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WO2024086549A1 - Alphavirus chimériques pour évolution dirigée - Google Patents

Alphavirus chimériques pour évolution dirigée Download PDF

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Publication number
WO2024086549A1
WO2024086549A1 PCT/US2023/077051 US2023077051W WO2024086549A1 WO 2024086549 A1 WO2024086549 A1 WO 2024086549A1 US 2023077051 W US2023077051 W US 2023077051W WO 2024086549 A1 WO2024086549 A1 WO 2024086549A1
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cell
synthetic
alphavirus
replicon
heterologous nucleic
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Daniel Dewran KOCAK
Bryan Roth
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University of North Carolina at Chapel Hill
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University of North Carolina at Chapel Hill
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    • C—CHEMISTRY; METALLURGY
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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
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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    • C12N2770/00—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
    • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N2770/36141—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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36144—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N2770/36152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • This invention relates chimeric alphavirus genomes, alphavirus particles and compositions comprising the same, and their use in methods of directed evolution, such as methods of modify a heterologous nucleic acid, evolving a heterologous nucleic acid, modifying a synthetic product, evolving a synthetic product, providing populations of modified heterologous nucleic acids and/or evolved synthetic products. Also provided are methods of making the chimeric alphavirus genomes of the invention.
  • Directed evolution which adopts principles of natural evolution to the laboratory, has created an array of industrial products, therapeutic drugs, and molecular tools.
  • directed evolution has been used for the generation of the majority of approved therapeutic antibodies and those still in clinical development (Nobel work that galvanized an industry. Nat Biotechnol. 2018;36: 1023).
  • Computational protein design is beginning to mature, though it frequently relies on directed evolution to reach appropriate levels of activity (Silva et al. Nature. 2019;565(7738): 186-91; Cao et al. Nature. 2022;605(7910):551-560).
  • directed evolution Despite widespread use of directed evolution as a method in general, directed evolution in the mammalian cell has remained outside of reach, with standard of the art directed evolution platforms currently relying on systems that are performed in test tubes, bacteria or yeast.
  • the present invention overcomes shortcomings in the art by providing chimeric alphavirus genomes, methods of making the same, and methods of using the same for directed evolution in mammalian cells.
  • One aspect of the present invention provides a chimeric alphavirus genome comprising: a heterologous nucleic acid, a 5' UTR, a 3' UTR, and one or more open reading frame(s) (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3) (e.g.
  • an ORF encoding a functional mutagenic polymerase and an ORF encoding a Capsid protein and El, E2, and/or E3); wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP); and wherein the Capsid is an old world (OW) alphavirus Capsid.
  • NW new world alphavirus RNA-dependent RNA polymerase
  • the Capsid is an old world (OW) alphavirus Capsid.
  • the heterologous nucleic acid may comprise and/or encode a gene or fragment thereof, a DNA and/or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), or any combination thereof.
  • a DNA and/or RNA molecule e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR
  • the heterologous nucleic acid may encode a protein or fragment thereof.
  • alphavirus particles and populations of alphavirus particles encoded by the chimeric alphavirus genome of the present invention are also provided herein.
  • compositions comprising the chimeric alphavirus genome, alphavirus particle, and/or population of the present invention.
  • Another aspect of the present invention provides a method of modifying a heterologous nucleic acid, comprising: (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via vector, alphavirus particle and/or composition comprising the same) the chimeric alphavirus genome to a cell in
  • Another aspect of the present invention provides a method of evolving a heterologous nucleic acid, comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via vector, alphavirus particle and/or composition comprising the same) the chimeric alphavirus genome to a cell in a
  • Another aspect of the present invention provides a method of modifying a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising the synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering
  • Another aspect of the present invention provides a method of evolving a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising the synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via
  • Another aspect of the present invention provides a method of providing a population of modified heterologous nucleic acids, comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising a synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via vector, alphavirus particle and/or composition comprising the same)
  • Another aspect of the present invention provides a method of providing a population of evolved synthetic products (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising a synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g.
  • incubating the cell with the chimeric alphavirus genome comprising the heterologous nucleic acid in the culture may further comprise administering a selection agent to the culture comprising the cell (e.g., , wherein the cell comprises a detection/selection moiety) and the chimeric alphavirus genome (e.g., to apply selective pressure growth conditions).
  • a selection agent to the culture comprising the cell (e.g., , wherein the cell comprises a detection/selection moiety) and the chimeric alphavirus genome (e.g., to apply selective pressure growth conditions).
  • providing the chimeric alphavirus genome may comprise inserting a heterologous nucleic acid of interest (e.g., comprising and/or encoding a synthetic product of interest such as but not limited to a gene or fragment thereof, a protein or fragment thereof, a DNA and/or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), or any combination thereof) into a chimeric alphavirus genome comprising a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e
  • a further aspect of the present invention provides a method of producing a chimeric alphavirus genome (e.g., for use in directed evolution of a synthetic product), comprising: wherein providing the chimeric alphavirus genome comprises: (a) providing a chimeric alphavirus genome comprising a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid; and (b) inserting a heterologous nucleic acid (e.g., a heterologous nucleic acid of interest (e.g., comprising and/or encoding a synthetic product of interest
  • a further aspect of the invention provides a synthetic replicon comprising: an alphavirus backbone nucleic acid sequence comprising a 5' UTR, a 3' UTR, and one or more open reading frame(s) (ORF) encoding a functional mutagenic polymerase, wherein the alphavirus backbone nucleic acid is devoid of a nucleic acid sequence encoding a structural polyprotein precursor (SPP; e.g., devoid of encoding a Capsid protein or one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3)), a heterologous nucleic acid sequence, and a selection moiety; wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the replicon is capable of self-replication.
  • SPP structural polyprotein precursor
  • E Envelope glycoprotein(s)
  • particles and populations of particles comprising and/or encoded by the synthetic replicon of the present invention.
  • compositions comprising the synthetic replicon, particle, or population comprising the same of the present invention.
  • Another aspect of the invention provides a method of modifying a heterologous nucleic acid, comprising: (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional mutagenic polymerase mutates the heterologous nucleic acid of
  • Another aspect of the invention provides a method of evolving a heterologous nucleic acid, comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional mutagenic polymerase mutates the heterologous nucleic acid of the synthetic
  • Another aspect of the invention provides a method of modifying a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional
  • Another aspect of the invention provides a method of evolving a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing the synthetic replicon the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional muta
  • Another aspect of the invention provides a method of providing a population of modified heterologous nucleic acids, comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error- prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional mutagenic polymerase evolves the heterologous nucleic acid
  • Another aspect of the invention provides a method of providing a population of evolved synthetic products (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional
  • the cell of use in the invention may be a mammalian cell.
  • modified heterologous nucleic acids and/or evolved synthetic products produced by any one of the methods of the present invention.
  • populations e.g., a plurality, e.g., a pool, e.g., a library
  • populations e.g., a plurality, e.g., a pool, e.g., a library
  • FIG. 1 shows schematics, bar graphs and data plots from a screen of Togaviridae members for the capacity to activate gene circuits in mammalian cells.
  • FIG. 1 panel A Representative structure of the RNA genome of a member of the Togaviridae family.
  • FIG. 1 panel B Unrooted phylogenetic tree of 4,617 RNA viruses, adapted from reference 18, red dot is expanded in the next panel.
  • FIG. 1 panel C Members of the Togaviridae family, clustered on a rooted phylogenetic tree.
  • FIG. 1 panel D Structure of the viral replicons used in the following panels.
  • FIG. 1 panel E RNA gel demonstrating purity of viral replicons after in vitro transcription.
  • FIG. 1 panel A Representative structure of the RNA genome of a member of the Togaviridae family.
  • FIG. 1 panel B Unrooted phylogenetic tree of 4,617 RNA viruses, adapted from reference 18, red dot is expanded in the next panel.
  • FIG. 1 panel F % of BHK21 cells that were GFP+ after transfection of EGFP viral replicons.
  • FIG. 1 panel G % of BHK21 cells that were GFP+ after transfection of tTA viral replicons.
  • FIG. 1 panel H Mean values in panels F and G plotted against one another.
  • FIG. 2 shows a schematic and related bar graph of designed chimeric viruses with varying strategies for transgene expression.
  • FIG. 2 panel A A schema of viral genomes implementing different methods for the expression of EGFP.
  • FIG. 2 panel B RT-qPCR results from cell culture media after one passage, demonstrating that exemplified VEEV/CHIKV EGFP End" resulted in an active virus capable of propagation.
  • FIG. 3 shows a schematic and related data plots from a transgenic BHK21 cell line reporting on viral infection.
  • FIG. 3 panel A A VEEV/CHIKV chimeric virus that expresses the tetracycline transactivator "TetR" protein fused to VP 16.
  • FIG. 3 panel B A transposon encoding a gene circuit as a reporter and selective pressure.
  • FIG. 3 panel C The clonal BHK21 cell line is shown to be highly responsive to tTA expression when delivered on the VEEV/CHIKV transgenic strain.
  • FIG. 3 panel D Puromycin restricts viral growth, as determined by rt-qPCR. Puromycin and virus were applied to BHK21 cell simultaneously.
  • FIG. 3 panel A A VEEV/CHIKV chimeric virus that expresses the tetracycline transactivator "TetR" protein fused to VP 16.
  • FIG. 3 panel B A transposon encoding a gene circuit as a reporter and selective pressure.
  • 3 panel E An assessment of different time delays before adding puromycin after adding virus.
  • the 10 pM dox condition represents a viral pool that carries an inactive transgene. Viral samples were titered by counting GFP forming units on the transgenic cell line.
  • FIG. 4 shows images of blots and related bar graphs regarding passing VEEV/CHIV tTA End construct with and without selection.
  • FIG. 4 panel A shows a blot indicating recovery of viral genomes by RT-PCR after passage with and without selection.
  • FIG. 4 panel B shows viral titer over the course of the passaging as determined by rt-qPCR.
  • FIG. 4 panel C shows a bar graph indicating novel viral strains showing increased capacity for growth.
  • FIG. 5 shows schematics and related data plots regarding a control experiment for directed evolution, comprising selecting for doxycycline resistance in the tetracycline transactivator (tTA).
  • FIG. 5 panel A shows a schematic of the directed evolution campaign, wherein the virus is passaged, viral genomes with active tTA are selected using puromycin, and each round is subjected to increasing doxycycline concentrations.
  • FIG. 5 panel B shows rounds implemented and their details for the example experiment.
  • FIG. 5 panel C shows the frequency of unique clones observed in each round considered in isolation.
  • 5 panel D shows each unique clone identified in the evolution validated in a follow up experiment, wherein the plot indicates the range of qualitative behaviors observed: full resistance to doxycycline, partial resistance, no resistance (WT), and a clone harboring a deletion resulting in a defective transgene which has no activity.
  • FIG. 6 shows bar graphs indicating the diversity and speed of mutagenesis achieved by the methods of the invention.
  • FIG. 6 panel A shows a comparison of diversity as assessed in this study and other reported methods of directed evolution by determining the number of unique functional clones reported. Two criteria must be met for this metric: the clone must be functional (i.e., validated in a further experiment, not merely observed), and the clone must not have a mutation that overlaps with another clone (i.e., must be unique to other clones).
  • FIG. 6 panel B shows an analysis of speed of mutagenesis as assessed in each study by determining the total amount of time required to both generate the virus and passage it such that the viral pool has active members.
  • FIG. 7 shows an image of a schematic and two heatmaps, regarding Sequence diversity within the alphavirus family.
  • FIG. 7 panel A Structure of the alphavirus genome and mechanisms of transcriptional shutdown.
  • FIG. 7 panel B Multiple sequence alignment of the RdRp and SPP of alphavirus species. Open reading frames for each viral species were aligned using Clustal Omega.
  • FIG. 8 shows schematics and bar graphs regarding S2 Noncytopathic viral replicons allowing for transactivation of a mammalian gene circuit.
  • FIG. 8 panel A Schematic describing the mutations introduced to create noncytopathic replicons. For each variant, EGFP and tTA expressing replicons were created.
  • FIG. 8 panel B Transfecting replicons described in panel A and measuring transgene expression (EGFP replicons) and gene circuit activation (tTA replicons). Purified RNA was transfected into BHK21 -select cells and gene circuit activation was measured by flow cytometry.
  • FIG. 9 shows schematics and related data graphs regarding Directed evolution using a viral self-amplifying RNA.
  • FIG. 9 panel A Top, BHK21 -select, the clonal cell line used for directed evolution. Piggybac transposition was used to integrate a gene circuit that expresses both PuroR and mGreenLantern-Bsr under the control of a bidirectional promoter that harbors seven copies of the tet operator (TetO7).
  • TetO7 tet operator
  • FIG. 9 panel B VEEV replicon expressing dUnCasl2fl-VPR.
  • FIG. 9 panel C Experimental conditions of directed evolution campaign.
  • FIG. 9 panel D Gene circuit activation by viral replicons expressing the listed transgene, as measured by flow cytometry. BHK21 -select cells were transduced with lentivirus expressing a gRNA targeting the tet operator.
  • FIG. 9 panel E Gene circuit activation before and after selection as described in panel C.
  • FIG. 9 panel F Mutations detected across the transgene as determined by targeted deep-sequencing. Asterisks indicate that the mutation is located within or directly adjacent to a polyA tract.
  • FIG. 9 panel G Functional validation of mutations described in panel F.
  • FIG. 9 panel H Structure of the UnCasl2fl-gRNA-DNA complex (PDB:7C7L). Each UnCasl2fl monomer is depicted separately, functional mutations are highlighted in green, and boxed regions are expanded in the following panels. PAM, protospacer adjacent motif; TS, target strand; NTS, nontarget strand.
  • FIG. 9 panel I Depiction of nucleic acid interactions in panel H; SEQ ID NOs:6 (NTS) and 7 (gRNA) are shown.
  • FIG. 9 panel J Residue Q244 is in close proximity to both the TS and NTS.
  • FIG. 9 panel K Disruption of a salt-bridge between K217 and E206 could enable a new interaction with the NTS.
  • FIG. 9 panel L R66 interacts with the gRNA backbone. R66C could allow coordination of a nearby zinc ion.
  • FIG. 10 shows schematics and related data graphs regarding Replicon-based evolution of dox-resi stance in the tTA activator.
  • FIG. 10 panel A Schema of BHK21 -select cells, describing the gene circuit integrated in the genome using the piggybac transposon.
  • FIG. 10 panel B Schema of VEEV replicon used for evolution.
  • FIG. 10 panel C Selection scheme used for evolution. The viral replicon described in panel B was transfected into the clonal cell line described in panel A. The cellular pool was then passaged under increasing concentrations of puromycin.
  • FIG. 10 panel D Optimizing the doxycycline dose.
  • FIG. 10 panel E The cellular pool from panel D was selected for two days under lug/mL of puromycin. Cell survival was measured using flow cytometry and normalized to an unselected control.
  • FIG. 10 panel F Timeline of experiment.
  • FIG. 10 panel G Deep sequencing of the tTA transgene. Total RNA was harvested after 7 days of passage, RT-PCR was used to recover the transgene and was used as input for Nextera-based library production.
  • FIG. 11 shows bar graphs regarding Optimization of type V-F CRISPR systems for transactivation of the TetO7 promoter.
  • FIG. 11 panel A Comparing CasMINI, a UnlCasl2fl variant, and a Casl2fl variant from Acidibacillus sulfuroxidans for their ability to transactivate the TetO7 promoter. Both Cas effector and gRNA were delivered using plasmid transfection. GFP+ cells were measured using flow cytometry.
  • FIG. 10 panel G Deep sequencing of the tTA transgene. Total RNA was harvested after 7 days of passage, RT-PCR was used to recover the transgene and was used as input for Nextera-based library production.
  • FIG. 11 shows bar graphs regarding Optimization
  • FIG. 11 panel B Comparing different engineered gRNAs for UnlCasl2fl. Both Cas effector and gRNA were delivered using plasmid transfection. GFP+ cells were measured using flow cytometry.
  • FIG. 11 panel C A mutation at residue 297 reduces the activity of dUnlCasl2fl-VP64. Both Cas effector and gRNA were delivered using plasmid transfection. GFP+ cells were measured using flow cytometry.
  • FIG. 12 shows bar graphs regarding Long term culture of viral replicons reveals mutations in the viral replicon that improve cell viability.
  • FIG. 12 panel A Comparing the allele frequency between day 30 and 44 for three mutations that were validated as functionally improving UnCasl2fl activity.
  • FIG. 12 panel B Deep sequencing of the RdRp at day 44.
  • FIG. 12 panel C Testing the transactivation potential of replicon variants discovered in panel B. Replicons with the indicated mutations were generated and transfected into BHK21-cells. Gene circuit activation was measured by flow cytometry.
  • FIG. 12 panel D Testing the effect on cell growth of the replicon variants discovered in panel B. Replicons with the indicated mutations were generated and transfected into BHK21 -cells. Cell count was measured by flow cytometry.
  • FIG. 13 shows schematics and related data graphs regarding Gene-circuit based control of viral replication.
  • FIG. 13 panel A Transgenic stains of VEEV expressing tTA, based on the vaccine strain TC-3. Highlighted is a key region of the VEEV capsid (SEQ ID NO:8 as shown) that mediates its binding to nuclear transport receptors, ultimately occluding nuclear pores. The Capsid Mutant introduces mutations that disrupt these interactions (SEQ ID NOV as shown). The VEEV/CHIKV chimeric strain replaces all of the VEEV SPP with the CHIKV SPP, which does not block nuclear transport.
  • FIG. 13 panel A Transgenic stains of VEEV expressing tTA, based on the vaccine strain TC-3. Highlighted is a key region of the VEEV capsid (SEQ ID NO:8 as shown) that mediates its binding to nuclear transport receptors, ultimately occluding nuclear pores. The Capsid Mutant introduces mutations that disrupt
  • FIG. 13 panel B Titering the transgenic viral strains in A using BHK21 -select by measuring gene circuit activation.
  • FIG. 13 panel E Experimental scheme used to optimize time delay.
  • FIG. 13 panel F Measuring viral growth under selection. The 10 pM dox condition represents a viral population with nonfunctional transgene. The difference between conditions provides a measure of the efficiency of selection as a function of time delay.
  • FIG. 13 panel G Transgene stability across viral passage, both with and without selective pressure.
  • FIG. 13 panel H Validating mutations enriched during viral passage under selection. Mutations in the E2 and NSP2 genes confer increased viral fitness.
  • FIG. 14 shows a schematics and related bar graphs regarding Transgenic expression schemes for VEEV/CHIKV.
  • FIG. 14 panel A Schema of transgenic viral strains. Middle and end orientation used EGFP inserted either at the middle or end of the viral genome using a duplicated subgenomic promoter. P2A inserts EGFP at the end of the viral SPP separated by the P2A self-cleaving peptide.
  • FIG. 14 panel B Testing the viral strains in panel A for viral production. Viral genomes were transcribed in vitro, purified and transfected into BHK21 cells. Viral titer was measured in the supernatant using rt-qPCR on the indicated days post transfection.
  • FIG. 14 panel C Day2 harvests described in panel B were used to inoculate virus-naive cells at 10-fold dilution. Viral titer was measured in the supernatant using rt-qPCR 24 hours post infection.
  • FIG. 15 shows bar graphs regarding Viability of BHK21 -select cells as a function of viral and antibiotic dose.
  • FIG. 15 panel A BHK21 -select cells were treated with virus at the indicated MOI, 4 hours later they were treated with the indicated antibiotic. Cell viability was measured 24 hours post virus exposure via flow cytometry.
  • FIG. 15 panel B Normalizing the data in panel A to the antibiotic free condition.
  • FIG. 16 shows a schematic and related data graphs regarding Mutations in NSP2 and E2 of VEEV/CHIKV that increase viral fitness.
  • FIG. 16 panel A Schematic of viral mutations. Both coding and noncoding mutations are listed.
  • FIG. 16 panel B VEEV/CHIKV base was passaged under selection for 7 rounds and resulting supernatant from each round was tittered using BHK21 -select cells and measuring GFP forming units (gfu).
  • FIG. 17 shows schematics and related data graphs regarding Directed evolution using a chimeric alphavirus.
  • FIG. 17 panel A Schema of the evolutionary conditions using a transgenic VEEV/CHIKV strain expressing tTA and using BHK21 -select cells.
  • FIG. 17 panel B Dose response curves of representative tTA clones, demonstrating full resistance to doxycycline, partial resistance, and no resistance (WT).
  • FIG. 17 panel C The number of unique functional clones identified across rounds of evolution.
  • FIG. 17 panel D Validation of the tTA mutants recovered after evolution, experiments were completed as in panel B and replotted as a heatmap showing activity (Emax, shown as % of WT) and sensitivity to dox (IC50).
  • FIG. 17 panel A Schema of the evolutionary conditions using a transgenic VEEV/CHIKV strain expressing tTA and using BHK21 -select cells.
  • FIG. 17 panel B Dose response curves of representative
  • FIG. 17 panel E Cartoon representation of TetR bound to its ligand, Doxycycline (PDB: 2070). Mutated residues discovered during evolution and validated as functional are highlighted in blue. The orthosteric site is boxed and expanded in the next panel.
  • FIG. 17 panel F View of TetR ligand binding pocket, 90-degree clockwise rotation relative to view in panel E. Doxycycline shown in pink, magnesium ion shown in green.
  • FIG. 17 panel H Frequency of observed mutations generated by the VEEV RdRp.
  • FIG. 18 shows bar graphs regarding the mutational diversity and speed of the chimeric viral system of the invention ("NoVA") using the evolution of dox-resi stance in tTA.
  • FIG. 18 panel A Unique functional mutants validated in each study.
  • FIG. 18 panel B Speed with which viral pool was generated, including production and passage of the virus to the final functional pool.
  • a measurable value such as an amount or concentration and the like, is meant to encompass variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified value as well as the specified value.
  • "about X" where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measurable value may include any other range and/or individual value therein.
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
  • the transitional phrase “consisting essentially of'” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the term “consisting essentially of' when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.” Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise.
  • Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • nucleic acid encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA.
  • the nucleic acid may be double-stranded or single-stranded.
  • the nucleic acid may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • nucleic acid segment "nucleotide sequence,” “nucleic acid molecule,” or more generally “segment” will be understood by those in the art as a functional term that includes both genomic DNA sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, small regulatory RNAs, operon sequences and smaller engineered nucleotide sequences that express or may be adapted to express, proteins, polypeptides or peptides. Nucleic acids of the present disclosure may also be synthesized, either completely or in part, by methods known in the art.
  • sequence identity has the standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 45:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5: 151 (1989).
  • BLAST BLAST algorithm
  • WU-BLAST-2 WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • percent nucleic acid sequence identity is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • the percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotides.
  • sequence identity of sequences shorter than a sequence specifically disclosed herein will be determined using the number of nucleotides in the shorter sequence, in one embodiment.
  • percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.
  • identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations.
  • Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
  • polypeptide encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise.
  • a “fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame.
  • chimera refers to an amino acid sequence (e.g., polypeptide, e.g., a viral genome) generated non-naturally by deliberate human design comprising, among other components, an amino acid sequence of a protein of interest and/or a modified variant and/or active fragment thereof (a "backbone"), wherein the protein of interest comprises modifications (e.g., substitutions such as singular residues and/or contiguous regions of amino acid residues) from different wild type reference sequences (chimera).
  • the generated chimera may optionally be linked to other amino acid segments (fusion protein).
  • the different components of the designed chimera may provide differing and/or combinatorial function. Structural and functional components of the designed chimera may be incorporated from differing and/or a plurality of source material.
  • a "recombinant" nucleic acid, polynucleotide or nucleotide sequence is one produced by genetic engineering techniques.
  • operably linked refers to a functional linkage between two or more nucleic acids.
  • a promoter sequence may be described as being “operably linked” to a heterologous nucleic acid sequence because the promoter sequence initiates and/or mediates transcription of the heterologous nucleic acid sequence.
  • the operably linked nucleic acid sequences are contiguous and/or are in the same reading frame.
  • a "recombinant" polypeptide is produced from a recombinant nucleic acid, polypeptide or nucleotide sequence.
  • an “isolated” polynucleotide e.g., an “isolated nucleic acid” or an “isolated nucleotide sequence” means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the "isolated" polynucleotide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration).
  • the isolated polynucleotide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
  • an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • the "isolated" polypeptide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty -fold, one-hundred- fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration).
  • the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
  • an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier.
  • endogenous refers to a component naturally found in an environment, i.e., a gene, nucleic acid, miRNA, protein, cell, or other natural component expressed in the subject, as distinguished from an introduced component, i.e., an "exogenous” component.
  • heterologous refers to a nucleotide/polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • heterologous nucleotide sequence and “heterologous nucleic acid molecule” are used interchangeably herein and refer to a nucleic acid molecule and/or nucleotide sequence that is not naturally occurring in the virus.
  • the heterologous nucleic acid may comprise an open reading frame that encodes a protein, protein fragment, peptide or nontranslated RNA of interest (e.g., for delivery to a cell or subject).
  • a heterologous nucleic acid of the invention may comprise and/or encode a synthetic product of interest such as but not limited to a gene or fragment thereof, a protein or fragment thereof, a DNA and/or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), or any combination thereof).
  • a synthetic product of interest such as but not limited to a gene or fragment thereof, a protein or fragment thereof, a DNA and/or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), or any combination thereof).
  • fragment refers to a nucleic acid that is reduced in length relative to a reference nucleic acid and that comprises, consists essentially of and/or consists of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference nucleic acid.
  • a nucleic acid fragment may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • the nucleic acid fragment comprises, consists essentially of or consists of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, or more consecutive nucleotides.
  • the nucleic acid fragment comprises, consists essentially of or consists of less than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive nucleotides.
  • fragment refers to a polypeptide that is reduced in length relative to a reference polypeptide and that comprises, consists essentially of and/or consists of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference polypeptide.
  • a polypeptide fragment may be, where appropriate, included in a larger polypeptide of which it is a constituent.
  • the polypeptide fragment comprises, consists essentially of or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, or more consecutive amino acids.
  • the polypeptide fragment comprises, consists essentially of or consists of less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive amino acids.
  • the term "functional fragment” or “active fragment” refers to nucleic acid that encodes a functional fragment of a polypeptide.
  • the term "functional fragment” or “active fragment” refers to polypeptide fragment that retains at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of at least one biological activity of the full-length polypeptide (e.g., the ability to up- or down-regulate gene expression). In some embodiments, the functional fragment actually has a higher level of at least one biological activity of the full-length polypeptide.
  • modified refers to a sequence that differs from a wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof. Modified sequences may also be referred to as “modified variant(s).”
  • a "vector” refers to a compound used as a vehicle to carry foreign genetic material into another cell, where it can be replicated and/or expressed.
  • a cloning vector containing foreign nucleic acid is termed a recombinant vector. Examples of nucleic acid vectors are plasmids, viral vectors, cosmids, expression cassettes, and artificial chromosomes.
  • Recombinant vectors typically contain an origin of replication, a multicloning site, and a selectable marker.
  • the nucleic acid sequence typically consists of an insert (recombinant nucleic acid or transgene) and a larger sequence that serves as the "backbone" of the vector.
  • the purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell.
  • Expression vectors (expression constructs or expression cassettes) are for the expression of the exogenous gene in the target cell, and generally have a promoter sequence that drives expression of the exogenous gene. Insertion of a vector into the target cell is referred to transformation or transfection for bacterial and eukaryotic cells, although insertion of a viral vector is often called transduction.
  • the term "vector” may also be used in general to describe items to that serve to carry foreign genetic material into another cell, such as, but not limited to, a transformed cell or a nanoparticle.
  • replicon refers to a nucleic acid molecule or fragment thereof which independently replicates as a unit, e.g., replicated as a unit from a single origin of replication.
  • a replicon may also be referred to as a self-replicating nucleic acid molecule, such as but not limited to, a self-replicating RNA or self-replicating DNA.
  • a synthetic replicon e.g., a non-naturally occurring replicon
  • a synthetic replicon may be generated from a starting genomic backbone (e.g., a bacterial or viral genome, e.g., an alphaviral genome), from which one or more components are removed and/or modified via man-made mechanisms, e.g., in a laboratory setting.
  • a starting genomic backbone e.g., a bacterial or viral genome, e.g., an alphaviral genome
  • a component e.g., a viral genome, particle, modified heterologous nucleic acid and/or an evolved synthetic product of the invention
  • a component e.g., a viral genome, particle, modified heterologous nucleic acid and/or an evolved synthetic product of the invention
  • round of viral replication refers to a single duplication of a virus genome and/or particle, e.g., a single occurrence of the viral life cycle.
  • the term "passage” or “passaging” refers to the step(s) of inoculating and/or incubating a virus or population of viruses (e.g., chimeric viral genomes of the present invention) in a cell culture medium comprising a cell or population of cells (e.g., "host” cells), isolating the cell culture medium (e.g., comprising virus, e.g., replicated virus) from the cell(s), and introducing the cell culture medium to a new cell or population of cells.
  • a singular cycle of these steps may be referred to as "passaging,” e.g., for example wherein the virus is serially passaged across tissue culture plates.
  • One or more rounds of viral replication may occur per each passage of virus.
  • the term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold, and/or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more, or any value or range therein.
  • inhibitor or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).
  • a "subject" of the invention includes any animal which may benefit from the administration of a modified and/or evolved heterologous nucleic acid and/or synthetic product of the present invention, or from which a sample (e.g., a cell) may be beneficial in the application of the methods described herein.
  • a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.).
  • the subject is a primate subject, a non-human primate subject (e.g., a chimpanzee, baboon, monkey, gorilla, etc.) or a human.
  • a laboratory animal may include but is not limited to any standard laboratory mouse strain.
  • sample or biological sample of this invention can be any biological material, such as a biological fluid, an extract from a cell, an extracellular matrix isolated from a cell, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.
  • directed evolution refers to a term in the field of protein engineering describing a method of modifying a product of interest (e.g., a nucleic acid and/or protein) toward another state (e.g., a modified and/or "evolved" state), wherein the method mimics the process of natural selection.
  • the goal state may be a user- defined goal.
  • selective pressure may be applied in the method to exert modification toward the goal modified state.
  • a product modified via a method of direct evolution may be referred to as an "evolved" synthetic product, e.g., an evolved protein encoded by a heterologous nucleic acid of the present invention.
  • an evolved synthetic product comprises at least one or more modifications (e.g., substitutions, deletions, insertions, and the like) as compared to the parent (e.g., un evolved, e.g., original) product encoded by the parent (e.g., unevolved, e.g., original) heterologous nucleic acid.
  • an evolved synthetic product may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more modifications, or any value or range therein.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts may be referred to as "transcription products” and encoded polypeptides may be referred to as “translation products.”
  • Transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • the expression product itself e.g., the resulting nucleic acid or protein, may also be said to be “expressed.”
  • An expression product can be characterized as intracellular, extracellular, or secreted.
  • intracellular means something that is inside a cell.
  • extracellular means something that is outside a cell.
  • a substance is "secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
  • round of viral replication refers to a single duplication of a virus genome and/or particle, e.g., a single occurrence of the viral life cycle.
  • the term "passage” or “passaging” refers to the step(s) of inoculating and/or incubating a virus or population of viruses (e.g., chimeric viral genomes of the present invention) in a cell culture medium (e.g., a sample) comprising a cell or population of cells (e.g., "host” cells), isolating the cell culture medium (e.g., comprising virus, e.g., replicated virus) from the cell(s), and introducing the cell culture medium to a new cell or population of cells.
  • a singular cycle of these steps may be referred to as "passaging,” e.g., for example wherein the virus is serially passaged across tissue culture plates. One or more rounds of viral replication may occur per each passage of virus.
  • directed evolution in the mammalian cell has remained outside of reach. All robust directed evolution platforms currently rely on systems that are performed in test tubes, bacteria or yeast, and many directed evolution platforms result in biomolecules that function well in bacteria and yeast but fail when transplanted to human cells.
  • Existing methods for directed evolution in mammalian cells can be broadly categorized into non-viral and viral classes (Hendel & Shoulders 2021 Nat Methods 18(4):346-357; Molina and Mengiste 2022 Nat Rev Methods Primers 2(36); Xie et al. 2022 Trends Biochem Sci 47(5):403-416).
  • Non-viral methods may create genetic diversity using error-prone PCR (Banaszynski et al. 2006 cell 126(5):995-1004; Villette et al. 2019 Cell 179(7): 1590-1608; Piatkevich et al. 2018 Nat Chem Biol 14(4):352-360) or using cytidine deaminases (Wang et al. 2004 PNAS 101(48): 16745-16749; Hess et al. 2016 NatMethods 13(12): 1036-1042; Moore et al. 2018 J Am Chem Soc. 140(37): 11560-11564; Chen et al. 2020 Nat Biotechnol 38(2):165- 168).
  • Non- viral methods have been applied successfully to both create new molecular tools and understand gene function, but are limited in their ability to create diverse libraries and in their ability to implement selection in a manner that is high-throughput, rapid, simple, and iterative.
  • Viral methods may streamline mutagenesis and selection, creating diversity using error- prone viral polymerases and imposing selection by making viral replication contingent on the activity of a gene circuit.
  • Viral systems based on different viral species have been developed, including the retrovirus HIV-1 (Das et al. Q JBiol Chem 279(18): 18776-18782), the dsDNA adenovirus AdV (Berman et al. 2018 J Am Chem Soc 140(51): 18093-1813), and the (+)ssRNA Sindbis virus (English et al. 2019 Cell 178(3):748-761).
  • xCas9 reported in Hu et al. 2018 Nature 556(7699):57-63, was evolved in bacteria but was later found to have low activity in mammalian cells (Schmid-Burgk et al. 2020 Mol Cell 78(4): 794-800; Kim et al. 2020 Nat Biotechnol 38(11): 1328-1336; Sangree et al. 2022 Nat Commun 13(1): 1318) and evoCas9, reported in Casini et al. (2018 Nature Biotechnology 36(3):265-271), was evolved in yeast and was similarly inefficient.
  • RNA viruses are a highly diverse class of genetic entity that are unique in their ability to infect higher organisms. Of viral genera that infect prokaryotes and archaea, more than 99% are DNA viruses (Koonin et al. 2015 Virology 479-480:2-25). On the other hand, viral genera that infect plants and animals are majority RNA based, with positive stranded RNA viruses being the most prevalent. While not wishing to be bound to theory, that (+)ssRNA viruses so dominate the mammalian niche may be indicative of their evolutionary power and the unique advantages endowed by their replication strategy.
  • RNA viruses Their evolutionary power derives from two properties, 1) the polymerases of RNA viruses are highly mutagenic (Sanjuan et al. 2010 J Virol 84(19):9733-9748; Acevedo et al. 2014 Nature 505(7485)686-690) and 2) they grow to high titers, ⁇ 10 10 /mL, in tissue culture (Kulasegaran-Shylini et al. 2009 Virology 387(1):211-221; Meshram et al. 2019 Virology 534: 14-24).
  • RNA viruses are highly adapted for replication in mammalian cells and possess the two parameters that determine evolutionary power (i.e., fecundity and genetic variation) making them potentially useful for directed evolution applications.
  • the present invention is based in part on the adaptability and ability of alphavirus replication in mammalian cells, to provide chimeric alphavirus genomes for application in methods of directed evolution in cells such as mammalian cells.
  • alphavirus and/or “alphaviruses” refers to a genus of RNA viruses, which are the sole genus in the Togaviridae family.
  • Alphaviruses are positive-sense, single-stranded (ss+) RNA viruses which may naturally infect both vertebrates and invertebrates.
  • ss+ single-stranded
  • ORFs open reading frames
  • the first ORF expresses the RdRp, encoded by nonstructural proteins (NSP1-4) and the second ORF expresses the capsid and glycoproteins, encoded by the structural polyprotein (SPP: capsid, E3-E2-(6K/TF)-E1).
  • SPP structural polyprotein
  • the virus uses the positive sense genome to generate a full-length anti-sense transcript, which is then used to generate sense full-length and subgenomic transcripts using the 5’ UTR and subgenomic promoter, respectively (Strauss & Strauss, 1994 Microbiol Rev 58(3):491-562).
  • the RdRP and SPP are translated as polyproteins that mature via processing by viral and host proteases.
  • one aspect of the present invention provides a chimeric alphavirus genome comprising: a heterologous nucleic acid, a 5' UTR, a 3' UTR, and one or more open reading frame(s) (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s); wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP); and wherein the Capsid is an old world (OW) alphavirus Capsid.
  • ORF open reading frame(s)
  • ORF open reading frame(s)
  • E Envelope glycoprotein
  • the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP); and wherein the Capsid is an old world (OW) alphavirus Capsid.
  • 3 UTR refers to a section of a nucleic acid sequence (e.g
  • an mRNA molecule is transcribed from a DNA sequence and later translated into a peptide, polypeptide, or protein.
  • Several regions of sequence of an mRNA molecule are not translated into protein, including the 5’ untranslated region (5’ UTR) and 3’ UTR.
  • a 3’ UTR may contain regulatory regions that may influence gene expression post-transcriptionally.
  • the one or more E glycoproteins may comprise alphavirus glycoproteins El, E2, and/or E3.
  • a chimeric alphavirus genome of the present invention may comprise an ORF encoding a functional mutagenic polymerase, and an ORF encoding a Capsid protein and El, E2, and/or E3.
  • the Capsid may be a chimeric capsid wherein at least a portion of the Capsid comprises an old world alphavirus capsid.
  • the polymerase may be noncytopathic, i.e., wherein the polymerase does not cause cellular harm and/or death to the cell to which the chimeric alphavirus genome is introduced, delivered or, and/or otherwise comprised. In some embodiments, the polymerase does not cause cellular harm and/or death to the cell within which the chimeric alphavirus genome is comprised, optionally as measured by cellular transcription and/or translation functionality.
  • cellular transcription and/or translation of the cell may be reduced by no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% or less, or any value or range therein, for example, as compared to the same cell not comprising the chimeric alphavirus genome of the present invention.
  • the chimeric alphavirus genome of the present invention may lack (e.g., be devoid of) ability to inhibit transcription in a host cell, e.g., wherein transcription of the cell (cellular transcription) is retained in the presence of the chimeric alphavirus genome and/or a vector, composition and/or particle comprising the same.
  • the transcription of the cell is retained in the presence of the chimeric alphavirus genome and/or a vector, composition and/or particle comprising the same by about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any value or range therein, for example as compared to cellular transcription of the cell when not in the presence of the chimeric alphavirus genome and/or a vector, composition and/or particle comprising the same.
  • the term "functional mutagenic polymerase” refers to a polymerase which retains functionality to introduce mutations during viral replication (e.g., perform error- prone viral replication).
  • the functional mutagenic polymerase of the invention may have a mutation rate of about 10' 6 to about 10' 1 (e.g., about 10' 6 to about 1 O' 3 ) substitutions per nucleotide per cell infection (s/n/c), e.g., about 10' 6 , 10' 5 , 10' 4 , 10' 3 , 10' 2 , or 10' 1 or any value or range therein.
  • the metric substitutions per nucleotide per cell infection for RNA viruses is further described in Sanjuan, R.
  • the functional mutagenic polymerase of the invention may have a mutation rate of about 10' 6 to about 10' 1 s/n/c, about 10' 6 to about 10' 3 s/n/c, about 10' 3 to about 10' 1 s/n/c, about 10' 6 to about 10' 4 s/n/c, or about 10’ 1 , about 10' 3 , about 10' 6 , or about 10' 4 s/n/c.
  • the chimeric alphavirus genome may comprise a heterologous nucleic acid, a 5' UTR, a 3' UTR, an open reading frame (ORF) encoding a functional mutagenic polymerase, and an ORF encoding a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3); wherein the polymerase is noncytopathic; and wherein the Capsid and/or at least one of the one or more E glycoprotein(s) is an old world (OW) alphavirus Capsid and/or E glycoprotein.
  • E Envelope glycoprotein
  • the chimeric alphavirus genome may comprise a heterologous nucleic acid, a 5' UTR, a 3' UTR, an open reading frame (ORF) encoding a functional mutagenic polymerase, and an ORF encoding a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3); wherein the polymerase is noncytopathic; and wherein the Capsid is an old world (OW) alphavirus Capsid.
  • E Envelope glycoprotein(s)
  • the polymerase may be a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), e.g., comprising the alphavirus nonstructural proteins NSP1, NSP2, NSP3 and/or NSP4.
  • NW new world alphavirus RNA-dependent RNA polymerase
  • the chimeric alphavirus genome of the present invention may further comprise a subgenomic promoter ("SG"), a membrane protein (e.g., 6K) and/or any other alphavirus protein such as but not limited to those shown in FIG. 1 panel A.
  • SG subgenomic promoter
  • membrane protein e.g., 6K
  • any other alphavirus protein such as but not limited to those shown in FIG. 1 panel A.
  • a chimeric alphavirus genome of the invention may comprise a chimeric togavirus genome (i.e., a virus genome from the viral family Togaviridae) comprising a heterologous nucleic acid inserted into a togavirus genome backbone, the backbone comprising a 5' UTR, an ORF encoding a functional mutagenic RdRP (e.g., encoding NSP1, NSP2, NSP3, and/or NSP4, which form a RdRP), a subgenomic promoter, an ORF encoding a Capsid protein, a membrane protein (e.g., 6K) and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), and a 3' UTR, wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP); and wherein the Capsid is an old world (OW
  • a chimeric alphavirus genome of the invention may comprise a heterologous nucleic acid inserted into an alphavirus genome backbone, the backbone comprising a 5' UTR, an ORF encoding a functional mutagenic RdRP (e.g., encoding NSP1, NSP2, NSP3, and/or NSP4, which form a RdRP), a subgenomic promoter, an ORF encoding a Capsid protein, a membrane protein (e.g., 6K) and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), and a 3' UTR, wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP); and wherein the Capsid is an old world (OW) alphavirus Capsid.
  • a functional mutagenic RdRP e.g., encoding NSP1, NSP2, NSP3,
  • the backbone may be NW alphavirus genome backbone.
  • the backbone may be a OW alphavirus genome backbone.
  • the virus genome backbone (e.g., Togaviridae backbone, e.g., alphavirus backbone) may comprise, in 5' to 3' orientation, a 5' UTR; an ORF encoding NSP1, NSP2, NSP3, and NSP4 which form a functional mutagenic RdRP; a subgenomic promoter; an ORF encoding a Capsid protein, an E3 glycoprotein, an E2 glycoprotein, a 6K membrane protein, and an El glycoprotein; and a 3' UTR (e.g., as shown in FIG. 1 panel A).
  • a 5' UTR an ORF encoding NSP1, NSP2, NSP3, and NSP4 which form a functional mutagenic RdRP
  • a subgenomic promoter an ORF encoding a Capsid protein, an E3 glycoprotein, an E2 glycoprotein, a 6K membrane protein, and an El glycoprotein
  • a 3' UTR e.g., as
  • a noncoding element called the "subgenomic promoter," "SG”, or “SGI” located between NSP4 and the Capsid in a wildtype Togaviridae genome may allow the virus to separately regulate nucleic acid amplification (e.g., by polymerase expression) and particle generation (e.g., by Capsid expression).
  • a chimeric viral genome of the present invention may further comprise a second subgenomic promoter ("SG2"), e.g., to express a gene and/or gene product (e.g., a transgene) in a heterologous nucleic acid.
  • SG2 second subgenomic promoter
  • Such a second subgenomic promoter may be inserted anywhere along the chimeric genome, e.g., before, within, and/or after the 5' UTR, NSP1, NSP2, NSP4, NSP4, SGI, Capsid, E3, E2, El, 6K, and/or 3' UTR. In some embodiments, such a second subgenomic promoter may be comprised within the 5' and/or 3' UTR.
  • the polymerase is not from a Sindbis virus strain, such as but not limited to nsp2-P683S and nsp3-N24A mutations described in Akhrymuk et al. 2018 J. Virol 92(23):e01388-18).
  • At least one of the one or more E glycoproteins may be an OW alphavirus E glycoprotein.
  • at least the Capsid protein may be an OW alphavirus Capsid protein.
  • the Capsid and all of the one or more E glycoproteins are OW alphavirus Capsid and E glycoproteins.
  • an OW alphavirus Capsid and/or E glycoprotein may be substituted in the place of a NW Capsid and/or E glycoprotein in a NW alphavirus genome backbone.
  • an NW alphavirus RdRP may be substituted in the place of a OW RdRP in an OW alphavirus genome backbone.
  • the heterologous nucleic acid of the invention may be upstream of the one or more E glycoprotein(s) (e.g., directly upstream of E3), downstream of the one or more E glycoprotein(s) (e.g., directly downstream of El), and/or upstream of the 3' UTR.
  • the heterologous nucleic acid is upstream of the 3' UTR.
  • the heterologous nucleic acid may be directly upstream of the 3' UTR.
  • the OW and NW alphaviruses of the present invention may be any OW and/or NW alphavirus now known or later discovered.
  • the OW alphavirus may include, but is not limited to, Sindbis virus (SINV), Barmah Forest virus, Middelburg virus, Semliki Forest virus (SFV), and/or Chikungunya virus (CHIKV).
  • the OW alphavirus is not Sindbis virus.
  • the NW alphavirus may be, but is not limited to, Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and/or western equine encephalitis virus (WEEV).
  • VEEV Venezuelan equine encephalitis virus
  • EEEV eastern equine encephalitis virus
  • WEEV western equine encephalitis virus
  • the heterologous nucleic acid of the invention may comprise a coding region (e.g., a transgene), a promoter (e.g., a subgenomic promoter), an internal ribosome entry site (IRES), and/or any combination thereof.
  • a coding region e.g., a transgene
  • a promoter e.g., a subgenomic promoter
  • IRS internal ribosome entry site
  • the heterologous nucleic acid may comprise and/or encode a gene or fragment thereof, a DNA and/or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), and/or any combination thereof.
  • the heterologous nucleic acid encodes a protein or fragment thereof.
  • a chimeric alphavirus genome of the invention may further comprise a detectable moiety, e.g., a detection and/or selection moiety.
  • a detection and/or selection moiety of the present invention may be any moiety which allows for the detection and/or selection of the genome e.g., in an in vitro assay, e.g., in a scan performed in vivo.
  • Nonlimiting examples of detection/selection moi eties include, but are not limited to, molecular tags such as HA-Tag and/or FLAG-tag.
  • a chimeric alphavirus genome of the present invention may further comprise a selection moiety, e.g., an antibiotic resistance sequence.
  • antibiotic resistance sequences include sequences which confer resistance to blasticidin, neomycin, puromycin, tetracycline, and/or any combination thereof.
  • the detection and/or selection moiety e.g., an antibiotic resistance sequence
  • the detection and/or selection moiety may be comprised in (e.g., be expressed in) a host cell comprising an chimeric alphavirus genome of the present invention.
  • the present invention further provides an alphavirus particle encoded by the chimeric alphavirus genome of the present invention.
  • the particle may lack (e.g., be devoid of) ability to inhibit transcription in a host cell.
  • a population of alphavirus particles comprising the alphavirus particle of the present invention.
  • composition comprising the chimeric alphavirus genome, alphavirus particle, and/or population of the present invention.
  • the chimeric alphavirus genomes, alphaviruses particles, populations and/or compositions of this invention may be used for in vitro and/or in vivo research, therapeutic and diagnostic methods.
  • the chimeric alphavirus genomes, alphaviruses particles, populations and/or compositions of this invention may be advantageous in the use of methods of directed evolution.
  • one aspect of the present invention provides a method of modifying a heterologous nucleic acid, comprising: (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via vector, alphavirus particle and/or composition comprising the same) the chimeric alphavirus genome to a
  • RNA viruses comprise error-prone viral RNA polymerases (RdRP) which regulate viral replication, as well as viral mutagenesis due to the error-prone nature of the polymerases.
  • RdRP error-prone viral RNA polymerases
  • RNA viruses and in particular alphaviruses are highly mutagenic (Sanjuan et al. 2010 J Virol 84( 19):9733-9748; Acevedo et al. 2014 Nature 505(7485)686-690).
  • a single alphavirus particle of the present invention may produce a population of one or more alphavirus particles and/or one or more mutated chimeric alphavirus genomes, wherein the genomes comprised in the newly formed population of one or more alphaviruses (and the particles constructed therefrom) contain de novo mutations that differ from the sequence of the parent particle/genome, as introduced by the error-prone functional mutagenic polymerase.
  • Delivery of the chimeric alphavirus genome to the cell may be through any standard method in the art, including but not limited to directly contacting the cell and/or delivering to the cell via a vector, alphavirus particle and/or composition comprising the chimeric alphavirus genome.
  • Another aspect of the present invention provides a method of evolving a heterologous nucleic acid, comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via vector, alphavirus particle and/or composition comprising the same) the chimeric alphavirus genome to a cell in a
  • Another aspect of the present invention provides a method of modifying a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising the synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering
  • Another aspect of the present invention provides a method of evolving a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising the synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via
  • the methods of the present invention may further comprise isolating the modified heterologous nucleic acid and/or modified synthetic product after incubating the cell with the chimeric alphavirus genome comprising the heterologous nucleic acid for one or more rounds of viral replication (e.g., after incubating for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, e.g., after incubating for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more passages, e.g., after incubating for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of viral replication).
  • the modified heterologous nucleic acid and/or modified synthetic product after incubating the cell with the chimeric alphavirus genome comprising the heterologous nucleic acid for one or more rounds of viral replication (e.g., after incubating for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, e.g., after incubating
  • Another aspect of the present invention provides a method of providing a population of modified heterologous nucleic acids, comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising a synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g., delivering via vector, alphavirus particle and/or composition comprising the same)
  • Another aspect of the present invention provides a method of providing a population of evolved synthetic products (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing a chimeric alphavirus genome comprising a heterologous nucleic acid encoding and/or comprising a synthetic product, a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e.g., a chimeric alphavirus genome of the present invention); (b) delivering (e.g., directly contacting, e.g.
  • incubating the cell with the chimeric alphavirus genome comprising the heterologous nucleic acid in the culture further comprises administering a selection agent to the culture comprising the cell (e.g., wherein the cell comprises a detection/sel ection moiety) and the chimeric alphavirus genome (e.g., to apply selective pressure growth conditions).
  • a selection agent to the culture comprising the cell (e.g., wherein the cell comprises a detection/sel ection moiety) and the chimeric alphavirus genome (e.g., to apply selective pressure growth conditions).
  • incubating the cell with the chimeric alphavirus genome comprising the heterologous nucleic acid in the culture may further comprise administering a selection agent to the culture comprising the cell and the chimeric alphavirus genome to apply selective pressure growth conditions, e.g., wherein the chimeric alphavirus genome comprising the heterologous nucleic acid in the culture is incubated under selective pressure growth conditions, e.g., under conditions suitable for selective pressure.
  • the detection and/or selection moiety may be an antibiotic resistance sequence and the selection agent may be an antibiotic (e.g., not limited to blasticidin, neomycin, puromycin, tetracycline, and/or any combination thereof).
  • the dose of administered selection agent may be any dose required to apply selective pressure, and may be comprise one or more doses and/or escalating doses.
  • the selection agent is administered in an effective amount. In some embodiments, the selection agent may be administered in an amount of about 0.25 ,0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 ug/mL of cell culture medium, or any value or range therein.
  • providing the chimeric alphavirus genome may comprise inserting a heterologous nucleic acid of interest (e.g., comprising and/or encoding a synthetic product of interest such as but not limited to a gene or fragment thereof, a protein or fragment thereof, a DNA and/or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), or any combination thereof) into a chimeric alphavirus genome comprising a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid (e
  • transcription of the cell is retained in the presence of the chimeric alphavirus genome and/or alphavirus particle(s).
  • the cell may be incubated with the chimeric alphavirus genome and/or alphavirus particles comprising the same for one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) rounds of viral replication.
  • the chimeric alphavirus genome and/or alphavirus particles comprising the same for one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) rounds of viral replication.
  • the cell may be incubated with the chimeric alphavirus genome and/or alphavirus particles comprising the same for about 1 to about 24 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, e.g., about 2 to about 12 hours, etc.) per round of viral replication.
  • the chimeric alphavirus genome and/or alphavirus particles comprising the same for about 1 to about 24 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, e.g., about 2 to about 12 hours, etc.) per round of viral replication.
  • the cell may be incubated with the chimeric alphavirus genome for about 1 to about 12 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, e.g., about 2 to about 12 hours, etc.) per round of viral replication (e.g., incubated with the chimeric alphavirus genome in the culture under conditions lacking a selection agent), and then incubated with a selection agent for a further about 1 to about 12 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, e.g., about 2 to about 12 hours, etc.) per round of viral replication (e.g., for a cumulative total of about 24 hours).
  • a selection agent e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, e.g., about 2 to about 12 hours, etc.
  • incubating the cell with the chimeric alphavirus genome in the culture for one or more rounds of viral replication may comprise serially passaging the chimeric alphavirus genome in a culture comprising the cell (e.g., incubating the chimeric alphavirus genome in a culture comprising the cell to produce a population of one or more alphavirus particles, and then serially transferring at least a portion of the produced population of one or more alphavirus particles into a new culture comprising the cell to produce a new (second, third, fourth, etc.) population of one or more alphavirus particles).
  • incubating the cell with the chimeric alphavirus genome in the culture for one or more rounds of viral replication may comprise serially passaging the chimeric alphavirus genome for at least two, three, four, five, six, seven, eight, nine, or 10 or more passages (e.g., at least two, three, four, five, six, seven, eight, nine, 10 or more rounds of viral replication).
  • the mutagenic polymerase may introduce mutations at a rate of about 10' 6 to about 10' 1 (e.g., about 10' 6 , 10' 5 , 10' 4 , 10' 3 , 10' 2 , or 10' 1 or any value or range therein , e.g., about 10' 6 to about 10' 3 ) substitutions per nucleotide per cell infection (s/n/c).
  • Suitable cells for methods of the present invention may include but are not limited to mammalian cells.
  • mammalian cells includes, but is not limited to, humans, non-human primates, rodents, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.
  • Human cells include cells isolated from neonates, infants, juveniles, adults and geriatric subjects.
  • the cell is a mammalian cell.
  • the mammalian cell is an in vitro or ex vivo mammalian cell.
  • the mammalian cell is an immortalized cell line.
  • the culture may be a biological sample (e.g., a sample isolated from a subject, e.g., a patient sample, e.g., a blood sample, a serum sample, a bone marrow sample, a biopsy sample, etc.).
  • a biological sample e.g., a sample isolated from a subject, e.g., a patient sample, e.g., a blood sample, a serum sample, a bone marrow sample, a biopsy sample, etc.
  • modified heterologous nucleic acid produced by any one of the methods of the present invention.
  • a population e.g., a plurality, e.g., a pool, e.g., a library
  • a population e.g., a plurality, e.g., a pool, e.g., a library
  • modified heterologous nucleic acids produced by any one of the methods of the present invention.
  • Also provided herein is a population (e.g., a plurality, e.g., a pool, e.g., a library) of evolved synthetic products produced by any one of the methods of the present invention.
  • a population e.g., a plurality, e.g., a pool, e.g., a library
  • compositions comprising a modified heterologous nucleic acid, evolved synthetic product, and/or population of modified heterologous nucleic acids and/or evolved synthetic products.
  • a composition of the present invention may further comprise a pharmaceutically acceptable carrier, diluent, and/or adjuvant.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and will preferably be in solid or liquid particulate form.
  • the formulations may be conveniently prepared in unit dosage form and may be prepared by any of the methods well known in the art.
  • that pharmaceutically acceptable carrier can be a sterile solution or composition.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a modified heterologous nucleic acid, evolved synthetic product, and/or population of modified heterologous nucleic acids and/or evolved synthetic products, a pharmaceutically acceptable carrier, and, optionally, other medicinal agents, therapeutic agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc., which can be included in the composition singly or in any combination and/or ratio.
  • compositions of the present invention may be formulated by any means known in the art. Such compositions, especially vaccines, are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. Lyophilized preparations are also suitable.
  • the active ingredients are often mixed with excipients and/or carriers that are pharmaceutically acceptable and/or compatible with the active ingredient. Suitable excipients include but are not limited to sterile water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof, as well as stabilizers, e.g., HSA or other suitable proteins and reducing sugars.
  • the pharmaceutical compositions may contain minor amounts of auxiliary substances such as wetting and/or emulsifying agents, pH buffering agents, and/or adjuvants that enhance the effectiveness of the active ingredient or pharmaceutical composition.
  • Another aspect of the present invention provides a method of producing a chimeric alphavirus genome (e.g., for use in directed evolution of a synthetic product), comprising: wherein providing the chimeric alphavirus genome comprises: (a) providing a chimeric alphavirus genome comprising a 5' UTR, a 3' UTR, and one or more open reading frames (ORF) encoding a functional mutagenic polymerase, a Capsid protein and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the Capsid is an old world (OW) alphavirus Capsid; and (b) inserting a heterologous nucleic acid (e.g., a heterologous nucleic acid of interest (e.g., comprising and/or encoding a synthetic product of interest such
  • the chimeric alphavirus genome may further comprise a detection and/or selection moiety.
  • the cell contacted with the chimeric alphavirus genome of the present invention may further comprise a detection/ selection moiety.
  • providing the chimeric alphavirus genome may comprise providing a NW alphavirus genome backbone comprising a 5' UTR, an ORF encoding a functional mutagenic polymerase (e.g., encoding NSP1, NSP2, NSP3, and/or NSP4, which form a RdRP), a subgenomic promoter, an ORF encoding a Capsid protein, a membrane protein (e.g., 6K) and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), and/or a 3' UTR; and substituting an OW alphavirus Capsid and/or E protein in place of the corresponding backbone-encoded Capsid and/or E protein.
  • a functional mutagenic polymerase e.g., encoding NSP1, NSP2, NSP3, and/or NSP4, which form a RdRP
  • a subgenomic promoter e.g.,
  • providing the chimeric alphavirus genome may comprise providing a OW alphavirus genome backbone comprising a 5' UTR, an ORF encoding a functional mutagenic polymerase (e.g., encoding NSP1, NSP2, NSP3, and/or NSP4, which form aRdRP), a subgenomic promoter, an ORF encoding a Capsid protein, a membrane protein (e.g., 6K) and one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3), and/or a 3' UTR; and substituting a NW alphavirus functional mutagenic polymerase in place of the corresponding backbone-encoded polymerase.
  • a functional mutagenic polymerase e.g., encoding NSP1, NSP2, NSP3, and/or NSP4, which form aRdRP
  • a subgenomic promoter e.g., a subgenomic
  • Another aspect of the present invention provides a synthetic replicon comprising: an alphavirus backbone nucleic acid sequence comprising a 5' UTR, a 3' UTR, and one or more open reading frame(s) (ORF) encoding a functional mutagenic polymerase, wherein the alphavirus backbone nucleic acid is devoid of a nucleic acid sequence encoding a structural polyprotein precursor (SPP; e.g., devoid of encoding a Capsid protein or one or more Envelope (E) glycoprotein(s) (e.g., El, E2, and/or E3)), a heterologous nucleic acid sequence, and a selection moiety; wherein the polymerase is a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), and wherein the replicon is capable of self replication.
  • SPP structural polyprotein precursor
  • E Envelope glycoprotein(s)
  • RdRP new world alphavirus RNA-
  • a selection moiety of a synthetic replicon of the invention may be any moiety which allows for the detection and/or selection of the replicon e.g., in an in vitro assay, e.g., in a scan performed in vivo.
  • detection/selection moi eties include, but are not limited to, molecular tags such as HA-Tag and/or FLAG-tag.
  • a selection moiety of a synthetic replicon of the present invention may be an antibiotic resistance sequence.
  • antibiotic resistance sequences include sequences which confer resistance to blasticidin, neomycin, puromycin, tetracycline, and/or any combination thereof.
  • a selection moiety of a synthetic replicon of the present invention may be a ligand and/or a may be a nucleic acid sequence which encodes a ligand which may activate a gene product (e.g., a gene circuit) in a host cell.
  • a gene product e.g., a gene circuit
  • activation and/or expression of a gene product and/or gene circuit in a host cell may indicate the presence of a synthetic replicon of the invention in the cell.
  • the detection and/or selection moiety e.g., an antibiotic resistance sequence
  • the detection and/or selection moiety may be comprised in (e.g., be expressed in) a host cell comprising an chimeric alphavirus genome of the present invention.
  • the polymerase may be noncytopathic, i.e., wherein the polymerase does not cause cellular harm and/or death to the cell to which the synthetic replicon is introduced, delivered or, and/or otherwise comprised. In some embodiments, the polymerase does not cause cellular harm and/or death to the cell within which the synthetic replicon is comprised, optionally as measured by cellular transcription and/or translation functionality.
  • cellular transcription and/or translation of the cell may be reduced by no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% or less, or any value or range therein, for example, as compared to the same cell not comprising the synthetic replicon of the present invention.
  • the synthetic replicon of the present invention may lack (e.g., be devoid of) ability to inhibit transcription in a host cell, e.g., wherein transcription of the cell (cellular transcription) is retained in the presence of the synthetic replicon and/or a vector, composition and/or particle comprising the same.
  • the transcription of the cell is retained in the presence of the synthetic replicon and/or a vector, composition and/or particle comprising the same by about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any value or range therein, for example as compared to cellular transcription of the cell when not in the presence of the synthetic replicon and/or a vector, composition and/or particle comprising the same.
  • the functional mutagenic polymerase of the invention may have a mutation rate of about 10' 6 to about 10' 1 (e.g., about 10' 6 to about 10' 3 ) substitutions per nucleotide per cell infection (s/n/c), e.g., about 10' 6 , 10' 5 , 10' 4 , 10' 3 , 10' 2 , or 10' 1 or any value or range therein.
  • the functional mutagenic polymerase of the invention may have a mutation rate of about 10' 6 to about 10' 1 s/n/c, about 10' 6 to about 10" 3 s/n/c, about 10' 3 to about 10' 1 s/n/c, about 10' 6 to about 10' 4 s/n/c, or about 10’ 1 , about 10' 3 , about 10' 6 , or about 10' 4 s/n/c.
  • the polymerase may be a new world (NW) alphavirus RNA-dependent RNA polymerase (RdRP), e.g., comprising the alphavirus nonstructural proteins NSP1, NSP2, NSP3 and/or NSP4.
  • NW new world alphavirus RNA-dependent RNA polymerase
  • the viral backbone may be NW alphavirus genome backbone.
  • the NW alphaviruses of the present invention may be any NW alphavirus now known or later discovered.
  • the NW alphavirus may be, but is not limited to, Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and/or western equine encephalitis virus (WEEV).
  • VEEV Venezuelan equine encephalitis virus
  • EEEV eastern equine encephalitis virus
  • WEEV western equine encephalitis virus
  • the virus genome backbone (e.g., Togaviridae backbone, e.g., alphavirus backbone) may comprise, in 5' to 3' orientation, a 5' UTR; an ORF encoding NSP1, NSP2, NSP3, and NSP4 which form a functional mutagenic RdRP; a subgenomic promoter; an ORF encoding a Capsid protein, an E3 glycoprotein, an E2 glycoprotein, a 6K membrane protein, and an El glycoprotein; and a 3' UTR (e.g., as shown in FIG. 1 panel A).
  • a 5' UTR an ORF encoding NSP1, NSP2, NSP3, and NSP4 which form a functional mutagenic RdRP
  • a subgenomic promoter an ORF encoding a Capsid protein, an E3 glycoprotein, an E2 glycoprotein, a 6K membrane protein, and an El glycoprotein
  • a 3' UTR e.g., as
  • the virus genome backbone (e.g., Togaviridae backbone, e.g., alphavirus backbone) may be devoid of any one or more of a 5' UTR; an ORF encoding NSP1, NSP2, NSP3, and NSP4 which form a functional mutagenic RdRP; a subgenomic promoter; an ORF encoding a Capsid protein, an E3 glycoprotein, an E2 glycoprotein, a 6K membrane protein, and an El glycoprotein; and a 3' UTR (e.g., as shown in FIG. 1 panel A).
  • a 5' UTR an ORF encoding NSP1, NSP2, NSP3, and NSP4 which form a functional mutagenic RdRP
  • a subgenomic promoter an ORF encoding a Capsid protein, an E3 glycoprotein, an E2 glycoprotein, a 6K membrane protein, and an El glycoprotein
  • a 3' UTR e.g., as shown
  • the polymerase is not from a Sindbis virus strain, such as but not limited to nsp2-P683S and nsp3-N24A mutations described in Akhrymuk et al. 2018 J. Virol 92(23):e01388-18).
  • the heterologous nucleic acid of the invention may comprise a coding region (e.g., a transgene), a promoter (e.g., a subgenomic promoter), an internal ribosome entry site (IRES), and/or any combination thereof.
  • a coding region e.g., a transgene
  • a promoter e.g., a subgenomic promoter
  • IRS internal ribosome entry site
  • the heterologous nucleic acid may comprise and/or encode a gene or fragment thereof, a DNA and/or RNA molecule (e.g., mRNA, miRNA, dsRNA, RNAi, CRISPR), and/or any combination thereof.
  • the heterologous nucleic acid encodes a protein or fragment thereof.
  • the present invention further provides a particle encoded by and/or comprising the synthetic replicon of the present invention.
  • the particle may lack (e.g., be devoid of) ability to inhibit transcription in a host cell.
  • a population of alphavirus particles comprising the synthetic replicon of the present invention.
  • composition comprising the synthetic replicon, particle, and/or population of the present invention.
  • the synthetic replicons, particles, populations and/or compositions of this invention may be used for in vitro and/or in vivo research, therapeutic and diagnostic methods.
  • the synthetic replicons, particles, populations and/or compositions of this invention may be advantageous in the use of methods of directed evolution.
  • another aspect of the present invention provides a method of modifying a heterologous nucleic acid, comprising: (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error- prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional mutagenic polymerase mutates the heterologous nucleic
  • Another aspect of the invention provides a method of evolving a heterologous nucleic acid, comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional mutagenic polymerase mutates the heterologous nucleic acid of the synthetic
  • Another aspect of the invention provides a method of modifying a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional
  • Another aspect of the invention provides a method of evolving a synthetic product (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); and (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional mut
  • any one of the methods of the present invention may further comprises isolating the modified heterologous nucleic acid and/or modified synthetic product after incubating the cell with the synthetic replicon comprising the heterologous nucleic acid for one or more rounds of replicon replication (e.g., after incubating for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, e.g., after incubating for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more passages, e.g., after incubating for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of replicon replication).
  • the synthetic replicon comprising the heterologous nucleic acid for one or more rounds of replicon replication
  • Another aspect of the invention provides a method of providing a population of modified heterologous nucleic acids, comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error- prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional mutagenic polymerase evolves the heterologous nucleic acid
  • Another aspect of the invention provides a method of providing a population of evolved synthetic products (e.g., a synthetic protein or fragment thereof, a DNA and/or RNA molecule), comprising (a) providing the synthetic replicon of the present invention; (b) delivering (e.g., directly contacting, e.g., delivering via vector, particle and/or composition comprising the same) the synthetic replicon to a cell in a culture; (c) contacting the culture with an amount of selection agent (e.g., an antibiotic); (d) incubating the cell with the synthetic replicon in the culture in the presence of the selection agent and under conditions suitable for replicon replication for one or more rounds of replication (e.g., error-prone viral RdRP-driven "directed evolution") (e.g., thereby producing a population of two or more mutated replicons), wherein the selection moiety of the synthetic replicon activates an agent in the cell which counters the selection agent of step (c), and wherein the functional
  • condition suitable for replicon replication or “under conditions suitable for replicon replication” is used to refer to conditions such that transcription and/or translation of replicon-encoded products (e.g., nucleic acids, RNPs, and/or proteins; e.g., the functional mutagenic RdRP, e.g., the synthetic product comprised in and/or encoded by the heterologous nucleic acid, e.g., a synthetic protein) occurs.
  • RNA viruses comprise error-prone viral RNA polymerases (RdRP) which regulate viral replication, as well as viral mutagenesis due to the error-prone nature of the polymerases.
  • RdRP error-prone viral RNA polymerases
  • RNA viruses and in particular alphaviruses are highly mutagenic (Sanjuan et al. 2010 J Virol 84( 19):9733-9748; Acevedo et al. 2014 Nature 505(7485)686-690).
  • a single replicon of the present invention (and/or a particle constructed from a synthetic replicon of the present invention) may produce a population of one or more mutated replicons and/or one or more mutated particles, wherein the nucleic acid sequences comprised in the newly formed population of one or more replicons (and/or the particles constructed therefrom) contain de novo mutations that differ from the sequence of the parent replicon/particle, as introduced by the error-prone functional mutagenic polymerase.
  • Delivery of the synthetic replicon to the cell may be through any standard method in the art, including but not limited to directly contacting the cell and/or delivering to the cell via a vector, particle and/or composition comprising the synthetic replicon.
  • transcription of the cell is retained in the presence of the synthetic replicon.
  • the cell may be incubated with the synthetic replicon for one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) rounds of replicon replication.
  • the cell may be incubated with the synthetic replicon of the invention for about 1 to about 24 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, e.g., about 2 to about 12 hours, etc.) per round of replicon replication.
  • the synthetic replicon of the invention for about 1 to about 24 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, e.g., about 2 to about 12 hours, etc.) per round of replicon replication.
  • incubating the cell with the synthetic replicon in the culture for one or more rounds of replicon replication may comprise serially passaging the cell (e.g., incubating the cell in a culture in the presence of the selection agent and the synthetic replicon to produce a population of two or more cells, and then serially transferring at least a portion of the produced population of two or more cells into a new culture comprising the selection agent and the synthetic replicon to produce a new (second, third, fourth, etc.) population of two or more cells).
  • a method of the present invention may comprise serially passaging the cell for at least two, three, four, five, six, seven, eight, nine, or 10 or more passages (e.g., at least two, three, four, five, six, seven, eight, nine, 10 or more rounds of cellular and/or replicon replication).
  • the mutagenic polymerase may introduce mutations at a rate of about 10' 6 to about 10' 1 (e.g., about 10' 6 to about 10' 3 ) substitutions per nucleotide per cell infection (s/n/c).
  • Suitable cells for methods of the present invention may include but are not limited to mammalian cells.
  • mammalian cells includes, but is not limited to, humans, non-human primates, rodents, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.
  • Human cells include cells isolated from neonates, infants, juveniles, adults and geriatric subjects.
  • the cell may be a mammalian cell, e.g., an in vitro or ex vivo mammalian cell, e.g., an immortalized mammalian cell line.
  • Dosages of the synthetic replicon delivered to the cell will vary upon the type of cell, the mode of administration, and the like.
  • the culture may be a biological sample (e.g., a sample isolated from a subject, e.g., a patient sample, e.g., a blood sample, a serum sample, a bone marrow sample, a biopsy sample, etc.).
  • a biological sample e.g., a sample isolated from a subject, e.g., a patient sample, e.g., a blood sample, a serum sample, a bone marrow sample, a biopsy sample, etc.
  • modified heterologous nucleic acid produced by any one of the methods of the present invention.
  • a population e.g., a plurality, e.g., a pool, e.g., a library
  • a population e.g., a plurality, e.g., a pool, e.g., a library
  • modified heterologous nucleic acids produced by any one of the methods of the present invention.
  • Also provided herein is a population (e.g., a plurality, e.g., a pool, e.g., a library) of evolved synthetic products produced by any one of the methods of the present invention.
  • a population e.g., a plurality, e.g., a pool, e.g., a library
  • compositions comprising a modified heterologous nucleic acid, evolved synthetic product, and/or population of modified heterologous nucleic acids and/or evolved synthetic products.
  • a composition of the present invention may further comprise a pharmaceutically acceptable carrier, diluent, and/or adjuvant.
  • Example 1 Studies in a self-replicating RNA and a viral system for directed evolution in mammalian cells.
  • RNA viruses This study outlines a methodology for the domestication of RNA viruses and their application for directed evolution in mammalian cells.
  • a guiding principle is that the natural antagonism between RNA viruses and mammalian cells limits their ability to carry out molecular evolution (Agol VI. Trends Microbiol. 2012;20(12):570-576).
  • both the virus and cell must be engineered to remove their antagonism such that the virus can still replicate to high titers and the cell can still carry out essential signaling. This involves attenuating the cytopathic nature of the virus and compromising the innate immune system of the cell, which each stop the growth of the other.
  • the system disclosed herein may be used to solve outstanding problems in drug discovery.
  • the molecular system described herein may generally be applied to any molecular engineering platform for the development of molecular tools and therapeutics.
  • RNA viruses comprises a genetic structure that has two main components: nonstructural proteins (NSPs) which form the RNA-dependent RNA polymerase (RdRp) and a structural polyprotein (SPPs) which contains the capsid and glycoproteins (FIG. 1 panel A), as further described in Strauss & Strauss, Microbiol Rev. 1994;58(3):491-562, incorporated herein by reference.
  • NSPs nonstructural proteins
  • SPPs structural polyprotein
  • FIG. 1 panel A A recent large-scale classification of RNA viruses by their RdRp revealed 5 major clades (FIG. 1 panel B), including the Togaviridae family (FIG. 1 panel C), which has been well-studied and contains viral strains proven safe during passaging under BSL2 conditions (Strauss & Strauss; Wolf et al. mBio. 2018;9(6)).
  • transposase-based genome engineering was used.
  • the sleeping beauty transposon was used to deliver EGFP under the control of a TETO7 promoter.
  • cells were isolated into single cell clones and screened for their ability to respond to plasmid delivered tTA. A highly responsive clone was isolated.
  • the % of GFP+ cells can serve a measure of transgene expression (using the EGFP replicon) or transactivation (using the tTA replicon).
  • VEEV was the sole family member that can potently mediate transactivation with tTA (FIG. 1 panels E to H). After VEEV, EEEV was the second most active tTA replicon and was likely limited by low transgene expression.
  • RNA virus This study further focused on creating a safe version of an active RNA virus.
  • the live vaccine strain of VEEV, TC-83 has been safely passaged in labs around the world under BSL2 conditions, has been safely administered to humans to successfully immunize against wildtype VEEV, currently forms the base technology for many vaccines under development (Bloom et al., Gene Ther. 2021;28(3-4):l 17-129), and has been safely used in evolutionary studies in the laboratory (Kautz et al., Virus Evol. 2018;4(l):vey004; Kim et al., J Virol. 2011;85(9):4363- 4376).
  • Non-cytopathic VEEV/CHIKV strains were generated and tested under three different schemes for transgene expression on the viral genome: 1) adding an RNA promoter and transgene in the middle of the genome 2) adding an RNA promoter and transgene before the 3’ UTR 3) using P2A self-cleaving peptide to insert the transgene after glycoprotein El (FIG. 2 panel A).
  • Alphaviral replicons were generated where the SPP was replaced with a transgene of interest, creating a self-amplifying RNA (FIG. 1 panel D).
  • Six different species were selected in order to sample the diversity across the Togaviridae family (FIG. 1 panel C).
  • two replicons were created: an enhanced green fluorescent protein (EGFP) replicon to measure the efficiency of transgene expression and a tetracycline transactivator (tTA) replicon to measure the efficiency of gene circuit activation.
  • EGFP enhanced green fluorescent protein
  • tTA tetracycline transactivator
  • a cell line with a genomically integrated gene circuit was created, with EGFP under the control of a TetO7 promoter, thus reporting on tTA activity.
  • VEEV Venezuelan Equine Encephalitis Virus
  • RNA viruses are known to shutdown host cell transcription as a means of evading host innate immunity, and shutdown of host transcription is a property common to all tested alphaviruses.
  • each alphavirus species performs this task in a unique way (FIG. 7 panels A-C; Garmashova et al. 2007 J. Virol 81(5):2472-2484). It was speculated that the low activity of tTA when delivered using the Old-World alphaviruses may be due to the cytopathic nature of their RdRP, whose NSP2 subunit proteolyzes RNA polymerase II (Akhrymuk et al. 2012 J. Virol 86(13):7180-7191).
  • New-World alphaviruses like VEEV and EEEV, also inhibit host transcription but appear to do so using their capsid proteins (Atasheva et al. 2010 J. Virol 84(9):4158-4171).
  • the VEEV replicon expressing tTA was able to activate the gene circuit because it did not contain the cytopathic VEEV capsid; the corresponding SFV replicon was not able to activate the gene circuit because it contained the cytopathic NSP2 protease.
  • the EEEV replicon expressing tTA could only modestly transactivate, this is likely due to its overall low transgene expression.
  • a replicon-based system for directed evolution in mammalian cells' A system for directed evolution around the VEEV replicon was generated, wherein the VEEV RdRp could serve as an engine for genetic diversity and gene circuit activation could select diversity toward a desired phenotype.
  • a clonal BHK21 line, referred to as BHK-Select was generated with a genomically integrated gene circuit composed of a bidirectional TetO7 promoter expressing puromycin N-acetyltransferase and a fusion of GFP and blasticidin s-resistance gene (FIG. 8 panel A).
  • BHK21 cells were chosen because they have a compromised innate immune system, which is deficient in interferon signaling, yielding them permissive to viral growth and capable of indefinite passage with the viral replicon (Agapov et al. 1998 PNAS 95(22): 12989-12994).
  • VEEV replicons expressing the transgene were transfected into BHK-Select cells. Passing transfected cells under increasing antibiotic would give a replication advantage to those cells with active transgenes (FIG. 9 panels B and C).
  • cell growth and antibiotics act as selection mechanisms, where cell growth creates RNA turnover and antibiotic selects for the desired phenotype.
  • tTA a ligand inducible transcription factor
  • dox doxycycline
  • tTA dissociates from its target DNA, allowing dose dependent control of selective pressure. Passaging replicon-transfected-cells under increasing antibiotic and doxycycline concentrations would enrich for dox-resistant variants of tTA.
  • BHK-Select cells were transfected with a VEEV replicon encoding tTA under a range of dox concentrations (FIG. 10 panels A-D).
  • Puromycin was added 24 hrs post-transfection and cell count was measured 72 hrs posttransfection (FIG. 10 panel E). It was hypothesized that the 5 nM dox condition may be sufficiently selective given that it demonstrated 95% cell death compared to the 0 nM dox condition.
  • the 5 nM dox condition was passaged under puromycin for 7 days post transfection after which >90% cells were GFP positive. The transgene was recovered and subjected to targeted deep sequencing.
  • Casl2fl enzymes are highly compact (about 400-700 amino acids) but show limited efficacy for mammalian genome editing (Bigelyte et al. 2021 Nat Commun. 12(1):6191) without extensive protein- and guide RNA- engineering (Xu et al. 2021 Mol Cell 81(20):4333-4345; Wu et al. 2021 Nat Chem Biol. 17(11): 1132-1138; Kim et al. 2022 Nat Biotechnol. 40(l):94-102).
  • the cell population was harvested after 30 days of passage and the UnlCasl2fl transgene was subjected to deep sequencing (FIG. 9 panels E and F).
  • UnlCasl2fl transgene was subjected to deep sequencing (FIG. 9 panels E and F).
  • follow-up validation of the detected mutations demonstrated that 3 of the top 4 mutations increased UnlCasl2fl transcriptional activity.
  • the allele frequencies for each of the mutations correlated with their effect size (FIG. 9 panels F and G). Combining the mutations resulted in a variant with superior activity (FIG. 9 panel G).
  • the E24K mutation abolished activity of Casl2 and is proximal to a polyA region in the Casl2 gene, which may be the cause of the spurious enrichment of mutation.
  • Half of the top ten SNPs were proximal to a polyA tract, and these mutations did not improve transcriptional activity.
  • UnCasl2fl Structural analysis of UnCasl2fl revealed potential mechanisms underlying the observed activity increases.
  • Two structures of dUnCasl2fl were used in complex with guide and DNA target to evaluate mutations R66C, E206A, and Q244K.
  • the structural data suggest that all three mutations exert their effect through the same UnCasl2fl monomer (FIG. 9 panels H and I).
  • Q244 inserts itself between the target strand (TS) and non-target strand (NTS) (FIG. 9 panel J).
  • a Q244K mutation could enable hydrogen bonds with the backbone of either the TS or NTS.
  • E206 forms a salt-bridge with K217, a bond that would certainly be broken by E206A, potentially freeing K217 to interact with the NTS (FIG. 9 panel K).
  • R66 interacts with the guide RNA, forming hydrogen bonds at the backbone of nucleotide 156 (FIG. 9 panel L). While not wishing to be bound to theory, R66C may disrupt these bonds in order to coordinate Zn 2+ , which allows coordination numbers up to six (FIG. 9 panel L).
  • VEEV SPP While the VEEV replicon functioned well in isolation, the VEEV SPP may be unsuitable for packaging viral genomes given that the capsid antagonizes host cell transcription, which would compromise our ability to direct evolution. Creating a chimeric virus may bypass this issue: using noncoding and RdRp sequences from VEEV combined with SPP sequences from any Old-World alphavirus would result in a recombinant strain that retains host cell transcription and allows for selection of viral populations.
  • FIG. 14 panel A the viral genome was modified to test three different modes for transgenic expression. All three modes could induce EGFP expression upon transfection. Assaying viral growth demonstrated that the end orientation resulted in more efficient particle production, both in the initial transfection and after passage (FIG. 14 panels B and C). Using this expression scheme also yielded a viable tTA encoding virus with a viral titer of 10 7 gfu/mL (FIG. 13 panel B). Delivery of tTA using VEEV/CHIKV potently activated the gene circuit in BHK21 -select cells (FIG. 3 panel C; FIG. 13 panel D).
  • Transgenic alphaviruses can rapidly lose the transgene during passage and can generate parasitic RNAs that could interfere with transgene evolution.
  • antibiotics could potently restrict viral growth on unmodified BHK21 cells (FIG. 3 panel D; FIG. 13 panel C).
  • BHK21 -select cells were used to test the ability of a gene circuit to rescue viral growth under selection, using varied MOI and antibiotic concentration. It was found that 1 pg/mL puromycin and an MOI of 50 resulted in the improved viral growth and cell viability (FIG. 15 panels A and B).
  • the above-optimized system was used to carry out directed evolution by passaging the virus under selection while gradually increasing the concentration of (FIG. 17 panel A). Doxycycline resistance was observed as early as round 2, indicated by the presence of GFP+ cells. Transgenes were recovered from the viral pools of rounds 3-5 and sequenced. Allelic penetrance was high enough such that Sanger sequencing was sufficient to sample diversity and deep sequencing was not required as with evolution using the self-amplifying RNA. 80 clones were sequenced across the three rounds and unique clones were tested for function (FIG. 17 panels B and C). 65 of the 80 clones displayed an increased resistance to dox (FIG. 10 panel G). Most of the observed functional diversity was found in clones sequenced from round 3, which used the lowest dox concentration (FIG.
  • Plasmids encoding replicons of Chikungunya (CHIKV), Ross River (RRV), and Sindbis (SINV) viruses derive their sequences from specific viral strains as follows: CHIKV from strain SL15649 (Accession No. MK028838), RRV derives from strain 2975 (Accession No. GQ433360), SINV derives from the Girwood strain (Accession No. MF459683). Semliki Forest Virus (SFV) replicons were cloned using pSFVCs-lacZ (Addgene plasmid #92076) and was derived from SFV strain 4 (Accession No. KP699763).
  • SFV Semliki Forest Virus
  • VEEV Venezuelan Equine Encephalitis
  • EEEV Eastern Equine Encephalitis
  • VEEV replicons used the TC-83 strain (Accession No. L01443) and EEEV replicons used the North American strain (Accession No. NC_003899).
  • Plasmids encoding active viruses were created using the replicon plasmids and cloning the corresponding structural polyprotein (SPP) under the control of an additional subgenomic promoter. More specifically, to clone transgenic VEEV strains, the noncytopathic VEEV replicon expressing tTA was digested with Asci and/or Xbal and different SPPs were inserted using Gibson cloning. SPPs were synthesized as geneblocks from IDT. The VEEV capsid was derived from the TC-83 strain (Accession No.
  • a noncytopathic VEEV capsid was derived using the TC-83 strain and including previously described mutations (Atasheva et al. 2010 J. Virol 84(19): 10004-10015).
  • the VEEV/CHIKV strain used an SPP derived from the La Reunion strain of CHIKV (Accession No. KT449801).
  • Plasmids encoding piggybac transposons used for gene-circuit delivery were created as follows.
  • the TetO7 gene circuit were constructed using the Tet-On® 3G Bidirectional Inducible Expression System (Takara, 631337). Puromycin N-acetyl-transferase (PuroR) was cloned into the first multiple cloning site of the Tet-On vector.
  • mGreenLantem was cloned into the second multiple cloning site of the Tet-On vector using pcDNA3.1 -mGreenLantem (Addgene plasmid #161912), and P2A-Bsr was cloned immediately downstream of mGreenLantern using pLV-U6-gRNA-UbC-eGFP-P2A-Bsr (Addgene plasmid #83925).
  • PB-CA Additional piggybac terminal repeats were inserted stepwise to flank the gene circuit.
  • Plasmids encoding Cast 2 variants and gRNAs were obtained from Addgene and modified as follows. Wildtype UnCasl2fl, CasMINI, and optimized gRNA constructs were obtained (Addgene plasmids #176268, 176269, 176273), as well as a codon optimized UnCasl2fl variant and an optimized gRNA construct (Addgene plasmid #176544), AsCasl2fl and its gRNA (Addgene plasmids #171614, 171611).
  • CasMINI contained an unexpected mutation, G297C, that was propagated from a plasmid derived from an earlier study first describing UnCasl2fl (see Addgene plasmid # 112500, as described in Harringon et al. 2018 Science 362(6416):839-842). All Cas effectors were cloned into pcDNA3.1 and, if necessary, were modified to remove nuclease activity. Cas effectors were also fused to transcriptional activators (either VP64 or VPR). Similarly, all gRNAs were cloned into the FUGW plasmid, which allows for accurate comparisons between systems and enables subsequent lentivirus production. FUGW (Addgene plasmid #14883) and Lentivirus packaging plasmids pMD2G and psPax2 (Addgene plasmids # 12259, 12260) were obtained.
  • All PCRs were performed using either Primestar Max (Takara, R045B) or Q5 (NEB, M0493L). Q5 reactions were supplemented with 4% v/v DMSO. All plasmids were transformed using the NEB® Stable Competent E. coli (NEB, C3040H). Commercial stocks of NEB Stable were propagated in house and a standard protocol implementing rubidium chloride was used to create competent stocks for large scale use. Gibson assembly was used for all cloning procedures. All bacterial colonies were selected using carbenecillin on LB agar plates (Teknova, L1010). All plasmids were purified using silica-based column purification (Qiagen, 27106).
  • Sequence analysis of viral open reading frames To compare protein sequences within the alphavirus family, genomes of CHIKV, RRV, SINV, VEEV, and EEEV were downloaded using the aforementioned Accession numbers. The polymerases of each species were compared using their respective non- structural proteins (NSP1-4) which were combined as a single peptide. SINV, RRV, VEEV and EEEV contain a stop codon at the end of NSP3 which was removed for alignment. The structural polyproteins of each alphavirus (capsid and glycoproteins) were aligned using their native sequences, which exist as a single open reading frame.
  • IOUL reactions were assembled as follows: luL lOx reaction buffer, luL GTP, 5uL NTP/CTP, luL RNAse inhibitor (Invitrogen, AM2696), and 25-100ng of linearized template. If necessary, water was added to bring the reaction to IOuL. Reactions were incubated at 37C for one hour after which luL of TURBO DNAse (Invitrogen, AM2238) was added. Reactions were then incubated for 10 minutes to allow for removal of the template DNA.
  • BHK21 (clone 13) cells and HEK293T cells were obtained from the American Tissue Collection Center (ATCC) and were maintained in GibcoTM MEM a (ThermoFisher, 32571036), which contained nucleosides, GlutaMAXTM Supplement, 5% FBS, and 1% penicillin-streptomycin. Cells were grown at 37 °C with 5% CO2. HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Transfection efficiencies were routinely higher than 50%, as determined by fluorescence microscopy after delivery of a control eGFP expression plasmid.
  • HEK293T cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C with 5% CO2. HEK293T cells were also transfected with Lipofectamine 2000 and transfection and efficiencies were routinely higher than 80%. All transfections were performed in 96-well cell culture plates that were seeded with 20,000 cells per well on day 0. On day 1, cells were transfected in complete medium with 100 ng plasmid and 2 pl Lipofectamine 2000. On day 2 cells were collected for downstream analysis.
  • RNA genome for a given alphavirus was produced using in vitro transcription and purified as described above.
  • 100 ng of purified RNA viral genome was transfected into the cells using Lipofectamine MessengerMax (ThermoFisher, LMRNA015). Media was changed on day 2 and viral supernatant was harvested on day 3 (200uL harvest from a 96 well plate). The viral stock from the 96-well was then passaged once over a 10 cm dish of BHK21 to create a larger working stock.
  • Replication defective alphavirus production In order to create replication defective alphavirus particles, the alphaviral genome was split into two components and delivered simultaneously. VEEV replicons were transcribed and purified as described above. The structural polyprotein was delivered on a separate RNA that had a deletion spanning nucleotides 404-7314, which deletes all of NSP2-3 and removes the majority of NSP1 and NSP4. 1 pg of replicon and 1 ug of SPP were electroporated into 2 million BHK21 cells using a 100 pL tip (ThermoFisher, Neon Transfection System), and were plated into 10 cm dishes with 10 mL of complete media.
  • CHIKV SPP helper used VEEV sequence for UTRs and NSP1 and NSP4 regions.
  • Packaged particles were tittered by diluting the supernatant and measuring GFP forming units as well as rt-qPCR using purified RNA as a standard. To test propagation of these particles, particles were co-delivered with a helper SPP plasmid.
  • CDS SPP coding sequence
  • EGFP spread was measured by flow cytometry and was non-detectable.
  • Rt-qPCR was used to measure accumulation of particles in the supernatant, was non-detectable.
  • Lentivirus production 24-well cell culture plates were coated with a 1 : 10 dilution of poly-l-lysine (P8920 SIGMA). On day 0 200,000 HEK293T cells were plated per well in a 24- well plate. On day 1 transfection was carried out using Lipofectamine 2000 as follows, on a per well basis, 2 pL of lipofectamine, 0.2 pg of transfer vector, 0.2 pg pMD2G, and 0.6 pg psPAX2 were added. Components were combined in reduced serum media as per manufacturers instruction and added dropwise to each well of a 24-well plate. 12-14 hours after transfection, transfection medium was aspirated and replace with 0.5 ml fresh growth medium.
  • P8920 SIGMA poly-l-lysine
  • Lentiviral supernatant was harvested both 24 and 48 hours after this medium change. Depending on the number of viruses produced, the viral supernatant was pooled and then filtered through 0.45-pm cellulose acetate filters. Harvests were concentrated with LentiX (Takara, 631232) resuspended in PBS and stored at -80 °C.
  • BHK21 cells were genetically modified to insert a gene-circuit in their genome as follows.
  • a transposon harboring the gene circuit was cloned as described above.
  • cells were plated in a 24 well plate at a density of 200,000 cells per well.
  • one well of the 96 well plate was transfected using Lipofectamine 2000 to deliver the following plasmids: 400 ng of transposon, 50 ng of the piggybac transposase (SystemBiosciences, PB210PA-1), and 50 ng pcDNA3.1 encoding mGreenLantern. Fluorescence was then used for two purposes; first, to enrich for those cells that were efficiently transfected, and second, as marker whose loss indicates the loss of the transposase.
  • the cells were harvested, resuspended in RPMI-1640 without phenol red and supplemented with 2%FBS, 1% pen-strep, and 50 pg/ml gentamycin.
  • Cells were filtered (CelTrics, 04-0042-2316), and sorted on FACSAriall using purity mode and sorting any cell that was GFP+.
  • the bulk GFP+ sorted cell pool was then grown in complete media with the addition of 50 pg/mL gentamycin until a loss in GFP fluorescence was observed, indicating the loss of the transposase and genetic stability. This occurred approximately one week post transfection.
  • Single cell clones were then isolated by dilution, plating approximately 0.5 cells per well in a 96 well plate. Wells that showed a single defined cluster of cells were passed for testing. Each cluster was passed into two wells on separate plates. One plate was used to test gene circuit activity by delivering tTA by plasmid transfection and measuring GFP fluorescence by flow cytometry. One clone that had high gene-circuit activity and robust growth was chosen and the untransfected cell pool was expanded and banked in liquid nitrogen for long term storage.
  • Lentiviral titer was determined by using fluorescent functional assays: lentiviral constructs contained a constitutive UBC promoter expressing mCherry. Viral supernatant was diluted 1-1000 fold and fluorescence was measured using flow cytometry 48 hours post-transduction. Alphavirus titer was determined using two complementary methods that generally showed high concordance. The first is a functional assay that measures titer by measuring activity of the EGFP or tTA transgene. A 96 well plate was seeded with 20,000 BHK21-select cells per well.
  • Rt-qPCR was carried out directly on viral supernatant to determine titer. Primers and probes were ordered as a single PrimeTime mix through IDT as follows: FWD: CCTCTCGCTGAACAAGTCATAG (SEQ ID NO: 1);
  • REV CCTCTGGCACCACTACTTTAC (SEQ ID NO:2); Probe:/5HEX/TGGTATGGT/Zen/TCCACGGCATAACGC(SEQ ID NO:3)/3IABkFQ/.
  • This probe set spans residues 43-74 of NSP2 derived from TC-83.
  • the probe set was diluted in water, aliquoted, and stored at -20 °C, per manufacturer instructions.
  • TaqManTM Fast Virus 1-Step Master Mix (ThermoFisher, 4444434) was used to perform RT-qPCR, where 1 pL of probe, 1 pL of viral supernatant, 5 pL of FastVirus Mastermix, and 13 pL of water were mixed per reaction.
  • Reactions were carried out on a CFX96 Touch RT-PCR machine (BioRad), with fast cycling conditions as follows: 50 °C, 5 minutes; 95 °C, 20s; 40 cycles of (95 °C, 3s; 60 °C, 30s; read).
  • Replicon evolution Viral replicons were transcribed and purified as described above. Both the tTA and dUnCasl2fl-VPR evolution campaigns were performed similarly, however, for the Casl2 evolution, BHK21-cells were transduced with lentivirus expressing a guide RNA targeted to the tet operator. A 24-well plate was seeded with 200,000 BHK21 -select cells. 24hrs post plating, cells were transfected with 500 ng of purified replicon using Lipofectamine MessengerMax. Cells were then grown under gradually increasing concentrations of puryomycin and blasticidin.
  • Transgenes were recovered using RT-PCR run against the total RNA from the final cell pool.
  • SuperScriptTM IV One-Step RT- PCR System (ThermoFisher, 12594100) was used with 100 ng of total RNA as input into a 50 pL reaction and cycled per manufacturer’s protocol.
  • the following primers were used for recovery of the transgene, FWD: TGGCCATGACTACTCTAGCT (SEQ ID NO:4)
  • REV CGCCGCGAGTTCTATGTAAG (SEQ ID NO: 5).
  • PCR product was run on a gel to confirm purity of product.
  • PCR product was then purified and size selected using a 0.6x SPRI bead selection (AMPure XP, Beckman Coulter). Purified PCR product was then input into a Watchmaker DNA Library Prep Kit with Fragmentation (Watchmaker Genomics, 7K0019- 024), which randomly fragmented the PCR product and added illumina adapters. Libraries were quantified using fluorometry (ThermoFisher, Qubit HS DNA Assay) and average DNA fragment size was determined using TapeStation (Agilent). Deep sequencing and mutational analysis: Libraries were generated both from the final evolved cell pool and, as a control, from the in vitro transcribed replicons.
  • AMPure XP Beckman Coulter
  • VCF files were parsed using a custom python script to extract the read depth for reference and alternate alleles, the values of which were used to calculate alternate allele frequencies at each position. The alternate allele sequence was also extracted at each position.
  • a pipeline to create VCF files from fastq files is available from the authors as a jupyter notebook upon request; python scripts are also available upon request.

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Abstract

La présente invention concerne des génomes d'alphavirus chimériques, des particules d'alphavirus et des compositions les comprenant, ainsi que leur utilisation dans des procédés d'évolution dirigée, tels que des procédés de modification d'un acide nucléique hétérologue, impliquant un acide nucléique hétérologue, la modification d'un produit synthétique, l'évolution d'un produit synthétique, la fourniture de populations d'acides nucléiques hétérologues modifiés et/ou de produits synthétiques évolués. L'invention concerne également des procédés de fabrication des génomes d'alphavirus chimériques selon l'invention.
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WO2006076032A2 (fr) * 2004-05-25 2006-07-20 Novartis Vaccines And Diagnostics Inc. Constructions d'encapsidation de replicon d'alphavirus
US20090305344A1 (en) * 2001-05-31 2009-12-10 Novartis Vaccines And Diagnostics, Inc. Chimeric alphavirus replicon particles
US20200299724A1 (en) * 2016-03-21 2020-09-24 Biontech Rna Pharmaceuticals Gmbh Trans-Replicating RNA

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Publication number Priority date Publication date Assignee Title
US20090305344A1 (en) * 2001-05-31 2009-12-10 Novartis Vaccines And Diagnostics, Inc. Chimeric alphavirus replicon particles
WO2006076032A2 (fr) * 2004-05-25 2006-07-20 Novartis Vaccines And Diagnostics Inc. Constructions d'encapsidation de replicon d'alphavirus
US20200299724A1 (en) * 2016-03-21 2020-09-24 Biontech Rna Pharmaceuticals Gmbh Trans-Replicating RNA

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ENGLISH JUSTIN G.; OLSEN REID H.J.; LANSU KATHERINE; PATEL MICHAEL; WHITE KAROLINE; COCKRELL ADAM S.; SINGH DARSHAN; STRACHAN RYAN: "VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells", CELL, ELSEVIER, AMSTERDAM NL, vol. 178, no. 3, 4 July 2019 (2019-07-04), Amsterdam NL , pages 748, XP085747876, ISSN: 0092-8674, DOI: 10.1016/j.cell.2019.05.051 *

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