine composition is for use in immunising a patient against coronavirus infection.
  • the invention also provides a method of immunising a patient against coronavirus infection comprising administering a composition comprising any VLP described above.
  • the coronavirus infection is a betacoronavirus, such as HCoV-OC43 or HCoV-HKU1 or an alphacoronavirus, such as HCoV-NL63 or HCoV-229E.
  • the coronavirus infection is a Lineage C (Merbecovirus) betacoronavirus, such as MERS CoV.
  • the coronavirus infection is a Lineage B (Sarbecovirus) betacoronavirus, such as SARS-CoV, such as SARS- CoV-1 or SARS-CoV-2.
  • the invention provides a gene therapy composition comprising any VLP described above.
  • the invention also provides a method of delivering a nucleic acid to a cell, comprising contacting the cell with any VLP described above.
  • the invention also provides a gene therapy composition comprising any VLP described above for use in treating a disease.
  • the invention also provides a method of treating a disease comprising administering a gene therapy composition comprising any VLP described above
  • the invention provides a method of treating coronavirus infection comprising administering an agent that binds a nucleic acid packaging signal comprising the motif: a) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), b) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), c) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3), or d) 5'-URAU-(N) 8 -UGAU-3' (SEQ ID NO: 4).
  • the invention provides a method of treating Lineage B (Sarbecovirus) betacoronavirus or Lineage C (Merbecovirus) betacoronavirus infection comprising administering an agent that binds a nucleic acid packaging signal comprising the motif 5'-URRU-(N) 8 -URRU-3' (SEQ ID NO: 5).
  • the invention provides an agent for use in treating coronavirus infection, wherein the agent binds a nucleic acid packaging signal comprising the motif: a) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), b) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), c) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3), or d) 5'-URAU-(N) 8 -UGAU-3' (SEQ ID NO: 4).
  • a nucleic acid packaging signal comprising the motif: a) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), b) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), c) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3), or d) 5'-URAU-(N) 8
  • the invention provides an agent for use in treating Lineage B (Sarbecovirus) betacoronavirus or Lineage C (Merbecovirus) betacoronavirus infection, wherein the agent binds a nucleic acid packaging signal comprising the motif 5'-URRU-(N) 8 - URRU-3' (SEQ ID NO: 5).
  • the invention provides a method for identifying a candidate therapeutic agent for treating coronavirus infection comprising measuring the binding of an agent to a nucleic acid sequence comprising the motif: a) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), b) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), c) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3), or d) 5'-URAU-(N) 8 -UGAU-3' (SEQ ID NO: 4).
  • the invention provides a method for identifying a candidate therapeutic agent for treating Lineage B (Sarbecovirus) betacoronavirus or Lineage C (Merbecovirus) betacoronavirus infection comprising measuring the binding of an agent to a nucleic acid sequence comprising the motif 5'-URRU-(N) 8 -URRU-3' (SEQ ID NO: 5).
  • the invention provides a method of preparing a therapeutic composition comprising identifying a candidate therapeutic agent according to the methods above, and formulating the agent with pharmaceutically acceptable excipients. The method or agent of any of the preceding paragraphs, wherein the agent is a peptide, an RNA molecule, a DNA molecule or a low molecular weight organic compound.
  • the methods and agents for treating coronavirus infection treat a betacoronavirus, such as HCoV-OC43 or HCoV-HKU1 or an alphacoronavirus, such as HCoV-NL63 or HCoV-229E.
  • a betacoronavirus such as HCoV-OC43 or HCoV-HKU1
  • an alphacoronavirus such as HCoV-NL63 or HCoV-229E.
  • the coronavirus infection is a Lineage C (Merbecovirus) betacoronavirus, such as MERS CoV.
  • the coronavirus infection is a Lineage B (Sarbecovirus) betacoronavirus, such as SARS-CoV, such as SARS-CoV-1 or SARS-CoV-2.
  • the methods and agents are for treating a Lineage B (Sarbecovirus) coronavirus or Lineage C (Merbecovirus) coronavirus, and bind the motif: a) 5'-URAU-(N) 8 -URAU-3' (SEQ ID NO: 6), b) 5'-URGU-(N) 8 -URAU-3' (SEQ ID NO: 1), c) 5'-URAU-(N) 8 -URGU-3' (SEQ ID NO: 2), or d) 5'-URGU-(N) 8 -URGU-3' (SEQ ID NO: 3).
  • the methods and agents are for treating a Lineage B (Sarbecovirus) coronavirus or Lineage C (Merbecovirus) coronavirus VLP, and bind the motif: a) 5'-UAAU-(N) 8 -UAAU-3' (SEQ ID NO: 7), b) 5'-UGAU-(N) 8 -UAAU-3' (SEQ ID NO: 8), c) 5'-UAAU-(N) 8 -UGAU-3' (SEQ ID NO: 9), d) 5'-UGAU-(N) 8 -UGAU-3' (SEQ ID NO: 10), e) 5'-UAGU-(N) 8 -UAAU-3' (SEQ ID NO: 11), f) 5'-UGGU-(N) 8 -UAAU-3' (SEQ ID NO: 12), g) 5'-UAGU-(N) 8 -UGAU-3' (SEQ ID NO: 13), h) 5'-UGGU-(
  • the methods and agents are for treating coronavirus infection, and agent binds the motif: a) 5'-UAGU-(N) 8 -UAAU-3' (SEQ ID NO: 11), b) 5'-UGGU-(N) 8 -UAAU-3' (SEQ ID NO: 12), c) 5'-UAGU-(N) 8 -UGAU-3' (SEQ ID NO: 13), d) 5'-UGGU-(N) 8 -UGAU-3' (SEQ ID NO: 14), e) 5'-UAAU-(N) 8 -UAGU-3' (SEQ ID NO: 15), f) 5'-UGAU-(N) 8 -UAGU-3' (SEQ ID NO: 16), g) 5'-UAAU-(N) 8 -UGGU-3' (SEQ ID NO: 17), h) 5'-UGAU-(N) 8 -UGGU-3' (SEQ ID NO: 18), i) 5'-UAGU-(
  • RNA Packaging Signals Motifs in Coronaviruses RNA Packaging Signals Motifs in Coronaviruses.
  • a Previously determined secondary structures for the dominant PSs in HKU1 12 , OC43 12 , MHV 13 , and BCV 14 (SEQ ID NO: 629-632), each encompassing two copies of an 5'- URAU-(N) 8 -URAU-3' (SEQ ID NO: 6) motif along the 3' stem of a SL.
  • the -URRU- (SEQ ID NO: 27) submotifs are boxed, and nucleotides A, C, G, and U are indicated.
  • Fig. 2A Multiple Dispersed Packaging Signals in Human Coronaviruses.
  • Blue label at a filled dot indicates a site where two motifs combine into a nested motif with three 5'-URAU- 3' (SEQ ID NO: 25) submotifs.
  • Green label at a filled dot marks a site where a single copy of the 5'-URAU-(N) 8 -URAU-3' (SEQ ID NO: 6) motif can be extended into a double motif, provided that the search is extended to 5'-URRU-(N) 8 -URRU-3' (SEQ ID NO: 5).
  • the extended search motif reveals 28 additional sites in SARS-CoV-2, including a further nested site (filled dot with blue label) in the S gene.
  • Figure 2B Weblogo for Figure 2A
  • the weblogo indicating bias for specific nucleotide types, shows no preference for the 8nt spacer region between the submotifs; this is also the case for the extended search motif in the weblogos for, respectively from top to bottom, SARS-CoV-2, SARS-CoV-1, and MERS-CoV.
  • Figure 3 Packaging signal-mediated assembly in coronavirus. a, Cryo-EM and biochemical/biophysical assays reveal that the N protein self- associates, creating an octameric nucleocapsid complex (light & dark, adapted from Ref. 23), at least in vitro, that in turn self-assembles to generate several grooves lined with positively charged side chains.
  • Each repeat unit of 16 N proteins thus accommodates 424 nts for strands in both orientations.
  • the reportedly ⁇ 1000 N proteins in a coronavirus particle 4 could potentially form ⁇ 62.5 such units, and thus bind ⁇ 26,500 nucleotides, i.e. encompass the vast majority of the gRNA.
  • b Two alternative scenarios explaining how PSs may contact membrane protein M (maroon/balloon shape), thus organising several helical segments as in A inside the particle in a chromatin-like conformation.
  • the on average 34 PS-type sites across the SARS- CoV-2, SARS-CoV-1 and MERS-CoV genomes would be consistent with the reported ⁇ 1000 N proteins organising into 31 helical segments, each formed from, on average, four octamers as in a and spanning ⁇ 28 nm.
  • A-N Secondary structures of putative PSs in SARS-CoV-2. For each of the 15 matches of the 5'-URAU-(N) 8 -URAU-3' (SEQ ID NO: 6) to the SARS-CoV-2 gRNA in Fig. 2a, all genome fragments of length between 100 and160 nts that fully contain the motif were extracted (SEQ ID NO: 633-646). The secondary structure of each fragment was determined via Mfold, using the suboptimality 500% setting and limiting the number of folds to a maximum of 150.
  • the resulting secondary structures were then filtered to identify all folds presenting the motif in the 3' leg of a structure, such that at least one of the Rs in each URRU (SEQ ID NO: 27) submotif is single-stranded, i.e. is located in a 3' bulge.
  • the SL with the lowest free energy of formation is shown.
  • the search motif is indicated by boxes, and the nucleotide position of the first U indicated.
  • Fig. 5 Positions of the 5'-URRU-(N) 8 -URRU-3' search motif in Human Cold Coronaviruses.
  • VLPs comprising virus like particles
  • Virus-like particles comprise multiple capsid proteins that mimic the conformation of native viruses but generally lack the viral DNA or RNA and thus are unable to replicate in a host cell. The use of VLPs as a tool for the production of safe and efficient vaccines has been recognised and some VPL-based vaccines against human papilloma virus have been developed.
  • US8062642 discloses the production of papillomavirus capsid proteins and VLPs with antigenic characteristics similar to those of native infectious virus.
  • WO9913056 discloses methods of disassembly of papilloma VLPs.
  • the present disclosure relates to the formation of VLPs using nucleic acid packaging signals derived from coronaviruses and the design of nucleic acid sequences comprising packaging signals that provide a substrate for artificial VLP assembly and the use of artificial VLPs as vaccines and in the delivery of agents to cells, for example therapeutic or diagnostic agents.
  • the identification of coronavirus packaging signals allows the production of artificial, efficient RNA substrates for the efficient assembly of VLPs. The latter have similar properties to the natural virions formed by viruses.
  • VLP capsids retain the native immunological properties of those viruses as well as their cell tropism. They also retain many of the stability and mechanical properties of the original virus particle. VLPs have utility in a wide range of applications in relation to the cell specific delivery of agents and as safe, attenuated vaccines and vectors for targeted delivery of drugs and in gene therapy.
  • said virus like particle is immunogenic when administered to a subject.
  • said virus like particle provokes an immune response similar to an immune response to the cognate native virus.
  • said immune response is induction of an antibody response wherein said antibody response induces antibodies that specifically bind native virus particles.
  • a vaccine or immunogenic composition comprising a virus like particle according to the invention.
  • said vaccine or immunogenic composition further comprises an adjuvant and/or carrier.
  • Adjuvants immunomodulators
  • adjuvants have been used for decades to improve the immune response to vaccine antigens.
  • the incorporation of adjuvants into vaccine formulations is aimed at enhancing, accelerating and prolonging the specific immune response to vaccine antigens. Aluminium hydroxide and aluminium or calcium phosphate have been used routinely in human vaccines.
  • antigens incorporated into IRIV's immunosensing reconstituted influenza virosomes
  • vaccines containing the emulsion-based adjuvant MF59 have been licensed in countries.
  • the most commonly described adjuvant classes are gel-type, microbial, oil-emulsion and emulsifier-based, particulate, synthetic and cytokines. More than one adjuvant may be present in the final vaccine product. They may be combined together with a single antigen or all antigens present in the vaccine, or each adjuvant may be combined with one particular antigen.
  • Aluminium based adjuvants consist of simple inorganic compounds, PLG is a polymeric carbohydrate, virosomes can be derived from disparate viral particles, MDP is derived from bacterial cell walls; saponins are of plant origin, squalene is derived from shark liver and recombinant endogenous immunomodulators are derived from recombinant bacterial, yeast or mammalian cells.
  • adjuvants licensed for veterinary vaccines, such as mineral oil emulsions that are too reactive for human use.
  • complete Freund's adjuvant although being one of the most powerful adjuvants known, is not suitable for human use.
  • the nucleic acid sequence further comprises a transcription cassette comprising a nucleic acid molecule adapted to transcribe a nucleic acid encoding a polypeptide or a functional RNA.
  • said adaptation is the provision of a promoter sequence and termination sequence to enable expression of said nucleic acid molecule encoding said polypeptide or functional RNA.
  • said polypeptide is a therapeutic polypeptide, for example an antibody or antibody fragment.
  • Antibody fragments include nucleic acids encoding single chain antibody fragments.
  • said functional nucleic acid is an mRNA encoding a therapeutic polypeptide, an antisense oligonucleotide or a siRNA.
  • a technique to specifically ablate gene function which has broad acceptance is through the introduction of double-stranded RNA, also referred to as small inhibitory or interfering RNA (siRNA), into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA molecule.
  • the siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double-stranded RNA molecule.
  • the siRNA molecule is typically derived from exons of the gene which is to be ablated.
  • siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.
  • the coronavirus packaging signals identified in the present invention may be targeted to treat coronaviral infections and the invention provides agents targeting the packaging signals and methods for identifying therapeutic agents targeting the packaging signals. Any appropriate method for identifying agents targeting the packaging signals may be used.
  • Ligands targeting HBV packaging signals were successfully identified in WO2019/241631 and a similar approach may be used to develop agents targeting coronavirus packaging signals and effective for inhibiting replication of coronavirus.
  • a small molecule microarray library may be used to screen compounds which target the PSs of the invention.
  • SNR signal-to-noise ratio
  • antiviral agents already in use and libraries of agents that have been approved for clinical use.
  • the drugs may include agents used the for the treatment of, amongst other diseases, cancer, dementia, cardiovascular disorders, skin infections.
  • Suitable libraries for screening may include compounds having primary or secondary amines or primary alcohol functionalities (for covalent linkage to a microarray slide), purchased from (i) commercial vendors, (ii) the National Center for Advancing Translational Sciences Mechanism Interrogation PlatE library (a collection of FDA approved drugs, clinical candidates and well-annotated inhibitors, see Mathews Griner, et al., Proc. Natl. Acad. Sci.
  • the binding affinities of the compounds are calculated with the use of physics-based equations that quantify the interactions between the drug and its target.
  • the top-ranked compounds are then tested experimentally to see if they do indeed bind and have the required downstream effects (such as stopping viral infectivity) on cells and in animal models.
  • targets may be validated in further assays, that may involve screening on appropriate monolayer cell lines.
  • ALI 3D air liquid interface
  • a multistep approach may be used to identify compounds. The approach may involve statistical analysis, inspection of pharmacophore properties, selectivity, commercial availability and cell-based evaluation of top compounds.
  • a composite Z- score may be calculated based on increased fluorescence at that location on the array in the presence of a PS.
  • Compounds with a Z-score >3 may be further investigated. Visual inspection of array fluorescence signals and elimination of false positive compounds (e.g., dust particulates) may be used to identify compounds representing the most selective binders. Promiscuous binders may be excluded, defined as hits binding multiple RNAs in the library, such as more than three RNAs.
  • Agents useful for treating coronavirus infection may include small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, RNA interference, antisense, nucleic acid aptamers, etc.
  • small molecules which can inhibit interactions, between the PSs of the invention and viral proteins.
  • the small molecules encompassed by the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. (Lam, K. S. (1997) Anticancer Drug Des. 12: 145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and supplementary therapeutic agents.
  • the compositions of the invention can be administered by any conventional route, including injection. The administration may, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal or trans-epithelial.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount” is that amount of an agent that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of an agent according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the doses of agent administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment.
  • compositions may be employed to the extent that patient tolerance permits.
  • doses of nucleic acid therapeutics such as siRNA and antisense RNA are between 1nM - 1mM.
  • doses can range from 1nM-500nM, 5nM-200nM, and 10nM-100nM.
  • Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing.
  • the administration of compositions to mammals other than humans, e.g.
  • a subject is a mammal, preferably a human, and including a non- human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
  • compositions of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents (e.g. those typically used in the treatment of the specific disease indication).
  • the salts When used in medicine, the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically- acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • compositions containing agents according to the invention may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients.
  • Compositions containing agents according to the invention may be administered as aerosols and inhaled.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of agent, which is preferably isotonic with the blood of the recipient.
  • This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-Butanediol.
  • the acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • a combination of two or more therapeutic agents of the invention herein are co-administered to the patient in a suitable manner and in suitable doses; for example, simultaneously, sequentially, or cyclically.
  • a therapeutic agent, vaccine or gene delivery vector of the invention can be administered orally to a subject in need thereof.
  • a therapeutic agent, vaccine or gene delivery vector of the invention can be administered parenterally to a subject in need thereof.
  • the term “treat” does not necessarily imply complete elimination of coronavirus. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a benefit or therapeutic effect. For example, in a method of treating coronavirus, at least about 10% (e.g., at least about 20%, at least about 30%, or at least about 40%) of the symptoms of the coronavirus is reduced upon administration of an agent described herein.
  • At least about 50% (e.g., at least about 60%, at least about 70%, or at least about 80%) of the symptoms of the coronavirus is reduced upon administration of a compound described herein. More preferably, at least about 90% (e.g., at least about 95%, at least about 99%, or at least about 100%) of the symptoms of the coronavirus is reduced upon administration of a compound described herein.
  • the patient to be treated typically is a mammal. Mammals include, but are not limited to, the order Rodentia, such as mice, and the order Logomorpha, such as rabbits.
  • the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs), Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals are of the order Primates, Ceboids, or Simioids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal is a human.
  • the agents of the invention can be administered with another treatment.
  • the additional treatment can be an anti-viral treatment.
  • the additional treatment can be selected from remdesivir, lamivudine, tenofovir, entecavir, adefovir, telbivudine, and interferon alfa-2b.
  • the additional treatment can be administered simultaneously, sequentially, or cyclically, with the agent of the invention.
  • the additional treatment can be administered before an agent of the invention or after.
  • General It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
  • RNA packaging signal PS
  • the inventors developed a search strategy and sequence search motif based on these PS sites and identified multiple, homologous matches within the SARS-CoV-2 and other coronaviral genomes. It is expected that these PS sites mediate envelopment of the nucleocapsid based on the collective function of these multiple PS sites in analogy to other single-stranded RNA viruses 3 .
  • the PS sites identified show extensive conservation across all strain variants, including those of SARS-CoV, MERS-CoV, and the betacoronaviruses that cause common colds. This suggests that they would lend themselves as broad-spectrum drug targets for both existing and newly-emerging coronaviruses.
  • the inventors investigated an essential step within the viral life-cycle, the packaging of the viral genome inside the viral envelope.
  • the SARS-CoV-2 genome is a 29,903 nts long single-stranded (ss)RNA molecule, one of the largest genomes in any RNA virus. It therefore faces a particular challenge in packaging of its genome (gRNA) into the confines of the viral envelope.
  • gRNA genome
  • the gRNA is organised in a spool-like conformation around a helical core formed from nucleocapsid (N) protein 2 . It has been proposed that the N-gRNA complex is anchored to the viral membrane by a single PS- mediated contact between the N and M proteins 10 . A single-copy PS would only permit one such connection.
  • PSs could, however, mediate many such contacts, facilitating both selective gRNA packaging of the viral RNA and potentially providing some of the driving force for closure of the envelope around the nucleocapsid core 11 .
  • the concept of a single PS site in the coronaviral gRNA seems largely based on a similar concept for non-enveloped, spherical (ss)RNA viral genomes.
  • ss spherical
  • Such multiple PSs have widely differing affinities for viral structural proteins commonly leading to “identification” of a dominant PS, apparently solely responsible for assembly.
  • Such single PSs have been reported for a number of coronaviruses (Fig 1a), including betacoronaviruses (HKU1 & OC43) and animal coronaviruses 12 .
  • the latter include Mouse Hepatitis Virus (MHV) A59 13 , the first to have a PS identified, and Bovine Coronavirus (BCoV) 14 .
  • MHV Mouse Hepatitis Virus
  • BCoV Bovine Coronavirus
  • Each of these PSs are predicted to form extended stem-loops encompassing two copies of a near identical sequence motif along their 3' leg stems (Fig. 1a).
  • this motif is: 5'-UAAU-(N) 8 -UAAU-3' (SEQ ID NO: 7), where (N) 8 denotes a stretch of 8 unspecified nucleotides which are mostly base-paired. In the OC43 case this spacer is 11 nts long. Bases in the 5'-UAAU-3' (SEQ ID NO: 23) sub-motifs are mostly single-stranded 12 . In MHV, one sub-motif contains a purine substitution (5'-UGAU-3' (SEQ ID NO: 24)). Reverse genetics and secondary structure probing show that deletion of the purine bulges inhibits packaging 13 , implying that presentation of at least one purine in a bulge is important for recognition.
  • Each of the dominant PSs contains two copies of a 5'-URAU-(N) 8 -URAU-3' (SEQ ID NO: 6) motif, where R is a purine.
  • This arrangement separates the centres of the 5'-URAU- 3' (SEQ ID NO: 25) sub-motifs by ⁇ 12 nucleotides, roughly equivalent to a helical turn in A- form duplex RNA.
  • the two motifs within the PSs are separated by a single–stranded bulge, potentially allowing bending of the RNA around a molecular partner(s). These features are also present in PS sites identified in other Embecovirus genomes 10 .
  • Both viruses have motif matches in nsp3 and nsp15 at sites similar to those in SARS-CoV-2 (asterisks), and include a previously described PS that is known to bind MERS-CoV N-protein in vitro 8 .
  • SARS-CoV-1 shares its double motif (filled, labelled green) with SARS-CoV-2. Even though the locations of the E and M gene are further downstream and directly adjacent to the N gene for MERS-CoV, compared with a more upstream location in the other two genomes, all three gRNAs have a motif match in the E gene. There are also matches at similar positions in all three coronaviruses in the S gene.
  • nucleocapsid complex of gRNA-N a molecular interaction between the nucleocapsid complex of gRNA-N and the envelope would assist the encapsidation process.
  • An interesting scenario made possible by the multiple PS sites described above is that there are multiple, PS-mediated N-M contacts. These could aid enclosure of the nucleocapsid into the viral envelope and its budding out of the membrane.
  • the coronaviral nucleocapsid is believed to consist of ⁇ 1000 N protein 4 , organised as octamers, that can interact to form an extended spool on which to wrap ssRNA. Given estimates of the dimensions of the octameric complex 23 (Fig 3a), at least 26 kb of the genome ( ⁇ 89%) could be packaged this way.
  • RNA is organised in a series of helical segments akin to the “beads-on-a-string” arrangement of chromatin (Fig.3b), and that different PSs are associated with distinct helical units.
  • the on average 34 PS-type sites across the SARS- CoV-2, SARS-CoV-1 and MERS-CoV genomes would be consistent with N protein organising into about 31 helical segments formed from, on average, four octamers ( ⁇ 28 nm).
  • the PSs can also be used to generate artificial sequences and viral particles with improved packaging efficiencies for use in vaccines and as delivery agents.
  • the use of PSs in this manner has been shown to improve packaging efficiency in other viruses (Escors, 2003, J. Virology, 77(14)).
  • Studies with MHV showed that improper gRNA packaging triggers protective innate immune responses lowering viral titres 13 , suggesting that drugs targeting the SARS-CoV-2 PSs would be a promising novel avenue to explore, as an alternative to the repurposing of existing drugs 27-29 .
  • RNA molecules are relatively unusual drug targets, but these have been designed de novo against HIV 30 , or selected from small molecule libraries 31-35 .
  • Example 1 Tables Table 1: Positions and conservation of 5'-URRU-(N) 8 -URRU-3' motifs in SARS- CoV-2. Columns show: the nucleotide position of the first U in any match of the 5'-URRU- (N) 8 -URRU-3' (SEQ ID NO: 5) search motif, where (N) 8 denotes a stretch of 8 unspecified nucleotides, to the SARS-CoV-2 gRNA [NC_045512.2]; the sequence matching the search motif; conservation of the first URRU (SEQ ID NO: 27) submotif across all 5542 Sarbecoviruses (available from the NCBI at the time of analysis, 9 th June, and aligned using MAFFT); same for the second URRU (SEQ ID NO: 27) submotif; average conservation over both submotifs; conservation across all 16 nts of the search motif.
  • the 15 entries marked by an asterisk also fulfil the stricter search motif 5'-URAU-(N) 8 -URAU-3' (SEQ ID NO: 6).
  • Bold highlights indicate adjacent matches combining into a nested 5'-URRU-(N) 8 -URRU- (N) 8 -URRU-3' (SEQ ID NO: 30) motif
  • Underlined highlights indicate entries forming a double (tandem) 5'-URRU-(N) 8 -URRU-(N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 31) motif.
  • the nested and double motifs are also displayed contiguously in the additional subsequent rows.
  • Table 2 Codon information and reading frames for submotifs in URAU-8-URAU matches to the SARS-CoV-2 gRNA: Columns show: the start nucleotide position of each motif; the sequence matching the search motif; the nucleotide sequence of codons overlapping with submotif 1 (first U of submotif shown underlined); the position of the first U of submotif 1 within the first codon; the equivalent information for submotif 2.
  • Table 3 Positions of 5'-URRU-(N)8-URRU-3' motifs in SARS-CoV-1. Columns show: the nucleotide position of the first U in matches of the 5'-URRU-(N) 8 -URRU-3' (SEQ ID NO: 5) search motif, where (N) 8 denotes a stretch of 8 unspecified nucleotides (left), to the SARS- CoV-1 gRNA [NC_004718.3]; the sequence matching the search motif; conservation of the first URRU submotif across all 5542 Sarbecoviruses (available from the NCBI at the time of analysis, 9 th June, and aligned using MAFFT); same for the second URRU (SEQ ID NO: 27) submotif; average conservation over both submotifs; conservation across all 16 nts of the search motif.
  • Bold highlights indicate adjacent matches combining into a nested 5'-URRU- (N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 30) motif, and underlined highlights indicate entries forming a double (tandem) 5'-URRU-(N) 8 -URRU-(N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 32) motif.
  • the nested and double motifs are also displayed contiguously in the additional subsequent rows.
  • Table 4 Positions of 5'-URRU-(N)8-URRU-3' motifs in MERS-CoV. Columns show: the nucleotide position of the first U in matches of the 5'-URRU-(N) 8 -URRU-3' (SEQ ID NO: 5) search motif, where (N) 8 denotes a stretch of 8 unspecified nucleotides (left), to the MERS- CoV gRNA [NC_019843.3]; the sequence matching the search motif; conservation of the first URRU submotif across all 567 Merbecoviruses (available from the NCBI at the time of analysis, 9 th June, and aligned using MAFFT); same for the second URRU (SEQ ID NO: 27) submotif; average conservation over both submotifs; conservation across all 16 nts of the search motif.
  • Bold highlights indicate adjacent matches combining into a nested 5'-URRU- (N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 30) motif, and underlined highlights indicate entries forming a double (tandem) 5'-URRU-(N) 8 -URRU-(N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 31) motif.
  • the nested and double motifs are also displayed contiguously in the additional subsequent rows.
  • Table 5 Positions of 5'-URRU-(N)8-URRU-3' motifs in HCoV-HKU1. Columns show: the nucleotide position of the first U in matches of the 5'-URRU-(N) 8 -URRU-3' (SEQ ID NO: 5) search motif, where (N) 8 denotes a stretch of 8 unspecified nucleotides (left), to the HCoV- HKU1 gRNA [NC_006577]; the sequence matching the search motif; conservation of the first URRU (SEQ ID NO: 27) submotif across all 397 Embecoviruses (available from the NCBI at the time of analysis, 9 th June, and aligned using MAFFT); same for the second URRU submotif; average conservation over both submotifs; conservation across all 16 nts of the search motif.
  • Bold highlights indicate adjacent matches combining into a nested 5'-URRU-(N) 8 -URRU- (N) 8 -URRU-3' (SEQ ID NO: 30) motif, and underlined highlights indicate entries forming a double (tandem) 5'-URRU-(N) 8 -URRU-(N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 31) motif.
  • the nested and double motifs are also displayed contiguously in the additional subsequent rows.
  • Table 7 Positions of 5'-URRU-(N)8-URRU-3' motifs in HCoV-NL63. Columns show: the nucleotide position of the first U in matches of the 5'-URRU-(N)8-URRU-3' (SEQ ID NO: 5) search motif, where (N) 8 denotes a stretch of 8 unspecified nucleotides (left), to the HCoV- NL63 gRNA [NC_005831]; the sequence matching the search motif; conservation of the first URRU (SEQ ID NO: 27) submotif across all 1049 Alphacoronaviruses (available from the NCBI at the time of analysis, 9 th June, and aligned using MAFFT); same for the second URRU submotif; average conservation over both submotifs; conservation across all 16 nts of the search motif.
  • Bold highlights indicate adjacent matches combining into a nested 5'-URRU- (N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 30) motif, and underlined highlights indicate entries forming a double (tandem) 5'-URRU-(N) 8 -URRU-(N) 8 -URRU-(N) 8 -URRU-3' (SEQ ID NO: 31) motif.
  • the nested and double motifs are also displayed contiguously in the additional subsequent rows.
  • Example 2 As discussed above, the coronaviral nucleocapsid is believed to consist of ⁇ 1000 N proteins 4 .
  • Ribonucleoprotein complexes (RNPs) formed from gRNA and N protein have recently been imaged and are estimated to each contain ⁇ 12 N protein subunits and span ⁇ 800nts of gRNA (Yao et al., 2020, Cell 183:730–738). In a genome of ⁇ 30kb, there are thus around 37-38 such RNPs. It has been hypothesised that RNA-driven condensation plus interactions with M protein in the membrane drive RNA packaging into virions (Lu et al., 2021, Nat Commun 12, 502).
  • SHAPE data of gRNA structure in virio and in infected cells have been used to identify nucleotide positions that are less reactive in the packaged gRNA.
  • a difference map of reactivities in vitro and in virio was created on a nucleotide basis, with positive values indicating nucleotides that are more protected inside the virion. These values were offset (by -0.036) to generate a mean of zero across the sample.
  • the table shows any location of an URRU-(N)8-URRU motif (position indicating the first U, followed by its sequence).
  • RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promotes nucleocapsid assembly, Nature Microbiology 2, 17098 (2017).
  • Coronavirus vaccines five key questions as trials Begin Nature 579, 481- 481 (2020).

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Abstract

La présente invention concerne des particules de type virus, des vaccins et des vecteurs d'administration utilisant des signaux d'encapsidation de coronavirus, ainsi que des agents thérapeutiques ciblant de tels signaux d'emballage.
PCT/GB2021/051934 2020-07-27 2021-07-27 Signaux d'emballage coronaviraux Ceased WO2022023734A1 (fr)

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WO2023015232A1 (fr) * 2021-08-04 2023-02-09 The Regents Of The University Of California Particules pseudo-virales de sars-cov-2
WO2024262942A1 (fr) * 2023-06-19 2024-12-26 서울대학교산학협력단 Élément de signal d'encapsidation du coronavirus et son utilisation

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