WO2024097732A2 - Procédé de production de particules de type viral (vlp-na) autoréplicatives et chargées d'acide nucléique, et leurs utilisations - Google Patents

Procédé de production de particules de type viral (vlp-na) autoréplicatives et chargées d'acide nucléique, et leurs utilisations Download PDF

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WO2024097732A2
WO2024097732A2 PCT/US2023/078319 US2023078319W WO2024097732A2 WO 2024097732 A2 WO2024097732 A2 WO 2024097732A2 US 2023078319 W US2023078319 W US 2023078319W WO 2024097732 A2 WO2024097732 A2 WO 2024097732A2
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plant
vlp
protein
replicon
construct
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WO2024097732A3 (fr
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Qiang Chen
Huafang Lai
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Arizona State University ASU
Arizona State University Downtown Phoenix campus
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Arizona State University ASU
Arizona State University Downtown Phoenix campus
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    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/12011—Geminiviridae
    • C12N2750/12041—Use of virus, viral particle or viral elements as a vector
    • C12N2750/12043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2770/16011—Caliciviridae
    • C12N2770/16023—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
    • C12N2770/16011—Caliciviridae
    • C12N2770/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/16042—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Definitions

  • a Sequence Listing accompanies this application and is submitted as an XML file of the sequence listing named “112624.0143 l_SL_ST26.xml” which is 61,805 bytes in size and was created on October 30, 2023.
  • the sequence listing is electronically submitted via Patent Center with the application and is incorporated herein by reference in its entirety.
  • Plant biotechnology is currently limited by the cost, difficultly, and throughput of methods for probing and modifying plant genetics. To improve such methods, new methods for delivering polynucleotides to target plant cells that are more efficient, safer, and environmentally friendly are needed.
  • the present invention provides virus-like particles (VLPs) comprising a plant virus capsid protein and containing a replicon comprising a plant promoter operably linked to a heterologous polynucleotide.
  • VLPs virus-like particles
  • the 3’ end of the heterologous polynucleotide is flanked by a geminiviral short intergenic region (SIR) sequence, and the 5’ and 3’ ends of the replicon consist of a geminiviral LIR sequence.
  • SIR geminiviral short intergenic region
  • the present invention provides compositions comprising a VLP described herein and a carrier.
  • the present invention provides methods for producing a nucleic acid- loaded VLP. The methods comprise (a) introducing into a plant cell a first construct comprising a first replicon that comprises a plant promoter operably linked to a first polynucleotide encoding a plant virus capsid protein, wherein the 3’ end of the first polynucleotide is flanked by a geminiviral SIR sequence, and wherein the 5’ and 3’ ends of the first replicon consist of a geminiviral LIR sequence; (b) introducing into the plant cell a second construct comprising a second replicon that comprises a plant promoter operably linked to a second, heterologous polynucleotide, wherein the 3’ end of the heterologous polynucleotide is flanked by a geminiviral SIR sequence, and wherein the 5
  • the present invention provides methods of using a VLP described herein to deliver a heterologous polynucleotide to a plant cell.
  • the methods comprise delivering the VLP to a plant.
  • FIG. 1 demonstrates that plant-made Norwalk virus capsid protein (NVCP) virus-like particles (VLPs) efficiently bind and enter mammalian cells.
  • NVCP Norwalk virus capsid protein
  • VLPs virus-like particles
  • FIG. 2 demonstrates that smaller, deconstructed replicons result in more efficient protein expression in mammalian cells.
  • a plant-based geminiviral replicon comprising a mammalian green fluorescent protein (GFP) expression cassette i.e., pBY1050-GFP; -6973 bp
  • GFP expression was analyzed by microscopy.
  • FIG. 3 demonstrates that plants can be used to produce VLPs that are efficiently delivered into mammalian cells.
  • NVCP VLPs loaded with replicons encoding GFP were produced in N. benthamiana plants, purified, and incubated with Caco-2 cells for 48 hours. GFP expression in the Caco-2 cells was then analyzed by quantitating florescence intensity using flow cytometry.
  • FIG. 4 demonstrates that Rep/RepA expression enhances GFP expression from the viral replicon.
  • Caco-2 cells were transfected with a plasmid encoding the protein Rep/RepA (A).
  • Caco-2 cells that were not transfected with the Rep/RepA plasmid were used as a negative control (B).
  • 24 hours after transfection equal amounts (3 pg) of plant-produced replicon-loaded NVCP VLPs were incubated with the Caco-2 cells. GFP expression was observed 48 hours after VLP incubation using a fluorescent microscope.
  • FIG. 5 demonstrates that GFP replicon copy number is increased in the presence of Rep/RepA.
  • Caco-2 cells were transfected with a plasmid encoding the protein Rep/RepA (dots on right at each time point). Caco-2 cells that were not transfected with the Rep/RepA plasmid were used as a negative control (middle dots at each time point). 24 hours after transfection, equal amounts (3 pg) of plant-produced replicon-loaded NVCP VLPs were incubated with the Caco-2 cells. 48 hours after VLP incubation, quantitative PCR (qPCR) was performed using GFP-specific primers to analyze the copy number of GFP replicons in the Caco-2 cells. Caco-2 cells that were not incubated with VLPs were used as a negative control for PCR (left dots at each time point). Copy numbers are expressed as Ct values. (Note: A lower Ct value indicates that there is more DNA in sample.)
  • FIG. 6 demonstrates that genes delivered into mammalian cells by gene-encapsulating VLPs can result in the desired therapeutic effect.
  • NVCP VLPs that encapsulated genes of GFP or A10, a protein with inhibitive activity against SARS-CoV-2 (e.g., a viral inhibitory agent), were produced in plants by co-infiltration of gene constructs of NVCP + GFP or NVCP + A10. Vero cells were incubated with NVCP VLPs and then infected with SARS-CoV-2. Cells were then fixed and stained for the spike protein of SARS-CoV-2 and plates were imaged on an ELISPOT reader for viral foci. The percentage of inhibition on viral infection was measured by foci reduction. *** indicates p value ⁇ 0,0002.
  • FIG. 7A-7E provides maps of the vectors, referred to herein as (FIG. 7A) pBYR- 1118NVCP (SEQ ID NO:1), (FIG. 7B) pl060mRep (SEQ ID NO:2), (FIG. 7C) p!232mRepA (SEQ ID NO:3), (FIG. 7D) pBY1050-GFP (SEQ ID NO:4), and (FIG. 7E) pBY-1193GFP (SEQ ID NO: 5).
  • the present invention provides nucleic acid-loaded virus-like particles (VLPs) that are designed to deliver a nucleic acid into plant cells. Also provided are compositions comprising the nucleic acid-loaded VLPs, and methods of making and using the nucleic acid-loaded VLPs.
  • VLPs virus-like particles
  • the inventors describe a novel platform in which a DNA replicon encoding a polypeptide (e.g., inhibitory protein or vaccine) is loaded inside a VLP during its assembly in planta.
  • the polypeptide is placed under control of a constitutive promoter to allow for gene expression in a host cell after VLP uptake.
  • processes e.g., viral infection; gene therapy, delivery of gene editing reagents
  • the Examples are directed to mammalian VLPs for delivery to mammalian cells similar plant VLPs for delivery to plant cells are made using similar procedures.
  • the present invention provides virus-like particles (VLPs) comprising a plant virus capsid protein, such as an icosahedral or rod-shaped virus capsid protein, and containing a replicon comprising a plant promoter operably linked to a heterologous polynucleotide.
  • VLPs virus-like particles
  • the 3’ end of the heterologous polynucleotide is flanked by a geminiviral short intergenic region (SIR) sequence, and the 5’ and 3’ ends of the replicon consist of a geminiviral LIR sequence.
  • SIR geminiviral short intergenic region
  • virus-like particle refers to structures made of assembled viral proteins that are non-infectious because they lack some or all viral genetic content.
  • VLPs can be derived from enveloped or non-enveloped viruses.
  • VLPs can be naturally occurring or synthesized through the expression of viral structural proteins that can self-assemble into a virus-like structure.
  • the VLPs of the present invention are “nucleic acid-loaded,” meaning that they contain nucleic acids that are encapsulated in the viral protein structure.
  • capsid refers to the protein shell of a virus, which encloses its genetic material.
  • Viral capsids may consist of one or more proteins and are broadly classified according to their structure. The majority of viruses have capsids that have either a helical or an icosahedral structure.
  • the VLPs may comprise an icosahedral or helical capsid protein.
  • Icosahedral capsids which may comprise 20 equilateral triangular faces, approximate a sphere and are made up of subunits that self-assemble into a VLP even in the absence of the viral genome.
  • the icosahedral capsid protein used with the present invention maybe from a plant virus such that the VLPs can be used to deliver nucleic acids into plant cells.
  • Several viral capsid proteins are amenable to insertions of heterologous sequences, which allows one to display a protein (e.g., a targeting moiety) on the surface of the VLP.
  • the targeting moiety may allow a VLP comprising a mammalian capsid protein to target and invade a plant cell.
  • the capsid protein used with the present invention is preferably from a plant virus such that the VLPs can be used to deliver nucleic acids into plant cells.
  • Suitable plant virus capsid proteins for use in the VLPs include, without limitation, the cowpea mosaic virus (CPMV) coat proteins (i.e., large (L) and small (S) coat proteins), the cowpea chlorotic mottle virus (CCMV) coat protein, the brome mosaic virus (BMV) coat protein, the red clover necrotic mosaic virus (RCNMV) coat protein, the hibiscus chlorotic ringspot virus (HCRSV) coat protein, the tobacco mosaic virus (TMV) coat protein, and the potato virus X (PVX) coat protein.
  • the capsid proteins listed above are from nonenvelope plant viruses.
  • the capsid protein can be replaced with the glycoprotein (G protein) and matrix protein of the virus.
  • a “replicon” is a nucleic acid molecule that replicates from a single origin of replication.
  • the replicons used with the present invention include geminiviral genomic elements called long intergenic regions (LIRs), which allow them to replicate inside host cells in the presence of the proteins Rep and RepA via a rolling-circle mechanism that is described below.
  • LIRs long intergenic regions
  • the VLPs of the present invention are designed to deliver a replicon comprising a heterologous polynucleotide into plant cells. This replicon is referred to herein as the “cargo replicon”.
  • promoter refers to a DNA sequence that defines where transcription of a gene begins. RNA polymerase and the necessary transcription factors bind to the promoter to initiate transcription. Promoters are typically located directly upstream (i.e., at the 5' end) of the transcription start site. However, a promoter may also be located at the 3’ end, within a coding region, or within an intron of a gene that it regulates. Promoters may be derived in their entirety from a native or heterologous gene, may be composed of elements derived from multiple regulatory sequences found in nature, or may comprise synthetic DNA.
  • promoters may direct the expression of a gene in different tissues or cell types, at different stages of development, or in response to different environmental conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters,” whereas promoters that allow for controlled expression of a gene (e.g., under particular conditions or in the presence of a particular molecule) are referred to as “inducible promoters.” Suitable promoters for use with the present invention include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters. A promoter is “operably linked” to a gene if the promoter is positioned such that it can affect transcription of the gene.
  • the promoter included in the cargo replicon is a plant promoter.
  • a “plant promoter” is a promoter that drives gene expression in plant cells. Commonly used plant promoters include, without limitation, the 35 S promoter of the cauliflower mosaic virus, ubiquitin, tCUP cryptic constitutive promoter, the Rsyn7 promoter, the maize In2-2 promoter, and the tobacco PR- la promoter. Suitable plant promoters also include pathogen-inducible promoters, glucocorticoidinducible promoters, alcohol-inducible promoters, estrogen-inducible promoters, and tetracycline-inducible/repressible promoters.
  • polynucleotide and “nucleic acid” are used interchangeably to refer a polymer of DNA or RNA.
  • a polynucleotide may be single-stranded or double-stranded and may represent the sense or the antisense strand.
  • a polynucleotide may be synthesized or obtained from a natural source.
  • the term polynucleotide encompasses constructs, plasmids, vectors, and the like.
  • the VLPs of the present invention contain a cargo replicon that includes a heterologous polynucleotide.
  • a “heterologous polynucleotide” is a polynucleotide that is not naturally found in the viral genome from which the VLP is derived.
  • the heterologous polynucleotide included in the cargo replicon encodes a protein.
  • the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues connected to by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Proteins may include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs.
  • the heterologous polynucleotide may encode any protein of interest. Suitable proteins include, without limitation, protein-based drugs (e.g., antibodies), antigens, gene editing reagents (e.g., Cas9), and homing proteins.
  • protein-based drugs include the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibitory protein A10 (e g., a viral inhibitory agent) (SEQ ID NO:8 and SEQ ID NO:9).
  • antigen proteins include the spike (S) protein of SARS-CoV-2 and the envelope (E) protein of a flavivirus (e.g., West Nile virus, Dengue virus, Zika virus) er an alpha virus (e.g., Chikungunya virus), a Filovirus (e.g., Ebola virus), a Lentivirus (e.g., human immunodeficiency virus (HIV)), or an influenza virus (e.g., influenza virus A).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • E envelope protein of a flavivirus
  • an alpha virus e.g., Chikungunya virus
  • Filovirus e.g., Ebola virus
  • Lentivirus e.g.
  • a “a viral inhibitory agent” refers to any protein or nucleic acid capable of inhibiting the life cycle of a virus, resulting is reduced viral load, reduced viral infection symptoms, or prevention of viral infection.
  • Viral inhibitory agents may include but not be limited to antibodies that target viral proteins and nucleic acids that bind either viral proteins or viral genome sequences and RNAs.
  • homing protein refers to a protein that interacts with a tissue or cell type-specific surface protein. Expression of a homing protein on the surface of a VLP targets the VLP to particular cells. Thus, in some embodiments, the VLP expresses the protein encoded by the heterologous polynucleotide on its surface.
  • the heterologous polynucleotide encodes an RNA molecule.
  • the heterologous polynucleotide may encode a small interfering RNA (siRNA), short hairpin RNA (shRNA), anti-sense RNA, microRNA (miRNA), or guide RNA (gRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • gRNA guide RNA
  • the DNA replicon tested in the Examples is a deconstructed, single-stranded DNA plant virus replicon based on the bean yellow dwarf virus (BeYDV) of the Geminiviridae family.
  • the BeYDV genome contains a long intergenic region (LIR) and short intergenic region (SIR) with four protein-encoding genes lying therebetween.
  • the movement protein (MP) and the capsid protein (CP) genes occur on the sense (V) strand of the viral genome, while the replication initiator protein (Rep) and RepA protein genes lie on the complementary-sense (C) strand.
  • Alternative splicing of the C transcript yields mRNAs for Rep and RepA.
  • the LIR and SIR are the only cis-acting elements required for replication of the viral genome.
  • the LIR contains a bi-directional promoter and a stem-loop structure that is essential for initiation of rolling-circle replication.
  • the SIR is the origin of C-strand synthesis and contains transcription termination and polyadenylation signals. Geminiviral vectors are described in US Patent No. 10,125,373 and US Publication No. 20190336596, which are incorporated herein by reference.
  • Geminiviruses replicate DNA to high levels inside plant cells via a rolling-circle mechanism in which the Rep protein provides nicking and ligating functions.
  • viral single-stranded DNA enters the cell nucleus and is converted into double-stranded DNA, which serves as the template for viral transcription and further replication.
  • Rep nicks the positive sense (V) strand at a specific sequence within the LIR and then covalently binds to the 5' terminus. The 3'-OH terminus is used as the primer for the synthesis of the nascent plus strand.
  • DNA synthesis is accomplished by host replication proteins, including DNA polymerases. Completion of the nascent plus strand regenerates the origin of replication, which is nicked by the Rep protein again.
  • Rep protein then acts as a terminase to release the displaced plus strand and simultaneously ligates it into a circular form. In the process, Rep is transferred to the newly created 5' terminus. Late in the replication cycle single-stranded viral genomes are generated for encapsidation. RepA has an autoregulatory effect on Rep gene expression. It binds to a Rep binding site located within the LIR and is hypothesized to play a role in inhibiting complementary-sense gene expression and trans-activating sense gene expression. RepA has also been observed to bind the retinoblastoma-related protein (RBR), suggesting that it may help to arrest the infected plant cell in S-phase by interacting with RBR, thereby, creating conditions favorable for viral DNA replication. In plants, both Rep and RepA are required for replicons to self-replicate and the ratio of these proteins affects replication efficiency.
  • RBR retinoblastoma-related protein
  • the replicons used with the present invention comprise geminiviral long intergenic region (LIR) sequences at their 5’ and 3’ ends.
  • LIR geminiviral long intergenic region
  • a geminiviral long intergenic region (LIR) is a portion of a geminiviral genome that contains a Rep binding site that is capable of mediating excision and replication by a geminiviral Rep protein.
  • the LIRs define the portion of a construct that will be replicated as part of the replicon via the rolling-circle mechanism described above.
  • An exemplary geminiviral LIR sequence from bean yellow dwarf virus (BeYDV) is provided as SEQ ID N0:6.
  • the replicons may also comprise a geminiviral short intergenic region (SIR) sequence.
  • a geminiviral short intergenic region SIR
  • SIR is a portion of a geminiviral genome that contains bidirectional transcription terminator signals. SIRs are suspected to be the origin of complementary strand synthesis. In the cargo replicon, SIR flanks the heterologous polynucleotide on its 3’ end to aid in transcriptional termination.
  • An exemplary geminiviral SIR sequence from bean yellow dwarf virus (BeYDV) is provided as SEQ ID NO:7.
  • the replicons described herein can be made to be self-replicating by inclusion of a polynucleotide encoding Rep and RepA in the replicon.
  • Rep and RepA can be provided in trans (i.e., on a separate construct) from the heterologous polynucleotide.
  • the VLPs comprise a polynucleotide encoding both Rep and RepA.
  • Rep and RepA are encoded by two separate polynucleotides.
  • the polynucleotide(s) encoding Rep and/or RepA are operably linked to an inducible promoter such that replicon replication can be controlled.
  • construct refers to a recombinant polynucleotide.
  • a construct may be single-stranded or double-stranded and may represent the sense or the antisense strand.
  • a “recombinant polynucleotide” is an artificially constructed polynucleotide that includes polynucleotide sequences derived from at least two different sources. The constructs described herein may be prepared using standard techniques such as cloning, DNA and RNA isolation, amplification, and purification.
  • compositions for delivery into plant cells are:
  • compositions comprising a VLP described herein and a carrier for delivery into plant cells.
  • Carriers include, but are not limited to, diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), solubilizing agents (e.g., glycerol, polyethylene glycerol), emulsifiers, liposomes, and nanoparticles.
  • Carriers may be aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include isotonic solutions, alcoholic/aqueous solutions, emulsions, and suspensions, including saline and buffered media.
  • compositions of the present invention may further include additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), bulking substances, or tonicity modifiers (e.g., lactose, mannitol).
  • additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), bulking substances, or tonicity modifiers (e.g., lactose, mannitol).
  • compositions may be covalently attached to polymers (e.g., polyethylene glycol), complexed with metal ions, or incorporated into or onto particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels) or onto liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • polymeric compounds e.g., polylactic acid, polyglycolic acid, hydrogels
  • liposomes e.g., microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • lipophilic depots e.g., fatty acids, waxes, oils
  • compositions may be packaged into vials or other suitable containers, including individual or multi-dose ampoules.
  • compositions may be lyophilized before packaging, allowing them to be stored for extended periods of time without loss of viability at ambient temperatures.
  • lyophilization refers to freezing of a material at low temperature followed by dehydration by sublimation, usually under a high vacuum. Lyophilization is also known as freeze drying. Many freezing techniques can be used in lyophilization, such as tray-freezing, shelffreezing, spray-freezing, shell-freezing, and liquid nitrogen immersion. Each technique will result in a different rate of freezing.
  • the present invention provides methods for producing a nucleic acid- loaded VLP.
  • the methods comprise (a) introducing into a plant cell a first construct comprising a first replicon that comprises a plant promoter operably linked to a first polynucleotide encoding a plant virus capsid protein, wherein the 3’ end of the first polynucleotide is flanked by a geminiviral SIR sequence, and wherein the 5’ and 3’ ends of the first replicon consist of a geminiviral LIR sequence; (b) introducing into the plant cell a second construct comprising a second replicon that comprises a plant promoter operably linked to a second, heterologous polynucleotide, wherein the 3’ end of the heterologous polynucleotide is flanked by a geminiviral SIR sequence, and wherein the 5’ and 3’ ends of the second replicon consist of a geminiviral LIR sequence; (c) introducing
  • the present methods involve introducing at least three constructs into a plant cell.
  • the first construct includes a first replicon that drives the expression of a plant virus capsid protein in plant cells. This replicon is used to produce the VLP capsid in the plant cell and is, thus, referred to herein as the “packaging replicon”.
  • the second construct includes a second replicon that drives the expression of a gene product (i.e., an RNA or protein) encoded by the heterologous polynucleotide in plant cell.
  • This replicon is the cargo that is packaged into VLPs for delivery to a target plant cell and is, thus, referred to herein as the “cargo replicon.”
  • the cargo replicon will be preferentially included in the VLPs due to its small size relative to the packaging replicon.
  • the third construct drives the expression of Rep and RepA in plant cells, which allows the first and second replicons (which both include LIR sequences at their 5’ and 3’ ends) to be replicated in the plant cell.
  • the third construct comprises an inducible plant promoter such that Rep/RepA expression, and therefore replicon replication, can be controlled.
  • Rep and RepA are expressed from two different constructs (i.e., the third construct and an additional, fourth construct).
  • constructs may be introduced into the plant cell using any known method of plant cell transfection including, without limitation, Agrobacterium-mediated transformation and particle bombardment.
  • one or more of the first construct, the second construct, and the third construct are integrated into the genome of the plant cell.
  • the transfection is transient.
  • the plant cell is part of a plant.
  • plant includes plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants, as well as plant parts, such as embryos, pollen, ovules, flowers, glumes, panicles, leaves, stems, roots, root tips, anthers, and pistils.
  • the transfected plant cell is part of a leaf of the plant.
  • the plant cell used to produce the VLP may be from any species of plant that supports VLP production and replicon replication, including both dicotyledonous and monocotyledonous plants.
  • Suitable plants dicotyledonous plants include, without limitation, tobacco, potato, Nicotiana benthamiana, tomato, cotton, beans, spinach, alfalfa, and lettuce plants.
  • Monocotyledonous plants include, without limitation, rice, maize (corn), wheat, barley, oat, rye, sorghum, and millet.
  • Nicotiana benthamiana is a relative of tobacco that is a useful host for recombinant protein expression from viral vectors because it supports the replication of many different viruses.
  • the plant is Nicotiana benthamiana.
  • a “plant promoter” a promoter that drives gene expression in plant cells.
  • Suitable plant promoters for use in these constructs include, without limitation, the 35S promoter of the cauliflower mosaic virus promoter, ubiquitin promoter, tCUP cryptic constitutive promoter, Rsyn7 promoter, maize In2-2 promoter, and tobacco PR- la promoter.
  • Suitable plant promoters also include pathogen-inducible promoters, glucocorticoid-inducible promoters, alcohol-inducible promoters, estrogen-inducible promoters, and tetracycline-inducible/repressible promoters.
  • the VLPs produced by the plant cell may be harvested using any suitable method known in the art.
  • the VLPs are purified away from the plant material using centrifugation-based methods such as rate-zonal density, isopycnic, and gradient density centrifugation; or chromatography-based methods such as ion-exchange, affinity, hydrophobic interaction, or metal-chelation chromatography.
  • VLPs can also be purified by various filtration method such as diafiltration and ultrafiltration, and by precipitation method such as low pH precipitation.
  • the VLPs are harvested at least 3 days after the constructs are introduced into the plant cell.
  • VLPs After the VLPs have been harvested, they can be used to introduce the cargo replicon into a target plant cell.
  • the VLPs can be used to introduce nucleic acids into both cultured plant cells (i.e., by adding the VLPs to the culture) and plant cells in vivo (i.e., by delivering the VLPs to a plant and allowing them to infect any cells that are bound by the viral capsid protein).
  • any of the constructs used in these methods may be included together on one polynucleotide.
  • two of the constructs, three of the constructs, or all four of the constructs may be provided as a single polynucleotide.
  • these replicons can be linked in tandem such that both replicons share a geminivirus LIR positioned between the replicons.
  • the third construct is included on the same polynucleotide as the first construct or the second construct.
  • the third construct is included on the same polynucleotide as the first construct such that the second construct remains small and is preferentially incorporated into VLPs during assembly in plant cells.
  • the present invention provides methods of using a VLP described herein to deliver a heterologous polynucleotide to a plant cell.
  • the methods comprise delivering the VLP to a plant.
  • the methods are used to modulate the expression of a gene in the plant.
  • the heterologous polynucleotide encodes a gene product that modulates gene expression.
  • the heterologous polynucleotide may encode a microRNA (miRNA) or a small interfering RNA (siRNA) that is used to repress the expression of a gene via RNA interference.
  • RNA interference is a natural mechanism for sequencespecific gene silencing.
  • miRNAs or siRNAs can either (1) direct enzyme complexes to degrade messenger RNA (mRNA) molecules and thus prevent their translation, or (2) inhibit transcription of the mRNA molecules by directing an enzyme complex to catalyze DNA methylation at specific genomic positions.
  • mRNA messenger RNA
  • the heterologous polynucleotide may encode a gene editing reagent that is used to edit a gene.
  • Gene editing may involve the introduction of deletions or insertions into a native gene, integrations of exogenous DNA, gene correction, and/or gene mutation.
  • Gene editing can be used to introduce a heterologous polynucleotide into the genome; to silence, reduce, or increase the expression of a native gene; or to modify the gene product produced by a native gene.
  • the gene editing reagent is a zinc-finger nuclease, TALEN, or nucleic-acid guided nuclease.
  • a “nucleic acid-guided nuclease” is any nuclease that cleaves DNA, RNA or DNA/RNA hybrids, and which uses one or more guide nucleic acids (gNAs) to confer specificity.
  • a nucleic acid-guided nuclease can be a DNA-guided DNA nuclease, a DNA-guided RNA nuclease, an RNA-guided DNA nuclease, or an RNA-guided RNA nuclease.
  • a nucleic acid-guided nuclease can be an endonuclease or an exonuclease.
  • a nucleic acid-guided nuclease may be naturally occurring or engineered.
  • the nucleic acid-guided nuclease is selected from the group consisting of Cas9, Cpfl, Cas3, Cas8a-c, CaslO, Casl3, Casl4, Csel, Csyl, Csn2, Cas4, Csm2, Cm5, Csfl, C2c2, CasX, CasY, Casl4, and NgAgo.
  • the nucleic acid-guided nuclease can be from any bacterial or archaeal species.
  • the nucleic acid-guided nuclease is from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophiles, Treponema denticola, Francisella tularensis, Pasteurella multocida, Campylobacter jejuni, Campylobacter lari, Mycoplasma gallisepticum, Nitratifractor salsuginis, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria cinerea, Gluconacetobacter diazotrophicus, Azospirillum, Sphaerochaeta globus, Flavobacterium columnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasma mobile, Lactobacillus farciminis, Streptococcus pasteurianus, Lactobacillus johnsonii
  • the gene editing reagent is a guide nucleic acid.
  • a “guide nucleic acid (gNA)” is a nucleic acid that targets a nucleic acid-guided nuclease to a specific genomic sequence (i.e., the target sequence) via complementary base pairing.
  • the complementary portion of a gNA comprises at least 10 contiguous nucleotides, and often comprises 17-23 contiguous nucleotides that are complementary to the target sequence.
  • the complementary portion of the gNA may be partially or wholly complementary to the target sequence.
  • the gNA is from 20 to 120 bases in length, or more.
  • the gNA can be from 20 to 60 bases, 20 to 50 bases, 30 to 50 bases, or 39 to 46 bases in length.
  • the gNA may comprise DNA and/or RNA.
  • the gNA is a chemically modified gNA.
  • the gNA may be chemically modified to decrease a cell's ability to degrade the gNA.
  • Suitable gNA chemical modifications include 2'-fluoro (2' — F), 2'-O-methyl (2'-0 — Me), S-constrained ethyl (cEt), 2'-O-methyl (M), 2'-O-methyl-3'- phosphorothioate (MS), and/or 2'-O-methyl-3'-thiophosphonoacetate (MSP).
  • the gNA is composed of two molecules that base pair to form a functional gRNA: one comprising the region that binds to the nucleic acid-guided nuclease and one comprising a targeting sequence that binds to the target sequence.
  • the gNA may be a single molecule comprising both of these components, e.g., a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • delivering refers to the introduction of a substance into a plant. Suitable routes for delivering the VLPs described herein include cutting the plant, wounding the plant, vector-based transmission (e.g., aphid transmission), chemical treatment, vacuum infiltration, and particle bombardment.
  • vector-based transmission e.g., aphid transmission
  • chemical treatment e.g., vacuum infiltration, and particle bombardment.
  • the plant cell to which the heterologous polynucleotide is delivered may be from any species of plant and any part of a plant so long as the plant virus capsid protein can bind to the cell.
  • the target plant cell may be a cultured plant cell or a plant cell that is part of a plant or a plant part.
  • the methods of the present invention may be used to improve or introduce a trait of agronomic interest within the plant. Examples of such traits include, without limitation, herbicide resistance, resistance against a bacterial, fungal, or viral infection, insect resistance, pest resistance, resistance to extreme temperatures, drought resistance, male sterility, male fertility, enhanced nutritional quality, industrial usage, yield stability, higher seed yield, and yield enhancement. Many examples of genes that confer such traits have been described in the literature and are well known in the art.
  • the plant is a crop.
  • a “crop” is a plant that can be grown and harvested for profit or subsistence.
  • the methods may be used to increase the yield of the crop or improves the taste of the edible portion of the crop.
  • the plant produces a protein or gene product that may be a therapeutic such as an antibody.
  • the plant can be used to administer the therapeutic protein to a subject.
  • the subject may have a disease, and the heterologous polynucleotide encodes a gene product that treats the disease. Any disease that can be treated using a protein-based drug, RNA- based drug, or gene editing may be treated using the methods of the present invention.
  • treating describes the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treating includes means to prevent the onset of the symptoms or complications, to alleviate the symptoms or complications, or to eliminate the disease, condition, or disorder.
  • the plants generated herein are used to induce (e.g., cause) a therapeutic effect in the subject that is suffering from a disease, debilitating condition or injury.
  • Diseases may include any infectious disease, deficiency disease, or immune disease.
  • the disease may include a viral disease, such as influenza and SARS-CoV-2.
  • the VLPs may include a heterologous polynucleotide that encodes a viral inhibitory polypeptide.
  • the VLP may comprise an antibody, protein binding fragment, or antigen binding fragment that binds a viral component, causing inhibition of the virus.
  • the heterologous polynucleotide encodes A10, a polypeptide known to have inhibitory activity against SARS-CoV-2.
  • antibody refers to naturally occurring and synthetic immunoglobulin molecules and immunologically active portions thereof (i.e., molecules that contain an antigen binding site that specifically bind the molecule to which antibody is directed against).
  • antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments (e.g., antigen binding fragments) as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments.
  • antibody examples include: single chain Fvs (scFvs), Fab fragments, Fab’ fragments, F(ab’)2, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • the term “antigen” refers to a molecule that can initiate a humoral and/or a cellular immune response in a recipient.
  • antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, and antigens involved in autoimmune disease, allergy, and graft rejection.
  • the antigen is a disease-specific antigen, i.e., an antigen associated with or specific to a disease.
  • the antigen is a tumor antigen, i.e., an antigen that is preferentially expressed on the surface of a tumor cell and not expressed on normal, healthy cells.
  • a DNA replicon comprising a heterologous polynucleotide is loaded into plant-based virus-like particles (VLPs) during assembly in planta in Nicotiana benihamiana.
  • VLPs plant-based virus-like particles
  • the heterologous polynucleotide is placed under the control of a constitutive mammalian promoter to allow it to be expressed in the cells of an inoculated mammalian host following VLP uptake.
  • a similar system is under development for delivery to plants using a constitutive plant promoter to drive expression of a heterologous polynucleotide for expression in a plant without risk of infection by a plant virus.
  • the DNA replicon that was tested by the inventors is a deconstructed, single-stranded DNA plant viral replicon from the bean yellow dwarf virus of the Geminiviridae family. This plant virus replicates to high levels inside plant cells via a rolling circle mechanism. Its replication is dependent on two short gene sequences, i.e., the long intergenic region (LIR) and the short intergenic region (SIR), as well as at least one of the replication associated proteins, Rep and RepA.
  • LIR long intergenic region
  • SIR short intergenic region
  • Rep and RepA the replication associated proteins
  • NVCP Norwalk virus capsid protein
  • the plant-based expression vector used to produce the VLPs i.e., a vector containing the same LIR, SIR, and Rep/RepA genomic elements described above
  • a deconstructed replicon comprising a sequence encoding green fluorescent protein (GFP) under control of a constitutive mammalian promoter.
  • GFP green fluorescent protein
  • the smaller, deconstructed replicon pBY-1193GFP, SEQ ID NO:5
  • pBY1050-GFP, SEQ ID NO:4 when equal masses of DNA were transfected (FIG. 2).
  • Vero cells were also shown to be successfully transduced by the VLPs (FIG. 6). Furthermore, Vero cells transduced with VLPs containing a heterologous sequence encoding the SARS-CoV-2 viral inhibitory agent A10 showed increased viral inhibition over Vero cells transduced with VLPs containing a heterologous sequence encoding GFP when subsequently infected with SARS-CoV-2. These results suggest that the VLPs are capable of transducing mammalian cells and expressing in the mammalian cell a functional protein (e.g., encoded by the heterologous sequence) that is capable of inhibiting viral challenge. These results also suggest that the VLPs may be suitable for treatment of disease in a subject, such as for treatment or prevention of viral infections.
  • RNA replicons derived from deconstructed plant viruses are routinely used in our laboratory, our self-replicating, dual VLP-DNA vaccine platform can be easily adapted to use plants to produce VLPs that encapsulate RNA replicons. Therefore, we refer to this technology platform as a VLP -nucleic acid (NA) dual system.
  • NA nucleic acid
  • VLP self-replicating, dual VLP -NA system
  • Many diverse and multivalent vaccines, therapeutic polypeptides, and nucleic acids can be generated from this single VLP.
  • VLP could design the VLP to display a heterodimer of an antigen (that is known to form dimers) from two different viruses (e.g., ZUCV and WNV), while incorporating the antigen gene from a different virus (e.g., one of the DENV serotypes) as the DNA component to make a trivalent vaccine.
  • This type of multivalent vaccine would offer protection against multiple viruses or viral serotypes, and/or subvert safety concerns of antibody dependent enhancement of infection (ADE) in the case of DENV infection.
  • ADE antibody dependent enhancement of infection
  • the VLP-NA system could also be used for other applications, such as gene therapy delivery.
  • it can be used to repress gene expression in a mammalian host through RNA interference (RNAi).
  • RNAi RNA interference
  • This can be accomplished via DNA-based RNAi or direct encapsulation of the RNA construct during VLP assembly and production.
  • a sequence that will form a short hairpin RNA (shRNA) upon transcription can be introduced into a mammalian cell such that it is processed by host cellular machinery to generate a short interfering RNA (siRNA) that degrades a host mRNA to repress a gene of interest.
  • siRNA short interfering RNA
  • the VLP-NA can be designed to be self-replicating.
  • RNA virus encapsulated in the VLP can be utilized as a gene expression tool, so long as elements of the rationally designed mRNA construct interact with the appropriate host factors and drive expression of the gene of interest. This would make the system comparable to the recently popularized Pfizer and Moderna mRNA vaccines against COVID- 19.
  • VLP can be optimized to target specific tissue and cell types for delivery of the nucleic acid payload, which is important for gene therapy.
  • a VLP can have an inherent avidity for a cell or tissue type, or it can be genetically engineered to display a peptide or protein ligand to interact with a cell type-specific receptor. Fusing part (or all) of a specific AAV capsid for surface display in this system would allow for specific tropism to certain cell types and would be more favorable for regulatory authorities, in that AAVs have been well studied and a few AAV-based gene therapies have been approved by the FDA.
  • the self-replicating nature of our deconstructed plant virus-based replicon ensures a prolonged delivery of the gene of interest, as well as increased safety over AAVs by utilizing plant viral elements for gene therapy applications. Since we can display the desired ligand protein on the surface of VLPs on demand, our technology can also allow the specific delivery of the NA cargo to the target cells, thereby, (1) minimizing the off-target side effect, another impediment of gene therapy, and (2) eliminating any safety concerns associated with the use of AAV or any other animal viruses.
  • This system can also be used for delivery of protein-based drugs via the packaged nucleic acid.
  • Intracellular delivery of nucleic acid that encodes an antibody, antibody derivative, or antigen binding fragment is possible and many diseases would benefit from specific intracellular targeting and suppression of protein-protein interactions.
  • the high specificity and affinity for the target epitope offers “intrabodies” a unique option for therapeutic treatments.
  • intracellular antibody delivery has been around for decades, many technical hurdles, particularly an efficient delivery method of the intrabody (or DNA/RNA encoding it), have limited the potential of intrabody therapies.
  • the VLP-NA system along with other advances in intrabody development, can overcome these limitations and open many novel avenues for disease treatment.
  • this system has potential to deliver a targeted, substantial dose of the antibody drug in nucleic acid format to have a significant therapeutic effect on the disease state by interfering directly or indirectly with the pathogen.
  • Diseases that have potential to benefit from this include many different cancers, viral diseases (after viral entry to the host cell), tuberculosis, and malaria, among others.
  • This system can also be used to deliver nucleic acid-based tools into a target organism or cells to alter specific gene expression for multiple applications.
  • the VLPs can deliver CRISPR/cas9 in the format of DNA or RNA to target cells for gene editing. This will have multiple applications in treating various diseases including genetic disorders.
  • our system will (1) overcome the difficulty in delivering CRISPR/cas9 into cells, (2) make the delivery more specific to target cells by surface display of a specific ligand on the surface of VLP, reducing the off-target effect, and (3) address the safety concern of using animal virus vectors.
  • this technology can also be used for gene editing for scientific research purposes or to improve the traits of an organism. For example, it can be used to improve a plant’s resistance to drought, extreme temperatures, insects/pests, or infections by viruses, bacteria, or fungi. It also can be used to increase crop yield or make crops resistant to herbicides.
  • the replicon is based upon, encapsulation of the Rep/RepA gene on a separate replicon alongside a gene of interest in a single VLP allows for at least one round of replication of the replicon inside the target cell, enhancing the potency of the delivered nucleic acid.
  • This method for nucleic acid encapsulation and cellular delivery has many applications including vaccine development, gene therapy, RNAi, and gene editing, among others, and has potential to facilitate safer and more efficacious treatments for a wide range of diseases.
  • VLPs virus-like particles
  • the capsid protein gene of Norwalk virus was modified from to include restriction enzyme recognition sites and was subcloned into a geminiviral vector (pBYR2eAK2Mc) based on the bean yellow dwarf virus for plant expression.
  • pBYR2eAK2Mc geminiviral vector
  • transformed EHA 105 Agrobacterium tumefaciens containing the plant codon optimized NVCP construct (pBYR- 1118NVCP, SEQ ID NO: 1) or the deconstructed viral vector containing the mammalian GFP construct (pBY-1193GFP, SEQ ID NO:5) were grown in YenB media supplemented with 100 pg/mL of kanamycin and 0.5 pg/mL of rifampicin.
  • a 1 : 1 ratio (as measured by ODeoo) of the pBYR-1118NVCP and pBY-1193 GFP-containing A. tumefaciens strains (in combination, these two strains produce VLPs encapsulating a nucleic acid encoding GFP) or the pBYR-1118NVCP strain alone (which produces VLPs that lack nucleic acid cargo) were syringe infiltrated into N. benthamiana leaves as previously described.
  • NVCP-VLP alone or the NVCP-VLP harboring the pBY-1193 GFP replicon were purified by sucrose gradient centrifugation as previously described. Purified NVCP-VLPs were then characterized as described above.
  • Caco-2 cells were plated at 10,000 cells/well in 100 pl DMEM complete medium in 96- well tissue culture plate the day before transfection.
  • LipofectamineTM 3000 (Thermofisher) was used for transient transfection of plasmids encoding GFP, Rep, or RepA, according to the manufacturer’s instructions.
  • 0.1 pg of plasmid DNA was diluted in 5 pl Opti-MEM (Thermofisher) with 0.2 pl of P3000 reagent and then mixed with 0.2 pl of LipofectamineTM 3000 diluted in 5 pl Opti-MEM. After a 10-minute incubation at room temperature, 10 pl of the DNA/LipofectamineTM 3000 mixture was added to each well of Caco-2 cells.
  • GFP expression was visualized, and images were taken 24, 48, or 72 hours after transfection using the Evos Imaging System (Invitrogen).
  • NVCP Norwalk virus capsid protein
  • Caco-2 cells were plated at 0.5 million/well in 6 well plates the day before the experiment. The next day, the media in each well was replaced with 1 ml fresh complete culture media and then 100 pg/ml of Norwalk virus capsid protein (NVCP) in PBS was added to experimental wells, and an equal volume of PBS was added to negative control wells (Opg NVCP). The cells were incubated with NVCP for 1 hour at 37 °C. After the incubation, the cells were washed twice with PBS and then scraped from the wells using a cell scraper (VWR) and resuspended in PBS on ice.
  • VWR cell scraper
  • each sample was incubated with guinea pig anti-NVCP serum (1:500) for 1 hour and then washed twice with PBS and incubated with FITC goat anti-guinea pig (1:200) for 30 minutes. Finally, the samples were washed twice with PBS and resuspended in 400 pl PBS.
  • the samples were fixed with 4% paraformaldehyde (Electro Microscopy Sciences) in PBS at room temperature for 10 minutes and were then washed once with PBS and permeabilized with 0.1% saponin (Sigma) for 15 minutes. After that, samples were stained with guinea pig anti-NVCP serum and FITC goat anti-guinea pig as described above. Both NVCP on the Caco-2 cell surface and intracellular NVCP were detected by flow cytometry. Detection of GFP expression in Caco-2 cells incubated with NVCP
  • Caco-2 cells were plated at 0.5 million/well in 6 well plates the day before the experiment. The next day, the media in each well was replaced with 1 ml of fresh complete culture media and then 0, 25, 50, or 75 pl of purified NVCP was added to each well. The cells were incubated for 48 hours. After that, the cells were washed and scraped from the wells and resuspended in PBS. Then GFP expression was detected by flow cytometry.
  • Caco-2 cells were plated at 20,000 cells/well in a 48-well tissue culture plate on Day 1. On Day 2, the cells were transfected with either empty parental plasmid or plasmid comprising Rep or RepA cDNA. On Day 3, 3 pg of NVCP in PBS was added to each well. Then the cells were incubated for 48 hours. On Day 5, GFP expression in each well was detected using the Evos Cell Imaging System (ThermoFisher).
  • Vero-E6 cells were plated at 20,000 cells on day 1. Each well was transfected with 0.05 ug/well Rep and RepA on day 2. 200ug/ml NVCP VLPs that encapsulate either A10 or GFP (negative control) gene construct were added to each well 5 hours after Rep/A transfection. On day 3, the cells were infected with 2000 pfu/well SARS-Cov2 WA strain. On day 4, Vero-E6 cells were fixed and stained with an antibody (CR3022) against the spike protein of SARS-CoV-2, followed by an HRP-conjugated goat antihuman antibody. The plate was then incubated with KPL Trueblue substrate (Seracare LifeScience) and imaged on an AID ELISpot Reader.
  • KPL Trueblue substrate Seracare LifeScience
  • a virus-like particle comprising a plant virus capsid protein and containing a replicon comprising a plant promoter operably linked to a heterologous polynucleotide, wherein the 3’ end of the heterologous polynucleotide is flanked by a geminiviral short intergenic region (SIR) sequence, and wherein the 5’ and 3’ ends of the replicon consist of a geminiviral long intergenic region (LIR) sequence.
  • SIR geminiviral short intergenic region
  • VLP of embodiment 1 or 2 wherein the plant virus capsid protein is a cowpea mosaic virus capsid protein or a tobacco mosaic virus capsid protein.
  • VLP of embodiment 4 wherein the protein is a protein-based pesticide, a proteinbased means of herbicide resistance, a homing protein, or a gene editing reagent.
  • VLP of embodiment 4 or 5 wherein the VLP expresses the protein encoded by the heterologous polynucleotide on its surface.
  • RNA is a small interfering RNA (siRNA), short hairpin RNA (shRNA), anti-sense RNA, microRNA (miRNA), or guide RNA (gRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • gRNA guide RNA
  • VLP of any one of embodiments 1-8 further comprising a construct comprising a plant promoter operably linked to a polynucleotide encoding Rep and RepA.
  • a composition comprising the VLP of any one of the preceding embodiments and a carrier.
  • a method for producing a nucleic acid-loaded VLP comprising: a) introducing into a plant cell a first construct comprising a first replicon that comprises a plant promoter operably linked to a first polynucleotide encoding plant virus capsid protein, wherein the 3’ end of the first polynucleotide is flanked by a geminiviral SIR sequence, and wherein the 5’ and 3’ ends of the first replicon consist of a geminiviral LIR sequence; b) introducing into the plant cell a second construct comprising a second replicon that comprises a plant promoter operably linked to a second, heterologous polynucleotide, wherein the 3’ end of the heterologous polynucleotide is flanked by a geminiviral SIR sequence, and wherein the 5’ and 3’ ends of the second replicon

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Abstract

La présente invention concerne des particules de type viral (VLP) chargées d'acide nucléique qui peuvent être produites dans des plantes et qui sont conçues pour acheminer un acide nucléique dans des cellules de plantes. L'invention concerne également des compositions comprenant les VLP chargées d'acide nucléique, et des méthodes de production et d'utilisation des VLP chargées d'acide nucléique.
PCT/US2023/078319 2022-11-01 2023-11-01 Procédé de production de particules de type viral (vlp-na) autoréplicatives et chargées d'acide nucléique, et leurs utilisations Ceased WO2024097732A2 (fr)

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CA2736796A1 (fr) * 2008-08-27 2010-03-04 Arizona Board Of Regents For And On Behalf Of Arizona State University Systeme replicon pour production rapide et a haut rendement de vaccins et de traitements a base d'anticorps monoclonaux chez les plantes
US11285204B2 (en) * 2017-09-22 2022-03-29 The Government Of The United States, As Represen Tobamovirus-based virus-like particles and vaccines
US20240417698A1 (en) * 2021-11-01 2024-12-19 Arizona Board Of Regents On Behalf Of Arizona State University A method for production of self-replicating, nucleic acid-loaded, virus-like particles (vlp-na) and the uses thereof

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