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WO2023230675A1 - Method, use, and kit relating to an rna virus - Google Patents

Method, use, and kit relating to an rna virus Download PDF

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Publication number
WO2023230675A1
WO2023230675A1 PCT/AU2023/050485 AU2023050485W WO2023230675A1 WO 2023230675 A1 WO2023230675 A1 WO 2023230675A1 AU 2023050485 W AU2023050485 W AU 2023050485W WO 2023230675 A1 WO2023230675 A1 WO 2023230675A1
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nucleic acid
coding sequence
rna
gene
sequence
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French (fr)
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Minh Thuan Nguyen Tran
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Alphinia Pty Ltd
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Alphinia Pty Ltd
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Priority claimed from AU2022901529A external-priority patent/AU2022901529A0/en
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    • 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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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12N2310/00—Structure or type of the nucleic acid
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    • 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
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36151—Methods of production or purification of viral material
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    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36151—Methods of production or purification of viral material
    • C12N2770/36152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors

Definitions

  • the present invention provides nucleic acids sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest.
  • nucleic acid sequences of the invention have been developed primarily for use in evolution of biomolecules of interest and will be described hereinafter with reference to this application. It will, however, be appreciated that the invention is not limited to this particular field of use.
  • Directed evolution is an effective strategy for developing one or more gene products of a gene of interest with desirable characteristics.
  • a library of genetic variants is established via mutagenesis of an initial gene of interest.
  • expressed gene products of the gene of interest are selected and assayed for a specific activity and/or function.
  • RNA viruses In cases wherein attenuated RNA viruses are used with an inserted gene of interest, significant genomic instability and a tendency towards viral recombination occurs.
  • a gene of interest is often inserted into the viral genome, rendering it non-competent in the absence of a viral factor that has been segmented in trans. Viral propagation is therefore dependent on the reconstitution of the complete viral genome to mediate viral egress.
  • the viral factor can be supplied by the presence of a defective helper RNA genome, which cannot replicate on its own, but provides in trans the necessary components for a split, non-competent RNA virus to replicate.
  • DIPs Defective interfering particles
  • RNA-binding protein such as Cas13 effectors
  • DIPs RNA-binding protein
  • the present invention seeks to establish an inducible negative selection circuit for use in directed evolution platforms that leverages split, recombinant RNA viral genomes, allowing for counter-selection against unintended, bystander effects of a hypothetical gene of interest.
  • mutator RNA viruses as a basis for directed evolution and the serial passaging of the gene products thereof.
  • a gene of interest can be stably and recombinantly integrated into a viral vector via in-frame insertion of an open reading frame of the gene of interest with an aspect of the native but attenuated viral genome.
  • This configuration of the viral genome leveraging a split viral vector and an aspect of a viral factor that has been shown to robustly interact with the split viral vector, enables the serial passaging of the recombinant viral particle in an indel and recombination-averse manner.
  • the present invention discloses a method to modulate the synthesis of recombinant, non-competent RNA viruses for the purposes of evolving a gene of interest through use of nucleic acids.
  • the term “viral vector” should be understood to refer to a nucleic acid that includes a viral genome which, when introduced into a suitable host cell or used as a template for in vitro transcription, can produce viral RNA which can be replicated and packaged into viral particles. Such viral particles transfer the viral genome into another host cell. It will be appreciated that the term “viral vector” extends to at least part of a viral genome, i.e., truncated and/or partial viral genomes. In some embodiments, a viral vector is provided that lacks one or more gene encoding a protein essential for generation of an infectious viral particle.
  • viral particle should be understood to refer to viral nucleic acid(s) that encode one or more viral coat protein(s) and/or viral lipid envelope.
  • infectious viral particle should be understood to refer to a viral particle that can transport at least part of a viral nucleic acid into a suitable host cell.
  • the present invention relates to nucleic acid sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest.
  • a method of providing an RNA virus comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus, wherein: the first nucleic acid comprises
  • a method of evolution of a gene product of a gene of interest comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus; allowing maturation and egress of one or more RNA virus(es) that include the gene of interest or a variant thereof from the population of suitable host cells; recovering the one or more RNA virus(es); introducing
  • a kit comprising: a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub- genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’
  • a kit comprising: a 5’ portion of a first nucleic acid; a 3’ portion of a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; one or more instruction(s) for, in use, inserting a gene of interest selectively between the 5’ portion of a first nucleic acid and the 3’ portion of a first nucleic acid to provide a first nucleic acid; wherein: the first nucleic acid, in use, comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non- structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second
  • This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • Figure 1 is a schematic representation outlining the arrangement of nucleic acids comprising defective interfering particle (DIP).
  • the DIP sequence comprises a 5’UTR region, a non-structural protein 1 (nsp1) protein coding sequence, an E1 protein coding sequence, and a 3’UTR, which are sequences of a Togaviridae virus.
  • Figure 2 is a schematic representation outlining the preferential replication of defective interfering particle (DIP) mediated by a split, non-competent, recombinant RNA virus in a suitable host cell. The preferential replication of the DIP results in the eventual reduction in viral titre leading to extinction of the viral population in the gene pool.
  • DIP defective interfering particle
  • FIG. 3 is a heatmap overview outlining various species of defective interfering particle (DIPs) and their effects on repressing viral populations according to the invention.
  • SIN2A-EGFP-ENV was serially passaged in the presence of its viral complement component and a DIP variant.
  • CHIK3B refers to an inert RNA-species that was used as a negative control and did not lead to a decline in viral titres (genome count per pL (gc/pl)), as measured by qPCR against the enhanced green fluorescent protein (EGFP) transgene.
  • Girdwood refers to the engineering of a DIP variant that is specific to the Sindbis viral strain used for SIN2A- EGFP-ENV.
  • Figure 4 is a heatmap demonstrating that the removal of the Dg-Girdwood variant from the gene pool enables an increase in viral titres, indicating that introduction of the Dg- Girdwood variant does not result in gene fixation.
  • Orthogonal passaging refers to the serial passaging of repressed viral populations in the absence of Dg-Girdwood or defective interfering particle (DIP) under conditions of genetic drift.
  • Viral titres as measured by qPCR against the enhanced green fluorescent protein (EGFP) transgene, are represented by genome count per pL (gc/pl).
  • Figure 5 are bar graphs demonstrating the effects of nucleofected PspCas13b and its cognate spacer targeting the viral complement that provides viral factors in trans needed for viral maturation and egress.
  • SpCas9 is nucleofected, which has no known RNA-targeting capabilities, as a point of comparison.
  • BHK-21 cells were first nucleofected with the Cas effector and the spacer, followed by application of SIN2A-EGFP- ENV virus bearing an enhanced green fluorescent protein (EGFP) transgene reporter at a high MOI of between 50 to 100.
  • EGFP enhanced green fluorescent protein
  • the present invention employs methods and techniques that are known to a person having ordinary skill in the art. Such methods and techniques as employed in the present invention include conventional molecular biology, microbiology, and recombinant DNA methods and techniques as disclosed and explained fully in the relevant literature, for example only, Becker’s World of the Cell, 9 th edition, Hardin, J., et al., Pearson (2015); Essential Cell Biology, 5 th edition, Alberts, B, et al., T&F/Garland (2019); Freshney's Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Freshney, R.I., and Capes-Davis, A., Wiley-Blackwell (2021); Gel Electrophoresis: Nucleic Acids, Martin, R., Garland Science (2020); Karp’s Cell and Molecular Biology, 9 th edition, Karp, G
  • biomolecule biological macromolecule
  • biological macromolecule biological macromolecule
  • Nucleic acids also termed “polynucleotides”, as used herein should be understood to include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), artificial nucleic acid analogs, and any combination thereof. Artificial nucleic acid analogs include, for example, glycol nucleic acid, locked nucleic acid, morpholino nucleic acid, peptide nucleic acid, threose nucleic acid, and any combination of such analogs. Nucleic acids should also be understood to include, for example, modified nucleobases and artificial nucleobases.
  • nucleic acid and “nucleic acid sequence” and variations of each term should be understood as used herein to interchangeably refer to a nucleic acid molecule or to a series of letters that indicate the order of nucleotides in a nucleic acid as appropriate in the context of the relevant term.
  • gene of interest should be understood to refer to a nucleotide sequence encoding a gene product of interest. Such a gene product of interest should be understood to refer to a gene product intended to be evolved in a circuitous evolution process as disclosed herein. It will be appreciated that the term “gene of interest” includes variations of the gene of interest that are a result of the circuitous evolution process disclosed herein. A person of ordinary skill will appreciate that a gene of interest could be any nucleic acid encoding a gene product to be evolved.
  • protein and “peptide” and derivatives thereof as used herein should be understood to refer to, for example, molecules including amino acids, and analogs, and derivatives of amino acids.
  • protein as used herein should be understood to mean proteins, polypeptides, and peptide of any size, structure, or function.
  • the term as used herein should be understood to refer to a plurality of amino acids linked by peptide bonds.
  • Proteins, as understood herein, include naturally occurring and non-naturally occurring amino acids. Such naturally occurring and non-naturally occurring amino acids may also be modified by addition of one or more chemical entity.
  • proteins may include naturally occurring and non-naturally occurring amino acids analogs and/or derivatives. A person of ordinary skill will understand that proteins may be a single molecule or a plurality of molecules in a complex. Proteins as understood herein include protein fragments, naturally occurring molecules, synthetic molecules, recombination molecules, and any combination of the afore mentioned molecules.
  • lipid should be understood as used herein to refer to, for example, diglycerides, fat-soluble vitamins (such as vitamins A, D, E, and K), fatty acids, glycerolipids, glycerophospholipids, monoglycerides, phospholipids, polyketides, prenols, saccharolipids, sphingolipids, sterols, triglycerides, waxes, and analogs and derivatives of the afore mentioned.
  • fat-soluble vitamins such as vitamins A, D, E, and K
  • fatty acids such as vitamins A, D, E, and K
  • glycerolipids such as vitamins A, D, E, and K
  • fatty acids such as vitamins A, D, E, and K
  • glycerolipids such as vitamins A, D, E, and K
  • fatty acids such as vitamins A, D, E, and K
  • glycerolipids such as vitamins A, D, E, and K
  • fatty acids such
  • carbohydrate should be understood as used herein to refer to, for example, monosaccharides, disaccharides, oligosaccharides, polysaccharides, and analogs and derivatives of the afore mentioned.
  • the present invention relates to one or more nucleic acid sequence selected from the group consisting of: a nucleic acid sequence that includes 5’ to 3’: at least part of a 5’ UTR sequence, at least part of non-structural protein 1 (nsp1) coding sequence, at least part of an E1 coding sequence, and at least part of a 3’ UTR sequence, wherein the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence and the 3’ UTR sequence are sequences of a Togaviridae virus.
  • Promoters useful in the present invention include, but are not limited to, a polynucleotide motif related to the recruitment of VP64-p65-Rta elements, miniCMV promoter, a motif ten element and downstream promoter element, a polynucleotide motif encompassing a TATA box, a polynucleotide motif related to the recruitment of p65, a polynucleotide motif related to the recruitment of p65, a polynucleotide motif related to the recruitment of Rta, a polynucleotide motif related to the recruitment of VP64, SP6 promoter in a mammalian expression vector system, T3 promoter in a mammalian expression vector system, T7 promoter in a mammalian expression vector system, a tetracycline response element, and a tetracycline-inducible promoter response to tetracycline-responsive transactivator protein or reverse tetracycline-respons
  • the Togaviridae virus is an Alphavirus.
  • the Alphavirus is Sindbis virus.
  • An aspect of the invention provides a method of providing an RNA virus, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus, wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence
  • RNA virus should be understood to refer to mature, split, non-competent viral genome, which is used herein interchangeably to refer to a “recombinant RNA virus” encoding the gene of interest.
  • the “RNA virus” can be recombined to achieve self-competency.
  • the method further comprises: introducing to the suitable host cell a sixth nucleic acid; and enabling, in the suitable host cell, expression of the sixth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the sixth nucleic acid; wherein: the sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and expression of the sixth nucleic acid modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid modulates assembly of the one or more expression produces) of the sixth nucleic acid; and the 5’ UTR sequence; at least part of
  • the Togaviridae virus is an Alphavirus.
  • the Alphavirus is Sindbis virus.
  • the sixth nucleic acid is a defective interfering particle (DIP).
  • DIP defective interfering particle
  • DIP defective interfering particle
  • viral particle should be understood to refer to viral nucleic acid(s) that encode one or more viral coat protein(s) and/or viral lipid envelope.
  • infectious viral particle as herein should be understood to refer to a viral particle that can transport at least part of a viral nucleic acid into a suitable host cell.
  • viral titre should be understood to refer to the concentration of viral particles within a volume of liquid such as media or supernatant that can be measured using traditional qPCR methods using qPCR probes against the viral genome or measured via traditional plaque assays.
  • the RNA-binding protein is a ribonucleoprotein.
  • the RNA binding protein is an RNA-guided RNA endonuclease.
  • the RNA-guided RNA endonuclease is Cas13 or a Cas13 ortholog.
  • Cas13 is selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d protein.
  • the fourth nucleic acid is a first guide sequence capable of binding to the RNA-binding protein.
  • the first guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
  • the fifth nucleic acid is a second guide sequence capable of binding to the RNA-binding protein.
  • the second guide sequence capable of binding to the RNA- binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
  • the fourth and fifth nucleic acid can occur in a CRISPR array tiled against the first, second, third or sixth nucleic acid to further enhance Cas13’s antiviral effect.
  • the CRISPR array can have a varied direct repeat region based on the Cas13 ortholog, and that multiple guide sequences can be delivered to the host cell to mediate RNA interference.
  • the fourth and fifth nucleic acid can occur as a mature single-guide sequence, or as a precursor guide sequence (pre-crRNA).
  • the gene of interest encoded by the first nucleic acid can also encode an RNA-binding protein, such as a Cas13 ortholog, Cas12 ortholog, or a Cas9 ortholog, that can bind to the fourth and fifth nucleic acid.
  • the fourth and fifth nucleic acid can encode different direct repeat and tracRNA sequences for multiplex targeting against dsDNA or ssRNA.
  • spacer should be understood to refer interchangeably to “single guide RNA (sgRNA)” or “guide RNA” or “guide sequence” that describes a short RNA polynucleotide with a hair-pin motif encoded by a direct repeat sequence that binds to an RNA- guided endonuclease.
  • sgRNA single guide RNA
  • guide RNA guide sequence
  • the “spacer” can form part of a CRISPR array tiled against a target transcript or gene.
  • nucleic acid can be encoded by a mammalian expression vector that can be transiently transfected or integrated into mammalian host cell via lentivirus.
  • the fourth and fifth nucleic acid can be siRNA to mediate RNA interference of the first, second, third, and sixth nucleic acid.
  • the fourth and fifth nucleic acid can be delivered to the host cell via co-transfection of both the fourth and fifth nucleic acid or can be encoded on a single mammalian expression vector expressing one or both of the fourth and fifth nucleic acids.
  • the fourth and/or fifth nucleic acid binds to one or more of Cas9, Cas12, and Cas13 proteins.
  • the sixth nucleic acid comprises at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA.
  • the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA hairpin motif.
  • the RNA hairpin motif can be used synergistically to increase nuclear export of a nucleic acid.
  • the cis-acting element that promotes nuclear export of an incompletely spliced RNA is a constitutive transport element (CTE).
  • CTE constitutive transport element
  • the constitutive transport element is selected from the group consisting of simian retrovirus type 1 , Rous sarcoma virus, and Woodchuck hepatitis virus constitutive transport element.
  • the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA transport element (RTE).
  • RTE RNA transport element
  • At least one mammalian expression vector comprising at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid is introduced into the suitable host cell.
  • the mammalian expression vector is a mammalian expression plasmid.
  • the suitable host cell is a transgenic mammalian cell.
  • the transgenic mammalian cell comprises at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid.
  • Another aspect of the invention provides a method of evolution of a gene product of a gene of interest, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus; allowing maturation and egress of one or more RNA virus(es) that include the gene of interest or a variant thereof from the population of suitable host cells; recovering the one or more RNA virus(es); introducing the one or more
  • the method further comprises: introducing to the suitable host cell a sixth nucleic acid; and enabling, in the suitable host cell, expression of the sixth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the sixth nucleic acid; wherein: the sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and expression of the sixth nucleic acid modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid modulates assembly of the one or more expression produces) of the sixth nucleic acid; and the 5’ UTR sequence; at least part of
  • the Togaviridae virus is an Alphavirus.
  • the Alphavirus is Sindbis virus.
  • the RNA-binding protein is a ribonucleoprotein.
  • the sixth nucleic acid is a defective interfering particle (DIP).
  • DIP defective interfering particle
  • the third nucleic acid encodes an RNA-binding protein.
  • the RNA binding protein is an RNA-guided RNA endonuclease.
  • the RNA-guided RNA endonuclease is Cas13 or a Cas13 ortholog.
  • Cas13 is selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d protein.
  • the fourth nucleic acid is a first guide sequence capable of binding to the RNA-binding protein.
  • the first guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
  • the fifth nucleic acid is a second guide sequence capable of binding to the RNA-binding protein.
  • the second guide sequence capable of binding to the RNA- binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
  • the fourth and fifth nucleic acid can occur in a CRISPR array tiled against the first, second, third or sixth nucleic acid to further enhance Cas13’s antiviral effect.
  • the CRISPR array can have a varied direct repeat region based on the Cas13 ortholog, and that multiple guide sequences can be delivered to the host cell to mediate RNA interference.
  • the fourth and fifth nucleic acid can occur as a mature single-guide sequence, or as a precursor guide sequence (pre-crRNA).
  • pre-crRNA precursor guide sequence
  • the gene of interest encoded by the first nucleic acid can also encode an RNA-binding protein, such as a Cas13 ortholog, Cas12 ortholog, or a Cas9 ortholog, that can bind to the fourth and fifth nucleic acid.
  • the fourth and fifth nucleic acid can encode different direct repeat and tracRNA sequences for multiplex targeting against dsDNA or ssRNA.
  • nucleic acid can be encoded by a mammalian expression vector that can be transiently transfected or integrated into mammalian host cell via lentivirus.
  • the fourth and fifth nucleic acid can be siRNA to mediate RNA interference of the first, second, third, and sixth nucleic acid.
  • the fourth and fifth nucleic acid can be delivered to the host cell via co-transfection of both the fourth and fifth nucleic acid or can be encoded on a single mammalian expression vector expressing one or both of the fourth and fifth nucleic acids.
  • the fourth and fifth nucleic acid can bind to one or more protein selected from the group consisting of Cas9, Cas12, and Cas13 proteins.
  • the sixth nucleic acid comprises at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA.
  • the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA hairpin motif.
  • the RNA hairpin motif can be used synergistically to increase nuclear export of a nucleic acid.
  • the cis-acting element that promotes nuclear export of an incompletely spliced RNA is a constitutive transport element (CTE).
  • CTE constitutive transport element
  • the constitutive transport element is selected from the group consisting of simian retrovirus type 1 , Rous sarcoma virus, and Woodchuck hepatitis virus constitutive transport element.
  • the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA transport element (RTE).
  • RTE RNA transport element
  • At least one mammalian expression vector comprising at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid is introduced into the suitable host cell.
  • the mammalian expression vector is a mammalian expression plasmid.
  • the suitable host cell is a transgenic mammalian cell.
  • the transgenic mammalian cell comprises at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid.
  • general negative selection is used for directed evolution leveraging split, non-competent recombinant RNA viruses encoding a gene of interest and mammalian host cells to counter select against undesirable properties of a selected gene of interest.
  • negative selection in the context of directed evolution is used to penalize a viral population upon its induction; therefore, negative selection in this context is understood to have a repressive effect on viral titre.
  • gene products of the gene of interest have improved or altered, increased, or reduced specificity, stability, fidelity, or enantioselectivity.
  • gene products of the gene of interest have broadened substrate specificity resulting from both positive and negative selection, for example, a gene product may have increased catalytic activity on both its native substrate and on non-native substrates.
  • negative selection alone is used to evolve the gene of interest.
  • alternating rounds of positive selection followed by negative selection, or negative selection followed by positive selection is used to evolve the gene of interest.
  • negative selection comprises use of a defective interfering particle (DIP), where the expression of the DIP occurs within the host cell.
  • DIP defective interfering particle
  • the DIP can be transiently transfected using RNA or DNA transfection methods using ordinary kits (i.e., LipofectamineTM MessengerMAXTM Transfection Reagent, Thermo Fisher Scientific, #LMRNA008 or LipofectamineTM 2000 Transfection Reagent, Thermo Fisher Scientific, #1 1668019).
  • the DIP can be introduced as an in vitro transcribed product from a template using ordinary kits (i.e., mMESSAGE mMACHINETM SP6 Transcription Kit, Thermo Fisher, #AM1340), ortransiently transfected as a plasmid encoding the polynucleotide sequence of the DIP into amenable host cells using ordinary kits (i.e., Mirus Bio TransIT 2020, Mirus Bio, #MIR 5405).
  • ordinary kits i.e., mMESSAGE mMACHINETM SP6 Transcription Kit, Thermo Fisher, #AM1340
  • Mirus Bio TransIT 2020 Mirus Bio, #MIR 5405
  • the expression of the DIP from lentiviral-integration or plasmid in an amenable host cell, such as BHK-21 cells can be under a constitutive promoter such as CMV promoter, or an inducible promoter such as tetracycline-responsive promoters. Kits
  • kits comprising: a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence;
  • kits comprising: a 5’ portion of a first nucleic acid; a 3’ portion of a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; one or more instruction(s) for, in use, inserting a gene of interest selectively between the 5’ portion of a first nucleic acid and the 3’ portion of a first nucleic acid to provide a first nucleic acid; wherein: the first nucleic acid, in use, comprises 5’- 3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protea
  • kits will further comprise one or more suitable host cell(s).
  • kits will further comprise one or more instruction (s) for cloning a gene of interest into the first nucleic acid.
  • methods of cloning to insert the gene of interest into the first nucleic acid are contemplated to include any method that facilitates the joining of DNA fragments, i.e., two or more DNA fragments. Examples of such methods include, but are not limited to, Gibson assembly, Golden Gate assembly, typical restriction digestion, ligation methods, and other assembly methods that may involve PCR and homology arms complementary to the insert and/or the destination vector.
  • a person skilled in the art will also appreciate that the typical screening methods to identify a correct vector may include restriction digestion, Sanger sequencing, and/or deep sequencing to identify the inserted gene of interest.
  • kits will further comprise one or more instruction (s) for inserting the gene of interest into the first nucleic acid.
  • kits will further comprise one or more instruction (s) for performing the evolution of a gene product of a gene of interest.
  • BHK-21 [C-13] cells (#CCL-10) (hereinafter BHK-21) were grown in a humidified 37°C (5% CO2) incubator in MEM a (ThermoFisher, #32571101) supplemented with 1 % Penicillinstreptomycin (% v/v), 5% Fetal Bovine Serum (FBS) (ThermoFisher Scientific, #16000044) (% v/v) and 10% tryptose phosphate broth (TPB) (ThermoFisher, #CM0283B) (% v/v), referred to as BHK-21 Full Medium.
  • MEM a ThermoFisher, #32571101
  • FBS Fetal Bovine Serum
  • TPB tryptose phosphate broth
  • Plasmids (SEQ ID NO. 5, 6, 7, 8) (3 pg) were linearized prior to mRNA synthesis by using Xbal restriction enzyme. Phenol-chloroform extraction following manufacturer’s protocol (Thermofisher, #15593031) was used to purify linearized plasmid, and 500 ng of input DNA was used to produce mRNA, with LiCI purification steps taken immediately after, using the mMESSAGE mMACHINETM SP6 Transcription Kit (ThermoFisher, #AM1340), as outlined English, J. G. et al. Cell 178, 748-761 ,e17 (2019). Next, mRNA was quantified via nanodrop spectrophotometer. mRNA integrity was assessed by gel electrophoresis.
  • a single round of a campaign includes four technical replicates run in parallel.
  • Naive BHK-21 cells were seeded in T25 flasks at 2.5 x 10 5 cells/flask and incubated for 24 hours. Subsequently, each T25 flask was transduced using 1 mL of viral supernatant or viral supernatant diluted in TM diluent was prepared as described “Current Protocols in Microbiology”, Coico (2005), with intermittent shaking every 15 minutes for 1 hour under “General Cell Culture” conditions. Following transduction, viral supernatant was aspirated entirely and 2 x DPBS wash was performed.
  • transfection was carried out using 5 pg of in vitro transcribed, defective helper RNA genome (SEQ ID NO. 9) and 5 pg of in vitro transcribed, DIP (SEQ ID NO. 5, 6, 7 or 8) each made up to a total volume of 500 pL with Opti-MEM Reduced Serum Medium (GlutaMAX Supplement) (ThermoFisher, #51985034) with 15 pL LipofectamineTM MessengerMAXTM Transfection Reagent (Thermo Fisher Scientific, #LMRNA008) according to the manufacturer’s recommendations. After 4 hours, transfection media was removed, and cells were recovered in 5 mL of BHK-21 Full Medium and incubated for 18 to 24 hours.
  • Opti-MEM Reduced Serum Medium GlutaMAX Supplement
  • GlutaMAX Supplement ThermoFisher, #51985034
  • LipofectamineTM MessengerMAXTM Transfection Reagent Thermo Fisher Scientific, #LMRNA008
  • Virus-containing supernatants were harvested via centrifugation at 500 g for 5 minutes to pellet cellular debris, followed by filtration using a 0.45 pm filter (Merck Millipore #SLHV033RS) and a sterile 10 mL Luer lock syringe (MicroAnalytix, #MS S3P10LL). Clarified supernatants were collected for titration and used for subsequent transduction experiments or stored at 4°C for up to 1 month.
  • reverse transcription was carried out under the following parameters: 50°C for 1 min, 51 °C for 1 min, 52°C for 1 min, 53°C for 1 min, 54°C for 1 min, 55°C for 3 mins, 56°C for 3 mins, 57°C for 3 mins, 58°C for 3 mins, 59°C for 3 mins, 60°C for 10 mins, 50°C for 30 mins, and denaturation at 98°C for 2 mins. Cycling PCR was performed using the following parameters: 98°C for 10 secs, 50°C for 10 secs, 72°C for 30s per 1 kb for 40 cycles, and 72°C for 5 mins.
  • DNA concentration was determined using the QubitTM 1X dsDNA HS Assay Kit (ThermoFisher, #Q32851) following purification or PCR-related steps. 300 ng of viral RNA was amplified using the SuperscriptTM IV One-Step RT-PCR System. Bands of interest were gel extracted with the Monarch DNA Gel Extraction Kit (NEB, #T 1020). For all subsequent PCRs, ultra-high-fidelity PlatinumTM SuperFi II PCR Master Mix (ThermoFisher, #12368050) was used following the manufacturer's instructions. Next, a limited 2-cycle PCR was performed on 100 ng of DNA template (SEQ ID NO. 13) using modified dual-UMI primers (SEQ ID NO.
  • Illumina-specific adapters (Illumina, #FC-131-2003) were attached using 15- cycles of PCR following 1.0x Ampure XP bead clean-up prior to sequencing using a NovaSeq 6000 S4 SP PE250 flow cell (Illumina, #20039236). Quantification of viral titre using E2-specific probes.
  • Plaque assays were performed in 12-well plates with viral supernatant stocks diluted to concentrations between 10 -1 to 10 -6 . Briefly, pre-sterilised methylcellulose (Spectrum, #ME136) was dissolved in BHK-21 Full Medium (1% w/v) at 4°C overnight with agitation (300 RPM) using a magnetic stirrer (referred to henceforth as Plaquing Media). One day prior to viral transduction, naive BHK-21 cells were plated at a density of 70,000 cells/well in a 12-well plate and incubated as described in “General Cell Culture” above.
  • transduced BHK-21 cells were RNA transfected using 1 pg of LiCI purified, in vitro transcribed defective helper RNA (SEQ ID NO. 9), and 1.5 pL of Lipofectamine 2000 (Thermo Fisher, # 11668019) to a total volume of 250 pL of Opti-MEM Reduced Serum Medium (GlutaMAX Supplement) (ThermoFisher, #51985034).
  • the transfection:RNA complex was then incubated at room temperature for 20 minutes, before being added to each well of priorly nucleofected and transduced BHK-21 cells.
  • the media was again aspirated, and washed once with 1 X DPBS, before being further incubated for 48 hours, with supernatant collection at 24 hours and at 48 hours for downstream qPCR using E2-specific probes, as described in “Quantification of viral titre using E2-specific probes.”
  • Linearized, purified plasmid DNA bearing polynucleotide sequences of the viral vector, defective helper RNA genome (SEQ ID NO. 9) and defective interfering particles (DIPs) (SEQ ID NO. 5, 6, 7 and 8) are separately used as inputs in an in vitro transcribed reaction to individually produce 5’ capped, polyadenylated, long non-coding RNAs (IncRNAs).
  • the resulting in vitro transcribed RNAs are LiCI purified and quantified via spectrophotometry.
  • IncRNAs viral vector, defective helper RNA genome, and DIPs
  • 5 pg of the three types of IncRNAs are cointroduced into naive BHK-21 cells in a T75 flask at density of 1 .0 x 10 6 cells/flask for a period of 24 hours, followed by subsequent clarification of the viral supernatant via centrifugation and filtration using a 0.45 pm filter.
  • Naive or recombinant mammalian cells are transduced with viral particles for one hour with intermittent shaking in a humidified 37°C (5% CO 2 ) incubator.
  • 5 pg of LiCI purified, in vitro transcribed RNA corresponding to the defective helper RNA genome and DIP are co-transfected into the transduced mammalian cells.
  • the transfection reagent is removed from the cell followed by 2 X DPBS.
  • the viral particles interact with the IncRNA of the defective helper RNA genome (SEQ ID NO. 9) and DIP (SEQ ID NO. 5, 6, 7, and 8).
  • the viral particle is inhibited in the presence of DIP.
  • the defective helper RNA genome favours viral maturation and egress.
  • the virus can be clarified and collected from the cell culture and purified to isolate the gene of interest.
  • Viral supernatants can be monitored for growth or decay of the population via qPCR- based methods of quantification, plaque assays, or using RNA biosensors.
  • a mammalian cell line may be engineered or transiently transfected to mediate RNA interference of a viral factor.
  • RNA interference can be targeted against the DIP or may be targeted against the viral vector or defective helper RNA genome.
  • RNA interference can be mediated by PspCas13b, LwaCas13a, or other variant of Cas13 effectors with a spacer (SEQ ID NO. 25, 26, 27, 28, 29, 30, 31 and 32) or siRNA targeting the viral vector (SEQ ID NO. 10), defective helper RNA genome (SEQ ID NO. 9) or DIP (SEQ ID NO.
  • RNA interference such as PspCas13b.
  • the gene of interest contained within the recombinant viral particle directly or indirectly mediates RNA interference and alters the balance of defective helper genome or DIP depending upon a basis of positive or negative selection.
  • Some embodiments of the gene of interest develop mutations as a result of being in the viral particle/viral vector. These mutations confer a benefit to these gene of interest variants by increasing viral titre indicating efficient viral maturation and egress.
  • viral titre can increase as a result of the gene of interest disrupting a method of RNA interference targeting the viral vector or defective helper RNA genome.
  • viral titre can increase as a result of the gene of interest inducing a method of RNA interference targeting the DIP.
  • the viral titre can decrease as RNA interference of the viral vector or defective helper genome is promoted via induction by the gene of interest.
  • Viral maturation and egress can be mediated by the presence of the defective helper RNA genome and the viral vector, and the absence of the DIP, thereby conferring an advantage to the viral particle encoding that particular variant of the gene of interest.
  • Evolved viral particles containing mutated gene of interest are able to enter the media. Following collection of cell media, the process can be repeated iteratively, or the gene of interest can be isolated and sequenced.
  • the T25 flask is incubated at 37°C for one hour, with intermittent shaking every 15 minutes to allow even coverage of the surface area of the cell monolayer. [135] Subsequently, the viral supernatant is aspirated, and 1X DPBS wash is performed twice to remove residual viral supernatant. Transfection of the defective helper RNA genome enabling viral maturation and egress, or DIP inhibiting viral maturation and egress is performed as an RNA transfection method using ordinary kits.
  • the defective helper RNA genome or DIP can be either LiCI-purified RNA or delivered as a plasmid mediating expression of these components. Culture media is replenished and the T25 flasks are returned to the incubator for a period of up to 1 to 2 days.
  • viral supernatant Media is harvested, hereafter referred to as ‘viral supernatant’, and clarified as described above.
  • the viral supernatant is stored at refrigerated conditions.
  • a method of modulating production of an RNA virus included co-transfection of an in vitro transcribed defective helper genome and an in vitro transcribed recombinant Sindbis virus vector into a suitable host cell.
  • the Sindbis virus vector encoded a gene of interest.
  • gene of interest for example, was a reporter-like GFP gene.
  • RNA interference using an RNA-binding protein targeted against the in vitro transcribed defective helper genome, the in vitro transcribed recombinant Sindbis virus vector, or in vitro transcribed DIP modulates production ofthe RNA virus.
  • Such modulation of the production of the RNA virus is positive, i.e., increases the viral titre of a viral particle including the gene of interest coding sequence relative to a population of viral particles modulated by the presence of DIPs or a method of RNA interference targeting defective helper genomes or the recombinant Sindbis virus vector.
  • the modulation of the production of the RNA virus is negative, i.e., decreases the viral titre of a viral particle including the gene of interest coding sequence relative to a population of viral particles modulated by a method of RNA interference targeting the presence of DIPs in the gene pool.
  • the in vitro transcribed recombinant Sindbis virus vector is linearized via a restriction digest and used as a template for in vitro transcription if the viral coding sequence is downstream of an SP6 or T7 promoter using ordinary kits (i.e., mMESSAGE mMACHINETM SP6 Transcription Kit, Thermo Fisher, #AM1340).
  • RNA interference is directed to either the in vitro transcribed defective helper genome or the in vitro transcribed recombinant Sindbis virus vector, which modulates production of the RNA virus.
  • modulation of the production of the RNA virus includes providing one or more nucleic acid(s) to mediate the RNA interference.
  • Such one or more nucleic acids are contemplated to include guide sequences that direct the RNA binding protein.
  • RNA interference increases the viral titre of the viral particle containing the gene-of-interest coding sequence and/or variants thereof relative to a population of viral particles modulated by the overexpression of DIPs.
  • RNA interference decreases the viral titre of the viral particle containing the gene-of-interest coding sequence and/or variants thereof relative to a population of viral particles replicating in the absence of a method of RNA interference wherein either the defective helper genome or viral vector is targeted.
  • decreasing viral titre included nucleofection of mammalian expression plasmids constitutively expressing PspCas13b with a CRISPR array tiled against a SINV capsid sequence into a naive BHK-21 cells to provide a nucleofected BHK-21 cells.
  • the nucleofected BHK-21 cell was subsequently incubated for 24 hours.
  • RNA viruses i.e., recombinant viral vector genomes expressing an EGFP transgene reporter, hereafter referred to as SIN2A-EGFP-ENV viruses
  • MOI multiplicity- of-infection
  • in vitro transcribed defective helper RNA which encodes the SINV capsid gene
  • the nucleofected BHK-21 cells including the SIN2A-EGFP-ENV virus, expresses PspCas13b with its CRISPR array component resulting in the knockdown of the defective helper RNA. In effect, this inhibits viral maturation of SIN2A-EGFP-ENV as the RNA virus is non-competent without its in trans provision of the capsid gene. Subsequently, the nucleofected cells were transfected with in vitro transcribed defective helper RNA to mediate viral maturation. As a negative control, the experiment was performed in parallel with a condition in which SpCas9 was nucleofected into naive BHK-21 cells, as opposed to PspCas13b.
  • SpCas9 has no known RNA-targeting capabilities in the context of a CRISPR array with direct repeats specific to PspCas13b.
  • the condition in which SpCas9 was used showed a higher viral titre at both 24 hour- and 48 hour-time points post-nucleofection as compared to the condition nucleofected with active PspCas13b.
  • the method results in a 1.1-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1 .2-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1 .3-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1 .4-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1 .5-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1 .6-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1 .7-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1 .8-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 1.9-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 2-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 4-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in an 8-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 16-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 32-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 64-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • the method results in a 124-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
  • a method of modulating production of an RNA virus included co-transfection of an in vitro transcribed defective helper genome and in vitro transcribed recombinant Sindbis virus vector encoding GFP, hereafter referred to as SIN2A-EGFP-ENV.
  • Co-transfection of an additional in vitro transcribed DIP modulates the assembly the RNA virus by decreasing the viral titre of the RNA virus relative to a population of viruses that are replicating in the absence of overexpressed DIPs.
  • the presence of the DIP reduces synthesis of the viral components provided by the in vitro transcribed defective helper genome, thereby inhibiting viral production.
  • Dwt-WT and Dg-WT Two confirmed DIP variants, referred to as Dwt-WT and Dg-WT, and engineered Girdwood strain counterparts, hereafter referred to as Dwt-Girdwood and Dg-Girdwood, respectively were serially passaged by clarifying the viral particles isolated from the previous round and applying the viral supernatant to naive BHK-21 cells transfected with in vitro transcribed DIPs and defective helper RNA for three rounds. Each condition used one of the four DIP variants (Dwt-WT, Dg-WT, Dwt-Girdwood or Dg-Girdwood), normalizing for the initial input RNA amount for virus generation.
  • SIN2A-EGFP-ENV was also serially passaged in the presence of a non-interacting RNA species of Chikungunya virus (CHIK3B) in the presence of its defective helper genome in mammalian BHK-21 cells.
  • All DIP variants were able to reduce viral titres by at least 10-orders of magnitude as measured by gPCR, with the Dg-Girdwood variant showing particularly repressive effects.
  • a viral titre of 863080 vg/pL was measured via qPCR against the EGFP transgene.
  • the SIN2A-EGFP-ENV virus co-transfected in the presence of the Dg-Girdwood DIP had a viral titre of 17720 vg/pL by round three of serially passage, equating to a 48-fold reduction in viral titre.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 1-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • the method of modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 2-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 3-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 5-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 10-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 15-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 20-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 25-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 30-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 40-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 50-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 60-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 70-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in an 80-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 90-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 100-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
  • An example of the present method involves the nucleofection of mammalian expression plasmids constitutively expressing PspCas13b with a CRISPR array tiled against the Dwt-variant of a DIP (hereafter referred to as “Dwt-wt”) into naive BHK-21 cells.
  • the RNA virus (hereafter referring to the recombinant, “SIN2A-EGFP-ENV” virus) requires the transfection of in vitro transcribed defective helper RNA, which provides the RNA virus with the viral factor by encoding the SINV capsid gene to facilitate viral maturation and egress.
  • the in vitro transcribed, co-transfection of the Dwt-wt inhibits the synthesis of SIN2A-EGFP-ENV, thereby decreasing viral titres as measured by qPCR.
  • the host cell expressing PspCas13b with its CRISPR array component targeting Dwt-wt would result in the knockdown of the Dwt- wt, which consequently promotes viral maturation of SIN2A-EGFP-ENV as RNA virus synthesis is de-repressed in the absence of Dwt-wt.
  • an inducible circuit can be constructed by placing the TETO7 promoter upstream of the coding sequence for PspCas13b or LwaCas13a or RfxCas13d, wherein induction of PspCas13b or LwaCas13a or RfxCas13d in the presence of a spacer complementary to the co-transfected DIP can be mediated by a gene of interest encoded by a viral particle, such as tetracyclineresponsive activator protein (tTA), which can modulate the provision of the RNA virus by mediating an increase in the viral titre of the system.
  • tTA tetracyclineresponsive activator protein
  • an inducible circuit can be constructed by placing the TETO7 promoter upstream of the coding sequence for PspCas13b or LwaCas13a or RfxCas13d, wherein induction of PspCas13b or LwaCas13a or RfxCas13d in the presence of a spacer complementary to the DIP can be mediated by a gene-of-interest encoded by a viral particle to facilitate an increase in the viral titre of the system.
  • R61X-tTA disrupted tetracycline-controlled transactivator protein
  • an inducible circuit can be constructed by placing the TETO7 promoter upstream of the coding sequence for PspCas13b or LwaCas13a or RfxCas13d, wherein induction of PspCas13b or LwaCas13a or RfxCas13d in the presence of a spacer complementary to eitherthe viral vector or defective helper RNA genome can be mediated by a gene of interest recombinantly introduced into a viral vector to mediate a reduction or increase in viral titre of the system.
  • a correction mediating Y100H in a doxycycline-resistant variant of the tTA gene inactivates the antiviral effects of PspCas13b, LwaCas13a or RfxCas13d, thereby allowing for viral maturation and egress.
  • Supplementation of the media with 1 nM to 20 pM of doxycycline generates a selective pressure to enrich for mutant variants of adenine base editors or prime editors that can efficiently install the doxycycline-sensitive Y100H mutation, thereby knocking down expression of PspCas13b or LwaCas13a or RfxCas13d, which had previously been transcriptionally induced. Downstream knock-down of PspCas13b or LwaCas13a or RfxCas13d leads to an increased cellular concentration of the defective helper RNA, thereby facilitating viral maturation.
  • plastic shall be construed to mean a general term for a wide range of synthetic or semisynthetic polymerization products, and generally consisting of a hydrocarbon-based polymer.

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Abstract

The present invention provides nucleic acids sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest. Using a split, non-competent viral vector, a gene of interest can be stably and recombinantly integrated into a viral vector via in-frame insertion of an open reading frame of the gene of interest with an aspect of the native but attenuated viral genome. This configuration of the viral genome, leveraging a split viral vector and an aspect of a viral factor that has been shown to robustly interact with the split viral vector, enables the serial passaging of the recombinant viral particle in an indel and recombination-averse manner. This allows for the steady accumulation of mutations in the gene of interest and allows for its gene products to have improved function and protein solubility.

Description

METHOD, USE, AND KIT RELATING TO AN RNA VIRUS
Field of the Invention
[1] The present invention provides nucleic acids sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest.
[2] The nucleic acid sequences of the invention have been developed primarily for use in evolution of biomolecules of interest and will be described hereinafter with reference to this application. It will, however, be appreciated that the invention is not limited to this particular field of use.
Background of the Invention
[3] Directed evolution is an effective strategy for developing one or more gene products of a gene of interest with desirable characteristics. During typical directed evolution, a library of genetic variants is established via mutagenesis of an initial gene of interest. Subsequently, expressed gene products of the gene of interest are selected and assayed for a specific activity and/or function.
[4] In cases wherein attenuated RNA viruses are used with an inserted gene of interest, significant genomic instability and a tendency towards viral recombination occurs. Here, a gene of interest is often inserted into the viral genome, rendering it non-competent in the absence of a viral factor that has been segmented in trans. Viral propagation is therefore dependent on the reconstitution of the complete viral genome to mediate viral egress. The viral factor can be supplied by the presence of a defective helper RNA genome, which cannot replicate on its own, but provides in trans the necessary components for a split, non-competent RNA virus to replicate.
[5] Defective interfering particles (DIPs) are a naturally arising subpopulation of parasitic genomes observed in vitro that compete for the replication machinery of the RNA virus due to cis-acting elements. The presence of these parasitic DIPs in the gene pool of a population of RNA viruses would have a repressive effect on viral titres and lead to eventual viral extinction by inhibiting the production of the viral components needed for efficient viral egress.
[6] Robust viral replication and maturation within the host cell can thus be inhibited by knockdown of the transcript encoding the viral factor by an RNA-binding protein, such as Cas13 effectors, or via the provision of DIPs within the gene pool. Both these mechanisms confer antiviral effects and can reduce the competency of the RNA virus. [7] The present invention seeks to establish an inducible negative selection circuit for use in directed evolution platforms that leverages split, recombinant RNA viral genomes, allowing for counter-selection against unintended, bystander effects of a hypothetical gene of interest.
[8] It is to be understood that, if any prior art information is referred to herein, such reference does not constitute an admission that the information forms part of the common general knowledge in the art, in Australia or any other country.
Summary of the Invention
[9] There is a need to ameliorate issue of low and non-dynamic mutation rates by using mutator RNA viruses as a basis for directed evolution and the serial passaging of the gene products thereof. Using a split, non-competent viral vector, a gene of interest can be stably and recombinantly integrated into a viral vector via in-frame insertion of an open reading frame of the gene of interest with an aspect of the native but attenuated viral genome. This configuration of the viral genome, leveraging a split viral vector and an aspect of a viral factor that has been shown to robustly interact with the split viral vector, enables the serial passaging of the recombinant viral particle in an indel and recombination-averse manner. This allows for the steady accumulation of mutations in the gene of interest and allows for its gene products to have improved function and protein solubility. Thus, the present invention discloses a method to modulate the synthesis of recombinant, non-competent RNA viruses for the purposes of evolving a gene of interest through use of nucleic acids.
[10] As used herein, the term “viral vector” should be understood to refer to a nucleic acid that includes a viral genome which, when introduced into a suitable host cell or used as a template for in vitro transcription, can produce viral RNA which can be replicated and packaged into viral particles. Such viral particles transfer the viral genome into another host cell. It will be appreciated that the term “viral vector” extends to at least part of a viral genome, i.e., truncated and/or partial viral genomes. In some embodiments, a viral vector is provided that lacks one or more gene encoding a protein essential for generation of an infectious viral particle. As used herein the term “viral particle” should be understood to refer to viral nucleic acid(s) that encode one or more viral coat protein(s) and/or viral lipid envelope. As used herein, the term “infectious viral particle” as herein should be understood to refer to a viral particle that can transport at least part of a viral nucleic acid into a suitable host cell.
[11 ] The present invention relates to nucleic acid sequences, viral particles, viruses, vectors systems, host cells, kits, apparatus, and methods of evolution of a gene product of a gene of interest. [12] According to an aspect of the invention, there is provided a method of providing an RNA virus, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus, wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in combination, encode the RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[13] According to another aspect of the invention there is provided, a method of evolution of a gene product of a gene of interest, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus; allowing maturation and egress of one or more RNA virus(es) that include the gene of interest or a variant thereof from the population of suitable host cells; recovering the one or more RNA virus(es); introducing the one or more RNA virus(es) and the one or more second nucleic acid, third nucleic acid, fourth nucleic acid, and fifth nucleic acid sequence(s) into a population of naive suitable host cells; allowing further maturation and egress of further one or more RNA virus(es) that comprise the gene of interest or a variant thereof from the population of suitable host cells; recovering the further one or more RNA virus(es); isolating one or more nucleic acid sequence(s) from the further one or more RNA virus(es) to provide one or more isolated nucleic acid sequence(s); and isolating the gene of interest or a variant thereof from the one or more isolated nucleic acid sequence(s); wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in combination, encode the RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[14] According to another aspect of the invention, there is provided a kit comprising: a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub- genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in use in combination, encode an RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest, in use, modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[15] According to another aspect of the invention, there is provided a kit comprising: a 5’ portion of a first nucleic acid; a 3’ portion of a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; one or more instruction(s) for, in use, inserting a gene of interest selectively between the 5’ portion of a first nucleic acid and the 3’ portion of a first nucleic acid to provide a first nucleic acid; wherein: the first nucleic acid, in use, comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non- structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the 5’ portion of a first nucleic acidgene of interest-3’ portion of a first nucleic sequence and the second nucleic acid, in use in combination, encode an RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest, in use, modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[16] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
[17] To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.
[18] Other aspects of the invention are also disclosed.
Brief Description of the Drawings
[19] Notwithstanding any other forms which may fall within the scope of the present invention, a preferred embodiment/preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:
[20] Figure 1 is a schematic representation outlining the arrangement of nucleic acids comprising defective interfering particle (DIP). In a 5’ to 3’ orientation, the DIP sequence comprises a 5’UTR region, a non-structural protein 1 (nsp1) protein coding sequence, an E1 protein coding sequence, and a 3’UTR, which are sequences of a Togaviridae virus. [21] Figure 2 is a schematic representation outlining the preferential replication of defective interfering particle (DIP) mediated by a split, non-competent, recombinant RNA virus in a suitable host cell. The preferential replication of the DIP results in the eventual reduction in viral titre leading to extinction of the viral population in the gene pool.
[22] Figure 3 is a heatmap overview outlining various species of defective interfering particle (DIPs) and their effects on repressing viral populations according to the invention. SIN2A-EGFP-ENV was serially passaged in the presence of its viral complement component and a DIP variant. CHIK3B refers to an inert RNA-species that was used as a negative control and did not lead to a decline in viral titres (genome count per pL (gc/pl)), as measured by qPCR against the enhanced green fluorescent protein (EGFP) transgene. Girdwood refers to the engineering of a DIP variant that is specific to the Sindbis viral strain used for SIN2A- EGFP-ENV.
[23] Figure 4 is a heatmap demonstrating that the removal of the Dg-Girdwood variant from the gene pool enables an increase in viral titres, indicating that introduction of the Dg- Girdwood variant does not result in gene fixation. Orthogonal passaging refers to the serial passaging of repressed viral populations in the absence of Dg-Girdwood or defective interfering particle (DIP) under conditions of genetic drift. Viral titres, as measured by qPCR against the enhanced green fluorescent protein (EGFP) transgene, are represented by genome count per pL (gc/pl).
[24] Figure 5 are bar graphs demonstrating the effects of nucleofected PspCas13b and its cognate spacer targeting the viral complement that provides viral factors in trans needed for viral maturation and egress. As a negative control, SpCas9 is nucleofected, which has no known RNA-targeting capabilities, as a point of comparison. BHK-21 cells were first nucleofected with the Cas effector and the spacer, followed by application of SIN2A-EGFP- ENV virus bearing an enhanced green fluorescent protein (EGFP) transgene reporter at a high MOI of between 50 to 100. Cells were incubated for 24 to 48 hours, with media removal for downstream qPCR using probes designed against the E2 glycoprotein of the virus, or the EGFP transgene, for the determination of viral titres. Each replicate was performed with both SpCas9 and PspCas13b in parallel (n=3 replicates), with qPCR performed in duplicates for each replicate.
Description of Embodiments
[25] Notwithstanding any other forms which may fall within the scope of the present invention, preferred embodiments of the invention will now be described, by way of example only. [26] The present invention employs methods and techniques that are known to a person having ordinary skill in the art. Such methods and techniques as employed in the present invention include conventional molecular biology, microbiology, and recombinant DNA methods and techniques as disclosed and explained fully in the relevant literature, for example only, Becker’s World of the Cell, 9th edition, Hardin, J., et al., Pearson (2015); Essential Cell Biology, 5th edition, Alberts, B, et al., T&F/Garland (2019); Freshney's Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Freshney, R.I., and Capes-Davis, A., Wiley-Blackwell (2021); Gel Electrophoresis: Nucleic Acids, Martin, R., Garland Science (2020); Karp’s Cell and Molecular Biology, 9th edition, Karp, G., et al., Wiley (2020); Lewin’s Genes, 12th edition, Krebs, J.E., et al., Jones & Bartlett Learning (2017); Molecular Biology of the Cell, 6th edition, Alberts, B., et al., Garland Science (2014); Molecular Biology of the Gene, 7th edition, Watson, J., et al., Pearson (2013); Molecular Biology, 5th edition, Weaver, R., McGraw-Hill Education (201 1); Molecular Biology: Principles of Genome Function, 2nd edition, Craig, N., et al., Oxford University Press (2014); Molecular Cell Biology, 8th edition, Lodish, H, W.H. Freeman (2016); Molecular Cloning: A Laboratory Manual, Volumes 1 , 2, and 3, 4th edition, Green, M.R., and Sambrook, J., Cold Spring Harbour Laboratory Press (2014); and Nucleic Acid Hybridization, Anderson, M.L.M., Garland Science (2020).
[27] Abbreviations of amino acids and nucleic acids, and analogs and derivatives of amino acids and nucleic acids will be known to a person having ordinary skill in the art. Such abbreviations may be found as published as the International Union of Pure and Applied Chemistry (IUPAC) and the International Union of Biochemistry and Molecular Biology (IUBMB) recommendation (see, for example only, Amino Acids and Peptides, 1985, 16, 387- 410; Arch. Biochem. Biophys. 1971 , 145, 425-436; Biochem. J., 1971 , 120, 449-454; Biochem. J., 1984, 219, 345-373; Biochem. J., 1985, 229, 281-286; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69, 109-114, and 122-126; Biochemistry, 1971 , 9, 4022-4027; Biochim. Biophys. Acta 1971 , 247, 1 -12; Eur. J. Biochem., 1970, 15, 203-208; 1972, 25, 1 ; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1 ; 1993, 213, 2; Eur. J. Biochem., 1985, 150, 1-5; Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1970, 245, 5171-5176; J. Biol. Chem., 1985, 260,14-42; J. Biol. Chem., 1986, 261 , 13-17; J. Mol. Biol., 1971 , 55, 299-310; Mol. Biol. Evol., 1986, 3, 99-108; Nucl. Acids Res., 1985, 13, 3021-3030; Proc. Nat. Acad. Sci. (U. S.), 1986, 83, 4-8; Pure Appl. Chem., 1974, 40, 277-290; and Pure Appl. Chem., 1984, 56, 595-624 with respect to abbreviations of this nature.
[28] The terms “biomolecule”, “biomacromolecule”, and “biological macromolecule” as used herein should be understood to refer to naturally occurring macromolecular compounds and synthetic macromolecular compounds such as nucleic acids, proteins, peptides, lipids, and carbohydrates.
[29] Nucleic acids, also termed “polynucleotides”, as used herein should be understood to include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), artificial nucleic acid analogs, and any combination thereof. Artificial nucleic acid analogs include, for example, glycol nucleic acid, locked nucleic acid, morpholino nucleic acid, peptide nucleic acid, threose nucleic acid, and any combination of such analogs. Nucleic acids should also be understood to include, for example, modified nucleobases and artificial nucleobases.
[30] The terms “nucleic acid” and “nucleic acid sequence” and variations of each term should be understood as used herein to interchangeably refer to a nucleic acid molecule or to a series of letters that indicate the order of nucleotides in a nucleic acid as appropriate in the context of the relevant term.
[31] The term “gene of interest” as used herein should be understood to refer to a nucleotide sequence encoding a gene product of interest. Such a gene product of interest should be understood to refer to a gene product intended to be evolved in a circuitous evolution process as disclosed herein. It will be appreciated that the term “gene of interest” includes variations of the gene of interest that are a result of the circuitous evolution process disclosed herein. A person of ordinary skill will appreciate that a gene of interest could be any nucleic acid encoding a gene product to be evolved.
[32] The terms “protein” and “peptide” and derivatives thereof as used herein should be understood to refer to, for example, molecules including amino acids, and analogs, and derivatives of amino acids.
[33] The term “protein” as used herein should be understood to mean proteins, polypeptides, and peptide of any size, structure, or function. The term as used herein should be understood to refer to a plurality of amino acids linked by peptide bonds. Proteins, as understood herein, include naturally occurring and non-naturally occurring amino acids. Such naturally occurring and non-naturally occurring amino acids may also be modified by addition of one or more chemical entity. Furthermore, proteins may include naturally occurring and non-naturally occurring amino acids analogs and/or derivatives. A person of ordinary skill will understand that proteins may be a single molecule or a plurality of molecules in a complex. Proteins as understood herein include protein fragments, naturally occurring molecules, synthetic molecules, recombination molecules, and any combination of the afore mentioned molecules.
[34] The term “lipid” should be understood as used herein to refer to, for example, diglycerides, fat-soluble vitamins (such as vitamins A, D, E, and K), fatty acids, glycerolipids, glycerophospholipids, monoglycerides, phospholipids, polyketides, prenols, saccharolipids, sphingolipids, sterols, triglycerides, waxes, and analogs and derivatives of the afore mentioned.
[35] The term “carbohydrate” should be understood as used herein to refer to, for example, monosaccharides, disaccharides, oligosaccharides, polysaccharides, and analogs and derivatives of the afore mentioned.
Nucleic Acid Sequences
[36] The present invention relates to one or more nucleic acid sequence selected from the group consisting of: a nucleic acid sequence that includes 5’ to 3’: at least part of a 5’ UTR sequence, at least part of non-structural protein 1 (nsp1) coding sequence, at least part of an E1 coding sequence, and at least part of a 3’ UTR sequence, wherein the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence and the 3’ UTR sequence are sequences of a Togaviridae virus.
[37] Promoters useful in the present invention include, but are not limited to, a polynucleotide motif related to the recruitment of VP64-p65-Rta elements, miniCMV promoter, a motif ten element and downstream promoter element, a polynucleotide motif encompassing a TATA box, a polynucleotide motif related to the recruitment of p65, a polynucleotide motif related to the recruitment of p65, a polynucleotide motif related to the recruitment of Rta, a polynucleotide motif related to the recruitment of VP64, SP6 promoter in a mammalian expression vector system, T3 promoter in a mammalian expression vector system, T7 promoter in a mammalian expression vector system, a tetracycline response element, and a tetracycline-inducible promoter response to tetracycline-responsive transactivator protein or reverse tetracycline-response transactivator protein.
[38] Preferably, the Togaviridae virus is an Alphavirus.
[39] Preferably, the Alphavirus is Sindbis virus.
Method of Providing an RNA Virus
[40] An aspect of the invention provides a method of providing an RNA virus, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus, wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in combination, encode the RNA virus; the third nucleic acid encodes an RNA- binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non- structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[41] As used herein, the term “RNA virus” should be understood to refer to mature, split, non-competent viral genome, which is used herein interchangeably to refer to a “recombinant RNA virus” encoding the gene of interest. In some embodiments, the “RNA virus” can be recombined to achieve self-competency.
[42] In some embodiments, the method further comprises: introducing to the suitable host cell a sixth nucleic acid; and enabling, in the suitable host cell, expression of the sixth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the sixth nucleic acid; wherein: the sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and expression of the sixth nucleic acid modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid modulates assembly of the one or more expression produces) of the sixth nucleic acid; and the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[43] In some embodiments, the Togaviridae virus is an Alphavirus.
[44] In some embodiments, the Alphavirus is Sindbis virus.
[45] In some embodiments, the sixth nucleic acid is a defective interfering particle (DIP).
[46] As used herein, the term “defective interfering particle (DIP)” should be understood to refer to a nucleic acid that includes part of a viral genome which, when introduced into a suitable host cell or used as a template for in vitro transcription, can produce a non-competent viral RNA particle which can decrease the viral titre of a split or non-split RNA viral particle.
[47] As used herein the term “viral particle” should be understood to refer to viral nucleic acid(s) that encode one or more viral coat protein(s) and/or viral lipid envelope. As used herein, the term “infectious viral particle” as herein should be understood to refer to a viral particle that can transport at least part of a viral nucleic acid into a suitable host cell.
[48] As used herein, the term “viral titre” should be understood to refer to the concentration of viral particles within a volume of liquid such as media or supernatant that can be measured using traditional qPCR methods using qPCR probes against the viral genome or measured via traditional plaque assays.
[49] In some embodiments, the RNA-binding protein is a ribonucleoprotein.
[50] In some embodiments, the RNA binding protein is an RNA-guided RNA endonuclease.
[51] In some embodiments, the RNA-guided RNA endonuclease is Cas13 or a Cas13 ortholog.
[52] In some embodiments, Cas13 is selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d protein.
[53] In some embodiments, the fourth nucleic acid is a first guide sequence capable of binding to the RNA-binding protein.
[54] In some embodiments, the first guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
[55] In some embodiments, the fifth nucleic acid is a second guide sequence capable of binding to the RNA-binding protein. [56] In some embodiments, the second guide sequence capable of binding to the RNA- binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
[57] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can occur in a CRISPR array tiled against the first, second, third or sixth nucleic acid to further enhance Cas13’s antiviral effect. A person skilled in the art will also appreciate that the CRISPR array can have a varied direct repeat region based on the Cas13 ortholog, and that multiple guide sequences can be delivered to the host cell to mediate RNA interference.
[58] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can occur as a mature single-guide sequence, or as a precursor guide sequence (pre-crRNA).
[59] A person skilled in the art will appreciate that the gene of interest encoded by the first nucleic acid can also encode an RNA-binding protein, such as a Cas13 ortholog, Cas12 ortholog, or a Cas9 ortholog, that can bind to the fourth and fifth nucleic acid. In some embodiments, the fourth and fifth nucleic acid can encode different direct repeat and tracRNA sequences for multiplex targeting against dsDNA or ssRNA.
[60] As used herein, the term “spacer” should be understood to refer interchangeably to “single guide RNA (sgRNA)” or “guide RNA” or “guide sequence” that describes a short RNA polynucleotide with a hair-pin motif encoded by a direct repeat sequence that binds to an RNA- guided endonuclease. A person skilled in the art will appreciate that the “spacer” can form part of a CRISPR array tiled against a target transcript or gene.
[61] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can be encoded by a mammalian expression vector that can be transiently transfected or integrated into mammalian host cell via lentivirus.
[62] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can be siRNA to mediate RNA interference of the first, second, third, and sixth nucleic acid.
[63] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can be delivered to the host cell via co-transfection of both the fourth and fifth nucleic acid or can be encoded on a single mammalian expression vector expressing one or both of the fourth and fifth nucleic acids.
[64] In some embodiments, the fourth and/or fifth nucleic acid binds to one or more of Cas9, Cas12, and Cas13 proteins.
[65] In some embodiments, the sixth nucleic acid comprises at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA. [66] In some embodiments, the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA hairpin motif.
[67] In some embodiments, the RNA hairpin motif can be used synergistically to increase nuclear export of a nucleic acid.
[68] In some embodiments, the cis-acting element that promotes nuclear export of an incompletely spliced RNA is a constitutive transport element (CTE).
[69] In some embodiments, the constitutive transport element (CTE) is selected from the group consisting of simian retrovirus type 1 , Rous sarcoma virus, and Woodchuck hepatitis virus constitutive transport element.
[70] In some embodiments, the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA transport element (RTE).
[71] In some embodiments, at least one mammalian expression vector comprising at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid is introduced into the suitable host cell.
[72] In some embodiments, the mammalian expression vector is a mammalian expression plasmid.
[73] In some embodiments, the suitable host cell is a transgenic mammalian cell.
[74] In some embodiments, the transgenic mammalian cell comprises at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid.
Method of Evolution of a Gene Product of a Gene of interest
[75] Another aspect of the invention provides a method of evolution of a gene product of a gene of interest, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus; allowing maturation and egress of one or more RNA virus(es) that include the gene of interest or a variant thereof from the population of suitable host cells; recovering the one or more RNA virus(es); introducing the one or more RNA virus(es) and the one or more second nucleic acid, third nucleic acid, fourth nucleic acid, and fifth nucleic acid sequence(s) into a population of naive suitable host cells; allowing further maturation and egress of further one or more RNA virus(es) that comprise the gene of interest or a variant thereof from the population of suitable host cells; recovering the further one or more RNA virus(es); isolating one or more nucleic acid sequence(s) from the further one or more RNA virus(es) to provide one or more isolated nucleic acid sequence(s); and isolating the gene of interest or a variant thereof from the one or more isolated nucleic acid sequence(s); wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in combination, encode the RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[76] In some embodiments, the method further comprises: introducing to the suitable host cell a sixth nucleic acid; and enabling, in the suitable host cell, expression of the sixth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the sixth nucleic acid; wherein: the sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and expression of the sixth nucleic acid modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid modulates assembly of the one or more expression produces) of the sixth nucleic acid; and the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[77] In some embodiments, the Togaviridae virus is an Alphavirus.
[78] In some embodiments, the Alphavirus is Sindbis virus.
[79] In some embodiments, the RNA-binding protein is a ribonucleoprotein.
[80] In some embodiments, the sixth nucleic acid is a defective interfering particle (DIP).
[81] In some embodiments, the third nucleic acid encodes an RNA-binding protein.
[82] In some embodiments, the RNA binding protein is an RNA-guided RNA endonuclease.
[83] In some embodiments, the RNA-guided RNA endonuclease is Cas13 or a Cas13 ortholog.
[84] In some embodiments, Cas13 is selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d protein.
[85] In some embodiments, the fourth nucleic acid is a first guide sequence capable of binding to the RNA-binding protein.
[86] In some embodiments, the first guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
[87] In some embodiments, the fifth nucleic acid is a second guide sequence capable of binding to the RNA-binding protein.
[88] In some embodiments, the second guide sequence capable of binding to the RNA- binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid.
[89] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can occur in a CRISPR array tiled against the first, second, third or sixth nucleic acid to further enhance Cas13’s antiviral effect. A person skilled in the art will also appreciate that the CRISPR array can have a varied direct repeat region based on the Cas13 ortholog, and that multiple guide sequences can be delivered to the host cell to mediate RNA interference.
[90] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can occur as a mature single-guide sequence, or as a precursor guide sequence (pre-crRNA). [91] A person skilled in the art will appreciate that the gene of interest encoded by the first nucleic acid can also encode an RNA-binding protein, such as a Cas13 ortholog, Cas12 ortholog, or a Cas9 ortholog, that can bind to the fourth and fifth nucleic acid. In some embodiments, the fourth and fifth nucleic acid can encode different direct repeat and tracRNA sequences for multiplex targeting against dsDNA or ssRNA.
[92] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can be encoded by a mammalian expression vector that can be transiently transfected or integrated into mammalian host cell via lentivirus.
[93] A person skilled in the art will appreciate that the fourth and fifth nucleic acid can be siRNA to mediate RNA interference of the first, second, third, and sixth nucleic acid.
[94] In some embodiments, the fourth and fifth nucleic acid can be delivered to the host cell via co-transfection of both the fourth and fifth nucleic acid or can be encoded on a single mammalian expression vector expressing one or both of the fourth and fifth nucleic acids.
[95] In some embodiments, the fourth and fifth nucleic acid can bind to one or more protein selected from the group consisting of Cas9, Cas12, and Cas13 proteins.
[96] In some embodiments, the sixth nucleic acid comprises at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA.
[97] In some embodiments, the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA hairpin motif.
[98] In some embodiments, the RNA hairpin motif can be used synergistically to increase nuclear export of a nucleic acid.
[99] In some embodiments, the cis-acting element that promotes nuclear export of an incompletely spliced RNA is a constitutive transport element (CTE).
[100] In some embodiments, the constitutive transport element (CTE) is selected from the group consisting of simian retrovirus type 1 , Rous sarcoma virus, and Woodchuck hepatitis virus constitutive transport element.
[101] In some embodiments, the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA transport element (RTE).
[102] In some embodiments, at least one mammalian expression vector comprising at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid is introduced into the suitable host cell.
[103] In some embodiments, the mammalian expression vector is a mammalian expression plasmid. [104] In some embodiments, the suitable host cell is a transgenic mammalian cell.
[105] In some embodiments, the transgenic mammalian cell comprises at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid.
[106] In some embodiments, general negative selection is used for directed evolution leveraging split, non-competent recombinant RNA viruses encoding a gene of interest and mammalian host cells to counter select against undesirable properties of a selected gene of interest. A person skilled in the art will appreciate that negative selection in the context of directed evolution is used to penalize a viral population upon its induction; therefore, negative selection in this context is understood to have a repressive effect on viral titre. In some embodiments, gene products of the gene of interest have improved or altered, increased, or reduced specificity, stability, fidelity, or enantioselectivity.
[107] In some embodiments, gene products of the gene of interest have broadened substrate specificity resulting from both positive and negative selection, for example, a gene product may have increased catalytic activity on both its native substrate and on non-native substrates.
[108] In some embodiments, negative selection alone is used to evolve the gene of interest. In some embodiments, alternating rounds of positive selection followed by negative selection, or negative selection followed by positive selection is used to evolve the gene of interest.
[109] In some embodiments, negative selection comprises use of a defective interfering particle (DIP), where the expression of the DIP occurs within the host cell. A person skilled in the art will appreciate, for example, that the DIP can be transiently transfected using RNA or DNA transfection methods using ordinary kits (i.e., Lipofectamine™ MessengerMAX™ Transfection Reagent, Thermo Fisher Scientific, #LMRNA008 or Lipofectamine™ 2000 Transfection Reagent, Thermo Fisher Scientific, #1 1668019). A person skilled in the art will also appreciate, for example, that the DIP can be introduced as an in vitro transcribed product from a template using ordinary kits (i.e., mMESSAGE mMACHINE™ SP6 Transcription Kit, Thermo Fisher, #AM1340), ortransiently transfected as a plasmid encoding the polynucleotide sequence of the DIP into amenable host cells using ordinary kits (i.e., Mirus Bio TransIT 2020, Mirus Bio, #MIR 5405). A person skilled in the art will also appreciate, for example, that the expression of the DIP from lentiviral-integration or plasmid in an amenable host cell, such as BHK-21 cells, can be under a constitutive promoter such as CMV promoter, or an inducible promoter such as tetracycline-responsive promoters. Kits
[110] Another aspect of the invention provides a kit comprising: a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in use in combination, encode an RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest, in use, modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[111 ] Another aspect of the invention provides a kit comprising: a 5’ portion of a first nucleic acid; a 3’ portion of a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; one or more instruction(s) for, in use, inserting a gene of interest selectively between the 5’ portion of a first nucleic acid and the 3’ portion of a first nucleic acid to provide a first nucleic acid; wherein: the first nucleic acid, in use, comprises 5’- 3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the 5’ portion of a first nucleic acid-gene of interest-3’ portion of a first nucleic sequence and the second nucleic acid, in use in combination, encode an RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest, in use, modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus.
[112] In some embodiments, the kits will further comprise one or more suitable host cell(s).
[113] In some embodiments, the kits will further comprise one or more instruction (s) for cloning a gene of interest into the first nucleic acid.
[114] A person skilled in the art will appreciate that methods of cloning to insert the gene of interest into the first nucleic acid are contemplated to include any method that facilitates the joining of DNA fragments, i.e., two or more DNA fragments. Examples of such methods include, but are not limited to, Gibson assembly, Golden Gate assembly, typical restriction digestion, ligation methods, and other assembly methods that may involve PCR and homology arms complementary to the insert and/or the destination vector.
[115] A person skilled in the art will also appreciate that the typical screening methods to identify a correct vector may include restriction digestion, Sanger sequencing, and/or deep sequencing to identify the inserted gene of interest.
[116] In some embodiments, the kits will further comprise one or more instruction (s) for inserting the gene of interest into the first nucleic acid.
[117] In some embodiments, the kits will further comprise one or more instruction (s) for performing the evolution of a gene product of a gene of interest. EXAMPLES
General Cell Culture
[118] BHK-21 [C-13] cells (#CCL-10) (hereinafter BHK-21) were grown in a humidified 37°C (5% CO2) incubator in MEM a (ThermoFisher, #32571101) supplemented with 1 % Penicillinstreptomycin (% v/v), 5% Fetal Bovine Serum (FBS) (ThermoFisher Scientific, #16000044) (% v/v) and 10% tryptose phosphate broth (TPB) (ThermoFisher, #CM0283B) (% v/v), referred to as BHK-21 Full Medium. During transduction, cells were maintained in TM diluent as described “Current Protocols in Microbiology”, Coico (2005). mRNA synthesis
[119] Plasmids (SEQ ID NO. 5, 6, 7, 8) (3 pg) were linearized prior to mRNA synthesis by using Xbal restriction enzyme. Phenol-chloroform extraction following manufacturer’s protocol (Thermofisher, #15593031) was used to purify linearized plasmid, and 500 ng of input DNA was used to produce mRNA, with LiCI purification steps taken immediately after, using the mMESSAGE mMACHINE™ SP6 Transcription Kit (ThermoFisher, #AM1340), as outlined English, J. G. et al. Cell 178, 748-761 ,e17 (2019). Next, mRNA was quantified via nanodrop spectrophotometer. mRNA integrity was assessed by gel electrophoresis.
Packaging of SINV Particles (referred to as Round 0 (R0)) - mRNA Nucleofection
[120] 1 x 106 BHK-21 cells were nucleofected using a total of 20 pg of mRNA (1 :1 of pSinCapsid (SEQ ID NO. 9) and SIN2A-Envelope-EGFP (SEQ ID NO. 10) using the Amaxa SE Cell Line 4D-NucleofectorTM X Kit L (Lonza, #V4XC-1024) with the 4D-Nucleofector™ System (Lonza) on the C-034 program. Nucleofected cells were plated in BHK-21 Full Medium and were incubated as described above under “General Cell Culture” for 20-24 hours. Supernatant was harvested and centrifuged at 500 g for 5 minutes at room temperature and filtered using a 0.45 pm filter (Merck Millipore #SLHV033RS) and a sterile 10 mL Luer lock syringe (MicroAnalytix, #MS S3P1 OLL). Viral supernatant was stored at 4°C for up to 1 month.
Viral transduction for serial passaging
[121 ] Briefly, a single round of a campaign includes four technical replicates run in parallel. Naive BHK-21 cells were seeded in T25 flasks at 2.5 x 105 cells/flask and incubated for 24 hours. Subsequently, each T25 flask was transduced using 1 mL of viral supernatant or viral supernatant diluted in TM diluent was prepared as described “Current Protocols in Microbiology”, Coico (2005), with intermittent shaking every 15 minutes for 1 hour under “General Cell Culture” conditions. Following transduction, viral supernatant was aspirated entirely and 2 x DPBS wash was performed. Subsequently, to each transduced T25 flask, transfection was carried out using 5 pg of in vitro transcribed, defective helper RNA genome (SEQ ID NO. 9) and 5 pg of in vitro transcribed, DIP (SEQ ID NO. 5, 6, 7 or 8) each made up to a total volume of 500 pL with Opti-MEM Reduced Serum Medium (GlutaMAX Supplement) (ThermoFisher, #51985034) with 15 pL Lipofectamine™ MessengerMAX™ Transfection Reagent (Thermo Fisher Scientific, #LMRNA008) according to the manufacturer’s recommendations. After 4 hours, transfection media was removed, and cells were recovered in 5 mL of BHK-21 Full Medium and incubated for 18 to 24 hours.
[122] Virus-containing supernatants were harvested via centrifugation at 500 g for 5 minutes to pellet cellular debris, followed by filtration using a 0.45 pm filter (Merck Millipore #SLHV033RS) and a sterile 10 mL Luer lock syringe (MicroAnalytix, #MS S3P10LL). Clarified supernatants were collected for titration and used for subsequent transduction experiments or stored at 4°C for up to 1 month.
Transqene isolation
[123] In total, 500 pl of viral supernatant was isolated with the MagMAX™ Viral RNA Isolation Kit (ThermoFisher, #AM1939) as per the manufacturer’s recommendations. Transgenespecific primers (SEQ ID NO. 11 and 12) anchored to the 5’ and 3’ end of the gene of interest GFP were used for gene-specific RT-PCR using 300 ng of input viral RNA and the Superscript™ IV One-Step RT-PCR System (ThermoFisher, #12594025). Specifically, reverse transcription was carried out under the following parameters: 50°C for 1 min, 51 °C for 1 min, 52°C for 1 min, 53°C for 1 min, 54°C for 1 min, 55°C for 3 mins, 56°C for 3 mins, 57°C for 3 mins, 58°C for 3 mins, 59°C for 3 mins, 60°C for 10 mins, 50°C for 30 mins, and denaturation at 98°C for 2 mins. Cycling PCR was performed using the following parameters: 98°C for 10 secs, 50°C for 10 secs, 72°C for 30s per 1 kb for 40 cycles, and 72°C for 5 mins.
[124] For all subsequent steps, DNA concentration was determined using the Qubit™ 1X dsDNA HS Assay Kit (ThermoFisher, #Q32851) following purification or PCR-related steps. 300 ng of viral RNA was amplified using the Superscript™ IV One-Step RT-PCR System. Bands of interest were gel extracted with the Monarch DNA Gel Extraction Kit (NEB, #T 1020). For all subsequent PCRs, ultra-high-fidelity Platinum™ SuperFi II PCR Master Mix (ThermoFisher, #12368050) was used following the manufacturer's instructions. Next, a limited 2-cycle PCR was performed on 100 ng of DNA template (SEQ ID NO. 13) using modified dual-UMI primers (SEQ ID NO. 14 and 15) to append 36 nt UMIs with a branched NNNYR motif. Illumina-specific adapters (Illumina, #FC-131-2003) were attached using 15- cycles of PCR following 1.0x Ampure XP bead clean-up prior to sequencing using a NovaSeq 6000 S4 SP PE250 flow cell (Illumina, #20039236). Quantification of viral titre using E2-specific probes.
[125] Titration and quantification of viral particles were performed using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher, # 4444434) as outlined in English, J. G. et al. Cell 178, 748-761 ,e17 (2019). Specifically, 10 pL of clarified, viral supernatant was used with an E2- specific primer probe pair (SEQ ID NO. 16, 17, and 18) and quantified on Quantstudio™ 3 Real-Time PCR System (Thermo Fisher, # A28567).
Plaque assay for the determination of plaque-forming units per mL
[126] Plaque assays were performed in 12-well plates with viral supernatant stocks diluted to concentrations between 10-1 to 10-6. Briefly, pre-sterilised methylcellulose (Spectrum, #ME136) was dissolved in BHK-21 Full Medium (1% w/v) at 4°C overnight with agitation (300 RPM) using a magnetic stirrer (referred to henceforth as Plaquing Media). One day prior to viral transduction, naive BHK-21 cells were plated at a density of 70,000 cells/well in a 12-well plate and incubated as described in “General Cell Culture” above. Diluted or undiluted viral aliquots (200 pL applied to cells) were used to transduce naive, untransfected BHK-21 cells as described in “Viral transduction and serial passaging” above. Next, 1 mL of 37°C prewarmed Plaquing Media was applied to the transduced cell monolayer and the plate was returned to the incubator as described in “General Cell Culture” above and incubated for up to 48 hours without disturbance. After two days, the Plaquing Media was aspirated, and the cell monolayer was washed once with 1 X DPBS. The cell monolayer was then fixed with 10% neutral buffered formalin for 30 minutes before staining with 0.1% crystal violet (% w/v; Sigma Aldrich, #C0775-25G).
RNA interference of defective helper genomes using PspCas13b
[127] Nucleofection of naive BHK-21 cells at density of 1 .0 x 106 cells/well was performed in a tissue-culture treated 6 well plate with 1.5 pg of plasmid expressing SpCas9 (SEQ ID NO. 33) or 1.5 pg of plasmid expressing PspCas13b (SEQ ID NO. 34), with 1 pg of plasmid expressing tiled CRISPR array targeting the positive strand of the defective helper genome (SEQ ID NO. 32) for viral complementation. After 24 hours, media was aspirated and 100 pL of virus (clarified SIN2A-EGFP-ENV virus generated as described in “Packaging of SINV Particles (referred to as Round 0 (R0)) - mRNA Nucleofection”) was added to the cells with each well topped up with 900 pL of complete BHK media. Cells were transduced for 1 hour with intermittent shaking every 15 minutes under cell culture conditions as described in “General Cell Culture.” After 1 hour, cells were washed twice with 1 X DPBS, and media was replaced to a volume of 2 mL with complete BHK media. Subsequently, transduced BHK-21 cells were RNA transfected using 1 pg of LiCI purified, in vitro transcribed defective helper RNA (SEQ ID NO. 9), and 1.5 pL of Lipofectamine 2000 (Thermo Fisher, # 11668019) to a total volume of 250 pL of Opti-MEM Reduced Serum Medium (GlutaMAX Supplement) (ThermoFisher, #51985034). The transfection:RNA complex was then incubated at room temperature for 20 minutes, before being added to each well of priorly nucleofected and transduced BHK-21 cells. After four hours, the media was again aspirated, and washed once with 1 X DPBS, before being further incubated for 48 hours, with supernatant collection at 24 hours and at 48 hours for downstream qPCR using E2-specific probes, as described in “Quantification of viral titre using E2-specific probes.”
A method for co-introducinq DIP to a viral particle for decreased viral titre
[128] Linearized, purified plasmid DNA bearing polynucleotide sequences of the viral vector, defective helper RNA genome (SEQ ID NO. 9) and defective interfering particles (DIPs) (SEQ ID NO. 5, 6, 7 and 8) are separately used as inputs in an in vitro transcribed reaction to individually produce 5’ capped, polyadenylated, long non-coding RNAs (IncRNAs). The resulting in vitro transcribed RNAs are LiCI purified and quantified via spectrophotometry.
[129] Using a process of electroporation, nucleofection, or transient RNA transfection, 5 pg of the three types of IncRNAs (viral vector, defective helper RNA genome, and DIPs) are cointroduced into naive BHK-21 cells in a T75 flask at density of 1 .0 x 106 cells/flask for a period of 24 hours, followed by subsequent clarification of the viral supernatant via centrifugation and filtration using a 0.45 pm filter.
A method for modulating viral titre of a recombinant viral particle
[130] Naive or recombinant mammalian cells are transduced with viral particles for one hour with intermittent shaking in a humidified 37°C (5% CO2) incubator. Immediately following transduction, 5 pg of LiCI purified, in vitro transcribed RNA corresponding to the defective helper RNA genome and DIP are co-transfected into the transduced mammalian cells. After 4 hours to 1 day, the transfection reagent is removed from the cell followed by 2 X DPBS. The viral particles interact with the IncRNA of the defective helper RNA genome (SEQ ID NO. 9) and DIP (SEQ ID NO. 5, 6, 7, and 8). The viral particle is inhibited in the presence of DIP. Conversely, the defective helper RNA genome favours viral maturation and egress. The virus can be clarified and collected from the cell culture and purified to isolate the gene of interest.
[131] Viral supernatants can be monitored for growth or decay of the population via qPCR- based methods of quantification, plaque assays, or using RNA biosensors. A method of performing evolution of a gene of interest using nucleic acids
[132] A mammalian cell line may be engineered or transiently transfected to mediate RNA interference of a viral factor. RNA interference can be targeted against the DIP or may be targeted against the viral vector or defective helper RNA genome. RNA interference can be mediated by PspCas13b, LwaCas13a, or other variant of Cas13 effectors with a spacer (SEQ ID NO. 25, 26, 27, 28, 29, 30, 31 and 32) or siRNA targeting the viral vector (SEQ ID NO. 10), defective helper RNA genome (SEQ ID NO. 9) or DIP (SEQ ID NO. 5, 6, 7, and 8) under a constitutive or inducible basis depending upon the function of the gene product(s) of the gene of interest encoded by the recombinant viral particle. The viral particles containing the gene of interest are added to these amenable host cells. The DIP inhibits viral replication, whereas the defective helper RNA genome conversely promotes viral maturation and egress. Both components are transiently transfected to the transduced mammalian cell line expressing a method of RNA interference such as PspCas13b. The gene of interest contained within the recombinant viral particle directly or indirectly mediates RNA interference and alters the balance of defective helper genome or DIP depending upon a basis of positive or negative selection. Some embodiments of the gene of interest develop mutations as a result of being in the viral particle/viral vector. These mutations confer a benefit to these gene of interest variants by increasing viral titre indicating efficient viral maturation and egress. In some embodiments, viral titre can increase as a result of the gene of interest disrupting a method of RNA interference targeting the viral vector or defective helper RNA genome. In some embodiments, viral titre can increase as a result of the gene of interest inducing a method of RNA interference targeting the DIP. In some embodiments, the viral titre can decrease as RNA interference of the viral vector or defective helper genome is promoted via induction by the gene of interest. Viral maturation and egress can be mediated by the presence of the defective helper RNA genome and the viral vector, and the absence of the DIP, thereby conferring an advantage to the viral particle encoding that particular variant of the gene of interest. Evolved viral particles containing mutated gene of interest are able to enter the media. Following collection of cell media, the process can be repeated iteratively, or the gene of interest can be isolated and sequenced.
[133] Transfection of Cas13-mediated interference is performed on an amenable host cell in mutually exclusive T25 flasks. After a period of 6 hours to 1 day following this transfection event, media is aspirated and viral supernatant containing engineered, non-naturally occurring viral particles are introduced onto the cell at a multiplicity of infection (MOI) of 1 for transduction, or by applying 1 mL of undiluted viral supernatant.
[134] The T25 flask is incubated at 37°C for one hour, with intermittent shaking every 15 minutes to allow even coverage of the surface area of the cell monolayer. [135] Subsequently, the viral supernatant is aspirated, and 1X DPBS wash is performed twice to remove residual viral supernatant. Transfection of the defective helper RNA genome enabling viral maturation and egress, or DIP inhibiting viral maturation and egress is performed as an RNA transfection method using ordinary kits. The defective helper RNA genome or DIP can be either LiCI-purified RNA or delivered as a plasmid mediating expression of these components. Culture media is replenished and the T25 flasks are returned to the incubator for a period of up to 1 to 2 days.
[136] Media is harvested, hereafter referred to as ‘viral supernatant’, and clarified as described above. The viral supernatant is stored at refrigerated conditions.
[137] Viral supernatant is again applied to cells that have been transfected as described above is repeated.
Methods of modulating production of an RNA virus via co-transfection of an in vitro transcribed defective helper genome and an in vitro transcribed recombinant Sindbis virus vector
[138] A method of modulating production of an RNA virus included co-transfection of an in vitro transcribed defective helper genome and an in vitro transcribed recombinant Sindbis virus vector into a suitable host cell. The Sindbis virus vector encoded a gene of interest. Such gene of interest, for example, was a reporter-like GFP gene.
[139] In some examples of the present method, RNA interference using an RNA-binding protein targeted against the in vitro transcribed defective helper genome, the in vitro transcribed recombinant Sindbis virus vector, or in vitro transcribed DIP modulates production ofthe RNA virus. Such modulation of the production of the RNA virus is positive, i.e., increases the viral titre of a viral particle including the gene of interest coding sequence relative to a population of viral particles modulated by the presence of DIPs or a method of RNA interference targeting defective helper genomes or the recombinant Sindbis virus vector. Alternatively, the modulation of the production of the RNA virus is negative, i.e., decreases the viral titre of a viral particle including the gene of interest coding sequence relative to a population of viral particles modulated by a method of RNA interference targeting the presence of DIPs in the gene pool.
[140] In some examples of the present method, the in vitro transcribed recombinant Sindbis virus vector is linearized via a restriction digest and used as a template for in vitro transcription if the viral coding sequence is downstream of an SP6 or T7 promoter using ordinary kits (i.e., mMESSAGE mMACHINE™ SP6 Transcription Kit, Thermo Fisher, #AM1340). [141] In some examples of the present method, RNA interference is directed to either the in vitro transcribed defective helper genome or the in vitro transcribed recombinant Sindbis virus vector, which modulates production of the RNA virus. Such modulation of the production of the RNA virus includes providing one or more nucleic acid(s) to mediate the RNA interference. Such one or more nucleic acids are contemplated to include guide sequences that direct the RNA binding protein.
[142] Examples of the present method are contemplated where RNA interference increases the viral titre of the viral particle containing the gene-of-interest coding sequence and/or variants thereof relative to a population of viral particles modulated by the overexpression of DIPs.
[143] Examples of the present method are contemplated where RNA interference decreases the viral titre of the viral particle containing the gene-of-interest coding sequence and/or variants thereof relative to a population of viral particles replicating in the absence of a method of RNA interference wherein either the defective helper genome or viral vector is targeted.
[144] In an example of the present method, decreasing viral titre included nucleofection of mammalian expression plasmids constitutively expressing PspCas13b with a CRISPR array tiled against a SINV capsid sequence into a naive BHK-21 cells to provide a nucleofected BHK-21 cells. The nucleofected BHK-21 cell was subsequently incubated for 24 hours. Following incubation, RNA viruses, i.e., recombinant viral vector genomes expressing an EGFP transgene reporter, hereafter referred to as SIN2A-EGFP-ENV viruses, at a multiplicity- of-infection (MOI) of 50 to 100, and in vitro transcribed defective helper RNA, which encodes the SINV capsid gene, were introduced into the transfected BHK-21 cells. The SIN2A-EGFP- ENV viruses require the in vitro transcribed defective helper RNA to facilitate viral maturation and egress. The nucleofected BHK-21 cells, including the SIN2A-EGFP-ENV virus, expresses PspCas13b with its CRISPR array component resulting in the knockdown of the defective helper RNA. In effect, this inhibits viral maturation of SIN2A-EGFP-ENV as the RNA virus is non-competent without its in trans provision of the capsid gene. Subsequently, the nucleofected cells were transfected with in vitro transcribed defective helper RNA to mediate viral maturation. As a negative control, the experiment was performed in parallel with a condition in which SpCas9 was nucleofected into naive BHK-21 cells, as opposed to PspCas13b. SpCas9 has no known RNA-targeting capabilities in the context of a CRISPR array with direct repeats specific to PspCas13b. The condition in which SpCas9 was used showed a higher viral titre at both 24 hour- and 48 hour-time points post-nucleofection as compared to the condition nucleofected with active PspCas13b. For example, after 24 hours, the viral titre was reduced by 19.6% (corresponding to 11 ,541 gc/pL and 14,353 gc/pL for PspCas13b and SpCas9, respectively, n=3) when the capsid gene was targeted by PspCas13b, with a 33.0% reduction in viral titre observed after 48 hours (corresponding to 34,263 gc/pL and 51 ,127 gc/pL for PspCas13b and SpCas9, respectively, n=3).
[145] In some examples of the present method, the method results in a 1.1-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[146] In some examples of the present method, the method results in a 1 .2-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[147] In some examples of the present method, the method results in a 1 .3-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[148] In some examples of the present method, the method results in a 1 .4-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[149] In some examples of the present method, the method results in a 1 .5-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[150] In some examples of the present method, the method results in a 1 .6-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[151] In some examples of the present method, the method results in a 1 .7-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[152] In some examples of the present method, the method results in a 1 .8-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b. [153] In some examples of the present method, the method results in a 1.9-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[154] In some examples of the present method, the method results in a 2-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[155] In some examples of the present method, the method results in a 4-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[156] In some examples of the present method, the method results in an 8-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[157] In some examples of the present method, the method results in a 16-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[158] In some examples of the present method, the method results in a 32-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[159] In some examples of the present method, the method results in a 64-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b.
[160] In some examples of the present method, the method results in a 124-fold difference in the viral titre of an RNA virus using PspCas13b and a CRISPR array tiled against the RNA virus genome or defective helper RNA relative to an RNA virus that is not targeted by PspCas13b. Methods of Modulating RNA Virus Production Using a Defective Interfering Particle £DIP)
[161] A method of modulating production of an RNA virus included co-transfection of an in vitro transcribed defective helper genome and in vitro transcribed recombinant Sindbis virus vector encoding GFP, hereafter referred to as SIN2A-EGFP-ENV. Co-transfection of an additional in vitro transcribed DIP modulates the assembly the RNA virus by decreasing the viral titre of the RNA virus relative to a population of viruses that are replicating in the absence of overexpressed DIPs. The presence of the DIP reduces synthesis of the viral components provided by the in vitro transcribed defective helper genome, thereby inhibiting viral production. Two confirmed DIP variants, referred to as Dwt-WT and Dg-WT, and engineered Girdwood strain counterparts, hereafter referred to as Dwt-Girdwood and Dg-Girdwood, respectively were serially passaged by clarifying the viral particles isolated from the previous round and applying the viral supernatant to naive BHK-21 cells transfected with in vitro transcribed DIPs and defective helper RNA for three rounds. Each condition used one of the four DIP variants (Dwt-WT, Dg-WT, Dwt-Girdwood or Dg-Girdwood), normalizing for the initial input RNA amount for virus generation. As a negative control, SIN2A-EGFP-ENV was also serially passaged in the presence of a non-interacting RNA species of Chikungunya virus (CHIK3B) in the presence of its defective helper genome in mammalian BHK-21 cells. All DIP variants were able to reduce viral titres by at least 10-orders of magnitude as measured by gPCR, with the Dg-Girdwood variant showing particularly repressive effects. For example, relative to the serially passaged SIN2A-EGFP-ENV virus co-transfected in the presence of CHIK3B at round 3 of serial passaging, a viral titre of 863080 vg/pL was measured via qPCR against the EGFP transgene. Conversely, the SIN2A-EGFP-ENV virus co-transfected in the presence of the Dg-Girdwood DIP had a viral titre of 17720 vg/pL by round three of serially passage, equating to a 48-fold reduction in viral titre.
[162] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 1-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[163] In some examples of the present method, the method of modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 2-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[164] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 3-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[165] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 5-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[166] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 10-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[167] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 15-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[168] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 20-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[169] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 25-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[170] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 30-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[171] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 40-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[172] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 50-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP. [173] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 60-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[174] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 70-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[175] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in an 80-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[176] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 90-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[177] In some examples of the present method, modulating the components of the RNA virus via the co-transfection or expression of the DIP results in a 100-fold change in the viral titre of the RNA virus relative to the viral titre of the RNA virus that was serially passaged in the absence of the DIP.
[178] An example of the present method involves the nucleofection of mammalian expression plasmids constitutively expressing PspCas13b with a CRISPR array tiled against the Dwt-variant of a DIP (hereafter referred to as “Dwt-wt”) into naive BHK-21 cells. The RNA virus (hereafter referring to the recombinant, “SIN2A-EGFP-ENV” virus) requires the transfection of in vitro transcribed defective helper RNA, which provides the RNA virus with the viral factor by encoding the SINV capsid gene to facilitate viral maturation and egress. The in vitro transcribed, co-transfection of the Dwt-wt inhibits the synthesis of SIN2A-EGFP-ENV, thereby decreasing viral titres as measured by qPCR. The host cell expressing PspCas13b with its CRISPR array component targeting Dwt-wt would result in the knockdown of the Dwt- wt, which consequently promotes viral maturation of SIN2A-EGFP-ENV as RNA virus synthesis is de-repressed in the absence of Dwt-wt.
Methods of evolution of a gene product of a gene of interest
[179] In an example of a method of evolution for a gene product of a gene-of-interest, an inducible circuit can be constructed by placing the TETO7 promoter upstream of the coding sequence for PspCas13b or LwaCas13a or RfxCas13d, wherein induction of PspCas13b or LwaCas13a or RfxCas13d in the presence of a spacer complementary to the co-transfected DIP can be mediated by a gene of interest encoded by a viral particle, such as tetracyclineresponsive activator protein (tTA), which can modulate the provision of the RNA virus by mediating an increase in the viral titre of the system. This creates a selective pressure depending on the addition of doxycycline amount. In this hypothetical scenario of evolving a doxycycline-resistant variant of tTA, increasing amounts of doxycycline from 1 nM to 20 pM can be supplemented into the media, wherein tTA induction of PspCas13b induces the degradation of the DIP that is constitutively introduced into the cell via transfection. Therefore, it is to be understood that doxycycline- resista nt variants of tTA would arise over successive rounds of serial passaging.
[180] In another example of a method of evolution for a gene product of a gene-of-interest, an inducible circuit can be constructed by placing the TETO7 promoter upstream of the coding sequence for PspCas13b or LwaCas13a or RfxCas13d, wherein induction of PspCas13b or LwaCas13a or RfxCas13d in the presence of a spacer complementary to the DIP can be mediated by a gene-of-interest encoded by a viral particle to facilitate an increase in the viral titre of the system. In a hypothetical scenario of evolving an adenine base editor or a prime editor for improved or broadened catalytic DNA-targeting abilities, correction of a disrupted tetracycline-controlled transactivator protein (R61X-tTA), integrated into an amenable host cell, can correct the open reading frame of tTA, which then mediates induction of the downstream PspCas13b or LwaCas13a or RfxCas13d, which in turn leads to mRNA degradation of the DIP. This in turn promotes viral replication and maturation, leading to an enrichment of gene of interest variants that are proficient in derepressing the system.
[181 ] In another example of a method of evolution for a gene product of a gene-of-interest, an inducible circuit can be constructed by placing the TETO7 promoter upstream of the coding sequence for PspCas13b or LwaCas13a or RfxCas13d, wherein induction of PspCas13b or LwaCas13a or RfxCas13d in the presence of a spacer complementary to eitherthe viral vector or defective helper RNA genome can be mediated by a gene of interest recombinantly introduced into a viral vector to mediate a reduction or increase in viral titre of the system. In a hypothetical scenario of evolving an adenine base editor or a prime editor with improved catalytic activity, a correction mediating Y100H in a doxycycline-resistant variant of the tTA gene (H100Y) inactivates the antiviral effects of PspCas13b, LwaCas13a or RfxCas13d, thereby allowing for viral maturation and egress. Supplementation of the media with 1 nM to 20 pM of doxycycline generates a selective pressure to enrich for mutant variants of adenine base editors or prime editors that can efficiently install the doxycycline-sensitive Y100H mutation, thereby knocking down expression of PspCas13b or LwaCas13a or RfxCas13d, which had previously been transcriptionally induced. Downstream knock-down of PspCas13b or LwaCas13a or RfxCas13d leads to an increased cellular concentration of the defective helper RNA, thereby facilitating viral maturation.
General Interpretation
[182] Where features or aspects of the invention are described herein in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[183] Where method steps are described herein in sequence, the sequence does not necessarily mean that the steps are to be carried out in chronological order in that sequence, unless there is no other logical manner of interpreting the sequence.
[184] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.
[185] Similarly, it should be appreciated that in the above description of example embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the Description of Embodiments are hereby expressly incorporated into this Description of Embodiments, with each claim standing on its own as a separate embodiment of this invention.
[186] Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. [187] As used herein, unless otherwise specified the use of the ordinal adjectives “first”, “second”, “third”, etc., to describe a common object, merely indicate that different instances of like objects are being referred to and are not intended to imply that the objects so described must be in a given sequence, either temporally, spatially, in ranking, or in any other manner.
[188] In the description provided herein, numerous specific details are set forth. It is to be understood, however, that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail in order not to obscure an understanding of this description.
[189] In describing the preferred embodiment of the invention illustrated in the drawings, specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar technical purpose. Terms such as "forward", "rearward", "radially", "peripherally", "upwardly", "downwardly", and the like are used as words of convenience to provide reference points and are not to be construed as limiting terms.
[190] For the purposes of this specification, the term “plastic” shall be construed to mean a general term for a wide range of synthetic or semisynthetic polymerization products, and generally consisting of a hydrocarbon-based polymer.
[191 ] As used herein the term “and/or” means “and” or “or”, or both.
[192] As used herein “(s)” following a noun means the plural and/or singular forms of the noun.
[193] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” are used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
[194] Any one of the terms: including or which includes or that includes as used herein is also an open term that also means including at least the elements/features that follow the term, but not excluding others. Thus, including is synonymous with and means comprising.
[195] Thus, while there has been described what are believed to be the preferred embodiments of the invention, those skilled in the art will recognize that other and further modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modifications as fall within the scope of the invention. For example, any formulas given above are merely representative of procedures that may be used. Functionality may be added or deleted from the block diagrams and operations may be interchanged among functional blocks. Steps may be added or deleted to methods described within the scope of the present invention.
[196] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.
[197] It is apparent from the above, that the arrangements described are applicable to the applications such as molecular biology, medical research, veterinary research, agricultural research, and biotechnology research in general.

Claims

Claims The claims defining the invention are as follows:
1 . A method of providing an RNA virus, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus, wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in combination, encode the RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The method of providing an RNA virus according to claim 1 , the method further comprising: introducing to the suitable host cell a sixth nucleic acid; and enabling, in the suitable host cell, expression of the sixth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the sixth nucleic acid; wherein: the sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and expression of the sixth nucleic acid modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid modulates assembly of the one or more expression product(s) of the sixth nucleic acid; and the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The method of providing an RNA virus according to either claim 1 or claim 2, wherein the Togaviridae virus is an Alphavirus. The method of providing an RNA virus according to claim 3, wherein the Alphavirus is Sindbis virus. The method of providing an RNA virus according to claim 2, wherein the sixth nucleic acid is a defective interfering particle (DIP). The method of providing an RNA virus according to claim 1 , wherein the RNA-binding protein is a ribonucleoprotein. The method of providing an RNA virus according to claim 7, wherein the RNA binding protein is an RNA-guided RNA endonuclease. The method of providing an RNA virus according to claim 8, wherein the RNA-guided RNA endonuclease is Cas13 or a Cas13 ortholog. The method of providing an RNA virus according to claim 9, wherein Cas13 is selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d protein. The method of providing an RNA virus according to claim 1 , wherein the fourth nucleic acid is a first guide sequence capable of binding to the RNA-binding protein. The method of providing an RNA virus according to claim 11 , wherein the first guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid. The method of providing an RNA virus according to claim 1 , wherein the fifth nucleic acid is a second guide sequence capable of binding to the RNA-binding protein. The method of providing an RNA virus according to claim 13, wherein the second guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid. The method of providing an RNA virus according to claim 2, wherein the sixth nucleic acid comprises at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA. The method of providing an RNA virus according to claim 15, wherein the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA hairpin motif. The method of providing an RNA virus according to claim 16, wherein the RNA hairpin motif can be used synergistically to increase nuclear export of a nucleic acid. The method of providing an RNA virus according to claim 15, wherein the cis-acting element that promotes nuclear export of an incompletely spliced RNA is a constitutive transport element (CTE). The method of providing an RNA virus according to claim 18, wherein the constitutive transport element (CTE) is selected from the group consisting of simian retrovirus type 1 , Rous sarcoma virus, and Woodchuck hepatitis virus constitutive transport element. The method of providing an RNA virus according to claim 15, wherein the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA transport element (RTE). The method of providing an RNA virus according to claim 2, wherein at least one mammalian expression vector comprising at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid is introduced into the suitable host cell. The method of providing an RNA virus according to claim 20, wherein the mammalian expression vector is a mammalian expression plasmid. The method of providing an RNA virus according to claim 2, wherein the suitable host cell is a transgenic mammalian cell. The method of providing an RNA virus according to claim 22, wherein the transgenic mammalian cell comprises at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid. A method of evolution of a gene product of a gene of interest, the method comprising: providing a suitable host cell; introducing to the suitable host cell a first nucleic acid and a second nucleic acid; introducing to the suitable host cell one or more of a third nucleic acid, a fourth nucleic acid, and a fifth nucleic acid; enabling, in the suitable host cell: expression of the first nucleic acid and the second nucleic acid; and expression of one or more of the third nucleic acid, fourth nucleic acid, and fifth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the first nucleic acid and one or more expression product(s) of the second nucleic to provide the RNA virus; allowing maturation and egress of one or more RNA virus(es) that include the gene of interest or a variant thereof from the population of suitable host cells; recovering the one or more RNA virus(es); introducing the one or more RNA virus(es) and the one or more second nucleic acid, third nucleic acid, fourth nucleic acid, and fifth nucleic acid sequence(s) into a population of naive suitable host cells; allowing further maturation and egress of further one or more RNA virus(es) that comprise the gene of interest or a variant thereof from the population of suitable host cells; recovering the further one or more RNA virus(es); isolating one or more nucleic acid sequence(s) from the further one or more RNA virus(es) to provide one or more isolated nucleic acid sequence(s); and isolating the gene of interest or a variant thereof from the one or more isolated nucleic acid sequence(s); wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in combination, encode the RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The method of evolution of a gene product of a gene of interest according to claim 25, the method further comprising: introducing to the suitable host cell a sixth nucleic acid; and enabling, in the suitable host cell, expression of the sixth nucleic acid; and enabling, in the suitable host cell, assembly of one or more expression product(s) of the sixth nucleic acid; wherein: the sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and expression of the sixth nucleic acid modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid modulates assembly of the one or more expression product(s) of the sixth nucleic acid; and the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The method of evolution of a gene product of a gene of interest according to either claim 25 or claim 26, wherein the Togaviridae virus is an Alphavirus. The method of evolution of a gene product of a gene of interest according to claim 27, wherein the Alphavirus is Sindbis virus. The method of evolution of a gene product of a gene of interest according to claim 25, wherein the RNA-binding protein is a ribonucleoprotein. The method of evolution of a gene product of a gene of interest according to claim 26, wherein the sixth nucleic acid is a defective interfering particle (DIP). The method of evolution of a gene product of a gene of interest according to claim 25, wherein the third nucleic acid encodes an RNA-binding protein. The method of evolution of a gene product of a gene of interest according to claim 31 , wherein the RNA binding protein is an RNA-guided RNA endonuclease. The method of evolution of a gene product of a gene of interest according to claim 32, wherein the RNA-guided RNA endonuclease is Cas13 or a Cas13 ortholog. The method of evolution of a gene product of a gene of interest according to claim 33, wherein Cas13 is selected from the group consisting of Cas13a, Cas13b, Cas13c, and Cas13d protein. The method of evolution of a gene product of a gene of interest according to claim 25, wherein the fourth nucleic acid is a first guide sequence capable of binding to the RNA- binding protein. The method of evolution of a gene product of a gene of interest according to 35, wherein the first guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid. The method of evolution of a gene product of a gene of interest according to claim 25, wherein the fifth nucleic acid is a second guide sequence capable of binding to the RNA-binding protein. The method of evolution of a gene product of a gene of interest according to claim 37, wherein the second guide sequence capable of binding to the RNA-binding protein is at least partially complementary to at least one of the first nucleic acid, second nucleic acid, third nucleic acid, and sixth nucleic acid. The method of evolution of a gene product of a gene of interest according to claim 26, wherein the sixth nucleic acid comprises at least a part of a cis-acting element that promotes nuclear export of an incompletely spliced RNA. The method of evolution of a gene product of a gene of interest according to claim 39, wherein the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA hairpin motif. The method of evolution of a gene product of a gene of interest according to claim 40, wherein the RNA hairpin motif can be used synergistically to increase nuclear export of a nucleic acid. The method of evolution of a gene product of a gene of interest according to claim 39, wherein the cis-acting element that promotes nuclear export of an incompletely spliced RNA is a constitutive transport element (CTE). The method of evolution of a gene product of a gene of interest according to claim 42, wherein the constitutive transport element (CTE) is selected from the group consisting of simian retrovirus type 1 , Rous sarcoma virus, and Woodchuck hepatitis virus constitutive transport element. The method of evolution of a gene product of a gene of interest according to claim 39, wherein the cis-acting element that promotes nuclear export of an incompletely spliced RNA is an RNA transport element (RTE). The method of evolution of a gene product of a gene of interest according to claim 26, wherein at least one mammalian expression vector comprising at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid is introduced into the suitable host cell. The method of evolution of a gene product of a gene of interest according to claim 45, wherein the mammalian expression vector is a mammalian expression plasmid. The method of evolution of a gene product of a gene of interest according to claim 26, wherein the suitable host cell is a transgenic mammalian cell. The method of evolution of a gene product of a gene of interest according to claim 47, wherein the transgenic mammalian cell comprises at least one of the first nucleic acid, second nucleic acid, third nucleic acid, fourth nucleic acid, fifth nucleic acid, and sixth nucleic acid. A kit comprising: a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid, wherein: the first nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the first nucleic acid and the second nucleic acid, in use in combination, encode an RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest, in use, modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The kit according to claim 49, the kit further comprising a sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and wherein: expression of the sixth nucleic acid, in use, modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid, in use, modulates assembly of the one or more expression product(s) of the sixth nucleic acid; and the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The kit according to claim 50, wherein the Togaviridae virus is an Alphavirus. The kit according to claim 51 , wherein the Alphavirus is Sindbis virus. A kit comprising: a 5’ portion of a first nucleic acid; a 3’ portion of a first nucleic acid; a second nucleic acid; a third nucleic acid; a fourth nucleic acid; and a fifth nucleic acid; one or more instruction(s) for, in use, inserting a gene of interest selectively between the 5’ portion of a first nucleic acid and the 3’ portion of a first nucleic acid to provide a first nucleic acid, wherein: the first nucleic acid, in use, comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid coding sequence; at least part of a first protease cleavage signal coding sequence; a gene of interest coding sequence; at least part of a second protease cleavage signal coding sequence; at least part of an E3 coding sequence; at least part of an E2 coding sequence; at least part of a 6K coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the second nucleic acid comprises 5’-3’: at least part of a 5’ UTR sequence; at least part of one or more non-structural protein(s) coding sequence(s); at least part of a sub-genomic promoter coding sequence; at least part of a capsid protein coding sequence; at least part of an E3 coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; the 5’ portion of a first nucleic acid-gene of interest-3’ portion of a first nucleic sequence and the second nucleic acid, in use in combination, encode an RNA virus; the third nucleic acid encodes an RNA-binding protein; the fourth nucleic acid is complementary to at least one of the first, second, and third nucleic acid(s); the fifth nucleic acid is partially identical to at least one of the first, second, and third nucleic acids; expression of any one or more of the third nucleic acid, fourth nucleic acid, fifth nucleic acid, and gene of interest, in use, modulates assembly of the one or more expression product(s) of the first nucleic acid or the one or more expression product(s) of the second nucleic to provide the RNA virus; and the 5’ UTR sequence, the one or more non-structural protein(s) coding sequence, the capsid coding sequence, the E3 coding sequence, the E2 coding sequence, the 6K coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The kit according to claim 58, the kit further comprising a sixth nucleic acid comprising 5’-3’: at least part of a 5’ UTR sequence; at least part of a non-structural protein 1 (nsp1) coding sequence; at least part of an E1 coding sequence; and at least part of a 3’ UTR sequence; and wherein: expression of the sixth nucleic acid, in use, modulates assembly of the one or more expression product(s) of the 5’ portion of a first nucleic acid-gene of interest-3’ portion of a first nucleic sequence or the one or more expression product(s) of the second nucleic acid to provide the RNA virus; expression of any one or more of the gene of interest, third nucleic acid, fourth nucleic acid, and fifth nucleic acid, in use, modulates assembly of the one or more expression product(s) of the sixth nucleic acid; and the 5’ UTR sequence, the non-structural protein 1 (nsp1) coding sequence, the E1 coding sequence, and the 3’ UTR sequence are coding sequences of a Togaviridae virus. The kit according to claim either claim 58 or claim 59, wherein the Togaviridae virus is an Alphavirus. The kit according to claim 60, wherein the Alphavirus is Sindbis virus.
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