WO2023023597A1 - Virus-like particle - Google Patents
Virus-like particle Download PDFInfo
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- WO2023023597A1 WO2023023597A1 PCT/US2022/075142 US2022075142W WO2023023597A1 WO 2023023597 A1 WO2023023597 A1 WO 2023023597A1 US 2022075142 W US2022075142 W US 2022075142W WO 2023023597 A1 WO2023023597 A1 WO 2023023597A1
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- vlp
- viral
- rna
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- protein
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20023—Virus like particles [VLP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention relates to virus-like particles based on the SARS-CoV-2 virus and methods and systems thereof for delivery of a cargo molecule (e.g., nucleic acids) to a target cell (e.g., with low off-target effects).
- a cargo molecule e.g., nucleic acids
- the present invention is also related to interfering RNAs (e.g., siRNA and shRNA) and systems, compositions, and methods thereof for the treatment or prevention of viral infections (e.g., SARS-CoV-2 infection).
- CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos.
- virus-like particles comprising a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor and at least one additional viral structural protein.
- the at least one additional viral structural protein comprises: a viral envelope protein; a viral membrane protein; a viral nucleocapsid protein; or any combination thereof.
- the viral spike protein and at least one or all of the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from a coronavirus (e.g., SARS-CoV or SARS-CoV-2).
- the VLPs comprise an encapsulated cargo molecule or a nucleic acid encoding a cargo molecule.
- the cargo molecule or a nucleic acid encoding the cargo molecule may further comprise a packaging signal.
- the packaging signal is included upstream of the cargo molecule or the nucleic acid encoding the cargo molecule.
- the cargo molecule or the nucleic acid encoding the cargo molecule comprising a packaging signal is preferentially loaded into the VLP in comparison to a cargo molecule not comprising a packaging signal.
- the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof.
- the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof.
- the cargo molecule comprises a nucleic acid.
- the cargo molecule comprises an interfering RNA or a messenger RNA.
- the interfering RNA is configured to bind to and inhibit translation of a viral RNA.
- the viral RNA is from a respiratory virus (e.g., a coronavirus).
- the cargo molecule comprises a sequence encoding a gene.
- the cargo molecule comprises a sequence encoding a gene editing system (e.g., a CRISPR/Cas system).
- compositions comprising the disclosed VLPs.
- the compositions comprise a pharmaceutically acceptable carrier.
- the compositions are configured to induce an immune response.
- the compositions are vaccines comprising the VLP and an adjuvant. Also provided are methods and cells for producing the disclosed VLPs. Further disclosed are methods of using the VLPs.
- a viral infection in a cell, tissue, or organism comprising contacting the cell, tissue, or organism with an effective amount of a VLP, pharmaceutical formulation, vaccine, or other composition as described.
- the cell is in vitro.
- the cell is in vivo and the method comprises administering the disclosed VLP, pharmaceutical formulation, vaccine, or other composition to a subject.
- the methods may be adapted to treat or prevent any viral infection.
- the viral infection is caused by a respiratory virus (e.g., a coronavirus).
- methods for treating or preventing a disease or disorder in a subject comprising administering to the subject an effective amount of a VLP, a pharmaceutical formulation, a vaccine, or other composition as described.
- the disease or disorder comprises an infectious disease, cancer, a genetic disorder, or a combination thereof.
- Kits comprising any or all of the components of the VLPs or compositions or agents necessary for making or using the VLPs or compositions are also provided.
- interfering RNAs or nucleic acids encoding an interfering RNA comprise a nucleic acid sequence at least partially complementary to a target sequence of a virus genome.
- the virus is a respiratory virus.
- the virus is a coronavirus.
- the virus is SARS-CoV-2.
- the target sequence comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 3-17, or an RNA equivalent thereof. In some embodiments, the target sequence comprises a nucleic acid sequence with at least 80% identity to SEQ ID NOs: 3-17, or an RNA equivalent thereof. In some embodiments, the target sequence comprises a nucleic acid sequence of SEQ ID NOs: 3-17, or an RNA equivalent thereof.
- the interfering RNA is configured to prevent replication of the virus genome. In some embodiments, the interfering RNA is a small interfering RNA (siRNA).
- the interfering RNA is a small hairpin RNA (shRNA).
- shRNA small hairpin RNA
- the carrier comprises a delivery vehicle.
- the delivery vehicle may be selected from the group consisting of liposomes, viruses, virus-like particles, immunolipoplexes, cyclodextrins, micro- or nano-particles, aptamers, dendrimers, exosomes, chitosan, or derivatives thereof.
- the carrier is a virus or virus- like particle.
- the interfering RNA, or the nucleic acid encoding thereof further comprises a packaging signal.
- the packaging signal is included upstream of the interfering RNA, or the nucleic acid encoding thereof.
- the packaging signal is configured to facilitate loading of the interfering RNA or the nucleic acid encoding thereof into the virus or virus-like particle.
- the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof.
- the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof.
- the cell is in vitro.
- the cell is in vivo.
- the viral infection is caused by a respiratory virus.
- the respiratory virus is a coronavirus.
- the respiratory virus is SARS-CoV-2.
- the contacting comprises administering to a subject or a tissue thereof.
- the administering comprises systemic administration, administration to the lungs, including but not limited to either intranasal or aerosol delivery, or a combination thereof.
- Kits comprising any or all of the interfering RNA, the nucleic acid encoding an interfering RNA, or any components of the composition or system are additionally provided herein.
- Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.
- FIG. 1 is a schematic of an exemplary non-infectious virus-like particle based on SARS-CoV-2 and scenarios of treatment or prevention of viral infection.
- the VLP (blue) may be loaded with genomic cargo designed to interrupt translation of SARS-CoV-2 viral genome and infection by viral particles (green).
- FIG. 2 is a schematic of the generation of an exemplary VLP in which shRNAs targeting elements of the SARS-CoV-2 RNA genome is generated through transfection of a packaging cell line using either a single polycistronic construct containing the nucleocapsid (N), membrane (M), spike (S), envelope (E) structural proteins and genomic cargo of interest with upstream SARS-CoV-2 packaging signal sequence or a multi-plasmid transfection system.
- FIG. 3 is a schematic of the viral RNA of SARS-CoV-2 with the open reading frames coding and non-coding regions identified. Arrow heads indicate target locations of exemplary interfering RNA sequences.
- FIGS. 5A and 5B show production and loading of exemplary VLPs, as disclosed herein.
- FIG. 5A is an electron micrograph of a VLP.
- FIG. 5B is a graph of the relative mRNA expression from purified VLPs loaded with GFP mRNA using both the PS101 and PS582 packaging sequences, showing preferential loading of GFP mRNA with these leader sequences.
- FIGS. 6A-6B show protection of cells from SARS-CoV-2 toxicity with exemplary shRNAs.
- FIG. 6A is a graph of the percentage of an image area covered by cells acquired 48 hours after NSP1 transfection in either control (scramble RNA) expressing 293T cells or shRNA expressing 293T cells at 10x.
- FIG. 6B is a graph of relative transcript levels of NSP1 in cells 48 hours after NSP1 challenge.
- virus-like particles comprising a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor and at least one additional viral structural protein (e.g., envelope proteins, membrane proteins, nucleocapsid proteins, etc.), compositions comprising these VLPs, as well as methods for making and using these VLPs (e.g., for treatment or prevention of diseases or disorders (such as, viral infections)).
- ACE2 angiotensin-converting enzyme 2
- the disclosed interfering RNAs e.g., siRNA and shRNA
- systems, compositions, and methods thereof are suitable for the treatment and prevention of viral infections (e.g., SARS- CoV-2 infection).
- RNAs were designed to avoid unwanted off-target effects by comparing to known genome sequences for intended subjects (e.g., humans).
- Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
- Definitions The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures.
- comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide.
- nucleic acid or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L.
- the present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like.
- the polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
- the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
- a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem.
- nucleic acid or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double- stranded, and represent the sense or antisense strand.
- nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
- a “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds.
- the peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic.
- Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain.
- polypeptide and “protein,” are used interchangeably herein.
- complementary and complementarity refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson- Crick base-pairing or other non-traditional types of pairing.
- the degree of complementarity between two nucleic acid sequences can be indicated by the percentage of nucleotides in a nucleic acid sequence which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementary).
- Two nucleic acid sequences are “perfectly complementary” if all the contiguous nucleotides of a nucleic acid sequence will hydrogen bond with the same number of contiguous nucleotides in a second nucleic acid sequence.
- Two nucleic acid sequences are “substantially complementary” if the degree of complementarity between the two nucleic acid sequences is at least 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%.
- nucleic acid sequences hybridize under at least moderate, preferably high, stringency conditions.
- Exemplary moderate stringency conditions include overnight incubation at 37° C in a solution comprising 20% formamide, 5 ⁇ SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt’s solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 ⁇ SSC at about 37-50° C, or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook et al., infra.
- High stringency conditions are conditions that use, for example (1) low ionic strength and high temperature for washing, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50° C, (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1% Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42° C, or (3) employ 50% formamide, 5 ⁇ SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 ⁇ Denhardt’s solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at
- percent sequence identity refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity.
- nucleic acid according to the technology is longer than a reference sequence, additional nucleotides in the nucleic acid, that do not align with the reference sequence, are not taken into account for determining sequence identity.
- Methods and computer programs for alignment are well known in the art, including BLAST, Align 2, and FASTA.
- hybridization refers to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence.
- hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the T m of the formed hybrid. Hybridization methods involve the annealing of one nucleic acid to another, complementary nucleic acid, e.g., a nucleic acid having a complementary nucleotide sequence.
- a “double-stranded nucleic acid” may be a portion of a nucleic acid, a region of a longer nucleic acid, or an entire nucleic acid.
- a “double-stranded nucleic acid” may be, e.g., without limitation, a double-stranded DNA, a double-stranded RNA, a double-stranded DNA/RNA hybrid, etc.
- a single-stranded nucleic acid having secondary structure e.g., base- paired secondary structure
- higher order structure e.g., a stem-loop structure
- triplex structures are considered to be “double-stranded.”
- any base-paired nucleic acid is a “double-stranded nucleic acid.”
- the term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor of any of the foregoing.
- RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.
- a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or an RNA chain that has functional role to play in an organism.
- genes include regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
- a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.
- wild-type refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.
- a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene.
- mutant refers to a gene or gene product that displays modifications in sequence and or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
- variant refers to the exhibition of qualities that have a pattern that deviates from what occurs in nature. In some embodiments, a variant may also be a mutant.
- non-naturally occurring “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man.
- nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
- a “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.
- a cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell.
- the presence of the exogenous DNA results in permanent or transient genetic change.
- the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
- the transforming DNA may be maintained on an episomal element such as a plasmid.
- a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA.
- a “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such as a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein.
- mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
- non- mammals include, but are not limited to, birds, fish, and the like.
- the mammal is a human.
- treat means a slowing, stopping, or reversing of progression of a disease or disorder when provided a VLP, composition, or vaccine described herein to an appropriate control subject.
- “treating” means an application or administration of the methods, VLPs, or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease outcome, disease progression, or symptoms of the disease.
- the term “preventing” refers to partially or completely delaying onset of a disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
- the term “contacting” as used herein refers to bring or put in contact, to be in or come into contact.
- contact refers to a state or condition of touching or of immediate or local proximity.
- compositions to a target destination may occur by any means of administration known to the skilled artisan.
- a target destination such as, but not limited to, an organ, tissue, cell, or tumor
- the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the systems of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization of the system to a desired site.
- the systems can be administered by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.
- virus-like particle” and “VLP” are used interchangeably to refer to a structure that in at least one attribute resembles a virus but which has not been demonstrated to be infectious.
- interfering RNA refers to any nucleic acid molecule capable of mediating sequence specific RNAi, for example short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), translational silencing, and the like.
- siRNA short (or small) interfering RNA
- dsRNA double-stranded RNA
- miRNA micro-RNA
- shRNA short hairpin RNA
- ptgsRNA post-transcriptional gene silencing RNA
- translational silencing and the like.
- the interfering RNA include synthetic double stranded small interfering RNA (siRNA) and short hairpin RNA (shRNA).
- siRNA synthetic double stranded small interfering RNA
- shRNA short hairpin RNA
- Such molecules are constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos.5,898,031, 6,107,094, 6,506,559, 7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.
- VLPs Virus-Like Particles
- VLPs are structures that morphologically resemble a virus but are devoid of the genetic material required for viral replication and infection. Since they are non-replicative in nature, they are safe for administration to cells or subjects and can target physiologically relevant receptors with their surface proteins and, therefore, tissues in a subject, resulting in more effective delivery of cargo, antibody induction, or inhibition of infectious forms of viruses which target the same or similar tissues and receptors.
- virus-like particles comprising a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor and at least one additional viral structural protein.
- ACE2 angiotensin-converting enzyme 2
- the viral spike protein may be derived from any virus.
- the spike protein is derived fully or partially from a respiratory virus.
- Respiratory viruses include, but are not limited to: influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses.
- the spike protein may be derived from a natural viral spike protein with natural tropism for binding ACE2 receptors.
- the spike protein is derived from a coronavirus.
- the coronavirus family comprises 45 species distributed between four genera: alphacoronavirus, betacoronavirus, deltacoronavirus, and gammacoronavirus virus.
- the spike protein is derived from a betacoronavirus.
- the spike protein is derived from SARS-CoV or SARS-CoV- 2.
- the spike protein is wild-type spike protein from SARS-CoV-2 (Accession # QHD43416).
- the invention is not limited to this exemplary sequence.
- SARS-CoV-2 spike protein may comprise the wild-type amino acid sequence or variant having an amino acid sequence that is at least about 70% identical (e.g., 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%, about85%, 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%, about 99%, or about 100%) to the amino acid sequence of the wild- type spike protein.
- 70% identical e.g., 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%, about85%, about 86%, about 87%, about 88%
- the spike protein may, alternatively, be a chimera in which a viral spike protein is genetically modified to include a spike protein ACE2 receptor binding domain from a different virus or a receptor binding domain from another ACE2 binding protein.
- the spike protein may contain an ACE2 binding domain from a coronavirus, e.g., SARS-CoV or SARS-CoV-2.
- the at least one additional viral structural protein may be a nucleocapsid protein, an envelope protein, a membrane protein, or a fragment or complex thereof.
- viral structural proteins used for the present invention may consist of, consist essentially of, or comprise a nucleocapsid protein, an envelope protein, a membrane protein, and/or a fragment or complex thereof.
- the at least one additional viral structural protein comprises a viral envelope protein, a viral membrane protein, and nucleocapsid protein.
- the at least one additional viral structural protein may be derived from any virus, preferably a respiratory virus.
- Respiratory viruses include but are not limited to influenza viruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinoviruses, coronaviruses, adenoviruses, and bocaviruses.
- at least one or all of the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from a coronavirus.
- the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from SARS-CoV-2.
- the nucleocapsid protein is wild-type nucleocapsid protein from SARS-CoV-2 (Accession # QHD43423).
- the envelope protein is wild-type envelope protein from SARS-CoV-2 (Accession # QHD43418).
- the membrane protein is wild-type membrane protein from SARS-CoV-2 (Accession # QHD43419).
- the invention is not limited to these exemplary sequences.
- SARS-CoV-2 structural proteins may comprise the wild-type amino acid sequence or variant having an amino acid sequence that is at least about 70% identical (e.g., 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%, about85%, 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%, about 99%, or about 100%) to the amino acid sequence of the respective structural protein.
- 70% identical e.g., 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%, about85%, about 86%, about 87%, about 88%, about
- the nucleocapsid, the envelope, and/or the membrane protein may be from or derived from SARS-CoV-2 variants (e.g., alpha, gamma, beta, delta, and omicron variants) or subvariants or sublineages thereof.
- SARS-CoV-2 variants e.g., alpha, gamma, beta, delta, and omicron variants
- the SARS-CoV-2 variant is B.l.1.7 (Alpha), B.1.351 (Beta) and B.l.617.2 (Delta), and/or B.l.1.529 (Omicron).
- the N protein may be from or derived from lineage A Wuhan strain SARS-CoV-2, B.1.351 variant SARS-CoV-2, B.1.617.2 variant SARS-CoV-2 or B.1.1.529 variant SARS-CoV- 2.
- Any of the proteins described herein may comprise one or more amino acid substitutions as compared to the corresponding wild-type protein.
- amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence.
- Amino acids are broadly grouped as “aromatic” or “aliphatic.”
- An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp).
- Non- aromatic amino acids are broadly grouped as “aliphatic.”
- “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).
- the amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative.
- the phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property.
- a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).
- conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained.
- “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups.
- Non-conservative mutations involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.
- the VLPs of the invention may further comprise an encapsulated cargo molecule.
- Cargo molecules can include nucleic acid molecules, chemotherapeutic agents, imaging agents, and/or other agents.
- the cargo molecule comprises a nucleic acid.
- the cargo molecule may be an RNA molecule, such as, an interfering RNA or a messenger RNA.
- the cargo molecule comprises a nucleic acid encoding a gene (e.g., a messenger RNA).
- the messenger RNA encodes the interfering RNA.
- Interfering RNAs include synthetic double stranded small interfering RNA (siRNA) or short hairpin RNA (shRNA). Both mimic endogenous microRNAs (miRNAs) and carry out RNA interference (RNAi), a gene-silencing mechanism facilitated by small RNAs, which is highly dependent on gene sequences silencing genes of interest with high specificity.
- siRNA small interfering RNA
- shRNA short hairpin RNA interference
- short hairpin RNAs or “shRNA” refer to an RNA sequence comprising a double- stranded region and a loop region at one end forming a hairpin loop. The double-stranded region is typically about 19 to about 29 nucleotides in length, and the loop region is typically about 2 to about 10 nucleotides in length.
- shRNA are processed by the enzyme Dicer into an active RNAi species of about 21 nucleotides.
- “Small interfering RNA” or “siRNA” refer an RNA molecule comprising a double stranded region and, optionally, a 3 ⁇ overhang of nonhomologous residues at each end.
- the double-stranded region is typically about 18 to about 30 nucleotides in length, and the overhang may be of any length of nonhomologous residues, but a 2 nucleotide overhang is preferred.
- siRNA and shRNA have been demonstrated to be effective in in vitro and in vivo applications, each with their respective advantages.
- siRNA are structurally designed to promote efficient incorporation into the RNA Induced Silencing Complex (RISC).
- the interfering RNAs can be provided on a nucleic acid or vector which utilizes the host micro RNA biogenesis pathway to express the shRNA, allowing for modulation or regulation by promoters, as described elsewhere herein, or can be provided as a single nucleic acid.
- sequences for interfering RNAs e.g., shRNA
- the interfering RNA is configured to prevent replication of the viral genome.
- the interfering RNAs hybridize to target sequences of the viral genome of a respiratory virus.
- Respiratory viruses include but are not limited to influenza viruses, respiratory syncytial virus, parainfluenza viruses, rhinoviruses, coronaviruses, and adenoviruses.
- the respiratory virus comprises a coronavirus, for example, a betacoronavirus, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2.
- the respiratory virus is SARS-CoV-2.
- Interfering RNA suitable for use with the VLPs described herein include those which bind to and inhibit translation of viral RNA.
- the VLPs described herein may deliver interfering RNA to cells infected or at risk of being infected with a respiratory virus (e.g., a coronavirus).
- the interfering RNA targets, or fully or partially hybridizes to, coding sequences within the genome of a respiratory virus (e.g., SARS-CoV-2 viral RNA).
- the interfering RNA may hybridize with coding sequences for non-structural proteins or an RNA-dependent RNA polymerase.
- the interfering RNA included in the VLPs described herein inhibit translation of SARS-CoV-2 viral RNA (e.g., by targeting the nucleic acid encoding the viral replicase).
- the interfering RNA targets, or fully or partially hybridizes to, coding sequences within the genome of SARS-CoV-2 viral RNA.
- the interfering RNA may hybridize with coding sequences for non-structural proteins or an RNA- dependent RNA polymerase.
- the interfering RNA is fully or partially complementary to target sequences comprising a nucleic acid sequence with at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) to SEQ ID NOs: 3-17, or an RNA equivalent thereof.
- the interfering RNA comprises a sequence capable of hybridizing to target sequences comprising nucleic acid sequences with at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%) to SEQ ID NOs: 3-17 as shown in Table 1, or an RNA equivalent thereof.
- the interfering RNA comprises a sequence capable of hybridizing to target sequences comprising SEQ ID NOs: 3-17 as shown in Table 1.
- the interfering RNA sequence may comprise an exact reverse complement to the target sequence or, alternatively, may have sufficient homology to the reverse complement of the target sequence to result in efficient and stringent hybridization and/or efficient interference.
- the interfering RNA may comprise a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides different from that of the reverse complement to the target sequence.
- Table 1 The interfering RNAs may comprise additional nucleotides or residues in addition the sequence responsible for recognizing and binding the target sequence.
- shRNAs comprise a loop region separate from the region comprising the sequence which bind to the target nucleic acid sequence.
- the present disclosure also provides for DNA segments encoding the interfering RNAs disclosed herein, vectors containing these segments and cells containing the vectors.
- the vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment.
- the person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
- vectors of the present disclosure can drive the expression of the interfering RNAs in both prokaryotic and eukaryotic cells.
- the vectors of the present disclosure can drive the expression in mammalian cells using a mammalian expression vector.
- mammalian expression vectors examples include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference).
- the vector's control functions are typically provided by one or more regulatory elements.
- suitable expression systems and regulatory elements for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL.
- the cargo molecule comprises a nucleic acid sequence encoding a gene editing system.
- the gene editing system may comprise a CRISPR-Cas system.
- a “CRISPR-Cas system” refers collectively to transcripts and other elements involved in the expression of and/or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, Cas protein, a cr (CRISPR) sequence (e.g., crRNA or an active partial crRNA), or other sequences and transcripts from a CRISPR locus.
- one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. Any element of any suitable CRISPR/Cas gene editing system known in the art can be employed in the systems and methods described herein, as appropriate. CRISPR/Cas gene editing technology is described in detail in, for example, U.S. Patent Nos.
- the gene editing system may comprise one or more Cas proteins (e.g., Cas9) and at least one guide RNA directed to a target nucleic acid.
- Cas9 Cas9
- guide RNA RNA sequences employed in CRISPR/Cas systems
- gRNA guide RNA
- sgRNA single guide RNA
- guide RNA single guide RNA
- single guide RNA single guide RNA
- the terms “guide sequence,” “guide,” and “spacer,” are used interchangeably herein and refer to the nucleotide sequence within a guide RNA that specifies the target nucleic acid.
- the target nucleic acid is a nucleic acid endogenous to a target cell.
- the target nucleic acid is a genomic DNA sequence.
- genomic refers to a nucleic acid sequence (e.g., a gene or locus) that is located on a chromosome in a cell.
- the target nucleic acid is a DNA or RNA sequence from an infectious agent, for example an infectious bacteria or virus.
- the target nucleic acid encodes a gene or gene product.
- gene product refers to any biochemical product resulting from expression of a gene. Gene products may be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, micro RNA (miRNA), and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA).
- mRNA messenger RNA
- the target nucleic acid sequence encodes a protein or polypeptide. In some embodiments, the target nucleic acid sequence encodes a mutant protein.
- the target nucleic acid sequence encodes genes involves in the progression of cancer e.g., oncogenes or tumor suppressors or mutants thereof.
- the target nucleic acid sequence encodes a viral replicase.
- the cargo molecule further comprises a packaging signal (e.g., a nucleic acid encoding or comprising a psi sequence).
- Packaging signals, encapsulation signals, psi, or packaging sequence are used interchangeably in reference to the non-coding, cis-acting sequence required for encapsulation of a molecule (e.g., retroviral RNA strands) during viral particle formation.
- the packaging signal contains essential sequences that are responsible for packaging cargo (into VLPs), and optionally, non-essential nucleic acid sequences responsible for increasing the efficiency of packaging. Any packaging sequence that allows effective packaging of a cargo into the VLP is suitable for use with the disclosed VLP.
- the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof.
- the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof.
- the term “functional fragment” in relation to the packaging signal sequence refers to a fragment of the recited sequences which are able to efficiently mediate packaging of the cargo into the VLP in comparison to a cargo not having a packaging signal.
- the functional fragment may be at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotide, at least 45 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, or more.
- VLPs as disclosed herein may be produced by transformation of a suitable host cell with one or more nucleic acids encoding the viral spike protein and the at least one additional virus structure protein under conditions such that the VLPs assemble in the host cell.
- the methods further comprise expressing one or more nucleic acids encoding one or more cargo molecules, as described elsewhere herein, in the host cell.
- the methods further comprise introducing one or more cargo molecules or nucleic acids encoding a cargo molecule to the host cell.
- the one or more cargo molecules may further comprise a packaging signal.
- the nucleic acid encoding one or more cargo molecules further comprises a packaging signal sequence.
- the packaging sequence may be upstream or downstream of the cargo molecule or the nucleic acid encoding the cargo molecule. In select embodiments, embodiments, the packaging sequence is upstream of the cargo molecule or the nucleic acid encoding the cargo molecule.
- the packaging signal is configured to facilitate loading of the cargo molecule or the nucleic acid encoding one or more cargo molecules into the VLP. In some embodiments, the cargo or the nucleic acid encoding one or more cargo molecules comprising a packaging signal is preferentially loaded into the VLP in comparison to cargo not comprising a packaging signal.
- the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof. In some embodiments, the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof.
- the host may be a prokaryotic host (e.g., E.
- coli or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, mammalian cells, e.g., NIH 3T3, HeLa, COS cells, or plant cells, e.g., a cell from Arabidopsis thaliana, Taxus cuspidate, Catharanthus roseus, Nicotiana tabacum, Oryza sativa, Lycopersicum esculentum, and Glycine max).
- a eukaryotic host e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, mammalian cells, e.g., NIH 3T3, HeLa, COS cells, or plant cells, e.g., a cell from Arabidopsis thaliana, Taxus cuspidate, Catharanthus roseus, Nicotiana tabacum, Oryza sativa, Lyco
- Non limiting examples of insect cells are, Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells.
- Sf Spodoptera frugiperda
- yeast yeast host cells
- yeast yeast host cells
- yeast yeast host cells
- yeast yeast host cells
- Candida including C. albicans and C. glabrata
- Aspergillus nidulans Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica.
- mammalian cells examples include COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, CVl cells, HeLa cells, MDCK cells, Vero cells, and Hep-2 cells.
- Xenopus laevis oocytes, or other cells of amphibian origin may also be used.
- Prokaryotic host cells include bacterial cells, for example, E. coli, B. subtilis, and mycobacteria.
- a host cell strain may be chosen which modulates the expression of the sequences or modifies and processes the gene product of the sequences in a desired manner.
- Such modifications may be important for the generation of the VLP, the function of any of the proteins or polypeptides comprising the VLP, or the generation or function of the cargo molecule.
- Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
- the method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).
- the VLPs are produced by growing host cells transformed by an expression vector under conditions whereby the recombinant proteins are expressed and VLPs are formed.
- the selection of the appropriate growth conditions is within the skill or a person with skill of one of ordinary skill in the art.
- the VLPs are isolated and purified using methods that preserve the integrity thereof, such as by gradient centrifugation, e.g., cesium chloride, sucrose and iodixanol, as well as standard purification techniques including, e.g., ion exchange, gel filtration chromatography, and the like.
- the isolation and purification comprise a lysis step, including but not limited to ultrasonication, grinding with abrasives, and/or repeated freeze/thaw cycles.
- the present disclosure also provides for nucleic acids encoding the viral spike protein, the at least one additional virus structure protein, and the cargo molecules disclosed herein and vectors containing these segments.
- the vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment. The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
- vectors of the present disclosure can drive the expression of the viral spike protein, the at least one additional virus structure protein, and the cargo molecules in both prokaryotic and eukaryotic cells.
- the vectors of the present disclosure can drive the expression in mammalian cells using a mammalian expression vector.
- mammalian expression vectors include pcDNA, pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference).
- the vector's control functions are typically provided by one or more regulatory elements.
- Vectors of the present disclosure can comprise any of a number of promoters known to the art.
- a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns).
- promoter/regulatory sequences useful for driving constitutive expression of a gene include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta- globin splice acceptor), TRE (Tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like.
- CMV cytomegalovirus promoter
- EF1a human elongation factor 1 alpha promoter
- SV40 simi
- Additional promoters that can be used for expression of the components of the VLP described herein, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EF1- ⁇ ) promoter with or without the EF1- ⁇ intron.
- CMV cytomegalovirus
- a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (
- Additional promoters include any constitutively active promoter.
- any regulatable promoter may be used, such that its expression can be modulated within a cell.
- the vector may contain, for example, some or all of the following: a selectable marker gene for selection of stable or transient transfectants in cells.
- Selectable markers include neomycin resistance, chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, puromycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.
- Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acids or vectors into cells, tissues, or a subject.
- Delivery vehicles such as nanoparticle- and lipid-based mRNA delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1;459(1-2):70-83), incorporated herein by reference. The present disclosure additionally provides a cell for producing a VLP.
- RNP ribonucleoprotein
- the cell may be transfected with one or more nucleic acids encoding at least one or all of the viral spike protein and the at least one additional viral structural protein.
- the cell may further be transfected with one or more nucleic acids encoding one or more cargo molecules; in the instance of a nucleic acid cargo, the genetic sequence may be preceded by a packaging sequences (e.g., nucleic acid sequence at least 70% similar to SEQ ID NOs: 1 or 2, or a functional fragment thereof) to facilitate efficient loading into generated VLP.
- compositions Further disclosed herein are compositions comprising the disclosed VLPs. Also disclosed herein are compositions or systems for treatment or prevention of a viral infection.
- compositions or systems comprise an interfering RNA or nucleic acid encoding thereof, as disclosed herein, and a carrier (e.g., pharmaceutically acceptable carrier).
- a carrier e.g., pharmaceutically acceptable carrier
- the composition comprises a pharmaceutically acceptable carrier.
- pharmaceutically acceptable refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human).
- pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
- “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors) and does not negatively affect the subject to which the composition(s) are administered.
- Carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.
- materials which can serve as excipients and/or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
- compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). Carriers also encompass any agent capable of protecting the interfering RNA or nucleic acid encoding thereof from rapid elimination from the body and facilitating efficient systemic or targeted delivery to a cell, cell-type, or location of interest.
- compositions may be formulated for any particular mode of administration including for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).
- systemic administration e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral
- topical administration e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis
- Formulations suitable for intranasal delivery include liquids and dry powders.
- Formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, sucrose, trehalose, and chitosan.
- Mucosadhesive agents such as chitosan can be used in either liquid or powder formulations to delay mucocilliary clearance of intranasally- administered formulations.
- Sugars such as mannitol and sucrose can be used as stability agents in liquid formulations and as stability and bulking agents in dry powder formulations.
- the compositions are formulated for administration to the lungs, for example, intranasal or for use with nebulizers or aerosol generators.
- Suitable compositions for use in nebulizers comprise the VLPs in a liquid carrier.
- the carrier is water (and most preferably sterile, pyrogen-free water), a dilute aqueous buffered saline, or a dilute isotonic or hypertonic alcohol solution.
- Optional additives include preservatives, antioxidants, volatile oils, buffering agents and surfactants.
- Suitable compositions for use in aerosol generators may comprise or consist either solely of the VLPs or of a powder blend comprising the VLP a suitable powder diluent, such as lactose, and an optional surfactant.
- Suitable propellants for aerosol devices include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.
- the composition may additionally contain one or more co-solvents, for example, water, saline, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.
- the compositions are vaccines or an immunogenic composition comprising a VLP as described herein and an adjuvant or immunostimulant.
- Adjuvants and immunostimulants are compounds that either directly or indirectly stimulate the immune system’s response to a co-administered antigen.
- Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); mineral salts (for example, aluminum, silica, kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), and Al(OH) 3 ; salts of calcium (e.g., Ca 3 (PO 4 ) 2 ), iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polynucleotides (for example, poly IC and poly AU acids); polyphosphazenes; cyanoacrylates; polymerase-(DL-lactide-co
- Aminoalkyl glucosamine phosphate compounds can also be used (see, e.g., WO 98/50399, U.S. Pat. No. 6,113,918, and 6,355,257.
- adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants.
- Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule.
- immunostimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4',4-diaminodiphenylmethane-3,3'- dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids or carbohydrates.
- non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4',4-diaminodiphenylmethane-3,3'- dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365
- the protein or polypeptide adjuvants may be provided as a fusion protein with any of the proteins or polypeptides which make up the VLP (e.g., the envelope protein, the membrane protein, the nucleocapsid protein)
- the vaccines of the present disclosure may also contain other compounds, which may be biologically active or inactive.
- one or more immunogenic portions of other antigens may be present, either incorporated into a fusion polypeptide with a VLP polypeptide or as a separate compound, within the vaccine.
- the vaccines may generally be used for prophylactic and therapeutic purposes.
- the vaccines may be formulated for any appropriate manner of administration, and thus may be administered by various methods, including for example, topical, oral, nasal, intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, subcutaneous, intramuscular administration, or via inhalation.
- the vaccines may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
- buffers e.g., neutral buffered saline or phosphate buffered saline
- carbohydrates e.g., glucose, mannose, sucrose or dextrans
- mannitol proteins
- proteins polypeptides
- amino acids such as glycine
- antioxidants e.g., antioxidants, bacteriostats, chelating agents such as EDTA or glutathione,
- Methods of Use comprising contacting a cell with an effective amount of a VLP, composition, or vaccine, as described herein.
- the cell is a mammalian cell.
- the cell is a human cell.
- the methods may comprise administering to a cell an effective amount of the described VLP, composition, or vaccine.
- the cell is in a subject and administering comprises administering the described VLP, composition, or vaccine to a subject.
- the VLP comprises a cargo molecule comprising an interfering RNA which binds to and inhibits translation of viral RNA.
- the viral infection may be caused by a respiratory virus, as described elsewhere herein.
- the respiratory virus is a coronavirus, e.g., betacoronaviruses, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS- CoV, MERS-CoV, and SARS-CoV-2 .
- the respiratory virus is SARS- CoV-2.
- methods for treating a disease or disorder in a subject comprising administering to the subject an effective amount of the VLP, composition, or vaccine, as described herein.
- the disease or disorder may be an infectious disease, cancer, a genetic disorder, or a combination thereof.
- the disease or disorder comprises an infectious disease.
- the infectious disease may comprise a viral infection.
- the viral infection is caused by a respiratory virus, a coronavirus e.g., betacoronaviruses, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2.
- the respiratory virus is SARS-CoV-2.
- the VLP, composition, or vaccine is administered therapeutically, in response to a diagnosis or suspicion of a viral infection.
- Treating a viral infection includes, but is not limited to, reducing, inhibiting, or preventing one or more symptoms of the viral infection or exacerbations thereof, or decreasing viral replication or shedding or preventing viral entry into cells.
- the VLP, composition, or vaccine is administered prophylactically, such that it is used to prevent or delay the onset of or lessen the severity of a viral infection or the exacerbations of a viral infection.
- the VLP comprises a cargo molecule comprising an interfering RNA which binds to and inhibits translation of viral RNA.
- the viruses causing the infection will be unable to replicate within the cells.
- the disease or disorder comprises cancer, a genetic disorder, or a combination thereof.
- the disease or disorder comprises cancer.
- the disclosed VLPs, compositions, and methods may be useful to treat a wide variety of cancers including carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma.
- the cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, lymph nodes, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus.
- the cancer comprises a solid tumor.
- the cancer comprises a blood cancer or lymphoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the methods result in suppression or elimination of metastasis. In some embodiments, the methods result in decreased tumor growth. In some embodiments, the methods prevent tumor recurrence. In some embodiments, the disease or disorder comprises a genetic disorder. Genetic disorders include any disease caused by an abnormality in the genome. In some embodiments, a VLP or composition thereof comprises a messenger RNA for a gene to supplement cells with a wild-type copy of a gene for a gene which is defective in the genetic disorder, a “disease-associated” gene.
- a VLP or composition thereof comprises a gene editing system which may be used to correct one or more defects or mutations in a gene (referred to as “gene correction”).
- the target sequence for the gene editing system is a “disease-associated” gene.
- the term “disease-associated gene” refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease.
- a disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
- a disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
- the disease-associated gene may be associated with a genetic disorder or cancer (e.g., mutations in cell cycle proteins, oncogenes, or tumor suppressors).
- genes responsible for such “single gene” or “monogenic” diseases include, but are not limited to, adenosine deaminase, ⁇ -1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), ⁇ -hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate- regulating endopeptidase homologue, X-linked (PHEX), methyl- CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9
- the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes. Diseases caused by the contribution of multiple genes which lack simple (e.g., Mendelian) inheritance patterns are referred to in the art as a “multifactorial” or “polygenic” disease.
- multifactorial or polygenic diseases include, but are not limited to, asthma, cancer, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities also can be inherited in a multifactorial or polygenic pattern and include, for example, cleft lip/palate, congenital heart defects, and neural tube defects.
- the method of altering a target genomic DNA sequence can be used to delete nucleic acids from a target sequence in a cell by cleaving the target sequence and allowing the host cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule.
- a nucleic acid sequence in this manner can be used in a variety of applications, such as, for example, to remove disease-causing trinucleotide repeat sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research.
- the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof.
- the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
- the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject.
- the subject is a human.
- the VLP, composition, or vaccine may be administered by any desired route, including, but not limited to, inhalation, orally, intranasally, or parenteral administration (subcutaneous, intravenous, or intramuscular injection or infusion).
- the VLP, composition, or vaccine is administered to the lung.
- Methods of administering to the lungs include, for example, endotracheal administration, aerosolization and inhalation.
- the administration is by inhalation.
- inhalation includes, for example, both inhalation of a dry powder and inhalation of a wet aerosol.
- a nebulizer such as a jet nebulizer, ultrasonic nebulizer, vibrating mesh nebulizer, e.g., in the form of aqueous drug solutions or dispersions, may be used for the administration.
- an inhaler such as a metered dose inhaler, soft mist inhaler, an insufflator, or a dry powder inhaler may be used for the administration.
- the administration is through a mechanical means or along with respiratory support such as mechanical ventilation (conventional or high frequency ventilation), administration of supplemental oxygen, or continuous positive airway pressure (CPAP) including nasal CPAP (nCPAP) treatment, face mask, oxygen hood, or the like.
- CPAP continuous positive airway pressure
- nCPAP nasal CPAP
- the employment of some of the devices may involve the use of various respiratory gases, as would be appreciated by the skilled artisan.
- CPAP gas Respiratory gases used for noninvasive pulmonary respiratory therapy are sometimes referred to herein as “CPAP gas,” “CPAP air,” “nCPAP,” “ventilation gas,” “ventilation air,” or simply “air.”
- the specific dose level may depend upon a variety of factors including the age, body weight, and general health of the subject, time of administration, and route of administration.
- An “effective amount” is an amount that is delivered to a subject, either in a single dose or as part of a series, which achieves a medically desirable effect.
- the amount of the VLP, composition, or vaccine in each dose is an amount which induces a protective result without significant adverse side effects.
- a wide range of second therapies may be used in conjunction with the VLPs, compositions, or methods of the present disclosure.
- the second therapy may be administration of an additional therapeutic agent or may be a second therapy not connected to administration of another agent.
- Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy, convalescent blood plasma therapy, or an additional chemotherapeutic or anti- cancer agent.
- the second therapy e.g., an immunotherapy
- the second therapy may be administered at the same time as the initial therapy, either in the same composition or in a separate composition administered at substantially the same time as the first composition.
- the second therapy may precede or follow the treatment of the first therapy by time intervals ranging from hours to months.
- the second therapy includes immunotherapy.
- Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies).
- the second therapy may include administration of antimicrobial agents, analgesics, anti-inflammatories, steroids, antipyretics, and the like.
- the second therapy includes an antiviral agent.
- Antiviral agents include, but are not limited to, oseltamivir, zanamivir, peramivir, acyclovir, valacyclovir, famciclovir, penciclovir, remdesivir, molnupiravir, and AT527 or other nucleotide analogs. Also disclosed herein are methods for treating or preventing viral infection in a cell comprising contacting the cell with an interfering RNA, a nucleic acid encoding an interfering RNA, or a composition or system, as described herein.
- the cell is a mammalian cell.
- the cell is a human cell.
- the viral infection may be caused by a respiratory virus.
- the respiratory virus is a coronavirus.
- the coronavirus family comprises 45 species distributed between four genera: alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus.
- the respiratory virus is a betacoronavirus, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2.
- the respiratory virus is SARS-CoV-2.
- the cell is in a subject.
- the method may comprise administering to a subject an effective amount of the described interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system.
- the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system may be administered by any desired route, including, but not limited to, inhalation, orally, intranasally, or parenteral administration (subcutaneous, intravenous, or intramuscular injection or infusion).
- the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered systemically or to the lungs.
- the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered to the lung.
- Methods of administering to the lungs include, for example, endotracheal administration, aerosolization and inhalation.
- the administration is by inhalation.
- inhalation includes, for example, both inhalation of a dry powder and inhalation of a wet aerosol.
- a nebulizer such as a jet nebulizer, ultrasonic nebulizer, vibrating mesh nebulizer, e.g., in the form of aqueous drug solutions or dispersions, may be used for the administration.
- an inhaler such as a metered dose inhaler, soft mist inhaler, an insufflator, or a dry powder inhaler may be used for the administration.
- the administration is through a mechanical means or along with respiratory support such as mechanical ventilation (conventional or high frequency ventilation), administration of supplemental oxygen, or continuous positive airway pressure (CPAP) including nasal CPAP (nCPAP) treatment, face mask, oxygen hood, or the like.
- CPAP continuous positive airway pressure
- nCPAP nasal CPAP
- the employment of some of the devices may involve the use of various respiratory gases, as would be appreciated by the skilled artisan.
- CPAP gas Respiratory gases used for noninvasive pulmonary respiratory therapy are sometimes referred to herein as “CPAP gas,” “CPAP air,” “nCPAP,” “ventilation gas,” “ventilation air,” or simply “air.”
- CPAP gas Respiratory gases used for noninvasive pulmonary respiratory therapy
- CPAP air Respiratory gases used for noninvasive pulmonary respiratory therapy
- nCPAP nCPAP
- ventilation gas ventilation air
- air Air
- the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
- the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject.
- the subject is a human.
- the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered therapeutically, in response to a diagnosis or suspicion of a viral infection. Treating a viral infection, includes, but is not limited to, reducing, inhibiting, or preventing one or more symptoms of the viral infection or exacerbations thereof, or decreasing viral replication or shedding or preventing viral entry into cells.
- the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered prophylactically, such that it is used to prevent or delay the onset of or lessen the severity of a viral infection or the exacerbations of a viral infection.
- the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
- the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject.
- the subject is a human.
- a wide range of second therapies may be used in conjunction with the compounds of the present disclosure.
- the second therapy may be administration of an additional therapeutic agent or may be a second therapy not connected to administration of another agent.
- Such second therapies include, but are not limited to, surgery, immunotherapy, convalescent blood plasma therapy, or an additional antimicrobial agent.
- the second therapy (e.g., an immunotherapy) may be administered at the same time as the initial therapy, either in the same composition or in a separate composition administered at substantially the same time as the first composition.
- the second therapy may precede or follow the treatment of the first therapy by time intervals ranging from hours to months.
- the second therapy may include administration of antimicrobial agents, analgesics, anti-inflammatories, steroids, antipyretics, and the like.
- the second therapy includes an antiviral agent.
- Antiviral agents include, but are not limited to, oseltamivir, zanamivir, peramivir, acyclovir, valacyclovir, famciclovir, penciclovir, remdesivir, molnupiravir, and AT527 or other nucleotide analogs.
- the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system may be introduced into cells by methods known in the art.
- Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle.
- Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
- Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. Delivery systems also include virus-like particles, as described above. A variety of viral constructs may be used to deliver the interfering RNA to the targeted cells and/or a subject. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc.
- AAV recombinant adeno-associated virus
- AAV recombinant adeno-associated virus
- recombinant adenoviruses recombinant adenoviruses
- recombinant lentiviruses recombinant retroviruses
- the present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.
- Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a host cell whether or not any coding sequences are in fact expressed.
- Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome.
- transduction generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome. Any of the vectors comprising a nucleic acid sequence that encodes the interfering RNA is also within the scope of the present disclosure.
- Such a vector may be delivered into host cells by a suitable method.
- Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction.
- the vectors are delivered to host cells by viral transduction.
- Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment).
- the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell.
- the construct or the nucleic acid encoding the components of the present system is a DNA molecule.
- the nucleic acid encoding the components of the present system is an RNA molecule.
- delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used.
- delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics.
- RNP ribonucleoprotein
- lipid-based delivery system lipid-based delivery system
- gene gun hydrodynamic, electroporation or nucleofection microinjection
- biolistics Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm.2014 Jan 1;459(1-2):70-83), incorporated herein by reference.
- kits that include the VLP or one or more nucleic acids encoding the components of the VLP (e.g., the spike protein, the structural proteins, the cargo molecule), host cells, and/or transfection reagents.
- the present disclosure also provides kits that include an interfering RNA, nucleic acid encoding an interfering RNA, and/or a carrier (e.g., delivery vehicle), as described above.
- the kit may further comprise a device for holding or administering the interfering RNA, nucleic acid encoding the interfering RNA, and/or a carrier.
- the kits may include reagents for making or formulating a delivery vehicle with the interfering RNA.
- the kit may include instructions for use in any of the methods described herein.
- the instructions can comprise method for making and/or using the VLP.
- the instructions can comprise a description of transfection of a host and methods of purifying, isolating, and characterizing the VLP. These instructions generally include information as to transfection reagents, purification procedures and reagents, and host cell compatibility with the VLP components.
- the instructions can comprise a description of administration to a cell, tissue, or subject to achieve the intended effect. These instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
- the kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment.
- the kits provided herein are in suitable packaging.
- kits include, but is not limited to, vials, bottles, jars, flexible packaging, and the like.
- a kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
- the packaging may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above.
- Example 1 Specific Treatment for and/or Prevention of SARS-CoV-2 Infection
- the cargo-carrying VLP is generated through the expression of SARS-CoV-2 spike (S) protein with other structural proteins (e.g., envelope (E), membrane (M), and nucleocapsid (N)) and shRNA sequence(s) preceded by either a 582 bp or 101 bp SARS-CoV-2 psi/packaging element (SEQ ID NOs: 1 and 2).
- S SARS-CoV-2 spike
- other structural proteins e.g., envelope (E), membrane (M), and nucleocapsid (N)
- shRNA sequence(s) preceded by either a 582 bp or 101 bp SARS-CoV-2 psi/packaging element (SEQ ID NOs: 1 and 2).
- These components are cloned into separate plasmids and/or polycistronic plasmids containing various combinations of structural elements in addition to single or multiple shRNA-expression cassettes, and are subsequently transfected into a viral packaging cell line, including but not limited to HEK293T and VeroE6 cell lines, using a lipid transfection-based method at various molar ratios. For example, co-transfection of S, M, E, N, and shRNA single plasmids or polycistronic elements is performed at 8:6:8:3:8 molar ratios, followed by numerous iterations of molar ratios.
- SARS-CoV-2 virus-like particles containing viral spike protein with tropism for lung epithelial ACE2 receptors and packaged with multiple shRNAs targeting the viral genome and/or viral replicase-encoding mRNA are delivered to the lungs of SARS-CoV-2 infected individuals (humans or non-human animals).
- Virus-like particles bind specifically to lung epithelial cells, enter them and are uncoated, releasing anti-viral shRNAs screened and optimized to inhibit viral replication and therefore the subsequent packaging, budding and intercellular spread of SARS- CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells.
- VLPs loaded with a cocktail of shRNAs or other genomic elements that interrupt the SARS-CoV-2 virus life cycle through targeting of the viral RNA genome are delivered either via intranasal administration, or aerosol/nebulization or through intravenous injection to individuals, either human or non-human animals, without evidence of SARS-CoV-2 infection.
- this approach is designed with individuals with immunocompromise, potentially related to hematologic malignancy, bone marrow transplant, solid organ transplant, need for chronic immunosuppressive medication in the setting of a rheumatologic, allergic, or hematologic disorder, or those with a congenital immunodeficiency, amongst others.
- Individuals unable to undergo vaccination against SARS-CoV-2 are also appropriate for this approach.
- the VLP delivers a genomic cocktail, including, but not limited to shRNAs targeting elements of the SARS-CoV-2 viral genome to lung epithelium expressing the ACE II receptor, which is at risk for SARS-CoV-2 infection.
- Genomic cargo is released into the target cells, and upon subsequent infection by SARS-CoV-2, persistent genomic cargo interferes with the translation of viral RNA, interrupting the viral life cycle.
- SARS-CoV-2 virus-like particles containing viral spike protein with tropism for lung epithelial ACE2 receptors are generated, packaged with shRNAs designed to target endemic or emerging respiratory viruses, and screened and optimized.
- SARS-CoV-2 virus-like particles containing viral spike protein with tropism for ACE2 receptors which is highly expressed in lung and GI tissues, amongst others, is packaged with multiple shRNAs targeting the viral genome and/or viral replicase-encoding mRNA.
- the therapeutic is delivered to the lungs of infected individuals (humans or non-human animals).
- Virus-like particles bind specifically to lung epithelial cells, enter them and are uncoated, releasing anti-viral shRNAs, thereby inhibiting viral replication and the subsequent packaging, budding and intercellular spread of SARS-CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells.
- VLP containing a genomic cargo which targets the RNA genome of a defined respiratory virus may be administered prophylactically to prevent successful infection by the targeted virus.
- a cocktail of VLPs carrying genomic cargo against a cocktail of several different respiratory viruses may also be administered; this may be informed by predicted viral outbreaks driven by global public health/community surveillance.
- Example 3 Platform for Emerging Viral Threats Emerging viral threats are sequenced and shRNAs targeting the relevant viral genome or its expressed mRNAs are designed as described above. Virus-like particles containing viral spike protein with tropism for lung epithelial ACE2 receptors are generated as described and packaged with screened the shRNAs for delivery to the lungs of infected individuals (humans or non-human animals). Virus-like particles bind specifically to lung epithelial cells, enter them and are uncoated, releasing anti-viral shRNAs.
- Inhibitory RNAs inhibit viral replication and therefore the subsequent packaging, budding and intercellular spread of SARS-CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells.
- Example 4 Delivery of Gene Therapy Cargo Messenger RNA encoding a normal copy of the CFTR gene, both copies of which may be dysfunctional in cystic fibrosis patients, is delivered to the lung epithelium using virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium.
- mRNA encoding Cas9 and a guide RNA directing targeted gene editing of the CFTR gene are packaged into virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium.
- Virus-like particles are delivered to the lung, attach to lung epithelial cells, enter them and are uncoated, releasing these RNA entities, thereby resulting in expression of normally functioning CFTR in these cells.
- Example 5 Delivery of Cancer Therapies Messenger RNAs encoding Cas9 and guide RNA(s) directing targeted gene editing of mutated tumor-driving oncogenes or tumor suppressors, such as oncogenic RAS or p53, respectively are packaged into virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium.
- Virus-like particles are delivered to the lung epithelium of patients with lung malignancies, attach to lung epithelial cells, enter them and are uncoated, releasing these RNA entities, thereby restoration of the normal function of target genes in tumor cells.
- shRNAs targeting mRNAs that encode immune checkpoint proteins or immunosuppressive proteins are packaged into virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium.
- shRNAs targeting key tumor proteins such as (including, but not limited to) RAS, EGFR, ALK, ROS, RET may also be encapsulated in the VLP, with the combined product delivered either through aerosolization/nebulization or intravenous injection.
- Virus-like particles are delivered to the lung epithelium of patients with lung malignancies, attach to lung epithelial cells, enter them and are uncoated, releasing these RNA entities, resulting in de-repression of immune function in the tumor microenvironment and immune killing of tumor cells, while targeting of key oncogenes leads to reduced tumor growth and tumor cell apoptosis.
- Example 6 shRNA Design and Validation shRNAs were designed using both DSIR and Kay (Stanford) algorithms in silico with the SARS-CoV-2 protein coding sequence of viral orf1ab as target sequence input. Targeting the protein coding sequence of orf1ab.
- the top 30 shRNA sequences resulting from each of these algorithms were selected for further analysis based on efficacy and off-target prediction algorithms.
- Candidate sequences were culled based on sequence similarity to human protein coding genes. This final culling of candidates required ⁇ 17-base ID with non-coding genes.
- the resulting 15 shRNA sequences targeting coding regions for NSP1, NSP2, NSP3, NSP4, NSP 5, NSP 6 and RdRP (Table 1 and FIG. 3) were cloned into a pRB-puro-U6 plasmid containing a puromycin resistance marker.
- Each shRNA construct was transfected into HEK 293T cells, which were selected for stable expression of shRNAs using puromycin.
- Each stable cell line was challenged with overexpression of each relevant NSP tagged at the N terminus with GFP in a pReceiver vector. Effective depletion of NSPs was evaluated by quantitative measurement of GFP signal, NSP transcript level by RT-qPCR and phenotypic effect (viability and confluence). While shRNA treated cells demonstrate high levels of confluency, significant cell death can be seen by cell morphology with resultant decrease in confluency of scrambled shRNA (control) cells in response to viral RNA challenge (FIG. 4).
- Example 7 Treatment of Coronaviruses SARS-CoV-2-infected individuals, either human or non-human animals or models, are treated using shRNAs targeting the viral RNA genome and/or viral replicase-encoding viral mRNA delivered to the lung or by intravenous administration, including but not limited to (virus-like particles, nanoparticles, lipid particles, aerosol and/or nebulized particles). Interfering RNAs delivered to the airway epithelium inhibit the replication and therefore the subsequent packaging, budding and intercellular spread of SARS-CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells.
- shRNAs targeting the viral RNA genome and/or viral replicase-encoding viral mRNA delivered to the lung or by intravenous administration including but not limited to (virus-like particles, nanoparticles, lipid particles, aerosol and/or nebulized particles).
- Interfering RNAs delivered to the airway epithelium inhibit the replication and therefore the subsequent packaging
- Each shRNA target sequence has homology with sequences within the RNA genomes of multiple endemic betacoronaviruses, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1.
- SEQ ID NO: 13 has homology with bases 9182 to 9199 of Human coronavirus OC43 strain ATCC VR-759 (NC_006213.1).
- Individuals infected with endemic betacoronaviruses either human or non-human animals or models, are treated using shRNAs disclosed herein, targeting the viral RNA genome and/or viral replicase-encoding viral mRNA.
- Example 8 Prevention of SARS-CoV-2 Infection
- Interfering or shRNA targeting the SARS-CoV-2 genome can be administered by aersolization/nebulization or intravenous injection to prevent SARS-CoV-2 infection.
- This approach has particular use in individuals who are immunocompromised or are otherwise unable to undergo vaccination against SARS-CoV-2.
- the same approach could be used to prevent infection with other coronaviridae family members in individuals who are immunocompromised. In practice, this may take the form of an aersolized/nebulized formulation administered on a weekly or monthly basis under the direction of a physician.
- Example 9 Adaptation to Emerging Viral Pathogens The genomes of emerging viral pathogens are sequenced for the study of novel viruses and the development of interventions.
- VLPs were generated using a multiplasmid system, with cargo including packaging sequences optimized green fluorescence protein (GFP) mRNA (FIG. 6A). Following generation, the VLPs were collected from packaging cell line supernatant, lysed, and RNA extracted using Trizol. qPCR was subsequently performed to evaluate loading of GFP mRNA.
- GFP green fluorescence protein
- Control (scramble RNA expressing) 293T cells exhibit morphologic signs of cell death, with decreased confluence, suggesting significant toxicity from NSP1 (FIG. 7A).
- shRNA expressing cells by contrast demonstrate no morphologic signs of cell death and are confluent. Representative images were acquired 48 hours after NSP1 transfection in either control (scramble RNA) expressing 293T cells or shRNA expressing 293T cells at 10x. ImageJ was used to calculate the percentage of the image area covered by cells. As shown in FIG.
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Abstract
The present disclosure provides virus-like particles (VLPs) comprising a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor and at least one additional viral structural protein (e.g., envelope proteins, membrane proteins, nucleocapsid proteins, etc.), compositions comprising these VLPs, as well as methods for making and using these VLPs (e.g., for treatment or prevention of diseases or disorders (such as, viral infections)).
Description
VIRUS-LIKE PARTICLE FIELD The present invention relates to virus-like particles based on the SARS-CoV-2 virus and methods and systems thereof for delivery of a cargo molecule (e.g., nucleic acids) to a target cell (e.g., with low off-target effects). The present invention is also related to interfering RNAs (e.g., siRNA and shRNA) and systems, compositions, and methods thereof for the treatment or prevention of viral infections (e.g., SARS-CoV-2 infection). CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 63/234,478 and 63/234,479, filed August 18, 2021, the contents of each which are herein incorporated by reference in their entirety. SEQUENCE LISTING STATEMENT The contents of the electronic sequence listing titled NANOR-39718-601.xml (Size: 16,570 bytes; and Date of Creation: August 18, 2022) is herein incorporated by reference in its entirety. BACKGROUND Successful delivery of exogenous biological cargo (e.g., DNA, RNA, proteins) into a cells in a patient has major implications on the adoption of biologic or genetic therapies and therapeutics. Delivery systems allow cargo to cross biological barriers, increase cargo stability, and decrease toxicity of the therapy or therapeutic. Most importantly, to increase the clinical efficacy of the cargo and reduce unwanted side effects due to off-target interactions, exogenous biological cargo needs to be safely and efficiently delivered to the target cells. SUMMARY Provided herein are virus-like particles (VLPs) comprising a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor and at least one additional viral structural protein. In some embodiments, the at least one additional viral structural protein comprises: a viral envelope protein; a viral membrane protein; a viral nucleocapsid protein; or any combination thereof. In some embodiments, the viral spike protein
and at least one or all of the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from a coronavirus (e.g., SARS-CoV or SARS-CoV-2). In some embodiments, the VLPs comprise an encapsulated cargo molecule or a nucleic acid encoding a cargo molecule. In some embodiments, the cargo molecule or a nucleic acid encoding the cargo molecule may further comprise a packaging signal. In select embodiments, the packaging signal is included upstream of the cargo molecule or the nucleic acid encoding the cargo molecule. In some embodiments, the cargo molecule or the nucleic acid encoding the cargo molecule comprising a packaging signal is preferentially loaded into the VLP in comparison to a cargo molecule not comprising a packaging signal. In some embodiments, the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof. In some embodiments, the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof. In some embodiments, the cargo molecule comprises a nucleic acid. In some embodiments, the cargo molecule comprises an interfering RNA or a messenger RNA. In some embodiments, the interfering RNA is configured to bind to and inhibit translation of a viral RNA. In select embodiments, the viral RNA is from a respiratory virus (e.g., a coronavirus). In some embodiments, the cargo molecule comprises a sequence encoding a gene. In some embodiments, the cargo molecule comprises a sequence encoding a gene editing system (e.g., a CRISPR/Cas system). Additionally provided herein are compositions comprising the disclosed VLPs. In some embodiments, the compositions comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions are configured to induce an immune response. In some embodiments, the compositions are vaccines comprising the VLP and an adjuvant. Also provided are methods and cells for producing the disclosed VLPs. Further disclosed are methods of using the VLPs. Included are methods for preventing or treating a viral infection in a cell, tissue, or organism, comprising contacting the cell, tissue, or organism with an effective amount of a VLP, pharmaceutical formulation, vaccine, or other composition as described. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo and the method comprises administering the disclosed VLP, pharmaceutical formulation, vaccine, or other composition to a
subject. The methods may be adapted to treat or prevent any viral infection. Preferably the viral infection is caused by a respiratory virus (e.g., a coronavirus). Also included are methods for treating or preventing a disease or disorder in a subject comprising administering to the subject an effective amount of a VLP, a pharmaceutical formulation, a vaccine, or other composition as described. In some embodiments the disease or disorder comprises an infectious disease, cancer, a genetic disorder, or a combination thereof. Kits comprising any or all of the components of the VLPs or compositions or agents necessary for making or using the VLPs or compositions are also provided. Provided herein are interfering RNAs or nucleic acids encoding an interfering RNA. The interfering RNAs comprise a nucleic acid sequence at least partially complementary to a target sequence of a virus genome. In some embodiments, the virus is a respiratory virus. In some embodiments, the virus is a coronavirus. In select embodiments, the virus is SARS-CoV-2. In some embodiments, the target sequence comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 3-17, or an RNA equivalent thereof. In some embodiments, the target sequence comprises a nucleic acid sequence with at least 80% identity to SEQ ID NOs: 3-17, or an RNA equivalent thereof. In some embodiments, the target sequence comprises a nucleic acid sequence of SEQ ID NOs: 3-17, or an RNA equivalent thereof. In some embodiments, the interfering RNA is configured to prevent replication of the virus genome. In some embodiments, the interfering RNA is a small interfering RNA (siRNA). In some embodiments, the interfering RNA is a small hairpin RNA (shRNA).Also provided herein are compositions or system comprising an interfering RNA or a nucleic acid encoding thereof and a carrier. In some embodiments, the carrier comprises a delivery vehicle. The delivery vehicle may be selected from the group consisting of liposomes, viruses, virus-like particles, immunolipoplexes, cyclodextrins, micro- or nano-particles, aptamers, dendrimers, exosomes, chitosan, or derivatives thereof. In select embodiments, the carrier is a virus or virus- like particle. In some embodiments, the interfering RNA, or the nucleic acid encoding thereof, further comprises a packaging signal. In select embodiments, the packaging signal is included upstream of the interfering RNA, or the nucleic acid encoding thereof. In some embodiments, the packaging signal is configured to facilitate loading of the interfering RNA or the nucleic acid encoding thereof into the virus or virus-like particle.
In some embodiments, the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof. In select embodiments, the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof. Further provided are methods for treating or preventing a viral infection in a cell comprising contacting a cell, tissue, or subject with an effective amount of an interfering RNA, a nucleic acid encoding an interfering RNA, or a composition or system thereof, as described herein. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo. In some embodiments, the viral infection is caused by a respiratory virus. In some embodiments, the respiratory virus is a coronavirus. In select embodiments, the respiratory virus is SARS-CoV-2. In some embodiments, the contacting comprises administering to a subject or a tissue thereof. In some embodiments, the administering comprises systemic administration, administration to the lungs, including but not limited to either intranasal or aerosol delivery, or a combination thereof. Kits comprising any or all of the interfering RNA, the nucleic acid encoding an interfering RNA, or any components of the composition or system are additionally provided herein. Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic of an exemplary non-infectious virus-like particle based on SARS-CoV-2 and scenarios of treatment or prevention of viral infection. The VLP (blue) may be loaded with genomic cargo designed to interrupt translation of SARS-CoV-2 viral genome and infection by viral particles (green). FIG. 2 is a schematic of the generation of an exemplary VLP in which shRNAs targeting elements of the SARS-CoV-2 RNA genome is generated through transfection of a packaging cell line using either a single polycistronic construct containing the nucleocapsid (N), membrane (M), spike (S), envelope (E) structural proteins and genomic cargo of interest with upstream SARS-CoV-2 packaging signal sequence or a multi-plasmid transfection system.
FIG. 3 is a schematic of the viral RNA of SARS-CoV-2 with the open reading frames coding and non-coding regions identified. Arrow heads indicate target locations of exemplary interfering RNA sequences. FIG. 4 are images of HEK293T cells transfected with exemplary shRNA sequences (bottom row) or a scrambled shRNA control (top row). 24 hours after puromycin selection, cells were challenged with 2.5 ^g of RNA encoding SARS-CoV-2 non-structural protein 1 (left), non- structural protein 2 (middle), or non-structural protein 4 (right). Images were acquired 48 hours after viral RNA challenge, as indicated. FIGS. 5A and 5B show production and loading of exemplary VLPs, as disclosed herein. FIG. 5A is an electron micrograph of a VLP. FIG. 5B is a graph of the relative mRNA expression from purified VLPs loaded with GFP mRNA using both the PS101 and PS582 packaging sequences, showing preferential loading of GFP mRNA with these leader sequences. FIGS. 6A-6B show protection of cells from SARS-CoV-2 toxicity with exemplary shRNAs. FIG. 6A is a graph of the percentage of an image area covered by cells acquired 48 hours after NSP1 transfection in either control (scramble RNA) expressing 293T cells or shRNA expressing 293T cells at 10x. FIG. 6B is a graph of relative transcript levels of NSP1 in cells 48 hours after NSP1 challenge. DETAILED DESCRIPTION Described herein are virus-like particles (VLPs) comprising a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor and at least one additional viral structural protein (e.g., envelope proteins, membrane proteins, nucleocapsid proteins, etc.), compositions comprising these VLPs, as well as methods for making and using these VLPs (e.g., for treatment or prevention of diseases or disorders (such as, viral infections)). The disclosed interfering RNAs (e.g., siRNA and shRNA) and systems, compositions, and methods thereof are suitable for the treatment and prevention of viral infections (e.g., SARS- CoV-2 infection). The interfering RNAs were designed to avoid unwanted off-target effects by comparing to known genome sequences for intended subjects (e.g., humans). Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
Definitions The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other
kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double- stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein,” are used interchangeably herein. The terms “complementary” and “complementarity” refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson- Crick base-pairing or other non-traditional types of pairing. The degree of complementarity between two nucleic acid sequences can be indicated by the percentage of nucleotides in a nucleic acid sequence which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementary). Two nucleic acid sequences are “perfectly complementary” if all the contiguous nucleotides of a nucleic acid sequence will hydrogen bond with the same number of contiguous nucleotides in a second nucleic acid sequence. Two nucleic acid sequences are “substantially complementary” if the degree of complementarity between the two nucleic acid sequences is at least 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100%) over a region of at least 8
nucleotides (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides), or if the two nucleic acid sequences hybridize under at least moderate, preferably high, stringency conditions. Exemplary moderate stringency conditions include overnight incubation at 37° C in a solution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt’s solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C, or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook et al., infra. High stringency conditions are conditions that use, for example (1) low ionic strength and high temperature for washing, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50° C, (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1% Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42° C, or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt’s solution, sonicated salmon sperm DNA (50 ^g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C, with washes at (i) 42° C in 0.2×SSC, (ii) 55° C in 50% formamide, and (iii) 55° C in 0.1×SSC (preferably in combination with EDTA). Additional details and an explanation of stringency of hybridization reactions are provided in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York (1994). As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid according to the technology is longer than a reference sequence, additional nucleotides in the nucleic acid, that do not align with the reference sequence, are not taken into account for determining sequence identity. Methods and computer programs for alignment are well known in the art, including BLAST, Align 2, and FASTA.
The term “homology” and “homologous” refers to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence. As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the Tm of the formed hybrid. Hybridization methods involve the annealing of one nucleic acid to another, complementary nucleic acid, e.g., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and “anneal” or “hybridize” through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA, 46: 453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA, 46: 461 (1960), have been followed by the refinement of this process into an essential tool of modern biology. For example, hybridization and washing conditions are now well known and exemplified in Sambrook et al., supra. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. As used herein, a “double-stranded nucleic acid” may be a portion of a nucleic acid, a region of a longer nucleic acid, or an entire nucleic acid. A “double-stranded nucleic acid” may be, e.g., without limitation, a double-stranded DNA, a double-stranded RNA, a double-stranded DNA/RNA hybrid, etc. A single-stranded nucleic acid having secondary structure (e.g., base- paired secondary structure) and/or higher order structure (e.g., a stem-loop structure) may also be considered a “double-stranded nucleic acid.” For example, triplex structures are considered to be “double-stranded.” In some embodiments, any base-paired nucleic acid is a “double-stranded nucleic acid.” The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor of any of the foregoing. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Thus, a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or an RNA chain that has functional role
to play in an organism. For the purpose of this disclosure, it may be considered that genes include regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions. The term “wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified,” “mutant,” or “polymorphic” refers to a gene or gene product that displays modifications in sequence and or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. As used herein, the term “variant” refers to the exhibition of qualities that have a pattern that deviates from what occurs in nature. In some embodiments, a variant may also be a mutant. The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature. A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell. A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such as a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non- mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human. As used herein, “treat,” “treating” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a VLP, composition, or vaccine described herein to an appropriate control subject. As such, “treating” means an application or administration of the methods, VLPs, or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease outcome, disease progression, or symptoms of the disease. As used herein, the term “preventing” refers to partially or completely delaying onset of a disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination, such as, but not
limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan. As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the systems of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization of the system to a desired site. The systems can be administered by any appropriate route which results in delivery to a desired location in the cell, organism, or subject. As used herein, the terms “virus-like particle” and “VLP” are used interchangeably to refer to a structure that in at least one attribute resembles a virus but which has not been demonstrated to be infectious. Virus-like particles may or may not carry genetic information encoding for the proteins of the virus-like particle, but in general do not include the genetic materials required for viral replication and infection. As used herein, interfering RNA refers to any nucleic acid molecule capable of mediating sequence specific RNAi, for example short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), translational silencing, and the like. In some embodiments, the interfering RNA include synthetic double stranded small interfering RNA (siRNA) and short hairpin RNA (shRNA). Such molecules are constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos.5,898,031, 6,107,094, 6,506,559, 7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Virus-Like Particles (VLPs) VLPs are structures that morphologically resemble a virus but are devoid of the genetic material required for viral replication and infection. Since they are non-replicative in
nature, they are safe for administration to cells or subjects and can target physiologically relevant receptors with their surface proteins and, therefore, tissues in a subject, resulting in more effective delivery of cargo, antibody induction, or inhibition of infectious forms of viruses which target the same or similar tissues and receptors. Provided herein are virus-like particles (VLPs) comprising a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor and at least one additional viral structural protein. The viral spike protein may be derived from any virus. Preferably, the spike protein is derived fully or partially from a respiratory virus. Respiratory viruses include, but are not limited to: influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses. The spike protein may be derived from a natural viral spike protein with natural tropism for binding ACE2 receptors. In some embodiments, the spike protein is derived from a coronavirus. The coronavirus family comprises 45 species distributed between four genera: alphacoronavirus, betacoronavirus, deltacoronavirus, and gammacoronavirus virus. Preferably, the spike protein is derived from a betacoronavirus. In select embodiments, the spike protein is derived from SARS-CoV or SARS-CoV- 2. In select embodiments, the spike protein is wild-type spike protein from SARS-CoV-2 (Accession # QHD43416). However, the invention is not limited to this exemplary sequence. SARS-CoV-2 spike protein may comprise the wild-type amino acid sequence or variant having an amino acid sequence that is at least about 70% identical (e.g., 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%, about85%, 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%, about 99%, or about 100%) to the amino acid sequence of the wild- type spike protein. The spike protein may, alternatively, be a chimera in which a viral spike protein is genetically modified to include a spike protein ACE2 receptor binding domain from a different virus or a receptor binding domain from another ACE2 binding protein. In some embodiments, the spike protein may contain an ACE2 binding domain from a coronavirus, e.g., SARS-CoV or SARS-CoV-2.
The at least one additional viral structural protein may be a nucleocapsid protein, an envelope protein, a membrane protein, or a fragment or complex thereof. Thus, viral structural proteins used for the present invention may consist of, consist essentially of, or comprise a nucleocapsid protein, an envelope protein, a membrane protein, and/or a fragment or complex thereof. In some embodiments, the at least one additional viral structural protein comprises a viral envelope protein, a viral membrane protein, and nucleocapsid protein. The at least one additional viral structural protein may be derived from any virus, preferably a respiratory virus. Respiratory viruses include but are not limited to influenza viruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinoviruses, coronaviruses, adenoviruses, and bocaviruses. In some embodiments, at least one or all of the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from a coronavirus. In select embodiments, at least one or all of the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from SARS-CoV-2. In certain embodiments, the nucleocapsid protein is wild-type nucleocapsid protein from SARS-CoV-2 (Accession # QHD43423). In certain embodiments, the envelope protein is wild-type envelope protein from SARS-CoV-2 (Accession # QHD43418). In certain embodiments, the membrane protein is wild-type membrane protein from SARS-CoV-2 (Accession # QHD43419). However, the invention is not limited to these exemplary sequences. SARS-CoV-2 structural proteins (e.g., envelope, membrane, nucleocapsid) may comprise the wild-type amino acid sequence or variant having an amino acid sequence that is at least about 70% identical (e.g., 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%, about85%, 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%, about 99%, or about 100%) to the amino acid sequence of the respective structural protein. In some embodiments, the nucleocapsid, the envelope, and/or the membrane protein may be from or derived from SARS-CoV-2 variants (e.g., alpha, gamma, beta, delta, and omicron variants) or subvariants or sublineages thereof. In some embodiments, the SARS-CoV-2 variant is B.l.1.7 (Alpha), B.1.351 (Beta) and B.l.617.2 (Delta), and/or B.l.1.529 (Omicron).^For example, the N protein may be from or derived from lineage A Wuhan strain SARS-CoV-2,
B.1.351 variant SARS-CoV-2, B.1.617.2 variant SARS-CoV-2 or B.1.1.529 variant SARS-CoV- 2. Any of the proteins described herein may comprise one or more amino acid substitutions as compared to the corresponding wild-type protein. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg). The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative
mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. The VLPs of the invention may further comprise an encapsulated cargo molecule. Cargo molecules can include nucleic acid molecules, chemotherapeutic agents, imaging agents, and/or other agents. In select embodiments, the cargo molecule comprises a nucleic acid. The cargo molecule may be an RNA molecule, such as, an interfering RNA or a messenger RNA. In some embodiments, the cargo molecule comprises a nucleic acid encoding a gene (e.g., a messenger RNA). In some embodiments, the messenger RNA encodes the interfering RNA. a) Interfering RNAs Interfering RNAs include synthetic double stranded small interfering RNA (siRNA) or short hairpin RNA (shRNA). Both mimic endogenous microRNAs (miRNAs) and carry out RNA interference (RNAi), a gene-silencing mechanism facilitated by small RNAs, which is highly dependent on gene sequences silencing genes of interest with high specificity. “Short hairpin RNAs” or “shRNA” refer to an RNA sequence comprising a double- stranded region and a loop region at one end forming a hairpin loop. The double-stranded region is typically about 19 to about 29 nucleotides in length, and the loop region is typically about 2 to about 10 nucleotides in length. In vivo, shRNA are processed by the enzyme Dicer into an active RNAi species of about 21 nucleotides. “Small interfering RNA” or “siRNA” refer an RNA molecule comprising a double stranded region and, optionally, a 3ƍ overhang of nonhomologous residues at each end. The double-stranded region is typically about 18 to about 30 nucleotides in length, and the overhang may be of any length of nonhomologous residues, but a 2 nucleotide overhang is preferred. Both siRNA and shRNA have been demonstrated to be effective in in vitro and in vivo applications, each with their respective advantages. Most siRNA are structurally designed to promote efficient incorporation into the RNA Induced Silencing Complex (RISC). The interfering RNAs can be provided on a nucleic acid or vector which utilizes the host micro RNA biogenesis pathway to express the shRNA, allowing for modulation or regulation by promoters, as described elsewhere herein, or can be provided as a single nucleic acid. Provided herein are sequences for interfering RNAs (e.g., shRNA) for use in the treatment or prevention of viral infection which comprise a nucleic acid sequence at least
partially complementary to target sequences of the viral genome of a virus (e.g., a respiratory virus). In some embodiments, the interfering RNA is configured to prevent replication of the viral genome. The sequences limit off-target effects based on known sequences within the human genome. In some embodiments, the interfering RNAs hybridize to target sequences of the viral genome of a respiratory virus. Respiratory viruses include but are not limited to influenza viruses, respiratory syncytial virus, parainfluenza viruses, rhinoviruses, coronaviruses, and adenoviruses. In some embodiments, the respiratory virus comprises a coronavirus, for example, a betacoronavirus, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. In select embodiments, the respiratory virus is SARS-CoV-2. Interfering RNA suitable for use with the VLPs described herein include those which bind to and inhibit translation of viral RNA. For example, the VLPs described herein may deliver interfering RNA to cells infected or at risk of being infected with a respiratory virus (e.g., a coronavirus). In some embodiments, the interfering RNA targets, or fully or partially hybridizes to, coding sequences within the genome of a respiratory virus (e.g., SARS-CoV-2 viral RNA). For example, the interfering RNA may hybridize with coding sequences for non-structural proteins or an RNA-dependent RNA polymerase. In some embodiments, the interfering RNA included in the VLPs described herein inhibit translation of SARS-CoV-2 viral RNA (e.g., by targeting the nucleic acid encoding the viral replicase). In some embodiments, the interfering RNA targets, or fully or partially hybridizes to, coding sequences within the genome of SARS-CoV-2 viral RNA. For example, the interfering RNA may hybridize with coding sequences for non-structural proteins or an RNA- dependent RNA polymerase. In some embodiments, the interfering RNA is fully or partially complementary to target sequences comprising a nucleic acid sequence with at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) to SEQ ID NOs: 3-17, or an RNA equivalent thereof. The interfering RNA comprises a sequence capable of hybridizing to target sequences comprising nucleic acid sequences with at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%) to SEQ ID NOs: 3-17 as shown in Table 1, or an RNA equivalent thereof. In some embodiments, the interfering RNA comprises a sequence capable of hybridizing
to target sequences comprising SEQ ID NOs: 3-17 as shown in Table 1. The interfering RNA sequence may comprise an exact reverse complement to the target sequence or, alternatively, may have sufficient homology to the reverse complement of the target sequence to result in efficient and stringent hybridization and/or efficient interference. For example, the interfering RNA may comprise a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides different from that of the reverse complement to the target sequence. Table 1
The interfering RNAs may comprise additional nucleotides or residues in addition the sequence responsible for recognizing and binding the target sequence. For example, shRNAs comprise a loop region separate from the region comprising the sequence which bind to the target nucleic acid sequence.
The present disclosure also provides for DNA segments encoding the interfering RNAs disclosed herein, vectors containing these segments and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment. The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence. In certain embodiments, vectors of the present disclosure can drive the expression of the interfering RNAs in both prokaryotic and eukaryotic cells. In some embodiments, the vectors of the present disclosure can drive the expression in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the vector's control functions are typically provided by one or more regulatory elements. For other suitable expression systems and regulatory elements for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference. b) Gene Editing Systems In some embodiments, the cargo molecule comprises a nucleic acid sequence encoding a gene editing system. The gene editing system may comprise a CRISPR-Cas system. A “CRISPR-Cas system” refers collectively to transcripts and other elements involved in the expression of and/or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, Cas protein, a cr (CRISPR) sequence (e.g., crRNA or an active partial crRNA), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. Any element of any suitable CRISPR/Cas gene editing system known in the art can be employed in the systems and methods described herein, as appropriate. CRISPR/Cas gene editing technology is described in detail in, for example, U.S. Patent Nos. 8,546,553, 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,889,418; 8,895,308; 8,9066,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; 9,115,348; 9,149,049; 9,493,844; 9,567,603; 9,637,739; 9,663,782; 9,404,098; 9,885,026; 9,951,342; 10,087,431; 10,227,610; 10,266,850;
10,601,748; 10,604,771; and 10,760,064; and U.S. Patent Application Publication Nos. US2010/0076057; US2014/0113376; US2015/0050699; US2015/0031134; US2014/0357530; US2014/0349400; US2014/0315985; US2014/0310830; US2014/0310828; US2014/0309487; US2014/0294773; US2014/0287938; US2014/0273230; US2014/0242699; US2014/0242664; US2014/0212869; US2014/0201857; US2014/0199767; US2014/0189896; US2014/0186919; US2014/0186843; and US2014/0179770, each incorporated herein by reference. For example, the gene editing system may comprise one or more Cas proteins (e.g., Cas9) and at least one guide RNA directed to a target nucleic acid. Typically, the RNA sequences employed in CRISPR/Cas systems are referred to collectively as “guide RNA” (gRNA) or single guide RNA (sgRNA). Thus, the terms “guide RNA,” “single guide RNA,” and “synthetic guide RNA,” are used interchangeably herein and may refer to a nucleic acid sequence comprising a tracrRNA and a pre-crRNA array containing a guide sequence. The terms “guide sequence,” “guide,” and “spacer,” are used interchangeably herein and refer to the nucleotide sequence within a guide RNA that specifies the target nucleic acid. In some embodiments, the target nucleic acid is a nucleic acid endogenous to a target cell. In some embodiments, the target nucleic acid is a genomic DNA sequence. The term “genomic,” as used herein, refers to a nucleic acid sequence (e.g., a gene or locus) that is located on a chromosome in a cell. In some embodiments, the target nucleic acid is a DNA or RNA sequence from an infectious agent, for example an infectious bacteria or virus. In some embodiments, the target nucleic acid encodes a gene or gene product. The term “gene product,” as used herein, refers to any biochemical product resulting from expression of a gene. Gene products may be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, micro RNA (miRNA), and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA). In some embodiments, the target nucleic acid sequence encodes a protein or polypeptide. In some embodiments, the target nucleic acid sequence encodes a mutant protein. In some embodiments, the target nucleic acid sequence encodes genes involves in the progression of cancer e.g., oncogenes or tumor suppressors or mutants thereof. In some embodiments, the target nucleic acid sequence encodes a viral replicase. In some embodiments, the cargo molecule further comprises a packaging signal (e.g., a nucleic acid encoding or comprising a psi sequence). Packaging signals, encapsulation signals, psi, or packaging sequence are used interchangeably in reference to the non-coding, cis-acting
sequence required for encapsulation of a molecule (e.g., retroviral RNA strands) during viral particle formation. The packaging signal contains essential sequences that are responsible for packaging cargo (into VLPs), and optionally, non-essential nucleic acid sequences responsible for increasing the efficiency of packaging. Any packaging sequence that allows effective packaging of a cargo into the VLP is suitable for use with the disclosed VLP. In some embodiments, the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof. In select embodiments, the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof. As used herein, the term “functional fragment” in relation to the packaging signal sequence refers to a fragment of the recited sequences which are able to efficiently mediate packaging of the cargo into the VLP in comparison to a cargo not having a packaging signal. The functional fragment may be at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotide, at least 45 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, or more. SEQ ID NO: 1 (PSI 582) –
SEQ ID NO: 2 (PSI 101) -
In general, VLPs as disclosed herein may be produced by transformation of a suitable host cell with one or more nucleic acids encoding the viral spike protein and the at least one additional virus structure protein under conditions such that the VLPs assemble in the host cell. In some embodiments, the methods further comprise expressing one or more nucleic acids encoding one or more cargo molecules, as described elsewhere herein, in the host cell. In some embodiments, the methods further comprise introducing one or more cargo molecules or nucleic acids encoding a cargo molecule to the host cell. In some embodiments, the one or more cargo molecules may further comprise a packaging signal. In some embodiments, the nucleic acid encoding one or more cargo molecules further comprises a packaging signal sequence. The packaging sequence may be upstream or downstream of the cargo molecule or the nucleic acid encoding the cargo molecule. In select embodiments, embodiments, the packaging sequence is upstream of the cargo molecule or the nucleic acid encoding the cargo molecule. The packaging signal is configured to facilitate loading of the cargo molecule or the nucleic acid encoding one or more cargo molecules into the VLP. In some embodiments, the cargo or the nucleic acid encoding one or more cargo molecules comprising a packaging signal is preferentially loaded into the VLP in comparison to cargo not comprising a packaging signal. In some embodiments, the packaging signal comprises a nucleic acid sequence with at least 70% identity to SEQ ID NOs: 1 or 2, or a functional fragment thereof. In some embodiments, the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 or 2, or a functional fragment thereof. The host may be a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, mammalian cells, e.g., NIH 3T3, HeLa, COS cells, or plant cells, e.g., a cell from Arabidopsis thaliana, Taxus cuspidate, Catharanthus roseus, Nicotiana tabacum, Oryza sativa, Lycopersicum esculentum, and Glycine max). Non limiting examples of insect cells are, Spodoptera frugiperda (Sf) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells. Examples of fungi (including
yeast) host cells are S. cerevisiae, Kluyveromyces lactis (K lactis), species of Candida including C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, CVl cells, HeLa cells, MDCK cells, Vero cells, and Hep-2 cells. Xenopus laevis oocytes, or other cells of amphibian origin, may also be used. Prokaryotic host cells include bacterial cells, for example, E. coli, B. subtilis, and mycobacteria. In addition, a host cell strain may be chosen which modulates the expression of the sequences or modifies and processes the gene product of the sequences in a desired manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage or transport to the membrane) may be important for the generation of the VLP, the function of any of the proteins or polypeptides comprising the VLP, or the generation or function of the cargo molecule. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987). Depending on the expression system and host cell selected, the VLPs are produced by growing host cells transformed by an expression vector under conditions whereby the recombinant proteins are expressed and VLPs are formed. The selection of the appropriate growth conditions is within the skill or a person with skill of one of ordinary skill in the art. The VLPs are isolated and purified using methods that preserve the integrity thereof, such as by gradient centrifugation, e.g., cesium chloride, sucrose and iodixanol, as well as standard purification techniques including, e.g., ion exchange, gel filtration chromatography, and the like. In some embodiments, the isolation and purification comprise a lysis step, including but not limited to ultrasonication, grinding with abrasives, and/or repeated freeze/thaw cycles. As such, the present disclosure also provides for nucleic acids encoding the viral spike protein, the at least one additional virus structure protein, and the cargo molecules disclosed herein and vectors containing these segments. The vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment. The person of ordinary skill
in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence. In certain embodiments, vectors of the present disclosure can drive the expression of the viral spike protein, the at least one additional virus structure protein, and the cargo molecules in both prokaryotic and eukaryotic cells. In some embodiments, the vectors of the present disclosure can drive the expression in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pcDNA, pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the vector's control functions are typically provided by one or more regulatory elements. For other suitable expression systems and regulatory elements for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference. Vectors of the present disclosure can comprise any of a number of promoters known to the art. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter/regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta- globin splice acceptor), TRE (Tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the VLP described herein, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1-
alpha (EF1-Į) promoter with or without the EF1-Į intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell. Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene for selection of stable or transient transfectants in cells. Selectable markers include neomycin resistance, chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, puromycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae. Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acids or vectors into cells, tissues, or a subject. Delivery vehicles such as nanoparticle- and lipid-based mRNA delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1;459(1-2):70-83), incorporated herein by reference. The present disclosure additionally provides a cell for producing a VLP. The cell may be transfected with one or more nucleic acids encoding at least one or all of the viral spike protein and the at least one additional viral structural protein. The cell may further be transfected with one or more nucleic acids encoding one or more cargo molecules; in the instance of a nucleic acid cargo, the genetic sequence may be preceded by a packaging sequences (e.g., nucleic acid sequence at least 70% similar to SEQ ID NOs: 1 or 2, or a functional fragment thereof) to facilitate efficient loading into generated VLP. Compositions Further disclosed herein are compositions comprising the disclosed VLPs. Also disclosed herein are compositions or systems for treatment or prevention of a viral infection. In some embodiments, the compositions or systems comprise an interfering RNA or nucleic acid encoding thereof, as disclosed herein, and a carrier (e.g., pharmaceutically acceptable carrier).
In some embodiments, the composition comprises a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable,” as used in connection with compositions and/or cells of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors) and does not negatively affect the subject to which the composition(s) are administered. Carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and/or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). Carriers also encompass any agent capable of protecting the interfering RNA or nucleic acid encoding thereof from rapid elimination from the body and facilitating efficient systemic or targeted delivery to a cell, cell-type, or location of interest.
The compositions may be formulated for any particular mode of administration including for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis). Formulations suitable for intranasal delivery include liquids and dry powders. Formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, sucrose, trehalose, and chitosan. Mucosadhesive agents such as chitosan can be used in either liquid or powder formulations to delay mucocilliary clearance of intranasally- administered formulations. Sugars such as mannitol and sucrose can be used as stability agents in liquid formulations and as stability and bulking agents in dry powder formulations. In some embodiments, the compositions are formulated for administration to the lungs, for example, intranasal or for use with nebulizers or aerosol generators. Suitable compositions for use in nebulizers comprise the VLPs in a liquid carrier. In some embodiments, the carrier is water (and most preferably sterile, pyrogen-free water), a dilute aqueous buffered saline, or a dilute isotonic or hypertonic alcohol solution. Optional additives include preservatives, antioxidants, volatile oils, buffering agents and surfactants. Suitable compositions for use in aerosol generators may comprise or consist either solely of the VLPs or of a powder blend comprising the VLP a suitable powder diluent, such as lactose, and an optional surfactant. Suitable propellants for aerosol devices include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The composition may additionally contain one or more co-solvents, for example, water, saline, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents. In some embodiments the compositions are vaccines or an immunogenic composition comprising a VLP as described herein and an adjuvant or immunostimulant. Adjuvants and immunostimulants are compounds that either directly or indirectly stimulate the immune system’s response to a co-administered antigen. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); mineral salts (for example, aluminum, silica, kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4),
and Al(OH)3; salts of calcium (e.g., Ca3(PO4)2), iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polynucleotides (for example, poly IC and poly AU acids); polyphosphazenes; cyanoacrylates; polymerase-(DL-lactide-co- glycoside); biodegradable microspheres; liposomes; lipid A and its derivatives; monophosphoryl lipid A; wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella); bovine serum albumin; diphtheria toxoid; tetanus toxoid; edestin; keyhole-limpet hemocyanin; Pseudomonal Toxin A; choleragenoid; cholera toxin; pertussis toxin; viral proteins; and Quil A. Aminoalkyl glucosamine phosphate compounds can also be used (see, e.g., WO 98/50399, U.S. Pat. No. 6,113,918, and 6,355,257. In addition, adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants. Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule. Other known immunostimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4',4-diaminodiphenylmethane-3,3'- dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids or carbohydrates. In some embodiments, the protein or polypeptide adjuvants may be provided as a fusion protein with any of the proteins or polypeptides which make up the VLP (e.g., the envelope protein, the membrane protein, the nucleocapsid protein) The vaccines of the present disclosure may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other antigens may be present, either incorporated into a fusion polypeptide with a VLP polypeptide or as a separate compound, within the vaccine. The vaccines may generally be used for prophylactic and therapeutic purposes. The vaccines may be formulated for any appropriate manner of administration, and thus may be administered by various methods, including for example, topical, oral, nasal,
intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, subcutaneous, intramuscular administration, or via inhalation. The vaccines may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, vaccines may be formulated as a lyophilisate. Methods of Use Also disclosed herein are methods for preventing or treating a viral infection comprising contacting a cell with an effective amount of a VLP, composition, or vaccine, as described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. The methods may comprise administering to a cell an effective amount of the described VLP, composition, or vaccine. In some embodiments, the cell is in a subject and administering comprises administering the described VLP, composition, or vaccine to a subject. In some embodiments, the VLP comprises a cargo molecule comprising an interfering RNA which binds to and inhibits translation of viral RNA. Thus, the viruses causing the infection will be unable to replicate within the cells. The viral infection may be caused by a respiratory virus, as described elsewhere herein. In some embodiments, the respiratory virus is a coronavirus, e.g., betacoronaviruses, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS- CoV, MERS-CoV, and SARS-CoV-2 . In select embodiments, the respiratory virus is SARS- CoV-2. Also disclosed herein are methods for treating a disease or disorder in a subject comprising administering to the subject an effective amount of the VLP, composition, or vaccine, as described herein. The disease or disorder may be an infectious disease, cancer, a genetic disorder, or a combination thereof. In some embodiments, the disease or disorder comprises an infectious disease. The infectious disease may comprise a viral infection. In some embodiments, the viral infection is caused by a respiratory virus, a coronavirus e.g., betacoronaviruses, including but not limited to
HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. In select embodiments, the respiratory virus is SARS-CoV-2. In some embodiments, the VLP, composition, or vaccine is administered therapeutically, in response to a diagnosis or suspicion of a viral infection. Treating a viral infection, includes, but is not limited to, reducing, inhibiting, or preventing one or more symptoms of the viral infection or exacerbations thereof, or decreasing viral replication or shedding or preventing viral entry into cells. In some embodiments, the VLP, composition, or vaccine is administered prophylactically, such that it is used to prevent or delay the onset of or lessen the severity of a viral infection or the exacerbations of a viral infection. In some embodiments, the VLP comprises a cargo molecule comprising an interfering RNA which binds to and inhibits translation of viral RNA. Thus, the viruses causing the infection will be unable to replicate within the cells. In some embodiments, the disease or disorder comprises cancer, a genetic disorder, or a combination thereof. In some embodiments, the disease or disorder comprises cancer. The disclosed VLPs, compositions, and methods may be useful to treat a wide variety of cancers including carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, lymph nodes, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a blood cancer or lymphoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the methods result in suppression or elimination of metastasis. In some embodiments, the methods result in decreased tumor growth. In some embodiments, the methods prevent tumor recurrence. In some embodiments, the disease or disorder comprises a genetic disorder. Genetic disorders include any disease caused by an abnormality in the genome. In some embodiments, a VLP or composition thereof comprises a messenger RNA for a gene to supplement cells with a wild-type copy of a gene for a gene which is defective in the genetic disorder, a “disease-associated” gene. In some embodiments, a VLP or composition thereof comprises a gene editing system which may be used to correct one or more defects or
mutations in a gene (referred to as “gene correction”). Thus, the target sequence for the gene editing system is a “disease-associated” gene. The term “disease-associated gene” refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease. A disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The disease-associated gene may be associated with a genetic disorder or cancer (e.g., mutations in cell cycle proteins, oncogenes, or tumor suppressors). Examples of genes responsible for such “single gene” or “monogenic” diseases include, but are not limited to, adenosine deaminase, Į-1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), ȕ-hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate- regulating endopeptidase homologue, X-linked (PHEX), methyl- CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y). Other single gene or monogenic diseases are known in the art and described in, e.g., Chial, H. Rare Genetic Disorders: Learning About Genetic Disease Through Gene Mapping, SNPs, and Microarray Data, Nature Education 1(1):192 (2008); Online Mendelian Inheritance in Man; and the Human Gene Mutation Database (HGMD). In another embodiment, the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes. Diseases caused by the contribution of multiple genes which lack simple (e.g., Mendelian) inheritance patterns are referred to in the art as a “multifactorial” or “polygenic” disease. Examples of multifactorial or polygenic diseases include, but are not limited to, asthma, cancer, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities also can be inherited in a multifactorial
or polygenic pattern and include, for example, cleft lip/palate, congenital heart defects, and neural tube defects. In another embodiment, the method of altering a target genomic DNA sequence can be used to delete nucleic acids from a target sequence in a cell by cleaving the target sequence and allowing the host cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule. Deletion of a nucleic acid sequence in this manner can be used in a variety of applications, such as, for example, to remove disease-causing trinucleotide repeat sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research. As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. The VLP, composition, or vaccine may be administered by any desired route, including, but not limited to, inhalation, orally, intranasally, or parenteral administration (subcutaneous, intravenous, or intramuscular injection or infusion). In some embodiments, the VLP, composition, or vaccine is administered to the lung. Methods of administering to the lungs include, for example, endotracheal administration, aerosolization and inhalation. In certain embodiments, the administration is by inhalation. The term inhalation includes, for example, both inhalation of a dry powder and inhalation of a wet aerosol. In certain embodiments, a nebulizer such as a jet nebulizer, ultrasonic nebulizer, vibrating mesh nebulizer, e.g., in the form of aqueous drug solutions or dispersions, may be used for the administration. In certain embodiments, an inhaler such as a metered dose inhaler, soft mist inhaler, an insufflator, or a dry powder inhaler may be used for the administration. In certain embodiments, the administration is through a mechanical means or along with respiratory
support such as mechanical ventilation (conventional or high frequency ventilation), administration of supplemental oxygen, or continuous positive airway pressure (CPAP) including nasal CPAP (nCPAP) treatment, face mask, oxygen hood, or the like. The employment of some of the devices may involve the use of various respiratory gases, as would be appreciated by the skilled artisan. Respiratory gases used for noninvasive pulmonary respiratory therapy are sometimes referred to herein as “CPAP gas,” “CPAP air,” “nCPAP,” “ventilation gas,” “ventilation air,” or simply “air.” The specific dose level may depend upon a variety of factors including the age, body weight, and general health of the subject, time of administration, and route of administration. An “effective amount” is an amount that is delivered to a subject, either in a single dose or as part of a series, which achieves a medically desirable effect. For prophylaxis purposes, the amount of the VLP, composition, or vaccine in each dose is an amount which induces a protective result without significant adverse side effects. A wide range of second therapies may be used in conjunction with the VLPs, compositions, or methods of the present disclosure. The second therapy may be administration of an additional therapeutic agent or may be a second therapy not connected to administration of another agent. Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy, convalescent blood plasma therapy, or an additional chemotherapeutic or anti- cancer agent. The second therapy (e.g., an immunotherapy) may be administered at the same time as the initial therapy, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the second therapy may precede or follow the treatment of the first therapy by time intervals ranging from hours to months. In some embodiments, the second therapy includes immunotherapy. Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies). In some embodiments, the second therapy may include administration of antimicrobial agents, analgesics, anti-inflammatories, steroids, antipyretics, and the like. In some embodiments, the second therapy includes an antiviral agent. Antiviral agents include, but are
not limited to, oseltamivir, zanamivir, peramivir, acyclovir, valacyclovir, famciclovir, penciclovir, remdesivir, molnupiravir, and AT527 or other nucleotide analogs. Also disclosed herein are methods for treating or preventing viral infection in a cell comprising contacting the cell with an interfering RNA, a nucleic acid encoding an interfering RNA, or a composition or system, as described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. The viral infection may be caused by a respiratory virus. Common respiratory agents include influenza viruses, respiratory syncytial virus, parainfluenza viruses, rhinoviruses, coronaviruses, and adenoviruses. In some embodiments, the respiratory virus is a coronavirus. The coronavirus family comprises 45 species distributed between four genera: alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus. In some embodiments, the respiratory virus is a betacoronavirus, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. In select embodiments, the respiratory virus is SARS-CoV-2. In some embodiments, the cell is in a subject. Thus, the method may comprise administering to a subject an effective amount of the described interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system. The interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system may be administered by any desired route, including, but not limited to, inhalation, orally, intranasally, or parenteral administration (subcutaneous, intravenous, or intramuscular injection or infusion). In some embodiments, the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered systemically or to the lungs. In some embodiments, the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered to the lung. Methods of administering to the lungs include, for example, endotracheal administration, aerosolization and inhalation. In certain embodiments, the administration is by inhalation. The term inhalation includes, for example, both inhalation of a dry powder and inhalation of a wet aerosol. In certain embodiments, a nebulizer such as a jet nebulizer, ultrasonic nebulizer, vibrating mesh nebulizer, e.g., in the form of aqueous drug solutions or dispersions, may be used for the administration. In certain embodiments, an inhaler such as a metered dose inhaler, soft mist inhaler, an insufflator, or a dry
powder inhaler may be used for the administration. In certain embodiments, the administration is through a mechanical means or along with respiratory support such as mechanical ventilation (conventional or high frequency ventilation), administration of supplemental oxygen, or continuous positive airway pressure (CPAP) including nasal CPAP (nCPAP) treatment, face mask, oxygen hood, or the like. The employment of some of the devices may involve the use of various respiratory gases, as would be appreciated by the skilled artisan. Respiratory gases used for noninvasive pulmonary respiratory therapy are sometimes referred to herein as “CPAP gas,” “CPAP air,” “nCPAP,” “ventilation gas,” “ventilation air,” or simply “air.” As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered therapeutically, in response to a diagnosis or suspicion of a viral infection. Treating a viral infection, includes, but is not limited to, reducing, inhibiting, or preventing one or more symptoms of the viral infection or exacerbations thereof, or decreasing viral replication or shedding or preventing viral entry into cells. In some embodiments, the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system is administered prophylactically, such that it is used to prevent or delay the onset of or lessen the severity of a viral infection or the exacerbations of a viral infection. When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective
amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. A wide range of second therapies may be used in conjunction with the compounds of the present disclosure. The second therapy may be administration of an additional therapeutic agent or may be a second therapy not connected to administration of another agent. Such second therapies include, but are not limited to, surgery, immunotherapy, convalescent blood plasma therapy, or an additional antimicrobial agent. The second therapy (e.g., an immunotherapy) may be administered at the same time as the initial therapy, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the second therapy may precede or follow the treatment of the first therapy by time intervals ranging from hours to months. In some embodiments, the second therapy may include administration of antimicrobial agents, analgesics, anti-inflammatories, steroids, antipyretics, and the like. In some embodiments, the second therapy includes an antiviral agent. Antiviral agents include, but are not limited to, oseltamivir, zanamivir, peramivir, acyclovir, valacyclovir, famciclovir, penciclovir, remdesivir, molnupiravir, and AT527 or other nucleotide analogs. The interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system may be introduced into cells by methods known in the art. Conventional viral and non-viral based gene transfer methods can be used to introduce the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system into cells, tissues, or a subject. Such methods can be used to administer the interfering RNA, the nucleic acid encoding an interfering RNA, or the composition or system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. Delivery systems also include virus-like particles, as described above.
A variety of viral constructs may be used to deliver the interfering RNA to the targeted cells and/or a subject. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference. Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome. Any of the vectors comprising a nucleic acid sequence that encodes the interfering RNA is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid
encoding the components of the present system is a DNA molecule. In some embodiments, the nucleic acid encoding the components of the present system is an RNA molecule. Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles are disclosed elsewhere herein and include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm.2014 Jan 1;459(1-2):70-83), incorporated herein by reference. Kits Also within the scope of the present disclosure are kits that include the VLP or one or more nucleic acids encoding the components of the VLP (e.g., the spike protein, the structural proteins, the cargo molecule), host cells, and/or transfection reagents. The present disclosure also provides kits that include an interfering RNA, nucleic acid encoding an interfering RNA, and/or a carrier (e.g., delivery vehicle), as described above. The kit may further comprise a device for holding or administering the interfering RNA, nucleic acid encoding the interfering RNA, and/or a carrier. Optionally, the kits may include reagents for making or formulating a delivery vehicle with the interfering RNA. The kit may include instructions for use in any of the methods described herein. The instructions can comprise method for making and/or using the VLP. The instructions can comprise a description of transfection of a host and methods of purifying, isolating, and characterizing the VLP. These instructions generally include information as to transfection reagents, purification procedures and reagents, and host cell compatibility with the VLP components. The instructions can comprise a description of administration to a cell, tissue, or subject to achieve the intended effect. These instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. A kit may have a sterile access
port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The packaging may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above. Examples The following are examples of the present invention and are not to be construed as limiting. Example 1 Specific Treatment for and/or Prevention of SARS-CoV-2 Infection The cargo-carrying VLP is generated through the expression of SARS-CoV-2 spike (S) protein with other structural proteins (e.g., envelope (E), membrane (M), and nucleocapsid (N)) and shRNA sequence(s) preceded by either a 582 bp or 101 bp SARS-CoV-2 psi/packaging element (SEQ ID NOs: 1 and 2). These components (structural elements and shRNA + psi sequence) are cloned into separate plasmids and/or polycistronic plasmids containing various combinations of structural elements in addition to single or multiple shRNA-expression cassettes, and are subsequently transfected into a viral packaging cell line, including but not limited to HEK293T and VeroE6 cell lines, using a lipid transfection-based method at various molar ratios. For example, co-transfection of S, M, E, N, and shRNA single plasmids or polycistronic elements is performed at 8:6:8:3:8 molar ratios, followed by numerous iterations of molar ratios. 48-72 hours after transfection, the cell culture supernatant is collected and VLPs are harvested and subjected to concentration by either high speed centrifugation, or sucrose gradient centrifugation. SARS-CoV-2 virus-like particles containing viral spike protein with tropism for lung epithelial ACE2 receptors and packaged with multiple shRNAs targeting the viral genome and/or viral replicase-encoding mRNA are delivered to the lungs of SARS-CoV-2 infected individuals (humans or non-human animals). Virus-like particles bind specifically to lung epithelial cells,
enter them and are uncoated, releasing anti-viral shRNAs screened and optimized to inhibit viral replication and therefore the subsequent packaging, budding and intercellular spread of SARS- CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells. VLPs loaded with a cocktail of shRNAs or other genomic elements that interrupt the SARS-CoV-2 virus life cycle through targeting of the viral RNA genome are delivered either via intranasal administration, or aerosol/nebulization or through intravenous injection to individuals, either human or non-human animals, without evidence of SARS-CoV-2 infection. In particular, this approach is designed with individuals with immunocompromise, potentially related to hematologic malignancy, bone marrow transplant, solid organ transplant, need for chronic immunosuppressive medication in the setting of a rheumatologic, allergic, or hematologic disorder, or those with a congenital immunodeficiency, amongst others. Individuals unable to undergo vaccination against SARS-CoV-2 are also appropriate for this approach. In this scenario, the VLP delivers a genomic cocktail, including, but not limited to shRNAs targeting elements of the SARS-CoV-2 viral genome to lung epithelium expressing the ACE II receptor, which is at risk for SARS-CoV-2 infection. Genomic cargo is released into the target cells, and upon subsequent infection by SARS-CoV-2, persistent genomic cargo interferes with the translation of viral RNA, interrupting the viral life cycle. Example 2 Treatment for and/or Prevention of Respiratory Virus Infection SARS-CoV-2 virus-like particles containing viral spike protein with tropism for lung epithelial ACE2 receptors are generated, packaged with shRNAs designed to target endemic or emerging respiratory viruses, and screened and optimized. SARS-CoV-2 virus-like particles containing viral spike protein with tropism for ACE2 receptors, which is highly expressed in lung and GI tissues, amongst others, is packaged with multiple shRNAs targeting the viral genome and/or viral replicase-encoding mRNA. The therapeutic is delivered to the lungs of infected individuals (humans or non-human animals). Virus-like particles bind specifically to lung epithelial cells, enter them and are uncoated, releasing anti-viral shRNAs, thereby inhibiting viral replication and the subsequent packaging, budding and intercellular spread of SARS-CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells. Likewise, as illustrated in example 1, VLP containing a genomic cargo which targets the RNA genome of a defined respiratory virus may be administered prophylactically to prevent successful infection by
the targeted virus. A cocktail of VLPs carrying genomic cargo against a cocktail of several different respiratory viruses may also be administered; this may be informed by predicted viral outbreaks driven by global public health/community surveillance. In practice, this may be used as a tool to protect immunocompromised individuals from highly penetrant endemic respiratory viruses as well as emerging respiratory viruses. Example 3 Platform for Emerging Viral Threats Emerging viral threats are sequenced and shRNAs targeting the relevant viral genome or its expressed mRNAs are designed as described above. Virus-like particles containing viral spike protein with tropism for lung epithelial ACE2 receptors are generated as described and packaged with screened the shRNAs for delivery to the lungs of infected individuals (humans or non-human animals). Virus-like particles bind specifically to lung epithelial cells, enter them and are uncoated, releasing anti-viral shRNAs. Inhibitory RNAs inhibit viral replication and therefore the subsequent packaging, budding and intercellular spread of SARS-CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells. Example 4 Delivery of Gene Therapy Cargo Messenger RNA encoding a normal copy of the CFTR gene, both copies of which may be dysfunctional in cystic fibrosis patients, is delivered to the lung epithelium using virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium. Alternatively, mRNA encoding Cas9 and a guide RNA directing targeted gene editing of the CFTR gene are packaged into virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium. Virus-like particles are delivered to the lung, attach to lung epithelial cells, enter them and are uncoated, releasing these RNA entities, thereby resulting in expression of normally functioning CFTR in these cells. Example 5 Delivery of Cancer Therapies Messenger RNAs encoding Cas9 and guide RNA(s) directing targeted gene editing of mutated tumor-driving oncogenes or tumor suppressors, such as oncogenic RAS or p53, respectively are packaged into virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium. Virus-like particles are delivered to the lung epithelium of patients with lung
malignancies, attach to lung epithelial cells, enter them and are uncoated, releasing these RNA entities, thereby restoration of the normal function of target genes in tumor cells. Alternatively, shRNAs targeting mRNAs that encode immune checkpoint proteins or immunosuppressive proteins are packaged into virus-like particles that co-opt the SARS-CoV-2 tropism for lung epithelium. shRNAs targeting key tumor proteins such as (including, but not limited to) RAS, EGFR, ALK, ROS, RET may also be encapsulated in the VLP, with the combined product delivered either through aerosolization/nebulization or intravenous injection. Virus-like particles are delivered to the lung epithelium of patients with lung malignancies, attach to lung epithelial cells, enter them and are uncoated, releasing these RNA entities, resulting in de-repression of immune function in the tumor microenvironment and immune killing of tumor cells, while targeting of key oncogenes leads to reduced tumor growth and tumor cell apoptosis. Example 6 shRNA Design and Validation shRNAs were designed using both DSIR and Kay (Stanford) algorithms in silico with the SARS-CoV-2 protein coding sequence of viral orf1ab as target sequence input. Targeting the protein coding sequence of orf1ab. The top 30 shRNA sequences resulting from each of these algorithms (60 in total) were selected for further analysis based on efficacy and off-target prediction algorithms. Candidate sequences were culled based on sequence similarity to human protein coding genes. This final culling of candidates required ^17-base ID with non-coding genes. The resulting 15 shRNA sequences targeting coding regions for NSP1, NSP2, NSP3, NSP4, NSP 5, NSP 6 and RdRP (Table 1 and FIG. 3) were cloned into a pRB-puro-U6 plasmid containing a puromycin resistance marker. Each shRNA construct was transfected into HEK 293T cells, which were selected for stable expression of shRNAs using puromycin. Each stable cell line was challenged with overexpression of each relevant NSP tagged at the N terminus with GFP in a pReceiver vector. Effective depletion of NSPs was evaluated by quantitative measurement of GFP signal, NSP transcript level by RT-qPCR and phenotypic effect (viability and confluence). While shRNA treated cells demonstrate high levels of confluency, significant cell death can be seen by cell morphology with resultant decrease in confluency of scrambled shRNA (control) cells in response to viral RNA challenge (FIG. 4).
Example 7 Treatment of Coronaviruses SARS-CoV-2-infected individuals, either human or non-human animals or models, are treated using shRNAs targeting the viral RNA genome and/or viral replicase-encoding viral mRNA delivered to the lung or by intravenous administration, including but not limited to (virus-like particles, nanoparticles, lipid particles, aerosol and/or nebulized particles). Interfering RNAs delivered to the airway epithelium inhibit the replication and therefore the subsequent packaging, budding and intercellular spread of SARS-CoV-2 virions by depletion of the viral genome or replicase-encoding RNA in infected cells. Each shRNA target sequence has homology with sequences within the RNA genomes of multiple endemic betacoronaviruses, including but not limited to HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1. For example, SEQ ID NO: 13 has homology with bases 9182 to 9199 of Human coronavirus OC43 strain ATCC VR-759 (NC_006213.1). Individuals infected with endemic betacoronaviruses either human or non-human animals or models, are treated using shRNAs disclosed herein, targeting the viral RNA genome and/or viral replicase-encoding viral mRNA. Example 8 Prevention of SARS-CoV-2 Infection Interfering or shRNA targeting the SARS-CoV-2 genome, can be administered by aersolization/nebulization or intravenous injection to prevent SARS-CoV-2 infection. This approach has particular use in individuals who are immunocompromised or are otherwise unable to undergo vaccination against SARS-CoV-2. The same approach could be used to prevent infection with other coronaviridae family members in individuals who are immunocompromised. In practice, this may take the form of an aersolized/nebulized formulation administered on a weekly or monthly basis under the direction of a physician. Example 9 Adaptation to Emerging Viral Pathogens The genomes of emerging viral pathogens are sequenced for the study of novel viruses and the development of interventions. Emerging infections also involve previously known pathogens with known genomic sequences. These sequences are applied to methods for the design of RNA interference modalities. Screening methods are applied to the resulting shRNAs
to identify efficient shRNA entities, which are delivered to infected individuals to inhibit viral replication as described above. Example 10 Production and Cargo Loading of VLPs VLPs were generated using a multiplasmid system, with cargo including packaging sequences optimized green fluorescence protein (GFP) mRNA (FIG. 6A). Following generation, the VLPs were collected from packaging cell line supernatant, lysed, and RNA extracted using Trizol. qPCR was subsequently performed to evaluate loading of GFP mRNA. In VLPs produced by packaging cells transfected with PS101 and PS582, mRNA encoding GFP was readily detected, while no GFP mRNA was detected in mock transfected packaging cell line supernatant or in VLPs generated without co-transfection of GFP encoding message in the packaging lines, as shown in FIG. 6B for PS101. Example 11 shRNA Protection Against SARS-CoV-2 Toxicity 293T cells expressing either an shRNA cocktail targeting the SARS-CoV-2 genome (right) or scramble RNA control were subsequently challenged with plasmid encoding SARS- CoV-2 protein, NSP1. Control (scramble RNA expressing) 293T cells exhibit morphologic signs of cell death, with decreased confluence, suggesting significant toxicity from NSP1 (FIG. 7A). shRNA expressing cells, by contrast demonstrate no morphologic signs of cell death and are confluent. Representative images were acquired 48 hours after NSP1 transfection in either control (scramble RNA) expressing 293T cells or shRNA expressing 293T cells at 10x. ImageJ was used to calculate the percentage of the image area covered by cells. As shown in FIG. 7B, the analysis revealed significantly lower confluence in scramble expressing cells versus shRNA expressing cells 48 hours after NSP1 plasmid challenge, suggesting protection from cell death and/or protection from cell stress/decreased proliferative rates induced by NSP1 challenge. qPCR was performed on 293T cells expressing either scramble (control) RNA or shRNA targeting the SARS-CoV-2 genome after harvesting RNA from cells 48 hours after NSP1 challenge. Primers were developed and validated targeting NSP1. qPCR reveals significant (>85%) decrease in NSP1 transcript levels in shRNA expressing 293T cells (FIG. 7C), suggesting that the shRNAs effectively deplete SARS-CoV-2 genomic elements.
Sequences
The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that
any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.
Claims
CLAIMS What is claimed is: 1. A virus like particle (VLP) comprising: a viral spike protein configured to bind to an angiotensin-converting enzyme 2 (ACE2) receptor; and at least one additional viral structural protein.
2. The VLP of claim 1, wherein the viral spike protein is derived from SARS-CoV-2.
3. The VLP of claim 1 or 2, wherein the at least one additional viral structural protein comprises: a viral envelope protein; a viral membrane protein; a viral nucleocapsid protein; or any combination thereof.
4. The VLP of claim 3, wherein at least one or all of the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from a coronavirus.
5. The VLP of claim 3 or 4, wherein at least one or all of the viral envelope protein, the viral membrane protein, and the viral nucleocapsid protein are derived from SARS-CoV-2.
6. The VLP of any of claims 1-5, wherein the VLP further comprises an encapsulated cargo molecule.
7. The VLP of claim 6, wherein the cargo molecule comprises a nucleic acid.
8. The VLP of claim 6 or 7, wherein the cargo molecule comprises an RNA molecule.
9. The VLP of any of claims 6-8, wherein the cargo molecule comprises an interfering RNA or a messenger RNA.
10. The VLP of claim 9, wherein the interfering RNA is configured to bind to and inhibit translation of viral RNA.
11. The VLP of claim 10, wherein the viral RNA is from a respiratory virus.
12. The VLP of claim 11, wherein the respiratory virus is a coronavirus.
13. The VLP of claim 11 or 12, wherein the respiratory virus is SARS-CoV-2.
14. The VLP of any of claims 6-13, wherein the cargo molecule comprises a nucleic acid sequence encoding a gene.
15. The VLP of any of claims 6-14, wherein the cargo molecule comprises a nucleic acid sequence encoding a gene editing system.
16. The VLP of claim 15, wherein the gene editing system comprises Cas9 and at least one guide RNA directed to a target nucleic acid.
17. The VLP of any of claims 6-16, wherein the cargo molecule further comprises a nucleic acid sequence encoding a packaging signal.
18. The VLP of any of claims 6-17, wherein the packaging signal is configured to facilitate loading of the cargo molecule into the VLP.
19. The VLP of claim 17 or 18, wherein the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 and 2, or a functional fragment thereof.
20. A composition comprising the VLP of any of claims 1-19 and a pharmaceutically acceptable carrier.
21. A vaccine or an immunogenic composition comprising the VLP of any of claims 1-19 and an adjuvant.
22. A cell for producing a VLP of any of claims 1-19, the cell transfected with one or more nucleic acids encoding at least one or all of the viral spike protein and the at least one additional viral structural protein.
23. The cell of claim 22, wherein the cell is transfected with a nucleic acid encoding one or more cargo molecules.
24. The cell of claim 23, wherein the nucleic acid encoding one or more cargo molecules further comprises a packaging signal.
25. A method of producing a VLP of any of claims 1-19, the method comprising the steps of: expressing one or more nucleic acids encoding the viral spike protein and the at least one additional viral structural protein in a host cell under conditions such that the VLPs assemble in the host cell; and isolating the assembled VLPs from the host cell.
26. The method of claim 25, further comprising expressing one or more nucleic acids encoding one or more cargo molecules in the host cell or introducing one or more cargo molecules into the host cell.
27. The method of claim 26, wherein the cargo molecule further comprises a packaging signal.
28. The method of claim 27, wherein the packaging signal is configured to facilitate loading of the cargo molecule into the VLP.
29. The method of claim 28, wherein the cargo comprising a packaging signal is preferentially loaded into the VLP in comparison to cargo not comprising a packaging signal.
30. The method of any of claims 27-29, wherein the packaging signal comprises a nucleic acid sequence of SEQ ID NOs: 1 and 2, or a functional fragment thereof.
31. A method of preventing of treating viral infection in a cell comprising contacting the cell with an effective amount of a VLP of any of claims 1-19, a pharmaceutical formulation of claim 20, or a vaccine of claim 21.
32. The method of claim 31, wherein the VLP comprises a cargo molecule comprising an interfering RNA or a messenger RNA.
33. The method of claim 32, wherein the interfering RNA is configured to bind to and inhibit translation of viral RNA.
34. The method of claim 33, wherein the viral RNA is from a respiratory virus.
35. The method of claim 34, wherein the respiratory virus is a coronavirus.
36. The method of claim 35, wherein the respiratory virus is SARS-CoV-2.
37. The method of any of claims 31-36, wherein the cell is in a subject and contacting comprises administering to a subject.
38. The method of claim 37, wherein the administering comprises systemic administration, administration to the lungs, or a combination thereof.
39. A method of treating or preventing a disease or disorder in a subject comprising administering to the subject an effective amount of a VLP of any of claims 1-19, a pharmaceutical formulation of claim 20, or a vaccine of claim 21.
40. The method of claim 39, wherein the disease or disorder comprises an infectious disease, cancer, a genetic disorder, or a combination thereof.
41. The method of claim 40, wherein the infectious disease comprises a viral infection.
42. The method of claim 41, wherein the viral infection is caused by a respiratory virus.
43. The method of claim 42, wherein the respiratory virus is a coronavirus.
44. The method of claim 42 or 43, wherein the respiratory virus is SARS-CoV-2.
45. The method of any of claims 39-44, wherein the VLP comprises a cargo molecule comprising an interfering RNA, a messenger RNA, a nucleic acid sequence encoding a gene, a nucleic acid sequence encoding a gene editing system, or a combination thereof.
46. The method of claim 45, wherein the interfering RNA is configured to bind to and inhibit translation of viral RNA.
47. The method of claim 45, wherein the gene editing system comprises Cas9 and at least one guide RNA directed to a target nucleic acid.
48. The method of any of claims 39-47, wherein the administering comprises systemic administration, administration to the lungs, or a combination thereof.
49. Use of a VLP of any of claims 1-19, a pharmaceutical formulation of claim 20, or a vaccine of claim 21 for the treatment and prevention of a viral infection in a cell.
50. The use of claim 49, wherein the VLP comprises a cargo molecule comprising an interfering RNA or a messenger RNA.
51. The use of claim 50, wherein the interfering RNA is configured to bind to and inhibit translation of viral RNA.
52. The use of claim 51, wherein the viral RNA is from a respiratory virus.
53. The use of claim 52, wherein the respiratory virus is a coronavirus.
54. The use of claim 52 or 53, wherein the respiratory virus is SARS-CoV-2.
55. Use of a VLP of any of claims 1-19, a pharmaceutical formulation of claim 20, or a vaccine of claim 21 for treating or preventing a disease or disorder in a subject.
56. The use of claim 55, wherein the disease or disorder comprises an infectious disease, cancer, a genetic disorder, or a combination thereof.
57. The use of claim 56, wherein the infectious disease comprises a viral infection.
58. The use of claim 57, wherein the viral infection is caused by a respiratory virus.
59. The use of claim 58, wherein the respiratory virus is a coronavirus.
60. The use of claim 58 or 59, wherein the respiratory virus is SARS-CoV-2.
61. The use of any of claims 55-60, wherein the VLP comprises a cargo molecule comprising an interfering RNA, a messenger RNA, a nucleic acid sequence encoding a gene, a nucleic acid sequence encoding a gene editing system, or a combination thereof.
62. The use of claim 61, wherein the interfering RNA is configured to bind to and inhibit translation of viral RNA.
63. The use of claim 61, wherein the gene editing system comprises Cas9 and at least one guide RNA directed to a target nucleic acid.
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BOSON BERTRAND, LEGROS VINCENT, ZHOU BINGJIE, SIRET EGLANTINE, MATHIEU CYRILLE, COSSET FRANÇOIS-LOÏC, LAVILLETTE DIMITRI, DENOLLY : "The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 296, 1 January 2021 (2021-01-01), US , pages 100111, XP055963106, ISSN: 0021-9258, DOI: 10.1074/jbc.RA120.016175 * |
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