WO2022150293A1 - Particules d'administration de réplicon de coronavirus et procédés d'utilisation de celles-ci - Google Patents

Particules d'administration de réplicon de coronavirus et procédés d'utilisation de celles-ci Download PDF

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WO2022150293A1
WO2022150293A1 PCT/US2022/011115 US2022011115W WO2022150293A1 WO 2022150293 A1 WO2022150293 A1 WO 2022150293A1 US 2022011115 W US2022011115 W US 2022011115W WO 2022150293 A1 WO2022150293 A1 WO 2022150293A1
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cell
nucleic acid
replicon
cells
coronavirus
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PCT/US2022/011115
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Charles Rice
Inna RICARDO-LAX
Joseph M. Luna
Nadine EBERT
Jörg JORES
Fabien LABROUSSAA
Volker Thiel
Thi Nhu Thao TRAN
John T. POIRIER
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The Rockefeller University
New York University
The University Of Bern
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Publication of WO2022150293A1 publication Critical patent/WO2022150293A1/fr

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2770/20031—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
    • 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
    • C12N2770/20051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to coronavirus reporter replicons and method of use thereof and more specifically to trans-packaged coronavirus reporter replicons and method of use thereof.
  • SARS-CoV-2 the causative agent of COVID-19, is a member of the Coronaviridae family of positive sense ssRNA viruses. Similar to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), the pathogenicity of SARS-CoV-2 necessitates that molecular virology studies of infectious virus can only occur in high containment biological safety level 3 (BSL3) laboratory settings. While studies of viral components in BSL2 settings have been invaluable to establish initial lists of putative host factors and to refine viral enzymatic mechanisms, non-infectious systems permitting the study of intracellular replication have been lacking.
  • BSL3 containment biological safety level 3
  • RNAs Self-replicating RNAs, known as replicons, have provided an important model system to study numerous aspects of RNA virus life cycles.
  • Replicons are typically constructed using reverse genetics systems to replace one or more viral structural proteins with selectable or reporter genes.
  • the replicon RNA Upon transfection or electroporation of viral replicon RNA into cells, the replicon RNA is translated to produce viral enzymes and cofactors necessary to establish RNA replication factories, with reporter genes providing convenient readouts for replicon viability and for the selection of non-cytopathic variants.
  • reporter genes providing convenient readouts for replicon viability and for the selection of non-cytopathic variants.
  • RNA replication, translation, and functions of viral gene products proceed without producing infectious virus.
  • Such systems have been invaluable as molecular virology and high-throughput compound screening and drug development platforms, most notably with hepatitis C virus.
  • RNA replicons e.g., SARS-CoV-2 RNA replicons
  • trans-packaged replicons TPRs
  • RDPs replicon delivery particles
  • this disclosure provides a nucleic acid molecule comprising or encoding a coronavirus replicon.
  • the replicon comprises: (i) a genomic or subgenomic nucleotide sequence of a coronavirus, wherein the nucleotide sequence comprises at least one of the coding sequences of a membrane (M) protein of the coronavirus and an envelope (E) protein of the coronavirus and wherein the coding sequence of a spike (S) protein of the coronavirus is inactivated or deleted; and (ii) a second nucleotide sequence encoding a selectable marker suitable for selection, wherein the selectable marker is under the control of the RNA virus replication machinery.
  • the replicon lacks the Spike-coding sequence, which can be replaced by a reporter gene cassette to monitor replicon activity.
  • examples include Trans- packaged Spike deleted Nspl mutant replicon (TPR AS+) in which Nspl is modified to inhibit its activity, Trans-packaged Spike deleted Minus Accessory replicon (TPR AAcc) which lacks all accessory genes, Trans-packaged Spike deleted Minus SEM replicon (TPR ASEM) which lacks all structural genes, and Trans-packaged Spike deleted minimal replicon (miniTPR) which lacks all accessory and structural genes.
  • the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • the coding sequence of the spike protein or a portion thereof is replaced with the second nucleotide sequence.
  • the selectable marker is a gene that confers resistance to an antibiotic.
  • the nucleic acid molecule further comprises a reporter gene.
  • the reporter gene is selected from the group consisting of NeonGreen, gaussia luciferase (Glue), mScarlet, green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced
  • GFP GFP
  • EGFP enhanced CFP
  • EYFP enhanced YFP
  • GFPS65T Emerald, Venus, mOrange, Topaz
  • GFPuv destabilized EGFP
  • dEGFP destabilized ECFP
  • dEYFP destabilized EYFP
  • HcRed HcRed
  • t-HcRed DsRed
  • DsRed2 t-dimer2(12)
  • mRFPl pocilloporin
  • Renilla GFP Monster GFP
  • paGFP Kaede protein
  • Kaede protein a Phycobiliprotein
  • a biologically active variant or fragment of thereof a biologically active variant or fragment of thereof.
  • the reporter gene is operatively linked to a spike transcription regulating sequence (TRS). In some embodiments, the reporter gene is operatively linked to the TRS through a T2A self-cleaving sequence.
  • TRS spike transcription regulating sequence
  • the nucleic acid molecule has at least 80% sequence identity to SEQ ID NO: 1 or 3, or comprises the nucleic acid sequence of SEQ ID NO: 1 or 3.
  • this disclosure also provides a virus particle or a virus-like particle comprising a nucleic acid molecule described above.
  • the virus particle or virus-like particle comprises a vesicular stomatitis virus G (VSV-G) protein or a variant/fragment thereof.
  • this disclosure additionally provides a cell or cell line comprising a nucleic acid molecule described above.
  • the cell or cell line further comprises a second nucleic acid molecule comprising a coding sequence of a VSV-G protein or a variant/fragment thereof.
  • the VSV-G protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or comprises the amino acid sequence of SEQ ID NO: 2.
  • VSV-G protein sequence SEQ ID NO: 2
  • the cell or cell line further comprises a third nucleic acid molecule comprising a coding sequence of a Spike protein or a variant/fragment thereof
  • the Spike protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or comprises the amino acid sequence of SEQ ID NO: 4.
  • the cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the cell is a Huh-7.5 cell or a BHK-21 cell. In some embodiments, the cell is a lung organoid.
  • composition comprising a nucleic acid molecule or a cell or cell line, as described above, and a pharmaceutically acceptable carrier.
  • this disclosure provides a kit comprising a nucleic acid molecule, a cell or cell line, or a composition, as described above.
  • this disclosure further provides a method of preparing a coronavirus replicon-harbored cell.
  • the method comprises: (i) introducing a nucleic acid molecule described above to a cell; and (ii) culturing the cell in a cell culture medium to produce the coronavirus replicon-harbored cell.
  • the cell can contain a second nucleic acid comprising a coding sequence of the VSV-G protein or a variant/fragment thereof.
  • the method may further comprise introducing to the cell a second nucleic acid comprising a coding sequence of the VSV-G protein or a variant/fragment thereof.
  • the method may further comprise introducing to the cell a third nucleic acid comprising a coding sequence of the Spike protein or a variant/fragment thereof.
  • the cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the cell is a Huh-7.5 cell.
  • this disclosure further provides a method for screening for antiviral agents for a coronavirus.
  • the method comprises: (i) contacting a cell or cell line described above with a candidate agent; and (ii) determining an increase or decrease in replication or activity of the coronavirus virus replicon relative to a control cell or cell line harboring the same replicon, wherein the control cell or cell line has not been contacted with the candidate agent.
  • the coronavirus is SARS-CoV or SARS-CoV-2.
  • the step of determining comprises determining a level of production of a coronavirus protein or a coronavirus RNA transcript.
  • the candidate agent comprises an organic compound or an antisense nucleic acid.
  • FIGS. 1A, IB, 1C, ID, IE, IF, and 1G are a set of diagrams showing SARS-CoV-2 replicon design and launch optimization.
  • FIG. 1A is a schematic of a modular SARS-CoV-2 replicon design. Fragments from (18) are shown in orange, and fragments harboring mutations in nspl or the RdRP (nspl2) are shown in red. Fragments to encode a spike-replaced reporter gene cassette, accessory proteins, and flanking regions are shown in purple, green, or blue, respectively.
  • FIG. IB shows agarose gel of replicon DNA recovered from yeast or bacteria, before or after amplification with phi29 DNA polymerase.
  • FIG. 1C shows agarose gel of T7 RNA transcription reactions from DNA plasmids shown in FIG. IB. Arrow highlights the expected size of full-length RNA. Asterisk denotes a non-specific band.
  • FIG. IF shows a flowchart diagram of optimized RNA production for SARS-CoV-2 replicons. Viral fragments and reporter transgenes are cloned and assembled in yeast. Yeast derived plasmids can either be propagated in bacteria or in yeast, in which case they are treated with plasmid-safe DNAse to remove DNA contaminants. Subsequent phi29 MDA amplification ensures full-length DNA template availability for T7 transcription.
  • FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21, and 2J are a set of diagrams showing that SARS- CoV-2 replicons are sensitive to antiviral compounds, required host factor loss, and viral mutant phenotypes.
  • FIG. 2A shows cumulative timecourse measurements of gaussia luciferase (Glue) in the supernatants of Huh-7.5 cells electroporated with the Glue replicon and seeded with 100 nM remdesivir (Rem) or vehicle.
  • a polymerase deficient (pol-) replicon was used as negative control. Dashed line indicates the limit of detection. Error bars represent standard deviation of 3 biological repeats.
  • FIG. 1A shows cumulative timecourse measurements of gaussia luciferase (Glue) in the supernatants of Huh-7.5 cells electroporated with the Glue replicon and seeded with 100 nM remdesivir (
  • FIG. 2B shows qRT-PCR measurements for subgenomic N RNA in Huh-7.5 cells harboring Glue replicons electroporated with the Glue replicon and seeded with lOOnM remdesivir (Rem) or vehicle.
  • a polymerase deficient (pol-) replicon was used for normalization (dashed line). Error bars represent standard deviation of 3 biological repeats.
  • FIG. 2C shows that Huh-7.5 Cells were electroporated with the Glue replicon and seeded in 96-wells plates on a dose-curve of remdesivir.
  • GLuc signal was measured in sup (black circles), and cell viability was measured by cell-titer Glo assay (empty circles). Data was normalized to vehicle (DMSO)-treated cells. Error bars represent standard deviation of 5 biological repeats.
  • FIG. 2D is the same as FIG. 2C except that the cells were treated with Masitinib. Error bars represent standard deviation of 4 biological repeats.
  • FIG. 2E is the same as FIG. 2C except that the cells were treated with 27-hydroxycholesterol (27HC). Error bars represent standard deviation of 3 biological repeats.
  • FIG. 2F shows that WT or TMEM41B KO cells were electroporated with the GLuc replicon and seeded with lOOnM Remdesivir or vehicle.
  • GLuc was measured 24h post electroporation. Error bars represent standard deviation of 4 biological repeats.
  • FIG. 2G shows that cells in F were electroporated with SinRep-GFP Sindbis virus replicon RNA and seeded in 6-wells plates. Percent of GFP-positive cells was determined by flow cytometry 24h post electroporation. Error bars represent standard deviation from three biological replicates.
  • FIG. 2H is the same as FIG.
  • FIG. 21 shows that Huh7.5 cells were electroporated with either WT or NSP1 K164A/H165A mutant (NSPl ' ). Electroporated cells were seeded with lOOnM Remdesivir or vehicle, and GLuc levels on the supernatant were measured at the indicated time points. Error bars represent standard deviation of 4 biological repeats.
  • FIG. 2J is the same as FIG. 21 except that cell viability was measured at each time point by cell-titer Glo assay. Mock-electroporated cells were used as control for normal post-electroporation cell viability.
  • FIGS. 3A, 3B, 3C, 3D, 3E, and 3F are a set of diagrams showing kinetics and drug susceptibility of different replicons.
  • FIG. 3 A shows that Huh7.5 cells were electroporated with WT or NSP- replicons and seeded on a dose-curve of Interferon alpha (IFNa). GLuc activity (closed circles) and cell viability (open circles) were measured 24 hours post electroporation.
  • FIG. 3B is the same as FIG. 3A except that cells were treated with interferon beta (IFNp)
  • FIG. 3C is the same as FIG. 3A except that cells were treated with Remdesivir.
  • FIG. 3 A shows that Huh7.5 cells were electroporated with WT or NSP- replicons and seeded on a dose-curve of Interferon alpha (IFNa). GLuc activity (closed circles) and cell viability (open circles) were measured 24 hours post electroporation.
  • FIG. 3D is a schematic of the full replicon, containing most structural proteins excluding Spike and all the accessory proteins, and minireplicons, which are devoid of all accessory and structural proteins and encode only for the replicase and N genes.
  • FIG. 3E shows kinetics of GLuc expression of the full and mini replicons. Huh7.5 cells were electroporated with the full or mini replicons and seeded with 100 nM Remdesivir (open circles) or vehicle (closed circles). GLuc activity in the supernatant was measured at the indicated time points.
  • FIG. 3F is the same as FIG. 3E, except that cell viability was measured by cell titer Glo assay and normalized to day 1 post electroporation.
  • FIGS. 4A, 4B, 4C, and 4D are a set of diagrams showing replicon delivery by transpackaging with VSV-G.
  • FIG. 4 A shows an experimental scheme describing the trans-packaging of replicons with VSV-G. Briefly, BHK-21 cells were transfected with VGV-G, and the next day electroporated with the full SARS-CoV2 replicon. After 48h, the supernatant of the producer cells was collected and concentrated. Multiple cell types can be transduced with the resulting trans- packaged replicons (TRPs).
  • TRPs trans- packaged replicons
  • FIG. 4B shows that indicated cells were seeded in 96-well plates and transduced with TPR-NeonGreen, produced as described in FIG. 4A. Brightfield and fluorescent images were taken at xlO magnification 24h after transduction.
  • FIG. 4C shows that Huh7.5 cells were transduced with concentrated or 1:10 diluted TPR-NeonGreen, and percent of reporter positive cells was measured by flow cytometry. As negative controls, cells were transduced with similarly collected supernatants from cells that were electroporated with the minireplicon instead, or where VSV-G was replaced by a control plasmid.
  • FIG. 5E shows that TPRs are single-cycle infectious virions. Huh7.5 cells were transduced with IOOmI of TPRs per 2ml in a 6-well format (P0 supernatant). After lhr, inoculum was saved and concentrated with PEG (P0’ supernatant).
  • FIGS. 5A, 5B, and 5C are a set of photographs and diagrams showing replicon assembly and RNA validation (Related to FIG. 1).
  • FIG. 5A shows that six different replicons were assembled in yeast, and DNA from four colonies of each assembly were extracted and subjected to multiplex PCR with primer set #1 (see Table 1). Expected PCR product sizes are indicated on the right.
  • FIG. 5B shows that RNA was in-vitro transcribed from lug of the indicated phi-29 amplified DNA templates, using T7 RiboMAX Express Large Scale RNA Production System (Promega) or HiScribe T7 High Yield RNA Synthesis kit (NEB).
  • FIG. 5C shows the results of qRT-PCR measurements for N RNA in Huh-7.5 cells electroporated with the 5pg Glue replicon RNA plus 2pg N mRNA and seeded with lOOnM remdesivir (Rem) or vehicle. The signal from mock infected cells was used for normalization (dashed line). Error bars represent standard deviation of 3 biological repeats.
  • FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H are a set of diagrams showing susceptibility of the Spike-deleted and minireplicons to antiviral compounds (Related to FIG. 3).
  • FIGS. 6A, 6B, 6C, and 6D show that Huh7.5 cells were electroporated with GLuc spike-deleted replicon and seeded in 96-wells plate on a dose-curve of Remdesivir (FIG. 6A), IFNa (FIG. 6B) IFNb (FIG. 6C) or AM580 (FIG. 6D).
  • GLuc activity in the supernatant (closed circles) and cell viability (open circles) were measured 48h post electroporation. Error bars represent standard error from 3 biological repeats.
  • FIGS. 7A, 7B, 7C, 7D, 7E, and 7F are a set of diagrams showing trans-complementation of replicons with Spike yields single-cycle SARS-CoV-2.
  • FIG. 7A shows a scheme to trans complement replicons with ectopically expressed Spike for single-cycle virion production.
  • BHK- 21 cells are transfected with a Spike encoding plasmid, and 24 hours later electroporated with the full SARS-CoV2 replicon.
  • Supernatant from these producer cells (P0) is collected and passaged onto recipient cells (PI) yielding reporter activity.
  • a second round of passaging onto naive recipient cells (P2) fails to propagate the replicon.
  • FIG. 7A shows a scheme to trans complement replicons with ectopically expressed Spike for single-cycle virion production.
  • BHK- 21 cells are transfected with a Spike encoding plasmid, and 24 hours later
  • FIG. 7B shows a spike trans-packaged replicon consisting of the full Spike deleted replicon RNA alongside plasmid driven Spike expression. Nspl mutations relative to the WT sequence indicated.
  • FIG. 7C shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer cells (P0) with supernatants concentrated and subsequently passaged twice onto Huh-7.5 ACE2/TMPRSS2 (Huh-7.5 AT) cells (PI and P2). Immunofluorescence images at 4X of the mNeongreen signal (green) and N antibody staining (magenta) are shown. Scale bar, lOOpm.
  • FIG. 7D shows neongreen quantification per passage for the results in (FIG. 1C).
  • FIGS. 8A, 8B, 8C, 8D, and 8E are a set of diagrams showing ectopic Spike expression and quantification.
  • FIG. 8A shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer BHK-21 cells (P0) with supernatants subsequently passaged onto Huh-7.5 ACE2/TMPRSS2 (Huh-7.5 AT) cells (PI). Immunofluorescence images at 4X of the NeonGreen signal (green) and Spike C144 antibody staining (magenta) are shown. Scale bar, 100 pm.
  • FIG. 8A shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer BHK-21 cells (P0) with supernatants subsequently passaged onto Huh-7.5 ACE2/TMPRSS2 (Hu
  • FIG. 8C shows N immunofluorescence quantification per passage for the results in Figure 4C.
  • FIG. 8D shows immunofluorescence images N protein (magenta) in Huh-7.5 cells infected with SARS-CoV-2 (WA1/2020, left) or transduced with TPRs packaged with WT Spike (right). Images at 4X magnification, scale bar 500pm.
  • FIG. 8E shows quantification of results in (FIG. 8D), measuring approximate per cell area for the N protein signal between virus or TPR positive cells.
  • FIGS. 9A, 9B, and 9C are a set of diagrams showing neutralization assays with TPRs recapitulate authentic SARS-CoV-2 antibody phenotypes.
  • FIGS. 9A, 9B, and 9C show neutralization assays for SARS-CoV-2 TPRs or virus in the presence of increasing concentrations of antibodies.
  • WT Spike with Cl 44 antibody (FIG. 9A)
  • B.1.351 Spike with Cl 44 antibody (FIG. 9B)
  • B.1.351 with C135 antibody FIG. 9C) are shown.
  • N 3
  • error bars SEM.
  • RNA replicons e.g, SARS-CoV-2 RNA replicons
  • SARS-CoV-2 RNA replicons RNA replicons
  • the disclosed RNA replicons are broadly amenable to molecular virology studies and drug development screening efforts.
  • this disclosure provides a nucleic acid molecule comprising or encoding a coronavirus replicon.
  • the replicon comprises: (i) a genomic nucleotide sequence of a coronavirus, wherein the nucleotide sequence comprises at least one of the coding sequences of a membrane (M) protein of the coronavirus and an envelope (E) protein of the coronavirus and wherein the coding sequence of a spike (S) protein of the coronavirus is inactivated or deleted; and (ii) a second nucleotide sequence encoding a selectable marker suitable for selection, wherein the selectable marker is under the control of the RNA virus replication machinery.
  • the selectable marker is a gene that confers resistance to an antibiotic.
  • the coding sequence of the spike protein or a portion thereof is replaced with the second nucleotide sequence.
  • the nucleic acid molecule further comprises a reporter gene.
  • the reporter gene can be selected from the group consisting of NeonGreen, gaussia luciferase (Glue), green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Venus, mOrange, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t- HcRed, DsRed, DsRed2, t-dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein a Phycobiliprotein, and a biologically active variant or fragment
  • the reporter gene is operatively linked to a spike transcriptionregulating sequence (TRS).
  • TRS spike transcriptionregulating sequence
  • the reporter gene can be directly or indirectly linked to a linker or a cleavage site.
  • the reporter gene can be operatively linked to a TRS through a T2A self-cleaving sequence.
  • the nucleic acid molecule has at least 80% sequence identity to SEQ ID NO: 1 or 3 (listed below), or comprises the nucleic acid sequence of SEQ ID NO: 1 or 3.
  • gagtacactg actttgcaac atcagcttgt gttttggctg ctgaatgtac aatttttaaa
  • 11221 atattgggta gtgctttatt agaagatgaa tttacacctt ttgatgttgt tagacaatgc 11281 tcaggtgtta ctttccaaag tgcagtgaaa agaacaatca agggtacaca ccactggttg
  • gagtttttcc cacggtggat atttcttctt gcgctgagcg taagagctat ctgacagaac
  • RNA replicon or “replicon RNA” refer to RNA, which contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell.
  • the RNA molecule (1) encodes polymerase, replicase, or other proteins which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and (2) contain cis-acting
  • RNA sequences required for replication and transcription of the subgenomic replicon-encoded RNA may be bound during the process of replication to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components.
  • a coronavims-derived replicon RNA molecule typically contains the following ordered elements: 5’ viral or defective-interfering RNA sequence(s) required in cis for replication, sequences coding for biologically active coronavirus nonstructural proteins (e.g., nsPl, nsP2, nsP3, and nsP4), promoter for the subgenomic RNA, 3’ viral sequences required in cis for replication, and a polyadenylate tract.
  • RNA replicon generally refers to a molecule of positive polarity or “message” sense, and the RNA replicon may be of length different from that of any known, naturally-occurring coronavirus.
  • the RNA replicon does not contain the sequences of at least a spike protein.
  • the coding sequence of the spike protein can be substituted with heterologous sequences.
  • the RNA replicon may be packaged into a recombinant coronavirus particle, and it may include one or more sequences, such as packaging signals, which serve to initiate interactions with coronavirus structural proteins that lead to particle formation.
  • “subgenomic RNA” refers to an RNA molecule of a length or size which is smaller than the genomic RNA from which it was derived.
  • the coronavirus subgenomic RNA should be transcribed from an internal promoter, whose sequences reside within the genomic RNA or its complement. Transcription of a coronavirus subgenomic RNA may be mediated by the viral- encoded polymerase(s) associated with host cell-encoded proteins, ribonucleoprotein(s), or a combination thereof.
  • the subgenomic RNA is produced from a modified RNA replicon, as disclosed herein.
  • a part or the entire coding sequence for the spike protein is absent and/or modified in the nucleic acid molecules disclosed herein.
  • the modified coronavirus genome or RNA replicon can be devoid of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the sequence encoding the spike protein.
  • the modified coronavirus genome or RNA replicon is devoid of a substantial portion of or the entire sequence encoding the spike protein.
  • a “substantial portion” of a nucleic acid sequence comprises enough of the nucleic acid sequence encoding the viral structural protein to afford putative identification of that protein, either by manual evaluation of the sequence by one skilled in the art or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, Altschul SF el al. 1993, supra).
  • the modified coronavirus genome or RNA replicon is devoid of the entire sequence encoding the spike protein.
  • the nucleic acid molecules may include a modified coronavirus genome or RNA replicon containing one or more attenuating mutations so as to increase the safety of virus manipulation and/or administration.
  • attenuating mutation means a nucleotide mutation or an amino acid encoded in view of such mutation, which results in a decreased probability of causing disease in its host (i.e., a loss of virulence), in accordance with standard terminology in the art, whether the mutation is a substitution mutation or an in-frame deletion or insertion mutation. Attenuating mutations may be in the coding or non-coding regions of the coronavirus genome.
  • Attenuating mutation excludes mutations or combinations of mutations that would be lethal to the virus. Further, those skilled in the art will appreciate that some attenuating mutations may be lethal in the absence of a second-site suppressor mutation(s).
  • the nucleic acid molecules are recombinant nucleic acid molecules.
  • the term “recombinant” means any molecule (e.g., DNA, RNA, polypeptide) that results from human manipulation.
  • a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector.
  • a recombinant nucleic acid molecule (1) can be synthesized or modified in vitro , for example, using chemical or enzymatic techniques (e.g., by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site- specific recombination) of nucleic acid molecules; (2) may include conjoined nucleotide sequences that are not conjoined in nature; (3) can be engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and/or (4) may be manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.
  • chemical or enzymatic techniques
  • the nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis.
  • the nucleic acid molecules may include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and/or substituted in such a manner that such modifications provide the desired property in effecting a biological activity as described herein.
  • a nucleic acid molecule including a variant of a naturally-occurring nucleic acid sequence, can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et ah, In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).
  • sequence of a nucleic acid molecule can be modified with respect to a naturally-occurring sequence from which it is derived using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as but not limited to site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including chemical synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules, and combinations thereof.
  • classic mutagenesis techniques and recombinant DNA techniques such as but not limited to site-directed mutagenesis
  • chemical treatment of a nucleic acid molecule to induce mutations
  • Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or the replicon encoded by the nucleic acid molecule and/or by hybridization with a wild-type gene or fragment thereof, or by PCR using primers having homology to a target or wild-type nucleic acid molecule or sequence.
  • the modified coronavirus genome or RNA replicon is operably linked to a heterologous regulatory element.
  • regulatory element refers to a nucleotide sequence located upstream (5’), within, or downstream (3’) of a coding sequence such as, for example, a polypeptide-encoding sequence or a functional RNA-encoding sequence. Transcription of the coding sequence and/or translation of an RNA molecule resulting from transcription of the coding sequence is typically affected by the presence or absence of the regulatory element.
  • These regulatory elements may comprise promoters, cis-elements, enhancers, terminators, or introns.
  • the heterologous regulatory element is, or comprises, a promoter sequence.
  • the heterologous promoter sequence can be any heterologous promoter sequence, for example, a SP6 promoter, a T3 promoter, or a T7 promoter, or a combination thereof.
  • the promoter sequence includes a T7 promoter sequence.
  • the modified coronavirus genome or RNA replicon can include one or more heterologous transcriptional termination signal sequences.
  • transcriptional termination signal refers to a regulatory section of genetic sequence that causes RNA polymerase to cease transcription.
  • the heterologous transcriptional termination signal sequences can generally be any heterologous transcriptional termination signal sequences, and for example, a SP6 termination signal sequence, a T3 termination signal sequence, a T7 termination signal sequence, or a variant thereof.
  • the nucleic acid molecules can include at least one of the one or more heterologous transcriptional termination signal sequences selected from the group consisting of a SP6 termination signal sequence, a T3 termination signal sequence, a T7 termination signal sequence, or a variant thereof.
  • the transcriptional termination sequence includes a T7 termination signal sequence.
  • the nucleic acid molecules disclosed herein can include one or more expression cassettes.
  • the nucleic acid molecules can include at least two, at least three, at least four, at least five, or at least six expression cassettes.
  • expression cassette refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. The expression cassette may be inserted into a vector for targeting a desired host cell and/or into a subject.
  • expression cassette may be used interchangeably with the term “expression construct.”
  • expression cassette refers to a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and/or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene.
  • operably linked denotes a functional linkage between two or more sequences. For example, an operably linkage between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is a functional link that allows for expression of the polynucleotide of interest.
  • operably linked refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest.
  • operably linked denotes a configuration in which a regulatory sequence is placed at an appropriate position relative to a sequence that encodes a polypeptide or functional RNA such that the control sequence directs or regulates the expression or cellular localization of the mRNA encoding the polypeptide, the polypeptide, and/or the functional RNA.
  • a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence.
  • Operably linked elements may be contiguous or non-contiguous.
  • the techniques for operably linking two or more sequences of DNA together are familiar to the skilled worker, and such methods have been described in a number of texts for standard molecular biological manipulation (see, for example, Maniatis et ah, “Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Gibson et al, Nature Methods 6:343-45, 2009).
  • the disclosed nucleic acid molecules may include a codon- optimized sequence.
  • the nucleic acid sequence may be codon-optimized for expression in a eukaryote or eukaryotic cell.
  • the codon-optimized nucleic acid sequence is codon-optimized for operability in a eukaryotic cell or organism, e.g. , a yeast cell, or a mammalian cell or organism, including a mouse cell, a rat cell, and a human cell or non-human eukaryote organism.
  • codon optimization refers to a process of modifying a nucleic acid sequence to enhance expression in the host cells by substituting at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., el al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa ).
  • one or more codons in a sequence encoding a DNA/RNA-targeting IL-2 variant corresponds to the most frequently used codon for a particular amino acid.
  • codon usage in yeast reference is made to the online Yeast Genome database available at http://www.yeastgenome.org/community/codonusage.shtml, or Codon selection in yeast , Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31.
  • codon usage in plants including algae reference is made to Codon usage in higher plants, green algae, and cyanobacteria , Campbell and Gowri, Plant Physiol. 1990 January; 92(1): 1-11.; as well as Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan. 25; 17(2):477-98; or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton B R, J Mol Evol. 1998 April; 46(4):449-59.
  • Coronavirus refers to a genus in the family Coronaviridae, which family is in turn classified within the order Nidovirales.
  • the coronaviruses are large, enveloped, positive- stranded RNA viruses. They have the largest genomes of all RNA viruses and replicate by a unique mechanism that results in a high frequency of recombination.
  • the coronaviruses include antigenic groups I, II, and III.
  • coronaviruses include SARS coronavirus (i.e ., SARS-CoV, SARS-CoV-2), MERS coronavirus, transmissible gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, murine hepatitis virus, sialodacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as chimeras of any of the foregoing.
  • SARS coronavirus i.e ., SARS-CoV, SARS-CoV-2
  • MERS coronavirus transmissible gastroenteritis virus (TGEV)
  • human respiratory coronavirus porcine respiratory coronavirus
  • canine coronavirus canine coron
  • the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • this disclosure also provides a virus particle or a virus-like particle comprising a nucleic acid molecule described above.
  • the virus particle or virus-like particle comprises a vesicular stomatitis virus G (VSV-G) protein.
  • VSV-G vesicular stomatitis virus G
  • VLP refers to a nonreplicating, viral shell.
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art and discussed more fully below.
  • VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical and immunological characterizations, and the like. See , e.g., Baker el al. , Biophys. J. (1991) 60:1445-1456; Hagensee et al, J. Virol. (1994) 68:4503-4505.
  • VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding.
  • cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation and images recorded under appropriate exposure conditions. Additional methods of VLP purification include but are not limited to chromatographic techniques such as affinity, ion exchange, size exclusion, and reverse-phase procedures.
  • this disclosure further provides a cell or cell line comprising a nucleic acid molecule described above.
  • the cell or cell line further comprises a second nucleic acid molecule comprising a coding sequence of a VSV-G protein or a variant/fragment thereof.
  • the VSV-G protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or comprises the amino acid sequence of SEQ ID NO: 2.
  • the cell or cell line further comprises a third nucleic acid molecule comprising a coding sequence of a Spike protein or a variant/fragment thereof.
  • the Spike protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or comprises the amino acid sequence of SEQ ID NO: 4.
  • a representative amino acid sequence of the Spike protein is provided below (Accession ID: NC_045512.2; SEQ ID NO: 4):
  • the method may include introducing a nucleic acid molecule described above to a host cell, such as an animal cell.
  • the method may further include culturing the cell in a cell culture medium to obtain the coronavirus replicon-harbored cell.
  • the method further comprises introducing to the cell a second nucleic acid comprising a coding sequence of the YSV-G protein or a variant/fragment thereof.
  • the method may further comprise introducing to the cell a third nucleic acid comprising a coding sequence of the Spike protein or a variant/fragment thereof.
  • the terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the nucleic acid molecule is introduced into a host cell by an electroporation procedure or a biolistic procedure.
  • host cells can be genetically engineered (e.g, transduced or transformed or transfected) with, for example, a vector construct of this disclosure.
  • the vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the vector containing a polynucleotide sequence as described herein, e.g, a nucleic acid molecule comprising a modified coronavirus genome or RNA replicon, as well as, optionally, a selectable marker or reporter gene, can be employed to transform an appropriate host cell.
  • Suitable host cells can include, but are not limited to, algal cells, bacterial cells, heterokonts, fungal cells, chytrid cells, microfungi, microalgae, and animal cells.
  • the animal cells are invertebrate animal cells.
  • the vertebrate animal cells are mammalians cells.
  • Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.
  • the methods disclosed herein can be used with host cells from species that are natural hosts of coronaviruses, such as rodents, mice, fish, birds, and larger mammals such as humans, horses, pig, monkey, and apes, as well as invertebrates.
  • any animal species can be generally used and can be, for example, human, dog, bird, fish, horse, pig, primate, mouse, cattle, swine, sheep, rabbit, cat, goat, donkey, hamster, or buffalo.
  • suitable bird species include chicken, duck, goose, turkey, ostrich, emu, swan, peafowl, pheasant, partridge, and guinea fowl.
  • the fish species is a salmon species.
  • Primary mammalian cells and continuous/immortalized cells types are also suitable.
  • suitable animal host cells include, but not limited to, pulmonary equine artery endothelial cell, equine dermis cell, baby hamster kidney (BHK) cell, rabbit kidney cell, mouse muscle cell, mouse connective tissue cell, human cervix cell, human epidermoid larynx cell, Chinese hamster ovary cell (CHO), human HEK-293 cell, mouse 3T3 cell, Vero cell, Madin- Darby Canine Kidney Epithelial Cell (MDCK), primary chicken fibroblast cell, a HuT78 cell, a Huh-7 cell, A549 lung cell, HeLa cell, PER.C6® cell, WI-38 cell, MRC-5 cell, FRhL-2, and CEM T-cell.
  • pulmonary equine artery endothelial cell equine dermis cell
  • BHK baby hamster kidney
  • the host cell is a baby hamster kidney cell. In some embodiments, the baby hamster kidney cell is a BHK-21 cell. In some embodiments, the host cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the host cell is a Huh-7.5 cell. In some embodiments, the cell is a lung organoid.
  • composition comprising a nucleic acid molecule or a cell or cell line, as described above, and a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subj ect such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer
  • “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • this disclosure provides a kit comprising a nucleic acid molecule, a cell or cell line, or the composition, as described above.
  • the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative.
  • the composition can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution.
  • reconstitution generally is by the addition of a suitable solvent and acidulant.
  • the acidulant and solvent e.g., an aprotic solvent, sterile water, or a buffer
  • the kit may further include informational materials.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about the production of the composition, concentration, date of expiration, batch or production site information, and so forth.
  • RNA replicons can be used as a low-containment platform for molecular virology studies and drug development screening. Accordingly, this disclosure further provides a method for screening for antiviral agents for a coronavirus.
  • the method comprises: (i) contacting a cell or cell line described above with a candidate agent (e.g., test compound); and (ii) determining an increase or decrease in replication or activity of the coronavirus virus replicon relative to a control cell or cell line harboring the same replicon, wherein the control cell or cell line has not been contacted with the candidate agent.
  • a candidate agent e.g., test compound
  • the coronavirus is SARS-CoV or SARS-CoV-2.
  • the step of determining comprises determining a level of production of a coronavirus protein or a coronavirus RNA transcript.
  • antiviral agents can be an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate.
  • test compound examples include small organic or inorganic molecules, proteins, peptides, peptidomimetics, polysaccharides, nucleic acids, nucleic acid analogues and derivatives, or peptoids.
  • Candidate compounds to be screened e.g., proteins, peptides, peptidomimetics, peptoids, antibodies, small molecules, or other drugs
  • proteins, peptides, peptidomimetics, peptoids, antibodies, small molecules, or other drugs can be isolated from naturally occurring substances or obtained using any of the numerous approaches in combinatorial library methods known in the art.
  • Such libraries include: peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that is resistant to enzymatic degradation); spatially addressable parallel solid phase or solution phase libraries; synthetic libraries obtained by deconvolution or affinity chromatography selection; and the “one-bead one-compound” libraries. See, e.g., Zuckermann et al. 1994, J. Med. Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145.
  • a candidate compound/composition identified by the evaluation method can be further tested to confirm its therapeutic effect or modified to optimize its effect and limit any side effects, and then formulated as a therapeutic agent.
  • Therapeutic agents thus identified can be used in a therapeutic protocol to treat coronavirus infection.
  • the term “determining” means methods that include detecting the presence or absence or a level of marker(s) (e.g ., a coronavirus protein or a coronavirus RNA transcript) in the sample, quantifying the amount of marker(s) in the sample, and/or qualifying the type of biomarker. Measuring can be accomplished by methods known in the art and those further described herein.
  • the level of the one or more markers in a sample obtained from a subject may be determined by any of a wide variety of well-known techniques and methods, which transform a marker within the sample into a moiety that can be detected and quantified.
  • Non-limiting examples of such methods include analyzing the sample using immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods, immunoblotting, Western blotting, Northern blotting, electron microscopy, mass spectrometry, e.g., MALDI-TOF and SELDI-TOF, immunoprecipitations, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assays (ELISAs), e.g., amplified ELISA, quantitative blood-based assays, e.g., serum ELISA, quantitative urine-based assays, flow cytometry, Southern hybridizations, array analysis, and the like, and combinations or sub
  • the level of a marker in a sample can be determined by detecting a transcribed polynucleotide or portion thereof, e.g., mRNA, or cDNA, of a marker gene.
  • RNA may be extracted from cells using RNA extraction techniques, including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland).
  • Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton el al, (1984) Nuc. Acids Res. 12:7035-56), Northern blotting, in situ hybridization, and microarray analysis.
  • More than one antiviral agents can be tested at the same time for their ability to modulate the expression and/or activity of a marker in a screening assay.
  • screening assay refers to assays that test the ability of a plurality of compounds to influence the readout of choice rather than to tests that test the ability of one compound to influence a readout.
  • the assays may identify compounds not previously known to have the effect that is being screened for.
  • high throughput screening HTS can be used to assay for the activity of a compound.
  • Gene is used broadly to refer to any segment of a nucleic acid molecule that encodes a protein or that can be transcribed into a functional RNA.
  • Genes may include sequences that are transcribed but are not part of a final, mature, and/or functional RNA transcript, and genes that encode proteins may further comprise sequences that are transcribed but not translated, for example, 5’ untranslated regions, 3’ untranslated regions, introns, etc.
  • genes may optionally further comprise regulatory sequences required for their expression, and such sequences may be, for example, sequences that are not transcribed or translated.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • a “coding sequence” or a sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • nucleic acid molecule and “polynucleotide” are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules can have any three-dimensional structure. A nucleic acid molecule can be double- stranded or single-stranded (e.g., a sense strand or an antisense strand).
  • Non-limiting examples of nucleic acid molecules include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, tracrRNAs, crRNAs, guide RNAs, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes, and nucleic acid primers.
  • a nucleic acid molecule may contain unconventional or modified nucleotides.
  • the nomenclature for nucleotide bases as set forth in 37 CFR ⁇ 1.822 is used herein.
  • Nucleic acid molecules can be nucleic acid molecules of any length, including but not limited to, nucleic acid molecules that are between about 3 Kb and about 50 Kb, for example, between about 3 Kb and about 40 Kb, between about 3 Kb and about 40 Kb, between about 3 Kb and about 30 Kb, between about 3 Kb and about 20 Kb, between 5 Kb and about 40 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.
  • the nucleic acid molecules can also be, for example, more than 50 kb.
  • polynucleotides of the present disclosure can be “biologically active” with respect to either a stmctural attribute, such as the capacity of a nucleic acid to hybridize to another nucleic acid, or the ability of a polynucleotide sequence to be recognized and bound by a transcription factor and/or a nucleic acid polymerase.
  • a stmctural attribute such as the capacity of a nucleic acid to hybridize to another nucleic acid, or the ability of a polynucleotide sequence to be recognized and bound by a transcription factor and/or a nucleic acid polymerase.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), and translation termination sequences, and/or sequence elements controlling an open chromatin structure see e.g., McCaughan etal. (1995) PNAS USA 92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355.
  • the term “construct” is intended to mean any recombinant nucleic acid molecule such as an expression cassette, plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single- stranded or double- stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g. operably linked.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as “gene products.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • cells include the primary subject cells and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment); however, such altered progeny are included in these terms, so long as the progeny retain the same functionality as that of the originally transformed cell.
  • variant refers to a first molecule that is related to a second molecule (also termed a “parent” molecule).
  • the variant molecule can be derived from, isolated from, based on or homologous to the parent molecule.
  • a “functional variant” of a protein as used herein refers to a variant of such protein that retains at least partially the activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or replacement mutants), including polymorphs, etc. Also included within functional variants are fusion products of such protein with another, usually unrelated, nucleic acid, protein, polypeptide, or peptide. Functional variants may be naturally occurring or may be man-made.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the Cas protein with one or more conservative modifications may retain the desired functional properties, which can be tested using the functional assays known in the art.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • a homolog has a greater than 60% sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.
  • substantially identity as applied to polypeptides, means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75% sequence identity.
  • a peptide or polypeptide “fragment” as used herein refers to a less than full-length peptide, polypeptide or protein.
  • a peptide or polypeptide fragment can have at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids in length, or single unit lengths thereof.
  • fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length.
  • peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less than about 250 amino acids in length.
  • variants and homologs may have sequences with at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequences of transgenes described herein.
  • disease as used herein is intended to be generally synonymous and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
  • the term “modulate” is meant to refer to any change in biological state, i.e., increasing, decreasing, and the like.
  • the terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
  • “reduced,” “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example, a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • the terms “increased,” “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,” “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • a biological macromolecule such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide
  • an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • the activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • sample can be a sample of, serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells (e.g., antibody-producing cells) or tissue.
  • sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
  • sample and biological sample as used herein generally refer to a biological material being tested for and/or suspected of containing an analyte of interest such as antibodies.
  • the sample may be any tissue sample from the subject.
  • the sample may comprise protein from the subject.
  • inhibitor and “antagonize,” as used herein, mean to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely.
  • Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down-regulate a protein, a gene, and mRNA stability, expression, function and activity, e.g., antagonists.
  • in vitro' refers to events that occur in an artificial environment, e.g, in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multi-cellular organism, such as a non-human animal.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.
  • Table 1 contains the primers used for fragment cloning.
  • Table 1 List of primers for SARS-CoV-2 replicon construction - Organized by PCR reaction, final fragments for subcloning and yeast transformation are highlighted in bold, all others are intermediate overlap PCR templates.
  • DNA fragments 2-6 and 8 were the same as described in Thao et al. (PMID: 32365353). The rest of the fragments were PCR-amplified from SARS-CoV-2 clone 3.1 (Thao et al. PMID: 32365353), except fragment 7, which was amplified from cDNA obtained by RT-PCR of viral RNA extracted from isolate USA-WA1/2020 (BEI #NR-5281), grown in Vero-E6 cells. RNA was extracted from cells using Trizol (ThermoFisher #15596026) and RNeasy mini kit (Qiagen #74104), and cDNA was prepared using SuperscriptTM IV First-Strand Synthesis System with random primers (Thermo #18091050).
  • PCR amplification reactions were performed using KOD Xtreme Hot Start DNA Polymerase (EMD Millipore #71975). Accessory sequences, such as Neon Green, Glue, NeoR, were amplified from plasmids or purchased as synthetic DNA (IDT). PCR amplicons of fragments
  • SARS-CoV-2 nspl and nspl2 were done using the two-step PCR method on fragments 2 and 7, respectively, using the primers listed in Table 1.
  • Pol(-) mutant was created by mutating SARS-CoV-2 RdRp (nspl2) D760, D761 catalytic residues to N760, N761 (SDD/SNN) (41).
  • Nspl mutant that does not bind the 40S ribosome was created by mutating K164A/H165A (28, 29). The mutated fragments were used for replicon assembly, as detailed below.
  • DNA fragments for assembly were prepared by restriction digestion or PCR as detailed in Table 2, and agarose gel was extracted. Table 2 Preparation of fragments for yeast transformation associated recombination
  • Yeast assembly was performed according to the protocol in Thao eial., 2020 (42). Briefly, 50-100 ng of each fragment was mixed in an equimolar ratio and transformed into Saccharomyces cerevisiae (S. cerevisiae) strain VL6-48N. Transformed yeast was grown for 2-3 days on selective -HIS plates at 30°C. 4-10 colonies from each plate were picked, re-streaked on a new plate, and grown for 2 days at 30°C.
  • the plasmid prep was digested with BamHI-HF enzyme (NEB #R3136T), which does not have restriction sites in any of the pCCl-BAC-His3- replicon plasmids.
  • BamHI-HF enzyme NEB #R3136T
  • DNA was digested with Plasmid-SafeTM ATP -Dependent DNase (Lucigen #E3101K) for 24h and cleaned by extraction with Phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation.
  • Final DNA concentration was measured using Qbit dsDNA HS Assay (ThermoFisher #Q32851)
  • MPA Multiple displacement amplification
  • Amplified DNA was digested with Notl-HF enzyme (NEB #R3189S) and cleaned up with phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation.
  • Notl-HF enzyme NEB #R3189S
  • phenol-chloroform-isoamylalcohol SigmaAldrich #77617
  • Sindbis replicon RNA was in-vitro transcribed from a SinRep5-GFP plasmid linearized with Xhol (NEB) (6), using SP6 mMessage mMachine High Yield Capped RNA Transcription kit (ThermoFisher #AM1340) RNA transcripts were electroporated into Huh7.5 or BHK-21 cells using adapted protocols originally developed for launching HCV (43). Briefly, Huh7.5 or BHK-21 cells were trypsinized, washed twice with ice-cold phosphate-buffered saline (PBS) (Invitrogen), and resuspended at 1.5 x 10 7 cells/ml in PBS.
  • PBS ice-cold phosphate-buffered saline
  • SARS-CoV-2 replicon RNA and 2 pg of SARS- CoV2 N mRNA were mixed with 0.4 ml of cell suspension in a 2-mm cuvette (BTX #45-0125) and immediately pulsed using a BTX ElectroSquare Porator ECM 830 (860V, 99 ps, five pulses). Electroporated cells were incubated at room temperature for 10 min prior to resuspension in plating media.
  • BHK-21 cells ATCC CCL-10, M. auratus
  • MEM Minimum Essential Medium
  • Calu-3 cells ATCC® HTB-55TM, H. sapiens ; sex: male
  • EMEM Eagle’s Minimum Essential Medium
  • TMEM41B-KO and dox-inducible TMEM4 IB-reconstituted Huh-7.5 cells were previously described (45). TMEM41B expression was induced by Doxy cy cline at least 24h before electroporation.
  • Huh7.5 cells containing repSARS-CoV-2 Glue, minirepSARS-CoV-2 Glue, or repSARS- CoV-2 Glue pol- replicons were seeded onto 12-well plates in triplicate at 1 x 10 5 cells/well and treated with lOOnM remdesivir or DMSO vehicle. After incubating for 24 or 48 hours at 37 °C, supernatants were aspirated, cells were washed three times with PBS and subsequently lysed in 250 m ⁇ Tri-reagent (Zymo, cat. #R2050) per well. RNA was extracted using the Direct-zol RNA Miniprep Plus kit (Zymo Research, cat.
  • RPS11 forward: 5’- GCCGAGACTATCTGCACTAC-3’ (SEQ ID NO: 147) and reverse: 5’- ATGTCCAGCCTCAGAACTTC-3’ (SEQ ID NO: 148)
  • SARS-CoV-2 subgenomic N Leader forward: 5’-GTTTATACCTTCCCAGGTAACAAACC-3’ (SEQ ID NO: 149) and N reverse: 5’-GTAGAAATACCATCTTGGACTGAGATC-3’ (SEQ ID NO: 150)).
  • SARS-CoV-2 primers targeting genomic N are from Chu et a!., 2020.
  • the following PCR conditions were used: 50 °C for 2 min and 95 °C for 2 min (initial denaturation); 45 cycles 95 °C for 1 sec, 60 °C for 30 sec (PCR); followed by 95 °C for 15 sec, 65 °C for 10 sec, a slow increase to 95 °C (0.07 °C/sec) for a melt curve.
  • the data were analyzed by melt curve analysis for product specificity as well as AACT analysis for fold changes (after normalization to housekeeping genes) and graphed using Prism 8 (GraphPad).
  • Fluorescent and brightfield images were taken with Nikon Eclipse TE300 fluorescent microscope at xlO magnification, using NIS-Elements 4.10.01 software (Nikon).
  • Flow cytometry was performed on a minimum of 10,000 single cells/sample using LSRII Flow cytometer (BD Biosciences). Data analysis was done using FloJo software (BD Biosciences).
  • AM580 was purchased from Cayman Chemical (#15261), Remdesivir and Masitinib were purchased from MedChemExpress (#HY- 104077 and #HY- 10209 respectively), 27- hydroxycholesterol (27HC) was purchased from Sigma Aldrich (#SML2042), Human IFN Alpha A (Alpha 2a) and Human IFN Beta (la) were purchased from Pbl assay science (#11100-1 and #11410-2 respectively).
  • BHK-21 cells were transfected with VSV-G or control plasmid, using Lipofectamine 3000 (ThermoFisher #L3000001), using a reverse-transfection protocol. 24h post transfection, 6 million cells were electroporated with 5ug replicon and 2ug N protein mRNA as detailed above. Each three electroporation reactions were combined into a T175 flask. Medium was replaced after a few hours overnight to remove free-floating RNA and dead cells.
  • nspl-nspl6 non-structural proteins
  • S structural proteins-spike
  • M membrane
  • E envelope
  • N nucleocapsid-and eight accessory proteins (3a, 3b, 6, 7a, 7b, 8b, 9b and 14) expressed from sub-genomic RNAs (FIG. 1A) (77).
  • a modulatory design was adopted to assemble two versions: a “minimal” replicon consisting of viral 5’ and 3’UTRs, Orfla/b, and N encoding regions, and a “full” replicon consisting of all viral proteins with the exception of spike (S).
  • the spike transcription-regulating sequence TRS was used to drive expression of a gene cassette consisting of neomycin-resistance (NeoR) and a reporter gene (nuclear-localized NeonGreen, or secreted Gaussia luciferase) separated by a T2A self-cleaving sequence (FIG. 1A).
  • Both versions contain an upstream T7 promoter for in vitro transcription at the 5’ end and a self-cleaving HDV ribozyme at the 3’ end, which cleaves immediately after an encoded polyA sequence.
  • RNA virus reverse genetics system in yeast was utilized (18). This system leverages transformation-associated recombination (TAR) to efficiently and accurately assemble numerous, large overlapping DNA fragments (19). After transforming yeast with equimolar ratios of replicon fragments and confirming proper assembly with multiplex PCR, restriction digests of the resulting DNA were performed to determine plasmid integrity. Using a spike deleted NeonGreen reporter SARS-CoV-2 replicon for optimization, yeast-derived plasmids were contaminated with genomic DNA and did not reveal the expected Ndel digest pattern (FIG. IB). To circumvent this, plasmid safe (PS) DNAse treatment was used to remove contaminating yeast genomic DNA.
  • PS plasmid safe

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Abstract

La présente invention concerne des réplicons d'ARN (par exemple, des réplicons d'ARN de SARS-CoV-2) qui peuvent être trans-empaquetés pour une administration à cycle unique dans une large gamme de types de cellules et récapitulant toutes les activités enzymatiques majeures de la réplication virale intracellulaire. En tant que plate-forme de confinement faible, les réplicons d'ARN de l'invention sont largement aptes à des études de virologie moléculaire et à des efforts de criblage de développement de médicament.
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FERNANDES ET AL.: "Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses", VIRUSES, vol. 12, no. 6, 2020, pages 598, XP055883449, DOI: https://doi.org/10.3390/v12060598 *
RICARDO-LAX INNA, LUNA JOSEPH M., THAO TRAN THI NHU, LE PEN JÉRÉMIE, YU YINGPU, HOFFMANN H.-HEINRICH, SCHNEIDER WILLIAM M., RAZOOK: "Replication and single-cycle delivery of SARS-CoV-2 replicons", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 374, no. 6571, 26 November 2021 (2021-11-26), US , pages 1099 - 1106, XP055956151, ISSN: 0036-8075, DOI: 10.1126/science.abj8430 *

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