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METHOD FOR ISOLATING EXTRACELLULAR VESICLES

20220356269 · 2022-11-10

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention provides a gentle and low cost means to isolate extracellular vesicles, including exosomes, from a surrounding sample. In particular the invention relates to the use of a fusion protein and biopolymer beads in such methods. Methods, compositions and medical uses of such compositions are also provided.

    Claims

    1. A method of producing biopolymer particles coated with a fusion protein, wherein the method comprises the steps of: (i) providing a host cell that produces a) biopolymer particles; and b) a fusion protein capable of coating the biopolymer particles in the cells, wherein the fusion protein comprises a biopolymer particle binding domain and an extracellular vesicle binding domain and a sequence capable of being cleaved by a protease, optionally a site specific protease, optionally a TEV protease; (ii) cultivating the host cell under conditions suitable for the production of biopolymer particles coated with the fusion protein.

    2. The method of claim 1 wherein the method further comprises isolating the coated biopolymer particles from the host cell.

    3. The method of any one of claims 1 or 2 wherein the biopolymer particle comprises one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE).

    4. The method of any one of claims 1-3 wherein the PHA comprises poly(3-hydroxybutyrate) (P(3HB)), poly(4-hydroxybutyrate) (P(4HB)), polyhydroxyvalerate (PHV), poly(3-hydroxyhexanoate) (P(3HHx)), poly(3-hydroxyheptanoate) (P(3HH)), poly(3-hydroxyoctanoate) (P(3HO)), poly(3-hydroxynonanoate) (P(3HN)), poly(3-hydroxydecanoate) (P(3HD)), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), poly(3HB-co-3-hydroxyvalerate) (P(3HB-co-3HV)), poly(3HB-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), poly(3HB-co-3-hydroxy-4-methylvalerate) (P(3HB-co-3H4MV)), or poly(3HB-co-medium-chain-length-3HA) (P(3HB-co-mcl-3HA)).

    5. The method of any one of claims 1-4 wherein the biopolymer particle is a PHA-blended biopolymer particle comprising PHA and a further biopolymer, optionally where the further biopolymer is selected from the group consisting of a poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB), starch-PHA, poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE).

    6. The method of claim 5 wherein the PHA-blended biopolymer comprises poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB) or starch-PHA.

    7. The method of any of claims 1-6 wherein the host cell comprises i) A biopolymer particle production nucleic acid construct, optionally wherein the biopolymer particle production nucleic acid construct expresses one or more proteins that produce one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE), optionally wherein the biopolymer particle production nucleic acid construct is a PHA production nucleic acid construct that expresses a phaCAB operon, optionally comprising a constitutive promoter, a synthetic ribosome binding site linked to a polynucleotide encoding a phaC gene, and/or natural ribosome binding sites linked to a polynucleotide encoding a phaA gene and a polynucleotide encoding a phaB gene; and ii) A fusion protein production nucleic acid construct.

    8. The method of any of claims 1-7, wherein the host cell is: a bacterial cell, optionally a cyanobacterial cell; an archaeal cell, optionally a haloarchaeal cell; a fungal cell, optionally a yeast cell: or a plant cell.

    9. The method of claim 8, wherein the bacterial cell is selected from the genera Alcaligenes, Azotobacter, Bacillus, Chlorogloea, Cupriavidus, Escherichia, Gloeothece, Haloferax, Halomonas, Lactobacillus, Pseudomonas, Ralstonia, Spirulina, Synechococcus, or Thermus.

    10. The method of claim 8 or 9, wherein the bacterial cell is a cell selected from the group comprising Alcaligenes latus, Azotobacter chroococcum, Azotobacter vinelandii, Bacillus amyloliquefaciens DSM7, Bacillus laterosporus, Bacillus licheniformis, Bacillus macerans, Bacillus cereus, Bacillus circulans, Bacillus firmus G2, Bacillus subtilis I68, Bacillus subtilis K8, Bacillus sphaericus X3, Bacillus megaterium Y6, Bacillus coagulans, Bacillus brevis, Bacillus sphaericus ATCC 14577, Bacillus thuringiensis, Bacillus mycoides RLJ B-017, Bacillus sp. JMa5, Bacillus sp. INT005, Chlorogloea fritschii, Cupriavidus necator, Escherichia coli, Haloferax mediterraneis, Halomonas elongate, Halomonas species TD01, Halomonas sp. KM-1, Halomonas smyrnensis, Halomonas profundus, Pseudomonas aeruginosa, Pseudomonas mendocina PSU, Pseudomonas oleovorans, Pseudomonas putida, Ralstonia eutropha, or Thermus thermophilus.

    11. The method of claim 8, wherein the yeast cell is a Saccharomyces cerevisiae or Pichia pastoris cell, the fungal cell is a Fusarium solani Thom cell, or the plant cell is an Arabidopsis thaliana, Camelina sativa, Nicotiana tabacum or Saccharum officinarum cell.

    12. The method of any one of claims 1-11 wherein the biopolymer particles are isolated from the host cell by disrupting the cell and isolating the particles.

    13. The method of claim 12 wherein disrupting the cell is performed by physical disruption.

    14. The method of claim 13 wherein the physical disruption is performed by sonication, a cell press, detergent lysis, freeze-thawing, bead-beating, hypotonic cell disruption, or enzymatic disruption.

    15. The method of any one of claims 12-14 wherein the isolating the particles is performed using a cell sorter, centrifugation, gravity sedimentation, electrophoresis, filtration, size exclusion chromatography or affinity chromatography.

    16. The method of any one of claims 12-15 wherein the isolating the particles is performed using filtration.

    17. The method of any one of claims 1-16 wherein the mean diameter of the uncoated biopolymer particle is: a) between 50 nm and 1,500 nm, for example between 60 nm and 1,250 nm, 80 nm and 1,000 nm, 100 nm and 800 nm, 150 nm and 600 nm, 200 nm and 500 nm, 300 and 400 nm; b) less than 1,500 nm, 1,250 nm, 1,000 nm, 800 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, 100 nm, 80 nm, 60 nm, or less than 50 nm; and/or c) greater than 1,500 nm, 1,250 nm, 1,000 nm, 800 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, 100 nm, 80 nm, 60 nm, or greater than 50 nm.

    18. The method of any one of claims 1-17 wherein the mean diameter of the isolating uncoated biopolymer particle is between 500 nm and 1,500 nm.

    19. The method of claim 17 or 18 wherein biopolymer particle size is determined using dynamic light scattering or flow cytometry.

    20. The method of any one of the claims 1-19 wherein: a) between about 5% and 60% of the surface of the biopolymer particle is coated with the fusion protein, for example between 10% and 50%, 20% and 40%, for example 20% or 30% of the surface is coated with the fusion protein; b) at least 5%, 10%, 20%, 30%, 40%, 50% or at least 60% of the surface is coated with the fusion protein; and/or c) less than 60%, 50%, 40%, 30%, 20%, 10% or less than 5% of the surface is coated with the fusion protein.

    21. The method of any one of claims 1-20 wherein about 20% of the surface of the biopolymer particle is coated with the fusion protein.

    22. The method of claim 20 or 21 wherein the percentage of the biopolymer particles that is coated with the fusion protein is determined by transmission electron microscopy or proteolytic cleavage of the fusion protein followed by protein quantification.

    23. The method of any one of claims 1-22 wherein the host cell comprises: a) between about 5 and about 60 coated biopolymer particles, for example between about 10 and about 50, about 20 and about 40, about 30; b) at least about 5 coated biopolymer particles, at least about 10, 20, 30, 40, 50 or at least about 60; and/or c) less than about 60 coated biopolymer particles, less than about 50, 40, 30, 20, or less than about 5.

    24. The method of any of claims 1-23 wherein the host cell comprises at least 5 biopolymer particles, preferably wherein the host cell contains 32 biopolymer particles.

    25. The method of any one of claims 1-24 wherein: i) the biopolymer binding domain comprises a domain capable of binding to one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE); optionally comprises a) PhaR-derived binding domain (PBD), optionally comprises or consists of SEQ ID NO: 2 or SEQ ID NO 1 or a sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; b) a phasin, optionally a PhaR, a PhaP, a PhaQ, a PhaF, a PhaI, or an inactive PhaZ1; c) IbpA (HspA); or d) PhaC, and/or ii) the extracellular vesicle binding domain is selected from a protein, a protein fragment, a binding domain, a target-binding domain, a binding protein, a binding protein fragment, an affibody, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody, a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab′)2 antibody fragment, a Fab′ antibody fragment, a single-chain Fv (scFv) antibody fragment, a camelid antibody, an IgNAR Shark antibody, a DARPin, a nanobody, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, biotin, a biotin derivative, an avidin, a streptavidin, a substrate, an enzyme, an abzyme, a co-factor, a receptor, a receptor fragment, a receptor subunit, a receptor subunit fragment, a ligand, an inhibitor, a hormone, a lectin, a polyhistidine, a coupling domain, a DNA binding domain, a FLAG epitope, a cysteine residue, a library peptide, a reporter peptide, and an affinity purification peptide, or a combination thereof; and optionally wherein the fusion protein further comprises: iii) a functionalisation domain, optionally wherein the functionalisation domain is a membrane disrupting peptide or a cell targeting peptide,

    26. The method of any of claims 1-25 wherein the extracellular vesicle binding domain is capable of binding specifically to an extracellular vesicle-specific surface antigen.

    27. The method of any of claims 1-26 wherein the biopolymer particle binding domain is located at the N-terminus of the fusion protein and the extracellular vesicle binding domain is located at the C-terminus of the fusion protein.

    28. The method of any of claims 1-26 wherein the extracellular vesicle binding domain is located at the N-terminus of the fusion protein and the biopolymer particle binding domain is located at the C-terminus of the fusion protein.

    29. The method of any one of claims 1-28 wherein the biopolymer particle binding domain is fused to the extracellular vesicle binding domain via a linker peptide, optionally where the fusion protein comprises a functionalisation domain the biopolymer particle binding domain is fused to the functionalisation domain via a linker peptide.

    30. The method of claim 29 wherein the linker peptide is more than 12 amino acids in length, for example more than 15, 20, 25, 30, 35, 40, 45, 50 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 amino acids in length, and/or between around 12-112 amino acid residues in length, and optionally comprises small, non-polar and/or small, polar amino acids.

    31. The method of any of claims 1-30 wherein the fusion protein comprises a protease site, optionally a TEV protease site.

    32. The method of any one of claims 1-31 wherein the sequence capable of being cleaved by a protease, optionally a site specific protease, optionally a TEV protease is located in the linker peptide.

    33. The method of any one of claims 1-32 wherein the biopolymer particle binding domain comprises a PHA binding domain of a phasin repressor protein, PhaR.

    34. The method of claim 33 wherein the PHA binding domain of a phasin repressor protein, PhaR, lacks DNA-binding activity.

    35. The method of any one of claims 1-34 wherein the extracellular vesicle binding domain is selected from a protein, a protein fragment, a binding domain, a target-binding domain, a binding protein, a binding protein fragment, an affibody, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody, a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab′)2 antibody fragment, a Fab′ antibody fragment, a single-chain Fv (scFv) antibody fragment, a camelid antibody, an IgNAR Shark antibody, a DARPin, a nanobody, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, biotin, a biotin derivative, an avidin, a streptavidin, a substrate, an enzyme, an abzyme, a co-factor, a receptor, a receptor fragment, a receptor subunit, a receptor subunit fragment, a ligand, an inhibitor, a hormone, a lectin, a polyhistidine, a coupling domain, a DNA binding domain, a FLAG epitope, a cysteine residue, a library peptide, a reporter peptide, and an affinity purification peptide, or a combination thereof.

    36. The method of any one of claims 1-35 wherein the extracellular vesicle binding domain is an affibody.

    37. The method of any one of claims 1-36 wherein the method produces at least two different fusion protein coated biopolymer particles.

    38. The method according to claim 38 wherein the at least two different fusion protein coated biopolymer particles comprise different biopolymer particles.

    39. The method according to claim 37 or 38 wherein the at least two different fusion protein coated biopolymer particles comprise different fusion proteins.

    40. A fusion protein as defined in any of claims 1-39.

    41. A nucleic acid encoding the fusion protein of claim 40.

    42. A nucleic acid construct comprising: i) a nucleic acid sequence encoding a fusion protein as defined in claim 40; ii) a nucleic acid sequence encoding a further entity that is capable of binding to an extracellular vesicle-specific surface antigen; and optionally a nucleic acid encoding a biopolymer synthase operably linked to at least one promoter.

    43. The nucleic acid of claim 41 or 42 wherein the promoter is an inducible promoter, a synthetic promoter, a viral promoter or a phage promoter.

    44. A nucleic acid construct comprising: i) A biopolymer particle production nucleic acid construct as defined in any of the preceding claims, and/or ii) A fusion protein production nucleic acid construct as defined in any of the preceding claims.

    45. A biopolymer particle coated with: i) one or more fusion proteins according to claim 40; or ii) one or more fusion proteins as defined in any of claim 40 and further comprising the further entity that is capable of binding specifically to an extracellular vesicle-specific surface antigen.

    46. The coated biopolymer particle according to claim 45 wherein the biopolymer particle was produced according to the method of any of claims 1-39.

    47. The coated biopolymer particle according to claim 45 wherein the biopolymer particle was produced by a cell-free method comprising the steps of: (i) providing a solution comprising a) at least one biopolymer particle production nucleic acid construct, that comprises a first promoter, optionally a first inducible or repressible promoter suitable for the production of biopolymer particles; and b) at least one fusion protein production nucleic acid construct that comprises a second promoter, optionally a second inducible or repressible promoter (ii) maintaining the solution under conditions suitable for expression of the at least one biopolymer particle production nucleic acid construct and the at least one fusion protein production nucleic acid construct, and for formation of biopolymer particles coated with the fusion protein; and optionally (iii) isolating the coated biopolymer particles from the solution.

    48. The coated biopolymer particle according to claim 45 wherein the biopolymer particle was produced by a method comprising the steps of: (i) providing a biopolymer particle; (ii) providing a fusion protein capable of coating the biopolymer particle; and (iii) contacting the biopolymer particle with the fusion protein under conditions suitable for formation of biopolymer particles coated with the fusion protein.

    49. A host cell as defined in any of claims 1-39.

    50. A host cell that comprises: i) A biopolymer particle production nucleic acid construct, and ii) A fusion protein production nucleic acid construct.

    51. A host cell comprising a nucleic acid according to any of claims 41-44, optionally operably linked to an inducible promoter, wherein the host cell optionally comprises one or more nucleic acids that drive production of the biopolymer particle, optionally under the control of an inducible promoter, optionally wherein the inducible promoter that is operably linked to the nucleic acid according to any of claims 41-44 and the inducible promoter that drives production of the biopolymer particle are induced by different inducers.

    52. A kit comprising any one or more of: i) an expression construct comprising a biopolymer production module, optionally wherein the biopolymer production module produces one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE), optionally wherein the biopolymer production module is a PHA production module that expresses a phaCAB operon, optionally comprising a constitutive promoter, a synthetic ribosome binding site linked to a polynucleotide encoding a phaC gene, and/or natural ribosome binding sites linked to a polynucleotide encoding a phaA gene and a polynucleotide encoding a phaB gene; ii) a nucleic acid according to any of claims 41-44; iii) a fusion protein according to claim 40; iv) a biopolymer particle or particles; v) a coated biopolymer particle or particles according to any of claims 45-48; and/or vii) an expression construct encoding a further entity that is capable of binding to an extracellular vesicle-specific surface antigen; viii) a host cell according to any of claims 49 or 51.

    53. A method for isolating extracellular vesicles from a sample, the method comprising: (i) providing a composition of coated biopolymer particles, the biopolymer particles being coated with a fusion protein; (ii) contacting the composition comprising the coated biopolymer particles with the sample comprising extracellular vesicles under conditions which allow the formation of a coated biopolymer particle-extracellular vesicle complex; and (iii) isolating the coated biopolymer particle-extracellular vesicle complex.

    54. A method for functionalising the surface of extracellular vesicles, the method comprising: (i) providing a composition of coated biopolymer particles, the biopolymer particles being coated with a fusion protein that comprises a functionalisation domain; (ii) contacting the composition comprising the coated biopolymer particles with a sample comprising extracellular vesicles under conditions which allow the formation of a coated biopolymer particle-extracellular vesicle complex; and (iii) isolating the coated biopolymer particle-extracellular vesicle complex.

    55. The method according to any of claims 53 or 54 wherein the method comprises releasing the extracellular vesicles from the coated biopolymer particle-extracellular vesicle complex, optionally wherein the releasing does not involve the use of chelating agents.

    56. The method according to claim 55 wherein the fusion protein comprises a sequence capable of being cleaved by a protease, optionally a site specific protease, optionally a TEV protease, and said releasing involves cleavage of said sequence.

    57. The method according to claim 53-56 further comprising: (iv) processing the coated biopolymer particle-extracellular vesicle complex so as to provide a) a functionalisation domain-associated extracellular vesicle and b) a fusion protein portion-associated biopolymer particle, optionally wherein the fusion protein comprises a sequence capable of being cleaved by a protease, optionally a site specific protease, optionally a TEV protease, said processing involves cleavage of said sequence.

    58. The method according to any of claims 53-57 wherein the coated biopolymer particles have been made according to the method of any of claims 1-39.

    59. The method of any of claims 53-58 wherein the extracellular vesicle is a microvesicle, an apoptotic body, an ectosome, an exosome, an exomere, a small oncosomes, a large oncosome or an exosome mimetic.

    60. The method of any one of claims 53-59 wherein the extracellular vesicle is an exosome.

    61. The method of any one of claims 53-60 wherein the contacting the composition comprising the coated biopolymer particles with a sample comprising extracellular vesicles, occurs in aqueous solution, at a temperature between 4° C.-60° C., at a pH between 6.0-8.5.

    62. The method of any one of claims 53-57 or 59-61 wherein the biopolymer particles have been formed by a cell-free method comprising the steps of: (i) providing a solution comprising a) at least one biopolymer particle production nucleic acid construct, that comprises a first promoter, optionally a first inducible or repressible promoter suitable for the production of biopolymer particles; and b) at least one fusion protein production nucleic acid construct that comprises a second promoter, optionally a second inducible or repressible promoter (ii) maintaining the solution under conditions suitable for expression of the at least one biopolymer particle production nucleic acid construct and the at least one fusion protein production nucleic acid construct, and for formation of biopolymer particles coated with the fusion protein; and optionally (iii) isolating the coated biopolymer particles from the solution.

    63. The method of any one of claims 53-57 or 59-61 wherein the coated biopolymer particles have been formed by a method comprising the steps of: (i) providing a biopolymer particle; (ii) providing a fusion protein capable of coating the biopolymer particle; and (iii) contacting the biopolymer particle with the fusion protein under conditions suitable for formation of biopolymer particles coated with the fusion protein.

    64. A method of isolating disease-specific extracellular vesicles from a sample obtained from a subject, wherein the method comprises the method of isolating extracellular vesicles from a sample according to any of claims 53-63 and wherein the fusion protein comprises an extracellular vesicle binding domain that can bind to a disease-specific antigen located on the disease-specific extracellular vesicles.

    65. A method of diagnosing a disease in a subject or providing an indication that the subject likely has the disease, where the disease results in the production of disease-specific extracellular vesicles, wherein the method comprises isolating the disease-specific extracellular vesicles according to claim 64 and wherein where disease-specific extracellular vesicles are isolated, the subject is diagnosed with the disease or is determined to likely have the disease.

    66. The method according to claim 65 wherein the number of, or relative number of, disease-specific extracellular vesicles isolated is quantified.

    67. A method of diagnosing a disease in a subject or providing an indication that the subject likely has the disease, where the disease results in the an increase or decrease in the number of extracellular vesicles, optionally disease specific extracellular vesicles, wherein the method comprises isolating extracellular vesicles according to any of claims 53-63, optionally disease-specific extracellular vesicles according to claim 64 and quantifying the isolated extracellular vesicles.

    Description

    FIGURE LEGENDS

    [0279] FIG. 1—Examples of current techniques to isolate exosomes, along with advantages and disadvantages of each, compared to the methods of the present invention.

    [0280] FIG. 2—Descriptions of the fusion protein constructs designed for the present invention. PBD is a PHA binding domain from PhaR. IbpA/HspA is an E. coli heat shock protein. 112L denotes a linker composed of 112 amino acids. TEV site refers to a proteolytic cleave site for the Tobacco Etch Virus (TEV) protease. Superfolder green fluorescent protein (sfGFP). AffiEGFR and AffiZHER 2 are epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) binding Affibodies, respectively.

    [0281] FIG. 3—A) Schematics of PBD-based exosome capture fusion protein designs. PBD is a PHA binding domain from PhaR. GFP is superfolder green fluorescent protein (sfGFP); B) Schematics of IbpA/HspA-based exosome capture fusion protein designs. IbpA/HspA is an E. coli heat shock protein. GFP is superfolder green fluorescent protein (sfGFP); C) Schematics of PBD-based exosome capture fusion protein designs that incorporate a HIS-tag. PBD is a PHA binding domain from PhaR.

    [0282] FIG. 4—Dynamic Light Scattering (DLS) analysis of control (C104) and exosome-capture PHAs beads (1-8), n=3. The average sizes of the indicated PHAs beads are shown.

    [0283] FIG. 5—HspA-HEP [6] functionalised PHAs beads [A] Flow cytometry analysis of non-functionalised control beads (C104) and three batches of HspA-HEP [6] beads that incorporate sfGFP as part of its design. [B] Visual inspection of batches of pelleted HspA-HEP [6] beads on a blue transilluminator.

    [0284] FIG. 6—Analysis of HIS-tag surface display on exosome capture beads. [A] Fusion-protein schematics of constructs [7] PBD-HIS-AffiEGFR1907 and [8] PBD-AffiEGFR1907-HIS indicating the location of HIS-tags displayed on PHAs bead surface. [B] Flow cytometry analysis of unlabeled and labeled (Anti-HIS-PE) PHAs beads. Comparison of constructs [7] and [8] against non-functionalised control beads (C104), n=3, *p<0.05, ***p<0.0001.

    [0285] FIG. 7—Flow cytometry analysis of captured extracellular vesicles. [A] Generic schematic of exosome capture beads and EV antibody labelling. [B] Control (C104) and exosome capture beads were incubated with HEK293 cell conditioned media and then stained with either control (IgG-PE) antibody or a cocktail of EV surface marker targeting antibodies (Anti-CD9, CD63 and CD81-PE). Antibody labelled samples were analysed on an Attune flow cytometer (YL1-A, Excitation 561 nm/Emission 578 nm), n=3.

    [0286] FIG. 8—TEV removes sfGFP from fusion protein functionalised PHAs beads. Visual inspection of batches of pelleted beads on a blue transilluminator.

    [0287] FIG. 9 Analysis of ultracentrifugation and PHA bead captured EVs. [A] Dynamic Light Scattering (DLS) analysis of captured EVs. Exosome capture beads (PBD-HIS-AffiEGFR1907 [7]) were incubated with the indicated batches of HEK293 conditioned media and washed. AcTEV was used to release the EVs. These released EVs were subsequently analysed with Dynamic Light Scattering (DLS) to determine EV sizes (diameters [nm]). EVs isolated, using ultracentrifugation, from the same batch of HEK293 conditioned media were used for comparison. [B] Qubit analysis of PHA Capture and ultracentrifugation isolated EV samples. Qubit 3.0 (Invitrogen) and the Qubit protein assay (Invitrogen) were used to analyse protein concentration.

    [0288] FIG. 10 qNano gold analysis of PHA captured EVs. [A] Histogram of analysed particle diameters and concentrations. n=1886 individual particles analysed. qNano gold (IZON Science) is based upon Tunable Resistive Pulse Sensing (TRPS) and enables single particle (EV) analysis. [B] qNano analysis parameters.

    [0289] FIG. 11—Optimisation of Polyhydroxyalkanoates (PHAs) production in engineered Escherichia coli. [A] Plasmid map of the optimised C104 phaCAB biosynthetic operon. [B] Optimised C104 biosynthetic operon enhances P(3HB) and P(3HB-co-3HV) production in engineered E. coli. Comparison against E. coli engineered with the Native Cupriavidus necator phaCAB operon.

    [0290] FIG. 12—Part 1 of 3—Schematics of PhaC-based exosome capture fusion protein designs. C104 is a control design where PhaC (PHA synthase) has not been engineered as a fusion protein. GFP is superfolder green fluorescent protein (sfGFP); Vn96 is heat shock binding peptide (including those that are EV-associated) and MT1-Af7p is an MMP14 binding peptide. Part 2 of 3—Dynamic Light Scattering (DLS) analysis of control (C104) and exosome-capture PHAs beads (PhaC fusions: MT1-Af7p and Vn96), n=3. The average sizes of the indicated PHAs beads are shown. Part 3 of 3—Flow cytometry analysis of captured extracellular vesicles. Control (C104) and exosome capture beads (PhaC fusions with MT1-Af7p or Vn96 peptides) were incubated with HEK293 cell conditioned media and then stained with an EV surface marker targeting antibody (Anti-CD63-PE). [Top left] Generic schematic of PhaC exosome/EV capture beads and EV antibody labelling. [Top right] Flow cytometry histogram of control (C104) and EV capture beads (PhaC fusions: MT1-Af7p and Vn96). [Bottom] Captured and antibody labelled samples were analysed on an Attune flow cytometer (GFP: BL1-A Excitation 488 nm/Emission 530-30; Anti-CD63 PE: YL1-A, Excitation 561 nm/Emission 578 nm), n=3.

    [0291] FIG. 13—High-throughput workflow for capture of extracellular vesicles/exosomes. [A] Schematic of workflow. [B] Flow cytometry analysis of PHAs captured EVs from HEK293 conditioned media—sfGFP (Attune BL1-A Ex. 488 nm/Em. 530-30 nm) and cell mask orange (CMO) stained EVs (Attune YL1-A Ex. 561 nm/Em. 5 85-16 nm). Nile red (PHAs content measurement) was carried out on a BMG CLARIOstar (Ex. 560-15 nm/Em. 610-20. Columns 1 and 2—C104 control beads, columns 3 and 4 PhaC-MT1-Af7p beads, columns 5 and 6 PhaC-Vn96 beads, columns 7 and 8 pre-stained EVs only, columns 9 and 10 flow cytometry calibration beads (1 □m) and columns 11 and 12 PBS only.

    [0292] FIG. 14—Optimisation of flexible amino acid linkers. [A] Schematics of different flexible amino acid linker lengths (12, 22 or 112 amino acids) within PhaC-fusion proteins. [B] Analysis of 12aa, 22aa and 112aa flexible linker control (C) and TEV site containing (T) PhaC-fusion protein designs. Functionalised PhaC-fusion PHAs beads were treated with units (10 U) of Tobacco Etch Virus (TEV) protease. Proteolytically released sfGFP in supernatant samples were analysed using a CLARIOstar plate reader (483-14 nm/530-nm) and these fluorescence data were normalised against untreated controls of the same PHAs bead batch. PHAs beads were analysed using flow cytometry and PHAs bead geometric mean (Attune BL1-A, 488 nm/530-30 nm) of TEV treated beads were normalised against untreated controls of the same biosensor batch. Error bars denote standard error of the mean, n=4-8, Student t-test *P<0.05, **P<0.01,****P<0.0001 or not statistically significant (ns).

    [0293] FIG. 15—EV capture array. PLA films were coated with either control (PhaC from C104 operon) or PHAs Binding Domain (PBD)-based exosome capture fusion proteins [constructs 1, 2, 3 or 7]. Coated PLA films were incubated with HEK293 conditioned media and captured EVs were stained with either control (IgG-PE) antibody or a EV surface marker targeting antibody (Anti-CD81-PE). Coated PLA films and captured EVs were well scanned using a CLARIOstar plate reader (sfGFP: Ex. 483-14 nm/Em. 530-30 nm; PE: Ex. 496-15 nm/Em. 578-20 nm). Whole-well scanned data was averaged and displayed as a heatmap.

    REFERENCES

    [0294] Armstrong, J. P. K., et al 2017. Re-Engineering Extracellular Vesicles as Smart Nanoscale Therapeutics. ACS Nano 11, 69-83. doi:10.1021/acsnano.6b07607 [0295] Bebelman, M. P., et al., 2018. Biogenesis and function of extracellular vesicles in cancer. Pharmacol. Ther. 188, 1-11. doi:10.1016/j.pharmthera.2018.02.013 [0296] Cheng, Y., et al., 2019. Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes. Protein Cell 10, 295-299. doi:10.1007/s13238-018-0529-4 [0297] Colao, I. L., et al 2018. Manufacturing Exosomes: A Promising Therapeutic Platform. Trends Mol. Med. 24, 242-256. doi:10.1016/j.molmed.2018.01.006 [0298] Du, J., Rehm, B. H. A., 2018. Purification of therapeutic proteins mediated by in vivo polyester immobilized sortase. Biotechnol. Lett. 40, 369-373. doi:10.1007/s10529-017-2473-4 [0299] Gonzalez-Miro, M., et al 2019. Polyester as Antigen Carrier toward Particulate Vaccines. Biomacromolecules acs.biomac.9b00509. doi:10.1021/acs.biomac.9b00509 [0300] Kelwick, R., et al 2015. A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli. PLoS One 10, e0117202. doi:10.1371/journal.pone.0117202 [0301] Kelwick, R., et al 2018. Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics. Synth. Biol. 3. doi:10.1093/synbio/ysy016 [0302] Kim, Y.-S., et al 2018. The potential theragnostic (diagnostic+therapeutic) application of exosomes in diverse biomedical fields. Korean J. Physiol. Pharmacol. 22, 113-125. doi:10.4196/kjpp.2018.22.2.113 [0303] Kim, S. M., et al 2017. Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting. J. Control. Release 266, 8-16. doi:10.1016/j.jconrel.2017.09.013 [0304] Konoshenko, M. Y., et al 2018. Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. Biomed Res. Int. 2018, 1-27. doi:10.1155/2018/8545347 [0305] Lener, T., et al 2015. Applying extracellular vesicles based therapeutics in clinical trials—an ISEV position paper. J. Extracell. Vesicles 4, 30087. doi:10.3402/jev.v4.30087 [0306] Li, S., et al 2018. Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools. Acta Pharmacol. Sin. 39, 542-551. doi:10.1038/aps.2017.178 [0307] Ng, K. S., et al 2019. Bioprocess decision support tool for scalable manufacture of extracellular vesicles. Biotechnol. Bioeng. 116, 307-319. doi:10.1002/bit.26809 [0308] Patel, D. B., et al 2018. Towards rationally designed biomanufacturing of therapeutic extracellular vesicles: impact of the bioproduction microenvironment. Biotechnol. Adv. 36, 2051-2059. doi:10.1016/j.biotechadv.2018.09.001 [0309] Raposo, G., Stahl, P. D., 2019. Extracellular vesicles: a new communication paradigm?Nat. Rev. Mol. Cell Biol. 20, 509-510. doi:10.1038/s41580-019-0158-7 [0310] Roy, S., et al 2018. Extracellular vesicles: the growth as diagnostics and therapeutics; a survey. J. Extracell. Vesicles 7, 1438720. doi:10.1080/20013078.2018.1438720 [0311] Théry, C., et a 2018. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750. doi:10.1080/20013078.2018.1535750 [0312] Wang, J., et al 2017. Exosome-Based Cancer Therapy: Implication for Targeting Cancer Stem Cells. Front. Pharmacol. 7, 533. doi:10.3389/fphar.2016.00533 [0313] Watson, D. C., et al 2018. Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes. J. Extracell. Vesicles 7. doi:10.1080/20013078.2018.1442088 [0314] Wiklander, O. P. B., et al 2015. Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. J. Extracell. Vesicles 4, 26316. doi:10.3402/jev.v4.26316 [0315] Willis, G. R., et al 2017. Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency. Front. Cardiovasc. Med. 4. doi:10.3389/fcvm.2017.00063 [0316] Zhang, Y., et a 2019. Exosomes: biogenesis, biologic function and clinical potential. Cell Biosci. 9, 19. doi:10.1186/s13578-019-0282-2 [0317] Zhang, D., et a 2018. Exosome-Mediated Small RNA Delivery: A Novel Therapeutic Approach for Inflammatory Lung Responses. Mol. Ther. 26, 2119-2130. doi:10.1016/j.ymthe.2018.06.007

    EXAMPLES

    [0318] Development of Exosome Capture PHAs Beads

    [0319] During our previous projects we used model-guided design and cell-free prototyping strategies to optimise the microbial production of PHAs-based biopolymers, at a range of production scales (Kelwick et al., 2015; 2018). These projects enabled us to engineer and develop phaCAB operons that produce relatively high levels of PHAs in phaCAB-engineered Escherichia coli (FIG. 11). Essentially, phaCAB-engineered E. coli convert Acetyl-CoA into poly-3-hydroxybutyrate (P(3HB)). Acetyl-CoA is enzymatically processed by PhaA (3-ketothiolase) to form acetoacetyl-CoA. Then, PhaB (acetoacetyl-CoA reductase) reduces acetoacetyl-CoA to form (R)-3-hydroxybutyl-CoA ((R)-3HB-CoA), which is finally polymerised by PhaC (PHA synthase) to form the final PHAs polymer—P(3HB) (Kelwick et al., 2015; 2018). Several studies have noted that PhaC remains bound to PHAs beads (Du et al., 2018; Gonzalez-Miro et al., 2019).

    [0320] Initially, we engineered phaCAB operons that incorporated PhaC-fusion proteins that once produced would be capable of producing functionalised PHAs granules that could capture extracellular vesicles (including exosomes). These PhaC-fusion constructs included PhaC, a twelve amino acid linker, sfGFP, an additional twelve amino acid linker and either a heat shock binding peptide (Vn96; Ghosh et al., 2014) or an MMP14 binding peptide (MT1-Af7p; Zhu et al., 2011) (FIGS. 12-13). These PhaC-fusion were designed as IDT gblocks and then cloned into C104 vector and were termed C104-Vn96 and C104-MT1-Af7p (FIG. 12; Table 1). C104-Vn96 and C104-MT1-Af7p functionalised PHAs granules were produced in engineered E. coli and were analysed using Dynamic Light Scattering and were typically ˜1.2 μm in size (FIG. 12). These PhaC-fusion based extracellular vesicle-capture particles were incubated with HEK293 cell conditioned media and then stained with a PE-conjugated antibody that targeted an EV surface marker (CD81-PE). Flow cytometry analysis of these antibody stained, extracellular vesicle capture particles revealed that C104-Vn96 and C104-MT1-Af7p PHAs beads stained positive for EV markers, indicating EV capture (FIG. 12). These PhaC-fusion based extracellular vesicle-capture particles also captured cell mask orange (CMO) stained HEK293 EVs within a high-throughput plate-based assay (FIG. 13). Three phaCAB operons (C104) that incorporated PhaC-fusion constructs with optimised flexible amino acid linkers and Tobacco Etch Virus proteolytic recognition motifs were also cloned from IDT gblocks (FIG. 14). These constructs were used to optimise the proteolytic release of fusion protein and upstream components (e.g. captured EVs) from the PHAs granules (FIGS. 8 and 14).

    [0321] To develop an improved scalable exosome capture technology a novel strategy was devised that is optimised for extracellular vesicle (such as exosome) capture. These new constructs separate the phaCAB-operon from the extracellular vesicle-binding fusion protein, enabling their expression levels to be independently controlled and fine-tuned. This enables better control over the surface coating of the PHAs particles with the extracellular vesicle-binding fusion protein. The extracellular vesicle-binding fusion proteins can include either PhaR-derived binding domains (PBD [10.69 kDa], for example comprises or consists of SEQ ID NO: 2) or E. coli heat shock protein HspA (IbpA [16 kDa])-fusion proteins that also incorporate interchangeable (modular) extracellular vesicle-binding peptides or affibodies (FIG. 2). The PHAs-binding domains in these novel fusion proteins were engineered to be ˜4-6× smaller than PhaC (˜64.38 kDa), enabling greater coverage of the PHAs particles. Since, like PhaC, these fusion proteins remain bound to PHAs-based biopolymer particles, even post-purification, these fusion proteins can bind extracellular vesicles to their biopolymer particle. Thus, we can rapidly generate libraries of biopolymer extracellular vesicle-capture particles that are designed to capture specific extracellular vesicles, such as specific exosomes (FIG. 2, Table 1). Captured extracellular vesicles such as exosomes can then be processed using gravity sedimentation, low-speed centrifugation, flow cytometry and other methods. Alternatively, extracellular vesicles such as exosomes can be released without damaging them using an AcTEV protease (or metalloproteinase) to cleave-off the exosome from the biopolymer particle. Since we can design where the protease cleavage is positioned this also enables us to display an engineered peptide or protein (e.g. sfGFP, His-tag or cell targeting peptide) on the surface of the extracellular vesicle, such as an exosome.

    TABLE-US-00003 TABLE 1 strain table Strain Relevant features Reference JM109 endA1, recA1, gyrA96, thi, hsdR17 (r.sub.k.sup.−, m.sub.k.sup.+), Promega UK relA1, supE44, Δ(lac-proAB), [F′ traD36, proAB, laqI.sup.qZΔM15] EV104 JM109 pSB1C3 [EV104]; J23104 promoter and B0034 Kelwick et al., RBS; BBaK608002; CamR 2015 C104 JM109 pSB1C3-phaC-phaA-phaB [C104]; phaCAB Kelwick et al., operon under the control of the J23104 promoter; CamR 2015 C104-Vn96 JM109 pSB1C3-phaC_fusion-phaA-phaB [C104]; This study phaCAB operon under the control of the J23104 promoter; PhaC fusion: PhaC, 12aa Linker, sfGFP, 12aa Linker, and Vn96 peptide; CamR C104-MT1-Af7p JM109 pSB1C3-phaC_fusion-phaA-phaB [C104]; This study phaCAB operon under the control of the J23104 promoter; PhaC fusion: PhaC, 12aa Linker, sfGFP, 12aa Linker, and MMP14-binding peptide-MT1-Af7p; CamR C104-12aa JM109 pSB1C3-phaC_fusion-phaA-phaB [C104]; This study phaCAB operon under the control of the J23104 promoter; PhaC fusion: PhaC, 12aa Linker, TEV site sfGFP; CamR C104-22aa JM109 pSB1C3-phaC_fusion-phaA-phaB [C104]; This study phaCAB operon under the control of the J23104 promoter; PhaC fusion: PhaC, 22aa Linker, TEV site sfGFP; CamR C104-112aa JM109 pSB1C3-phaC_fusion-phaA-phaB [C104]; This study phaCAB operon under the control of the J23104 promoter; PhaC fusion: PhaC, 112aa Linker, TEV site sfGFP; CamR [1] PBD- JM109 pSB103-C104; Secondary module with J23104 This study AffiEGFR1907 promoter, B0034 RBS, PhaR derived PHA binding domain (PBD), 112aa Linker, TEV site, sfGFP, 12aa Linker and EGFR binding affibody-1907; CamR [2] PBD- JM109 pSB1C3-C104; Secondary module with J23104 This study AffiZHER2-342 promoter, B0034 RBS, PhaR derived PHA binding domain (PBD), 112aa Linker, TEV site, sfGFP, 12aa Linker, and HER2 binding affibody342; CamR [3] PBD-HEP JM109 pSB1C3-C104; Secondary module with J23104 This study promoter, B0034 RBS, PhaR derived PHA binding domain (PBD), 112aa Linker, TEV site, sfGFP, 12aa Linker and Heparin binding peptide; CamR [4] HspA- JM109 pSB1C3-C104; Secondary module with J23104 This study AffiEGFR1907 promoter, B0034 RBS, Escherichia coli heat shock protein A, 112aa Linker, TEV site, sfGFP, 12aa Linker and EGFR binding affibody-1907; CamR [5] HspA- JM109 pSB1C3-C104; Secondary module with J23104 This study AffiZHER2-342 promoter, B0034 RBS, Escherichia coli heat shock protein A, 112aa Linker, TEV site, sfGFP, 12aa Linker and HER2 binding affibody-342; CamR [6] HspA-HEP JM109 pSB1C3-C104; Secondary module with J23104 This study promoter, B0034 RBS, Escherichia coli heat shock protein A, 112aa Linker, TEV site, sfGFP, 12aa Linker and Heparin Binding peptide; CamR [7] PBD-HIS- JM109 pSB1C3-C104; Secondary module with J23101 This study AffiEGFR1907 promoter, B0034 RBS, PhaR derived PHA binding domain (PBD), 112aa Linker, TEVsite, 4aa Linker, 6xHIS tag, 5aa Linker and EGFR binding affibody-1907. [8] PBD- JM109 pSB1C3-C104; Secondary module with J23101 This study AffiEGFR1907-HIS promoter, B0034 RBS, PhaR derived PHA binding domain (PBD), 112aa Linker, TEVsite, 9aa Linker, EGFR binding affibody-1907 and 6xHIS tag.

    [0322] In total eight extracellular vesicle-capture fusion protein constructs were designed as IDT gblocks and then cloned into C104 vector as a separate expression module with their own regulatory elements (promoter and RBS). These constructs are termed [1] PBD-AffiEGFR1907, [2] PBD-AffiZHER2-342, [3] PBD-HEP, [4] HspA-AffiEGFR1907, [5] HspA-AffiZHER2-342, [6] HspA-HEP, [7] PBD-HIS-AffiEGFR1907 and [8] PBD-AffiEGFR1907-HIS. Once these strains were verified by sequencing PHAs production cultures were setup and PHAs particles were isolated, as described in materials and methods. Dynamic Light Scattering (DLS) analysis of isolated particles revealed that these PHAs particles were typically ˜1 μm in size (FIG. 3). Visual and Flow cytometry analysis of HspA-HEP [6] particles revealed that, as designed, their surfaces were fluorescently labelled with sfGFP (FIG. 4). Functionalised PHAs particles from constructs 1-5 weren't particularly fluorescent in comparison to control, non-functionalised beads (C104) which we hypothesise relates to likely misfolding of sfGFP within the complex fusion protein designs that we engineered. Therefore constructs 7 and 8 were designed without sfGFP, but instead incorporate HIS-tags that enable labeling with an Anti-HIS antibody. As expected, flow cytometry analysis of Anti-HIS-PE conjugated antibody labelling of constructs 7 and 8 revealed that they are bound to the surface of their respective PHAs beads (FIG. 5).

    [0323] Functionalised PHAs particles from constructs 1-7 were screened in an extracellular vesicle binding assay (see materials and methods). Essentially, these extracellular vesicle-capture particles were incubated with HEK293 cell conditioned media and then stained with either a PE-conjugated control antibody or a cocktail of antibodies targeting EV surface markers (CD9, CD63, CD81). Flow cytometry analysis of these antibody stained, extracellular vesicle capture particles revealed that several constructs: [1], [4], [6] and [7] stained positive for EV markers, indicating EV capture (FIG. 6).

    [0324] Additionally, we demonstrated that TEV treatment removes sfGFP from the PHAs particle surface, as indicated visually by pelleted PHAs particles (FIG. 7). Thus, these constructs enable extracellular vesicle release from the PHAs-particle surface.

    [0325] Furthermore, we have also demonstrated that PBD-based EV capture constructs ([1-3] and [7]) can also bind onto poly lactic acid (PLA) films and then be used to immobilise EVs (FIG. 15). Thus, these data demonstrate the flexibility and utility of our engineered fusion protein designs.

    Example 2—Materials and Methods

    [0326] Bacterial Strains and General Growth Conditions

    [0327] The constructs and strains used in this study are listed in Table 1. Escherichia coli JM109 was used for both cloning and production of exosome capture beads. For plasmid recovery E. coli strains were grown in Luria-Bertani (LB) media supplemented with 34 μg/ml Chloramphenicol (final concentration) and cultured at 37° C. with shaking (220 rpm). During PHAs bead production E. coli strains were grown in Terrific-Broth (TB) supplemented with 34 μg/ml Chloramphenicol (final concentration) and 3% glucose (w/v), cultured at 37° C. with shaking (220 rpm).

    [0328] Construct Assembly

    [0329] E. coli JM109 pSB1C3 EV104 (EV104) is an empty vector control plasmid that has been used previously (Kelwick et al., 2015; 2018). E. coli JM109 pSB1C3 C104 (C104-(BBa_K1149052) strain harbours a phaCAB-operon under the control of a strong constitutive promoter (J23104) and an engineered RBS (B0034) that is used to generate non-functionalised PHAs particles (Kelwick et al., 2015; 2018). Constructs C104-Vn96, C104-MT1-Af7p, C104-12aa, C104-22aa, C104-112aa, [1] PBD-AffiEGFR1907, [2] PBD-AffiZHER2-342, [3] PBD-HEP, [4] HspA-AffiEGFR1907, [5] HspA-AffiZHER2-342, [6] HspA-HEP, [7] PBD-HIS-AffiEGFR1907 and [8] PBD-AffiEGFR1907-HIS were designed as, codon optimised, IDT gBlocks and then cloned using In-fusion (Takara/Clontech) into C104 vector.

    [0330] Vn96, peptide targeting heat shock proteins sequence sourced from: [0331] Ghosh, A., Davey, M., Chute, I. C., Griffiths, S. G., Lewis, S., Chacko, S., et al. (2014). Rapid Isolation of Extracellular Vesicles from Cell Culture and Biological Fluids Using a Synthetic Peptide with Specific Affinity for Heat Shock Proteins. PLoS One 9, e110443. doi:10.1371/journal.pone.0110443.

    [0332] MT1-Af7p, peptide targeting MMP14 sequence sourced from: [0333] Zhu, L., Wang, H., Wang, L., Wang, Y., Jiang, K., Li, C., et al. (2011). High-affinity peptide against MT1-MMP for in vivo tumor imaging. J. Control. Release 150, 248-255. doi:10.1016/j.jconrel.2011.01.032.

    [0334] AffiEGFR1907, EGFR targeting Affibody sequence sourced from: [0335] Friedman, M., Orlova, A., Johansson, E., Eriksson, T. L. J., Höidén-Guthenberg, I., Tolmachev, V., Nilsson, F. Y., Ståhl, S., 2008. Directed Evolution to Low Nanomolar Affinity of a Tumor-Targeting Epidermal Growth Factor Receptor-Binding Affibody Molecule. J. Mol. Biol. 376, 1388-1402. doi:10.1016/j.jmb.2007.12.060

    [0336] AffiZHER2-342, HER2 targeting affibody sourced from: [0337] Eigenbrot, C., Ultsch, M., Dubnovitsky, A., Abrahmsen, L., Hard, T., 2010. Structural basis for high-affinity HER2 receptor binding by an engineered protein. Proc. Natl. Acad. Sci. 107, 15039-15044. doi:10.1073/pnas.1005025107

    [0338] Heparin binding peptide sequence sourced from: [0339] Morris, J., Jayanthi, S., Langston, R., Daily, A., Kight, A., McNabb, D. S., Henry, R., Kumar, T. K. S., 2016. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins. Protein Expr. Purif. 126, 93-103. doi:10.1016/j.pep.2016.05.013

    [0340] The DNA sequences of all cloned inserts/constructs were verified using the sequencing service provided by Eurofins Genomics GmbH (Ebersberg, Germany), which typically provided sequencing reads of >800 bp. Sequencing chromatograms were analysed using SnapGene software (v4.1), to ensure quality, and sequencing results were aligned using Serial Cloner (v2-6-1) alignment tool against reference sequences in order to confirm that there were no discrepancies between cloned and reference sequences. Inserts were fully sequenced.

    [0341] Production and Purification of Exosome Capture Beads

    [0342] Briefly, glycerol stocks of E. coli JM109 strains engineered with either a negative control plasmid (EV104), a phaCAB-operon (C104) or an extracellular vesicle-capture construct ([1-8]) were used to inoculate flasks containing Terrific Broth supplemented with 3% (w/v) glucose and 34 μg/ml chloramphenicol (Cam) and then these were cultured at 37° C. for 24 h, with shaking at 200 rpm.

    [0343] Post PHAs production, these strains were pelleted using centrifugation (4000 rpm, Eppendorf) and washed three times with PBS (1×). Cell pellets were re-suspended in 1 ml PBS per gram of cell pellet and sonicated. Samples were sonicated on ice, using a Vibra-cell VCX130 sonicator (SONICS, Newtown, USA) with 6 mm diameter probe. Sonication settings were (3×40 s with 1-min cooling interval; output frequency: 20 kHz; amplitude: 50%). Post-lysis samples were centrifuged (6000×g) and re-suspended with vortexing (5 seconds). The samples were then sonicated a second time on ice. Sonication settings were 2×20 s with 1-min cooling interval: Output frequency: 20 KHz, Amplitude: 50%. Post-lysis samples were centrifuged (6000×g) and the supernatant was discarded, then washed twice with PBS. Finally, the particles were re-suspended as 20% slurry (pellet w/v PBS) with 2 μl kanamycin (25 ug/ml).

    [0344] DLS

    [0345] PHAs bead size was analysed using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) DLS system. Harvested PHAs beads were diluted 100-fold in PBS (1×) prior to DLS analysis. At least three replicates were measured per sample at 25° C.

    [0346] Flow Cytometry

    [0347] PHAs bead fluorescence and PE antibody detection was measured using an Attune NxT Flow Cytometer (Thermo Fisher Scientific, MA, USA). ˜10,000 events per sample were measured (GFP: BL1-A detector, Excitation 488 nm/Emission 530/30 nm, perCP Cy5.5 Excitation 488 nm/Emission 695/40 nm, Cell Mask Orange/: YL1-A Excitation 561 nm/Emission 585-16 nm or PE: YL1-A, Excitation 561 nm/Emission 578 nm). At least three replicates for each sample were used. Data analysis was performed using FlowJo (vX 10.4.1) software. The gate strategy was based upon standard 1 μm diameter beads (Flow Cytometry Sub-Micron Size Reference Kit, Thermo Fisher Scientific, MA, USA).

    [0348] Exosome Capture

    [0349] HEK293F cells (Thermo #R79007) were cultured in Freestyle expression medium (Thermo #12338001) within 1L suspension flasks at 37° C. with 8% CO.sub.2 and shaking at 110 rpm for 48 h. At which time cell density had reached 2.61×10.sup.7 cells/ml. Conditioned media was harvested and then centrifuged 300 g for 10 min. The supernatant was removed and further centrifuged 2,500 g for 10 min at room temperature. Subsequently, the supernatant was filtered (0.2 μm filter) as a final step to aid in the removal of cells and large debris. 2 μl of harvest PHAs beads were incubated with 298 μl HEK conditioned media, gentle vortexed (for 5 seconds) and incubated on a carousel mixer for 60 min at room temperature.

    [0350] In order to analyse captured EVs these EV/PHAs-bead mixtures were treated with either 5 μl of CD63-PerCP Cy5.5 (BD #565426), 3 μl of Mouse IgG1-PE Kappa isotype control (MACS Miltenyi biotec #130-113-200) or a cocktail consisting of 1 μl each of the following EV marker antibodies: PE-CD9, human (MACS Miltenyi biotec #130-103-955), PE-CD63, human (MACS Miltenyi biotec #130-100-153) and PE-CD81, human (MACS Miltenyi biotec #130-118-342). Samples were gently vortexed (for 5 seconds) and incubated for 30 minutes at room temperature. Post-incubation the samples were centrifuged (6000 g) for 10 min and the supernatant was removed. The beads were washed with 1 ml PBS (1×) and then analysed on an Attune flow cytometer, as described above.

    [0351] In Detail:

    [0352] Initially, PHAs EV capture beads were washed with DPBS (1×) and then pelleted using centrifugation—14,000×g for 5 minutes. Post-centrifugation, the DPS supernatant was removed and the pelleted EV capture beads were re-suspended in 2 ml of cell conditioned media (the media of the EV producing cell line). EV capture beads were incubated in cell conditioned media on a rotating carousel for 1 hour at room temperature. Post-incubation, PHA captured EVs were pelleted using centrifugation (14,000×g for 5 minutes) and the cell conditioned media was removed. As before, the PHAs beads were subsequently washed with PBS. Captured EVs were released from PHAs beads using AcTEV protease. Briefly, post-washing, PHA capture beads were re-suspended in 100 ul TEV assay reactions—1 μl AcTEV (10U; Life Technologies, CA, USA), 1 μl of dithiothreitol (DTT), 5 μl of TEV reaction buffer (20×; 1M Trix-HCl pH8.0, 10 mM EDTA), 93 μl of PBS (1×) and then incubated at 30° C. with shaking at 500 rpm (Eppendorf Thermo Mixer C) for 2 hours. PHAs beads were subsequently separated from PHAs beads using centrifugation (14,000×g for 5 minutes) or gravity sedimentation. EV containing supernatants were stored at −80° C. for downstream processing or analysis.

    [0353] High-Throughput EV-Capture Assay

    [0354] Control (C104) and PHAs-based EV capture strains (C104-Vn96, C104-MT1-Af7p) were inoculated, from glycerol stocks, into wells on a 96-well plate containing 100 μl Terrific Broth supplemented with 3% (w/v) glucose and 34 μg/ml chloramphenicol (Cam) and then these were cultured at 37° C. for 24 h, with shaking at 200 rpm. PHAs beads were isolated from these strains using a plate sonicator (QSonica #Q800R3; Sonication settings were 3×40 s with 1-min cooling interval and Programme 4). Post-sonication, the 96-well plates were centrifuged (2250 g for 10 min at 4° C.). Cell lysate supernatants were removed, and isolated PHAs-beads were washed 3× with 100 μl PBS (1×). During each wash PHAs-based were re-pelleted using centrifugation (2250 g for 10 min at 4° C.). Pre-stained (Cell Mask Orange) HEK293 EVs were mixed within 100 μl PBS (1×) and added to appropriate wells for EV binding. Control samples (e.g. EVs or PBS only and flow cytometry calibration beads) were also setup in appropriate wells. Plates were subsequently incubated at room temperature with shaking (200 rpm) and then analysed using flow cytometry as described above. Bead samples were also pipetted into a fresh 96-well plate and stained with Nile Red (0.5 μg/ml final concentration) and analysed for PHAs content on a CLARIOstar plate reader (BMG, Labtech, Ex. 560-15 nm/Em. 610-20).

    [0355] EV Capture on Functionalised Poly Lactic Acid Film

    [0356] C104 and PBD-based construct strains [1-3 and 7] were cultured overnight in 6 ml Terrific Broth supplemented with 34 μg/ml chloramphenicol (Cam) at 37° C. for 24 h, with shaking at 220 rpm. Glucose was excluded from these cultures to minimise PHAs bead production and to ensure free fusion proteins were produced. Post-culture, these overnights were centrifuged (2200 g for 10 minutes at 4° C.) to form cell pellets. Once pelleted, media supernatant was removed, and the cell pellets were washed with 5 ml PBS (1×). Cell pellets were then lysed within 300 μl Bugbuster (Merck/Sigma Aldrich, #70923-3) and incubated at room temperature for 10 min. Post-lysis, cell lysates were transferred to 2 ml tubes and centrifuged (14000 g for 10 min at room temperature) and the resultant lysate-supernatants were stored on ice. Poly lactic acid (PLA) film (Sigma, Aldrich #GF27769304) was cut into several discs (0.6 cm in diameter, 0.05 mm thickness) and placed into appropriate wells in a 96-well plate. These PLA film discs were washed with 100 μl PBS (1×) and then blocked with 100 μl PBS with 5% BSA (w/v) for 10 minutes at room temperature with shaking (Setting 85 Stuart SSM1 mini orbital shaker). Post-incubation, blocking solution was removed and PLA film discs were washed with 200 μl PBS (1×). 50 μl of appropriate cell lysates (see above) were applied to PLA film discs within appropriate plate wells and were incubated, to facilitate PBD-fusion protein binding, for 30 minutes at room temperature with shaking (Setting 85 Stuart SSM1 mini orbital shaker). Then 150 μl PBS (1×) was added to each well and the samples were incubated for a further 30 minutes at room temperature with shaking (Setting 85 Stuart SSM1 mini orbital shaker). Post-incubation, these PBS/cell lysate solutions were removed and 50 μl A549 EV solutions (20 μg total protein of A549 EVs reconstituted into 50 μl PBS [1×]; HansaBioMed/Newmarket scientific, #HBM-A549-100) were pipetted onto PLA discs in appropriate wells. These samples were subsequently incubated, to facilitate EV-binding to PLA-film bound PBD-fusion proteins, for 30 minutes at room temperature with shaking (Setting 85 Stuart SSM1 mini orbital shaker). 50 μl of antibody solutions (PBS [1×] with either 1 μl of IgG-PE (MACS Miltenyi biotec #130-113-200) or 1 μl of CD81-PE ((MACS Miltenyi biotec #130-118-342) were pipetted onto PLA discs in appropriate wells. Samples were subsequently incubated for 30 minutes at room temperature with shaking (Setting 85 Stuart SSM1 mini orbital shaker). Post-incubation, 100 μl PBS [1×]) were pipetted onto PLA discs in appropriate wells and these samples were incubated for an additional 10 minutes at room temperature with shaking (Setting 85 Stuart SSM1 mini orbital shaker). Post-incubation PLA discs were washed 3× with 200 μl PBS [1×]). EV-capture PLA discs were subsequently measured (whole-well scanning and averaging) on a CLARIOstar plate reader (sfGFP Ex. 483-14 nm/Em. 530-30; PE Ex. 496-15 nm/Em. 578-20).

    CONCLUSIONS

    [0357] These functionalised PHAs beads and in particular the fusion protein designs represent a novel way to isolate extracellular vesicles. We envision that their modular nature are enabling us to automate the cloning and generation of libraries of exosome capture beads that incorporate many different affibody/peptide sequences. Likewise, the shift from PhaC towards a PhaR-derived binding domain (PBD) affords greater flexibility in that PhaR can bind to many other polymers beyond PHAs including poly(L-lactide) (PLLA), polyethylene (PE), and polystyrene (PS). Thus, enabling different form factors for different exosome isolation applications.

    [0358] The following numbered paragraphs describe further embodiments of the invention:

    1. A method for isolating extracellular vesicles from a sample, the method comprising: [0359] (i) providing a composition of coated biopolymer particles, the biopolymer particles being coated with a fusion protein; [0360] (ii) contacting the composition comprising the coated biopolymer particles with the sample comprising extracellular vesicles under conditions which allow the formation of a coated biopolymer particle-extracellular vesicle complex; and [0361] (iii) isolating the coated biopolymer particle-extracellular vesicle complex.
    2. A method for functionalising the surface of extracellular vesicles, the method comprising: [0362] (i) providing a composition of coated biopolymer particles, the biopolymer particles being coated with a fusion protein that comprises a functionalisation domain; [0363] (ii) contacting the composition comprising the coated biopolymer particles with a sample comprising extracellular vesicles under conditions which allow the formation of a coated biopolymer particle-extracellular vesicle complex; and [0364] (iii) processing the coated biopolymer particle-extracellular vesicle complex so as to provide a) a functionalisation domain-associated extracellular vesicle and b) a fusion protein portion-associated biopolymer particle.
    3. The method of Paragraph 1 or 2 wherein the extracellular vesicle is a microvesicle, an apoptotic body, an ectosome, an exosome, an exomere, a small oncosomes, a large oncosome or an exosome mimetic.
    4. The method of any one of Paragraphs 1-3 wherein the extracellular vesicle is an exosome.
    5. The method of any one of Paragraphs 1-4 wherein the contacting the composition comprising the coated biopolymer particles with a sample comprising extracellular vesicles, occurs in aqueous solution, at a temperature between 4° C.-60° C., at a pH between 6.0-8.5.
    6. The method of any one of Paragraphs 1-5 wherein the biopolymer particle comprises one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE).
    7. The method of any one of Paragraphs 1-6 wherein the PHA comprises poly(3-hydroxybutyrate) (P(3HB)), poly(4-hydroxybutyrate) (P(4HB)), polyhydroxyvalerate (PHV), poly(3-hydroxyhexanoate) (P(3HHx)), poly(3-hydroxyheptanoate) (P(3HH)), poly(3-hydroxyoctanoate) (P(3HO)), poly(3-hydroxynonanoate) (P(3HN)), poly(3-hydroxydecanoate) (P(3HD)), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), poly(3HB-co-3-hydroxyvalerate) (P(3HB-co-3HV)), poly(3HB-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), poly(3HB-co-3-hydroxy-4-methylvalerate) (P(3HB-co-3H4MV)), or poly(3HB-co-medium-chain-length-3HA) (P(3HB-co-mcl-3HA)).
    8. The method of any one of Paragraphs 1-7 wherein the biopolymer particle is a PHA-blended biopolymer particle comprising PHA and a further biopolymer, optionally where the further biopolymer is selected from the group consisting of a poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB), starch-PHA, poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE).
    9. The method of Paragraph 8 wherein the PHA-blended biopolymer comprises poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB) or starch-PHA.
    10. The method of any one of Paragraphs 1-9 wherein the coated biopolymer particles have been formed by a method comprising the steps of: [0365] (i) providing a host cell that produces [0366] a) biopolymer particle; and [0367] b) a fusion protein capable of coating the biopolymer particles in the cells; [0368] (ii) cultivating the host cell under conditions suitable for the production of biopolymer particles coated with the fusion protein; and [0369] (iii) isolating the coated biopolymer particles from the host cell.
    11. The method of Paragraph 10 wherein the host cell comprises [0370] i) A biopolymer particle production nucleic acid construct, [0371] optionally wherein the biopolymer particle production nucleic acid construct expresses one or more proteins that produce one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE), [0372] optionally wherein the biopolymer particle production nucleic acid construct is a PHA production nucleic acid construct that expresses a phaCAB operon, optionally comprising [0373] a constitutive promoter, [0374] a synthetic ribosome binding site linked to a polynucleotide encoding a phaC gene, and/or [0375] natural ribosome binding sites linked to a polynucleotide encoding a phaA gene and a polynucleotide encoding a phaB gene;
    and [0376] ii) A fusion protein production nucleic acid.
    12. The method of any one of Paragraphs 1-9 wherein the biopolymer particles have been formed by a cell-free method comprising the steps of: [0377] (i) providing a solution comprising [0378] a) at least one biopolymer particle production nucleic acid construct, that comprises a first promoter, optionally a first inducible or repressible promoter suitable for the production of biopolymer particles; and [0379] b) at least one fusion protein production nucleic acid construct that comprises a second promoter, optionally a second inducible or repressible promoter [0380] (ii) maintaining the solution under conditions suitable for expression of the at least one biopolymer particle production nucleic acid construct and the at least one fusion protein production nucleic acid construct, and for formation of biopolymer particles coated with the fusion protein; and optionally [0381] (iii) isolating the coated biopolymer particles from the solution.
    13. The method of any one of Paragraphs 1-9 wherein the coated biopolymer particles have been formed by a method comprising the steps of: [0382] (i) providing a biopolymer particle; [0383] (ii) providing a fusion protein capable of coating the biopolymer particle; and [0384] (iii) contacting the biopolymer particle with the fusion protein under conditions suitable for formation of biopolymer particles coated with the fusion protein.
    14. The method of Paragraph 10 or 11, wherein the host cell is: a bacterial cell, optionally a cyanobacterial cell; an archaeal cell, optionally a haloarchaeal cell; a fungal cell, optionally a yeast cell; or a plant cell.
    15. The method of Paragraph 14, wherein the bacterial cell is selected from the genera Alcaligenes, Azotobacter, Bacillus, Chlorogloea, Cupriavidus, Escherichia, Gloeothece, Haloferax, Halomonas, Lactobacillus, Pseudomonas, Ralstonia, Spirulina, Synechococcus, or Thermus.
    16. The method of Paragraph 14 or 15, wherein the bacterial cell is a cell selected from the group comprising Alcaligenes latus, Azotobacter chroococcum, Azotobacter vinelandii, Bacillus amyloliquefaciens DSM7, Bacillus laterosporus, Bacillus licheniformis, Bacillus macerans, Bacillus cereus, Bacillus circulans, Bacillus firmus G2, Bacillus subtilis I68, Bacillus subtilis K8, Bacillus sphaericus X3, Bacillus megaterium Y6, Bacillus coagulans, Bacillus brevis, Bacillus sphaericus ATCC 14577, Bacillus thuringiensis, Bacillus mycoides RLJ B-017, Bacillus sp. JMa5, Bacillus sp. INT005, Chlorogloea fritschii, Cupriavidus necator, Escherichia coli, Haloferax mediterraneis, Halomonas elongate, Halomonas species TD01, Halomonas sp. KM-1, Halomonas smyrnensis, Halomonas profundus, Pseudomonas aeruginosa, Pseudomonas mendocina PSU, Pseudomonas oleovorans, Pseudomonas putida, Ralstonia eutropha, or Thermus thermophilus.
    17. The method of Paragraph 14, wherein the yeast cell is a Saccharomyces cerevisiae or Pichia pastoris cell, the fungal cell is a Fusarium solani Thom cell, or the plant cell is an Arabidopsis thaliana, Camelina sativa, Nicotiana tabacum or Saccharum officinarum cell.
    18. The method of any one of Paragraphs 10, 11, 13-17 wherein the biopolymer particles are isolated from the host cell by disrupting the cell and isolating the particles.
    19. The method of Paragraph 18 wherein disrupting the cell is performed by physical disruption.
    20. The method of Paragraph 19 wherein the physical disruption is performed by sonication, a cell press, detergent lysis, freeze-thawing, bead-beating, hypotonic cell disruption, or enzymatic disruption.
    21. The method of any one of Paragraphs 18-20 wherein the isolating the particles is performed using a cell sorter, centrifugation, gravity sedimentation, electrophoresis, filtration, size exclusion chromatography or affinity chromatography.
    22. The method of any one of Paragraphs 12, or 18-21 wherein the isolating the particles is performed using filtration.
    23. The method of any one of Paragraphs 1-22 wherein the mean diameter of the uncoated biopolymer particle is:
    a) between 50 nm and 1,500 nm, for example between 60 nm and 1,250 nm, 80 nm and 1,000 nm, 100 nm and 800 nm, 150 nm and 600 nm, 200 nm and 500 nm, 300 and 400 nm;
    b) less than 1,500 nm, 1,250 nm, 1,000 nm, 800 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, 100 nm, 80 nm, 60 nm, or less than 50 nm; and/or
    c) greater than 1,500 nm, 1,250 nm, 1,000 nm, 800 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, 100 nm, 80 nm, 60 nm, or greater than 50 nm.
    24. The method of any one of Paragraphs 1-23 wherein the mean diameter of the isolating uncoated biopolymer particle is between 500 nm and 1,500 nm.
    25. The method of Paragraph 23 or 24 wherein biopolymer particle size is determined using dynamic light scattering or flow cytometry.
    26. The method of any one of the Paragraphs 1-25 wherein: [0385] a) between about 5% and 60% of the surface of the biopolymer particle is coated with the fusion protein, for example between 10% and 50%, 20% and 40%, for example 20% or 30% of the surface is coated with the fusion protein; [0386] b) at least 5%, 10%, 20%, 30%, 40%, 50% or at least 60% of the surface is coated with the fusion protein; and/or [0387] c) less than 60%, 50%, 40%, 30%, 20%, 10% or less than 5% of the surface is coated with the fusion protein.
    27. The method of any one of Paragraphs 1-26 wherein about 20% of the surface of the biopolymer particle is coated with the fusion protein.
    28. The method of Paragraph 26 or 27 wherein the percentage of the biopolymer particles that is coated with the fusion protein is determined by transmission electron microscopy or proteolytic cleavage of the fusion protein followed by protein quantification.
    29. The method of any one of Paragraphs 10, 11 or 14-28 wherein the host cell comprises: [0388] a) between about 5 and about 60 coated biopolymer particles, for example between about 10 and about 50, about 20 and about 40, about 30; [0389] b) at least about 5 coated biopolymer particles, at least about 10, 20, 30, 40, 50 or at least about 60; and/or [0390] c) less than about 60 coated biopolymer particles, less than about 50, 40, 30, 20, or less than about 5.
    30. The method of Paragraph 10, 11 or 14-29 wherein the host cell comprises at least biopolymer particles, preferably wherein the host cell contains 32 biopolymer particles.
    31. The method of any one of Paragraphs 1-30 wherein the fusion protein comprises: [0391] i) a biopolymer particle binding domain, optionally wherein the biopolymer binding domain comprises a domain capable of binding to one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE); optionally comprises [0392] a) PhaR-derived binding domain (PBD), optionally comprises or consists of SEQ ID NO: 2 or SEQ ID NO 1 or a sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; [0393] b) a phasin, optionally a PhaR, a PhaP, a PhaQ, a PhaF, a PhaI, or an inactive PhaZ1; [0394] c) IbpA (HspA); or [0395] d) PhaC. [0396] and [0397] ii) an extracellular vesicle binding domain, optionally wherein the extracellular vesicle binding domain is selected from a protein, a protein fragment, a binding domain, a target-binding domain, a binding protein, a binding protein fragment, an affibody, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody, a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab′)2 antibody fragment, a Fab′ antibody fragment, a single-chain Fv (scFv) antibody fragment, a camelid antibody, an IgNAR Shark antibody, a DARPin, a nanobody, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, biotin, a biotin derivative, an avidin, a streptavidin, a substrate, an enzyme, an abzyme, a co-factor, a receptor, a receptor fragment, a receptor subunit, a receptor subunit fragment, a ligand, an inhibitor, a hormone, a lectin, a polyhistidine, a coupling domain, a DNA binding domain, a FLAG epitope, a cysteine residue, a library peptide, a reporter peptide, and an affinity purification peptide, or a combination thereof; [0398] optionally wherein the fusion protein further comprises: [0399] iii) a functionalisation domain, optionally wherein the functionalisation domain is a membrane disrupting peptide or a cell targeting peptide; and/or [0400] iv) a sequence capable of being cleaved by a protease, optionally a site specific protease, optionally a TEV protease.
    32. The method of Paragraph 31 wherein the extracellular vesicle binding domain is capable of binding specifically to an extracellular vesicle-specific surface antigen.
    33. A fusion protein comprising: [0401] i) a biopolymer particle binding domain; and [0402] ii) an extracellular vesicle binding domain, optionally wherein the extracellular vesicle binding domain is capable of binding specifically to an extracellular vesicle-specific surface antigen;
    optionally wherein the fusion protein further comprises: [0403] a functionalisation domain, optionally wherein the functionalisation domain is a membrane disrupting peptide or a cell targeting peptide; and/or [0404] a sequence capable of being cleaved by a protease, optionally a site specific protease, optionally a TEV protease.
    34. The method of any one of Paragraphs 31 or 32 or the fusion protein of Paragraph 33, wherein the biopolymer binding domain comprises a domain capable of binding to one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE).
    35. The method or the fusion protein of Paragraph 34, wherein the biopolymer binding domain is: [0405] a) PhaR-derived binding domain (PBD), optionally comprises or consists of SEQ ID NO: 2 or SEQ ID NO 1 or a sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; [0406] b) a phasin, optionally a PhaR, a PhaP, a PhaQ, a PhaF, a PhaI, or an inactive PhaZ1; [0407] c) IbpA (HspA); or [0408] d) PhaC.
    36. The method of any one of Paragraphs 31 or 32, 34 or 35, or the fusion protein of any one of Paragraphs 34-36, wherein the biopolymer particle binding domain is located at the N-terminus of the fusion protein and the extracellular vesicle binding domain is located at the C-terminus of the fusion protein.
    37. The method of any one of Paragraphs 31 or 32, 34 or 35, or the fusion protein of any one of Paragraphs 34-36, wherein the extracellular vesicle binding domain is located at the N-terminus of the fusion protein and the biopolymer particle binding domain is located at the C-terminus of the fusion protein.
    38. The method of any one of Paragraphs 31 or 32 or 34-37, or the fusion protein of any one of Paragraphs 34-37 wherein the biopolymer particle binding domain is fused to the extracellular vesicle binding domain via a linker peptide, [0409] optionally where the fusion protein comprises a functionalisation domain the biopolymer particle binding domain is fused to the functionalisation domain via a linker peptide.
    39. The method or the fusion protein of Paragraph 38 wherein the linker peptide is between around 12-112 amino acid residues in length and comprises small, non-polar and/or small, polar amino acids.
    40. The method of any of paragraphs 31 or 32, or 34-39 or the fusion protein of any of paragraphs 34-39 wherein the fusion protein comprises a cleavable site, optionally cleavable by the TEV protease.
    41. The method or the fusion protein of Paragraph 40 wherein the cleavable site is located in the linker peptide.
    42. The method of any one of Paragraphs 31 and 32 or 34-41, or the fusion protein of any one of Paragraphs 34-41, wherein the biopolymer particle binding domain comprises a PHA binding domain of a phasin repressor protein, PhaR.
    43. The method or the fusion protein of Paragraph 42 wherein the PHA binding domain of a phasin repressor protein, PhaR, lacks DNA-binding activity.
    44. The method of any one of Paragraphs 31 or 32 or 34-43 the fusion protein of any one of Paragraphs 34-43, wherein the extracellular vesicle binding domain is selected from a protein, a protein fragment, a binding domain, a target-binding domain, a binding protein, a binding protein fragment, an affibody, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody, a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab′)2 antibody fragment, a Fab′ antibody fragment, a single-chain Fv (scFv) antibody fragment, a camelid antibody, an IgNAR Shark antibody, a DARPin, a nanobody, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, biotin, a biotin derivative, an avidin, a streptavidin, a substrate, an enzyme, an abzyme, a co-factor, a receptor, a receptor fragment, a receptor subunit, a receptor subunit fragment, a ligand, an inhibitor, a hormone, a lectin, a polyhistidine, a coupling domain, a DNA binding domain, a FLAG epitope, a cysteine residue, a library peptide, a reporter peptide, and an affinity purification peptide, or a combination thereof.
    45. The method of any one of Paragraphs 31 or 32 or 34-44, or the fusion protein of any one of Paragraphs 34-44 wherein the extracellular vesicle binding domain is an affibody.
    46. A nucleic acid encoding the fusion protein of any one of paragraphs 34-45.
    47. A nucleic acid construct comprising: [0410] i) a nucleic acid encoding a fusion protein as defined in any one of Paragraphs 34-45; [0411] ii) a nucleic acid encoding a further entity that is capable of binding to an extracellular vesicle-specific surface antigen; and [0412] optionally a nucleic acid encoding a biopolymer synthase [0413] operably linked to at least one promoter.
    48. The nucleic acid of Paragraph 46 or 47 wherein the promoter is an inducible promoter, a synthetic promoter, a viral promoter or a phage promoter.
    49. A biopolymer particle coated with: [0414] i) one or more fusion proteins according to any of Paragraphs 34-45; or [0415] ii) one or more fusion proteins as defined in any of Paragraphs 34-45 and further comprising the further entity that is capable of binding specifically to an extracellular vesicle-specific surface antigen.
    50. A host cell comprising a nucleic acid according to any of paragraphs 46-48, optionally operably linked to an inducible promoter, [0416] wherein the host cell optionally comprises one or more nucleic acids that drive production of the biopolymer particle, optionally under the control of an inducible promoter, [0417] optionally wherein the inducible promoter that is operably linked to the nucleic acid according to any of paragraphs 46-48 and the inducible promoter that drives production of the biopolymer particle are induced by different inducers.
    51. A kit comprising any one or more of: [0418] i) an expression construct comprising a biopolymer production module, optionally wherein the biopolymer production module produces one or more of a polyhydroxyalkanoate (PHA), a poly(L-lactide) (PLLA), a polyethylene (PE), a polystyrene (PS), or a polythioester (PTE), [0419] optionally wherein the biopolymer production module is a PHA production module that expresses a phaCAB operon, optionally comprising [0420] a constitutive promoter, [0421] a synthetic ribosome binding site linked to a polynucleotide encoding a phaC gene, and/or [0422] natural ribosome binding sites linked to a polynucleotide encoding a phaA gene and a polynucleotide encoding a phaB gene; [0423] ii) a nucleic acid according to Paragraph 46; [0424] iii) a nucleic acid construct according to paragraph 47; [0425] iv) a fusion protein according to any of paragraphs 34-45; [0426] v) a biopolymer particle or particles; [0427] vi) a coated biopolymer particle or particles according to paragraph 49; and/or [0428] vii) an expression construct encoding a further entity that is capable of binding to an extracellular vesicle-specific surface antigen; [0429] viii) a host cell according to paragraph 50.
    52. A method of isolating disease-specific exosomes from a sample obtained from a subject, wherein the method comprises the method of isolating extracellular vesicles from a sample according to any of paragraphs 1, or 3-32, and wherein the fusion protein comprises an extracellular vesicle binding domain that can bind to a disease-specific antigen located on the disease-specific extracellular vesicles.
    53. A method of diagnosing a disease in a subject or providing an indication that the subject likely has the disease, where the disease results in the production of disease-specific extracellular vesicles, wherein the method comprises isolating the disease-specific exosomes according to paragraph 52 and wherein where disease-specific extracellular vesicles are isolated, the subject is diagnosed with the disease or is determined to likely have the disease.
    54. The method according to paragraph 53 wherein the number of, or relative number of, disease-specific extracellular vesicles isolated is quantified.
    55. A nucleic acid construct comprising: [0430] i) A biopolymer particle production nucleic acid construct as defined in any of the preceding paragraphs, and/or [0431] ii) A fusion protein production nucleic acid construct as defined in any of the preceding paragraphs.