US20230097907A1 - Non-naturally occurring vesicles comprising a chimeric vesicle localization moiety, methods of making and uses thereof - Google Patents

Non-naturally occurring vesicles comprising a chimeric vesicle localization moiety, methods of making and uses thereof Download PDF

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US20230097907A1
US20230097907A1 US17/795,857 US202117795857A US2023097907A1 US 20230097907 A1 US20230097907 A1 US 20230097907A1 US 202117795857 A US202117795857 A US 202117795857A US 2023097907 A1 US2023097907 A1 US 2023097907A1
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vesicle
moiety
chimeric
domain
exosome
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Terry Gaige
Colin David Gottlieb
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Mantra Bio Inc
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Mantra Bio Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • Extracellular vesicles can be membrane-based structures.
  • EVs can serve as vehicles that carry different types of cellular cargo-such as lipids, proteins, receptors and effector molecules-to the recipient cells.
  • Exosomes are a type of EV that can be released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. Exosome production has been described in cells, including B cells, T cells, and dendritic cells (DCs).
  • DCs dendritic cells
  • non-naturally occurring vesicles comprising a chimeric vesicle localization moiety for efficient EV biogenesis or localization.
  • the chimeric vesicle localization moiety may comprise a surface-and-transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety.
  • Such chimeric vesicle localization moiety may additionally comprise one or more tissue or cell targeting moieties for targeting exosomes to a tissue or a specific cell type.
  • the invention provides fusion proteins containing chimeric vesicle localization moieties, vectors comprising nucleic acid sequences encoding such fusion proteins, genetically modified cells comprising such vectors, methods of making the non-naturally occurring vesicles of the invention, pharmaceutical compositions and kits containing same.
  • FIG. 1 is a map of EV-localizing fusion proteins produced from expression vectors 91, 112, 135, 140 and 141. Numbers represent length in nucleotides for the marks on the line above. Arrangement of notable biological sequences are indicated by various arrows used to represent signal sequence, epitope sequence, affinity peptide, linkers, glycosylation site, and a vesicle localization moiety (vector #91 for LAMP2; vector #112 for CLSTN1) or a chimeric vesicle localization moiety comprising LAMP2 surface-and-transmembrane domain and cytosolic domain of PTGFRN or Prostaglandin F2 Receptor Inhibitor (vector #135), ITGA3 or Integrin Alpha 3 (vector #140), or IL3RA or Interleukin 3 Receptor Subunit Alpha (vector #141).
  • vector #91 for LAMP2 vector #112 for CLSTN1
  • a chimeric vesicle localization moiety comprising
  • the coding sequence for LAMP2 in vector #91 and that for CLSTN1 in vector #121 are for the respective mature protein which lacks the signal sequence (first 28 amino acid) present in the native LAMP2 nascent protein and native CLSTN1 nascent protein, respectively.
  • FIG. 2 is a map of EV-localizing fusion proteins produced from expression vectors 142, 143, 144 and 145. Numbers represent length in nucleotides for the marks on the line above. Arrangement of notable biological sequences are indicated by various arrows used to represent signal sequence, epitope sequence, affinity peptide, linkers, glycosylation site, and a chimeric vesicle localization moiety comprising LAM P2 surface-and-transmembrane domain and cytosolic domain of SELPLG or P-Selectin Glycoprotein Ligand 1 (vector #142), ITGBI1 or Integrin Beta-1 (vector #143), or CLSTN1 or Calsyntenin-1 (vector #144).
  • An expression vector serves as a control for ability for a truncated LAMP2 vesicle localization moiety retaining LAMP2 surface-and-transmembrane domain but lacking LAMP2 cytosolic domain to localize at an EV; the LAMP2 cytosolic domain has been replaced with a highly positive charged 4-amino acid peptide, KKPR (vector #145).
  • FIG. 3 provides the amino acid sequence of EV-localizing fusion proteins encoded by expression vector 91 (LAMP2) and produced when the expression vector is introduced into HEK293F cells along with the location of notable biological sequences.
  • the bold, regular text signifies a signal sequence (a portion of the translated sequence that helps the polypeptide be synthesized by the cell and inserted into a membrane, but is not present in the mature protein that gets incorporated into an EV).
  • the lowercase text signifies a glycosylation site.
  • the underlined text signifies an epitope sequence.
  • the boxed text signifies linker sequence.
  • the italicized text signifies a surface domain.
  • the italicized, bold text signifies a transmembrane domain.
  • the italicized, underlined text signifies a cytosolic domain (also considered to be lumenal domain when at an EV).
  • the highlighted text signifies an affinity peptide.
  • the signal sequence used here is a signal peptide sequence for optimal expression and secretion in human cells, and the epitope tag used here is 3xFLAG epitope tag.
  • FIG. 4 provides the amino acid sequence of EV-localizing fusion proteins encoded by expression vector 112 (CLSTN1) and produced when the expression vector is introduced into HEK293F cells along with the location of notable biological sequences.
  • the bold text signifies a signal sequence (a portion of the translated sequence that helps the polypeptide be synthesized by the cell and insert into a membrane, but is not present in the mature protein that gets incorporated into an EV).
  • the lowercase text signifies a glycosylation site.
  • the underlined text signifies an epitope sequence.
  • the boxed text signifies linker sequence.
  • the italicized text signifies a surface domain.
  • the italicized, bold text signifies a transmembrane domain.
  • the italicized, underlined text signifies a cytosolic domain (also considered to be lumenal domain when at an EV).
  • the highlighted text signifies an affinity peptide: THRPPMWSPVWP (SEQ ID NO.: 64).
  • the signal sequence used here is a signal peptide sequence for optimal expression and secretion in human cells, and the epitope tag used here is 3xFLAG epitope tag.
  • FIG. 5 provides the amino acid sequence of EV-localizing fusion proteins encoded by expression vector 135 (a chimeric vesicle localization moiety comprising LAMP2 surface-and-transmembrane domain and cytosolic domain of PTGFRN) and vector 140 (a chimeric localization moiety comprising LAMP2 surface-and-transmembrane domain and cytosolic domain of ITGA3) and produced when the expression vector is introduced into HEK293F cells along with the location of notable biological sequences.
  • the bold text signifies a signal sequence (a portion of the translated sequence that helps the polypeptide be synthesized by the cell and insert into a membrane, but is not present in the mature protein that gets incorporated into an EV).
  • the lowercase text signifies a glycosylation site.
  • the underlined text signifies an epitope sequence.
  • the boxed text signifies linker sequence.
  • the italicized text signifies a surface domain.
  • the italicized, bold text signifies a transmembrane domain.
  • the italicized, underlined text signifies a cytosolic domain (also considered to be lumenal domain when at an EV).
  • the highlighted text signifies an affinity peptide: THVSPNQGGLPS (SEQ ID NO.: 66).
  • the signal sequence used here is a signal peptide sequence for optimal expression and secretion in human cells, and the epitope tag used here is 3xFLAG epitope tag.
  • FIG. 6 provides the amino acid sequence of EV-localizing fusion proteins encoded by expression vector 141 (a chimeric vesicle localization moiety comprising LAMP2 surface-and-transmembrane domain and cytosolic domain of IL3RA) and vector 142 (a chimeric vesicle localization moiety comprising LAMP2 surface-and-transmembrane domain and cytosolic domain of SELPLG) and produced when the expression vector is introduced into HEK293F cells along with the location of notable biological sequences.
  • the bold text signifies a signal sequence (a portion of the translated sequence that helps the polypeptide be synthesized by the cell and insert into a membrane, but is not present in the mature protein that gets incorporated into an EV).
  • the lowercase text signifies a glycosylation site.
  • the underlined text signifies an epitope sequence.
  • the boxed text signifies linker sequence.
  • the italicized caps text a surface domain.
  • the italicized, bold text signifies a transmembrane domain.
  • the italicized, underlined text signifies a cytosolic domain (also considered to be lumenal domain when at an EV).
  • the highlighted text signifies an affinity peptide: THVSPNQGGLPS (SEQ ID NO.: 66).
  • the signal sequence used here is a signal peptide sequence for optimal expression and secretion in human cells, and the epitope tag used here is 3xFLAG epitope tag.
  • FIG. 7 provides the amino acid sequence of EV-localizing fusion proteins encoded by expression vector 143 (a chimeric vesicle localization moiety comprising LAMP2 surface-and-transmembrane domain and cytosolic domain of ITGB1) and vector 144 (a chimeric vesicle localization moiety comprising LAMP2 surface-and-transmembrane domain and cytosolic domain of CLSTN1) and produced when the expression vector is introduced into HEK293F cells along with the location of notable biological sequences.
  • the bold text signifies a signal sequence (a portion of the translated sequence that helps the polypeptide be synthesized by the cell and insert into a membrane, but is not present in the mature protein that gets incorporated into an EV).
  • the lowercase text signifies a glycosylation site.
  • the underlined text signifies an epitope sequence.
  • the boxed text signifies linker sequence.
  • the italicized text signifies a surface domain.
  • the italicized, bold text signifies a transmembrane domain.
  • the italicized, underlined text signifies a cytosolic domain (also considered to be lumenal domain when at an EV).
  • the highlighted text signifies an affinity peptide: THVSPNQGGLPS (SEQ ID NO.: 66).
  • the signal sequence used here is a signal peptide sequence for optimal expression and secretion in human cells, and the epitope tag used here is 3xFLAG epitope tag.
  • FIG. 8 provides the amino acid sequence of EV-localizing fusion proteins encoded by expression vector 145 (truncated LAMP2 having surface-and-transmembrane domain but lacking a LAMP2 cytosolic domain, which has been replaced with a positively charged 4-amino acid peptide, KKPR) and produced when the expression vector is introduced into HEK293F cells along with the location of notable biological sequences.
  • the bold text signifies a signal sequence (a portion of the translated sequence that helps the polypeptide be synthesized by the cell and insert into a membrane, but is not present in the mature protein that gets incorporated into an EV).
  • the lowercase text signifies a glycosylation site.
  • the underlined text signifies an epitope sequence.
  • the boxed text signifies linker sequence.
  • the italicized text signifies a surface domain.
  • the italicized, bold text signifies a transmembrane domain.
  • the italicized, underlined text signifies a cytosolic domain (also considered to be lumenal domain when at an EV).
  • the highlighted text signifies an affinity peptide: THVSPNQGGLPS (SEQ ID NO.: 66) in vector 145.
  • the signal sequence used here is a signal peptide sequence for optimal expression and secretion in human cells for vector 145.
  • the epitope tag used here is a 3xFLAG epitope tag.
  • FIG. 9 provides the amino acid sequences for the mature LAMP2 and CLSTN1 vesicle localization moieties in the fusion proteins produced from expression vectors #91 and #112, respectively.
  • the italicized text signifies a surface domain, topologically equivalent to an extracellular domain and is sometimes referred to as an extracellular domain of a transmembrane protein.
  • the three contiguous domains (surface, transmembrane and cytosolic domains) are indicated.
  • the italicized text signifies a surface domain, topologically equivalent to an extracellular domain and is sometimes referred to as an extracellular domain of a transmembrane protein.
  • the italicized, bold text signifies a transmembrane domain.
  • the italicized, underlined text signifies a cytosolic domain (also referred to as a lumenal domain when at an EV)
  • FIG. 10 provides the amino acid sequences of the mature chimeric vesicle localization moieties in the fusion proteins produced from expression vectors #135, #140 and #141.
  • the chimeric vesicle localization moieties share an amino acid sequence of the surface-and-transmembrane domain of LAMP2 at the amino-terminal end (indicated by italicized text for surface domain and italicized, bold text for a transmembrane domain) and amino acid sequences for the cytosolic domain of PTGFRN or Prostaglandin F2 Receptor Inhibitor (vector #135), ITGA3 or Integrin Alpha 3 (vector #140), IL3RA or Interleukin 3 Receptor Alpha (vector #141), indicated by italicized, underlined text and at the carboxyl-terminal end. Note that the cytosolic domain of LAMP2 has been replaced in these chimeric vesicle localization moieties.
  • FIG. 11 provides the amino acid sequences of the mature chimeric vesicle localization moieties in the fusion proteins produced from expression vectors #142 and #143.
  • the chimeric vesicle localization moieties share an amino acid sequence of the surface-and-transmembrane domain of LAMP2 at the amino-terminal end (indicated by italicized text for surface domain and italicized, bold text for a transmembrane domain) and amino acid sequences for the cytosolic domain of SELPLG or P-Selectin Glycoprotein Ligand 1 (vector #142) and ITGB1 or Integrin Beta-1 (vector #143), indicated by italicized, underlined text and at the carboxyl-terminal end. Note that the cytosolic domain of LAMP2 has been replaced in these chimeric vesicle localization moieties.
  • FIG. 12 provides the amino acid sequences of the mature chimeric vesicle localization moiety in the fusion protein produced from expression vector #144 and a mature truncated LAMP2 protein in the fusion protein produced from expression vector #145.
  • the amino acid sequences corresponding to the surface-and-transmembrane domain of LAMP2 is indicated by indicated by italicized text for surface domain and italicized, bold text for a transmembrane domain.
  • the cytosolic domain of LAMP2 has been replaced with the cytosolic domain of CLSTN1 or Calsyntenin-1 (vector #144) or a high positively charged tetrapeptide sequence, KKPR (vector #145), indicated by italicized, underlined text.
  • FIGS. 13 and 14 provide mean abundance of a recombinant or fusion protein on an EV, and fraction (or percent) of total EVs positive for the recombinant or fusion protein produced by the expression vector constructs of FIGS. 1 and 2 following transfection into HEK293F cells, respectively.
  • FIG. 13 shows EV populations isolated from cells transfected with the indicated vector number. Isolated EVs were stained with a mouse monoclonal antibody specific to an epitope sequence encoded in the EV surface domain of each recombinant or fusion protein.
  • the Y-axis represents the relative amount (on average) of antibody bound to each EV positively identified to comprise the recombinant or fusion protein and excludes those EVs not stained by the antibody, serving as an indirect measure of the abundance of recombinant or fusion protein incorporated into each EV which contains the recombinant or fusion protein.
  • the background signal associated with EVs from mock transfected cells has been subtracted from these values.
  • FIG. 14 shows the fraction of the total EV population displaying a detectable amount of the recombinant or fusion protein.
  • FIGS. 15 and 16 show fold increase in mean fusion protein abundance on EV surface, and fold increase in fraction (or percent) of total EVs positive for the recombinant or fusion protein relative to fusion protein produced by vector #91 construct (fusion protein with LAMP2 vesicle localization moiety lacking signal sequence following incorporation into an EV), respectively.
  • FIG. 15 shows enrichment of recombinant proteins in EVs: A) EV populations were isolated from cells transfected with the indicated vector numbers. Isolated EVs were stained with a mouse monoclonal antibody specific to an epitope sequence encoded in the EV surface domain of each recombinant protein.
  • the Y-axis represents the relative amount (on average) of antibody bound to each EV, serving as an indirect measure of the abundance of recombinant protein incorporated into each EV, relative to vector #91.
  • the background signal associated with EVs from mock transfected cells has been subtracted from these values.
  • FIG. 16 shows enrichment of recombinant proteins in EVs: EV populations were isolated from cells transfected with the indicated vector numbers. Isolated EVs were stained with a mouse monoclonal antibody specific to an epitope sequence encoded in the EV surface domain of each recombinant protein.
  • the Y-axis represents the fraction of the total EV population displaying a detectable amount of the recombinant protein on the EV surface, relative to vector #91.
  • the background signal associated with EVs from mock transfected cells has been subtracted from these values.
  • the fusion protein produced by vector #112 concentrates at a much lower level, about 25% the abundance of the LAMP2-vesicle localization moiety (compare values of #91 and #112 in FIG. 15 ).
  • the new chimeric vesicle localization moiety increases by about 2-fold the abundance of the fusion protein over its parental LAMP2 (compare values of #91 and #144) or over 8-fold the abundance of the fusion protein over its parental CLSTN1 (compare values of #112 and #144), indicative of synergistic interaction involving the surface-and-transmembrane domain of LAMP2 and the cytosolic domain of CLSTN1 that leads to increased EV localization.
  • Modified extracellular vesicles which comprise chimeric vesicle localization moieties (also referred to as “chimeric vesicle targeting moiety(ies)”) that enhance the efficient EV biogenesis or localization. Also provided are chimeric vesicle localization moieties that enhance the efficient EV biogenesis or localization. Recombinant plasmids that express chimeric vesicle localization moieties may be used to genetically modify mammalian cells with enhanced properties for enriching in extracellular vesicles, based on the nucleic acid encoding the chimeric vesicle localization moieties, disclosed herein.
  • the chimeric vesicle localization moieties may be produced in vitro or isolated from a cell and later introduced into an extracellular vesicle from an EV producer cell. Such chimeric vesicle localization moieties may additionally comprise one or more targeting moieties. Such targeting moiety(ies) can be engineered to be included on the vesicle surface.
  • the vesicles contemplated herein can include a payload. Such payload can preferably be one that is not naturally present in the vesicle. Such payload can be a natural or synthetic bioactive molecule for eliciting a phenotypic modification in the target cell or tissue of interest. In some instances, a payload is useful for the treatment of a condition. In some instances, a payload is a reporter for screening, detecting, and/or diagnosing a condition in a cell or a subject.
  • the targeting moieties provided herein can allow selective targeting or focused delivery of appropriate payloads to the cells of interest. This selective targeting or focused delivery can reduce delivery of therapeutics to off-target tissue and cell types, and/or reduce toxicity of the treatment.
  • An extracellular vesicle can be a membrane that encloses an internal space.
  • Extracellular vesicles can be cell-derived bubbles or vesicles made of the same material as cell membranes, such as phospholipids.
  • Cell-derived extracellular vesicles can be smaller than the cell from which they are derived and range in diameter from about 20 nm to 1000 nm (e.g., 20 nm to 1000 nm; 20 nm to 200 nm; 90 nm to 150 nm).
  • Such vesicles can be created through the outward budding and fission from plasma membranes, assembled at and released from an endomembrane compartment, or derived from cells or vesiculated organelles having undergone apoptosis, and can contain organelles. They can be produced in an endosome by inward budding into the endosomal lumen resulting in intraluminal vesicles of a multivesicular body (MVB) and released extracellularly as exosomes upon fusion of the multivesicular body (MVB) with the plasma membrane. They can be derived from cells by direct and indirect manipulation that may involve the destruction of said cells. They can also be derived from a living or dead organism, an explanted tissue or organ, and/or a cultured cell.
  • extracellular vesicles examples include exosomes, ectosome, microvesicle, microsome or other cell-derived membrane vesicles.
  • Other cell-derived membrane vesicles include a shedding vesicle, a plasma membrane-derived vesicle, and/or an exovesicle.
  • extracellular vesicle used here is produced by cells, and may comprise a phospholipid membrane bilayer enclosing a luminal space.
  • the membrane bilayer incorporates proteins and other macromolecules derived from the cell of origin.
  • the luminal space encapsulates lipids, proteins, organic molecules and macromolecules including nucleic acids and polypeptides.
  • Exosomes can be secreted membrane-enclosed vesicles that originate from the endosome compartment in cells.
  • the endosome compartment, or the multi-vesicular body can fuse with the plasma membrane of the cell, with ensuing release to the extracellular space of their vesicles as exosomes.
  • an exosome can comprise a bilayer membrane, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and/or spanning the membrane.
  • Cargo can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof.
  • Exosomes can range in size from about 20 nm to about 300 nm. Additionally, the exosome may have an average diameter in the range of about 50 nm to about 220 nm. Preferably, in a specific embodiment, the exosome has an average diameter of about 120 nm ⁇ 20 nm.
  • exosomes and other extracellular vesicles can be characterized and marked based on their protein compositions, such as integrins and tetraspanins.
  • Other protein markers that are used to characterize exosomes and other extracellular vesicles (EVs) include TSG101, ALG-2 interacting protein X (ALIX), flotillin 1, and cell adhesion molecules which are derived from the parent cells in which the exosome and/or EV is formed. Similar to proteins, lipids can be major components of exosomes and EVs and can be utilized to characterize them.
  • exosomes can originate from the endosome and can contain proteins such as heat shock proteins (Hsp70 and Hsp90), membrane transport and fusion proteins (GTPases, Annexins and flotillin), tetraspanins (CD9, CD63, CD81, and CD82) and proteins such as CD47.
  • heat shock proteins, annexins, and proteins of the Rab family can abundantly be detected in exosomes and can be involved in their intracellular assembly and trafficking.
  • Tetraspanins a family of transmembrane proteins, can also be detected in exosomes. In a cell, tetraspanins can mediate fusion, cell migration, cell-cell adhesion, and signaling.
  • integrins can be adhesion molecules that can facilitate cell binding to the extracellular matrix. Integrins can be involved in adhering the vesicles to their target cells. Certain proteins that can be found on the surface of exosomes, such as CD55 and CD59, can protect exosomes from lysis by circulating immune cells, while CD47 on exosomes can act as an anti-phagocytic signal that blocks the uptake of exosomes by immune cells. Other proteins that can be associated with exosomes include thrombospondin, lactadherin, ALIX (also known as PDCD6IP), TSG1012, and SDCB1.
  • Classes of membrane proteins that can naturally occur on the surface of exosomes and other extracellular vesicles include ICAMs, MHC Class I, LAMP2, lactadherin (C1C2 domain), tetraspannins (CD63, CD81, CD82, CD53, and CD37), Tsg101, Rab proteins, integrins, Alix, and lipid raft-associated proteins such as glycosylphosphatidylinositol (GPI)-modified proteins and flotillin.
  • ICAMs ICAMs
  • MHC Class I LAMP2
  • lactadherin C1C2 domain
  • tetraspannins CD63, CD81, CD82, CD53, and CD37
  • Tsg101 Tsg101
  • Rab proteins integrins
  • Alix lipid raft-associated proteins
  • GPI glycosylphosphatidylinositol
  • exosomes can also be rich in lipids, with different types of exosomes containing different types of lipids.
  • the lipid bilayer of exosomes can be constituted of cell plasma membrane types of lipids such as sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, monosialotetrahexosylganglioside (GM3), and phosphatidylinositol.
  • Other types of lipids that can be found in exosomes are cholesterol, ceramide, and phosphoglycerides, along with saturated fatty-acid chains.
  • exosomes can include nucleic acids such as micro RNA (miRNA), messenger RNA (mRNA), and non-coding RNAs.
  • Exosomes can also contain a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules.
  • An extracellular vesicle can have a longest dimension, such as a cross-sectional diameter, of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 nm and/or at most about 1000, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 nm.
  • a longest dimension of a vesicle can range from about 10 nm to about 1000 nm, about 20 nm to about 1000 nm, about 30 nm to about 1000 nm, about 10 nm to about 100 nm, about 20 nm to about 100 nm, about 30 nm to about 100 nm, about 40 nm to about 100 nm, about 10 nm to about 200 nm, about 20 nm to about 200 nm, about 30 nm to about 200 nm, about 40 nm to about 200 nm, about 10 nm to about 120 nm, about 20 nm to about 120 nm, such as about 30 nm to about 120 nm, about 40 nm to about 120 nm, about 10 nm to about 300 nm, about 20 nm to about 300 nm, about 30 nm to about 300 nm, about 40 nm to about 300 nm, about 50 nm to
  • the term “average” may be mean, mode or medium for a group of measurements.
  • a “vesicle localization moiety” may be a macromolecule that localizes at an extracellular vesicle.
  • the vesicle localization moiety is a polypeptide.
  • the vesicle localization moiety is a protein.
  • the protein is a single polypeptide chain.
  • the vesicle localization moiety is a protein that localizes at an extracellular vesicle.
  • the vesicle localization moiety is a membrane protein.
  • the vesicle localization moiety is a transmembrane protein comprising a surface domain, a transmembrane domain and a cytosolic domain. Localization of such a transmembrane protein at an extracellular vesicle results in the surface domain at the outer surface of the vesicle, the transmembrane domain with the lipid bilayer of the vesicle and the cytosolic domain in the lumen of the vesicle.
  • a surface domain may also be referred to as an extracellular domain, since the surface domain on the surface of an exosome shares the same topological state as plasma membrane bound transmembrane protein on the surface of a cell; similarly, a cytosolic domain may be referred to as a lumenal domain, since part of the cytoplasm where the cytosolic domain initially resides is incorporated into the lumen of a vesicle produced by inward budding of an endosomal membrane to eventually produce multiple intraluminal vesicles of a multivesicular body (MVB) prior to secretion of the vesicles as exosomes upon fusion of the MVB with the plasma membrane of an EV producer cell.
  • MVB multivesicular body
  • the vesicle localization moiety may be a single pass transmembrane protein.
  • the single pass transmembrane protein may comprise an amino-terminal surface domain and a carboxyl-terminal cytosolic domain (lumenal domain) joined by a transmembrane domain.
  • nascent or newly synthesized single pass transmembrane protein may additionally comprise a signal peptide preceding the surface domain, which is cleaved by a signal peptidase upon translocation of the nascent protein into a membrane, such as endoplasmic reticulum in eukaryotes or plasma membrane in prokaryotes.
  • the nascent or newly synthesized transmembrane protein may be processed to a mature transmembrane protein which lacks a signal peptide of the nascent or newly synthesized transmembrane protein.
  • the single pass transmembrane protein is a type I transmembrane protein.
  • the single pass, type I transmembrane protein comprises an amino-terminal surface domain and a carboxyl-terminal cytosolic domain (lumenal domain) joined by a transmembrane domain.
  • nascent or newly synthesized single pass, type I transmembrane protein additionally comprises a signal peptide preceding the surface domain, which is cleaved by a signal peptidase upon translocation of the nascent protein into a membrane, such as endoplasmic reticulum in eukaryotes or plasma membrane in prokaryotes.
  • the nascent or newly synthesized single pass, type I transmembrane protein is processed to a mature single pass, type I transmembrane protein which lacks a signal peptide of the nascent or newly synthesized single pass, type I transmembrane protein.
  • the nascent or newly synthesized single pass, type I transmembrane protein may be processed to a mature single pass, type I transmembrane protein which lacks a signal peptide of the nascent or newly synthesized single pass, type I transmembrane protein.
  • the vesicle localization moiety may have a surface domain, a transmembrane domain and a cytosolic domain.
  • Such protein domains are known in the art and are well annotated and defined for the proteins described, herein, in the figures and in annotations associated with Accession Numbers from publicly available databases, referred herein, such as UniProtKB (UniProt Release 2019_11 (11 Dec. 2019); The UniProt Consortium (2019) UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 47:D506-515) and Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13; GenBank assembly accession GCA_000001405.28 and RefSeq assembly accession GCF_000001405.39).
  • the vesicle localization moiety is produced in a eukaryotic cell, preferably a mammal and most preferably a human.
  • a vesicle localization moiety may be linked to a targeting moiety either covalently in a fusion protein comprising the vesicle localization moiety and a targeting moiety or non-covalently through a pair of interacting domains or surfaces shared between a polypeptide comprising the vesicle localization moiety and a second polypeptide comprising a targeting moiety.
  • a “chimeric vesicle localization moiety” is a vesicle localization moiety which may be produced by substituting one vesicle localization domain with another vesicle localization domain, so as to produce a chimeric vesicle localization moiety.
  • a chimeric vesicle localization moiety may be obtained by combining one or more functional domains of one vesicle localization moiety with one or more functional domains of another, different vesicle localization moiety.
  • the combination comprises portion(s) of at least two vesicle localization moieties, so as to obtain a chimeric vesicle localization moiety which is superior in its association with an EV than either of the parental vesicle localization moiety, as quantified by mean recombinant protein density on EV surface and/or fraction (or percent) of total EVs positive for the recombinant protein.
  • the chimeric vesicle localization moiety comprises a surface domain, a transmembrane domain and a lumenal or cytosolic domain of a transmembrane protein or the two parental transmembrane proteins from which it is derived.
  • the chimeric vesicle localization moiety has the same arrangement of surface domain, transmembrane domain and lumenal or cytosolic domain as described for the vesicle localization moiety, described above.
  • a chimeric vesicle localization moiety comprising a surface-and-transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety may interact synergistically to increase accumulation at an extracellular vesicle. This not only may improve EV localization but may also change the composition of EVs.
  • the chimeric vesicle localization moiety can be a single pass transmembrane protein.
  • the chimeric vesicle localization moiety can be a type I transmembrane protein, albeit a chimeric type I transmembrane protein.
  • the chimeric vesicle localization moiety can be a single pass, type I transmembrane protein, albeit a chimeric single pass, type I transmembrane protein.
  • the chimeric vesicle localization moiety comprises an amino-terminal surface domain and a carboxyl-terminal cytosolic domain (lumenal domain) joined by a transmembrane domain.
  • nascent or newly synthesized chimeric vesicle localization moiety additionally comprises a signal peptide preceding the surface domain, which is cleaved by a signal peptidase upon translocation of the nascent protein into a membrane, such as endoplasmic reticulum in eukaryotes or plasma membrane in prokaryotes.
  • the nascent or newly synthesized chimeric vesicle localization moiety is processed to a mature form which lacks a signal peptide of the nascent or newly synthesized transmembrane protein.
  • the nascent or newly synthesized chimeric vesicle localization moiety is processed to a mature transmembrane protein which lacks a signal peptide of the nascent or newly synthesized transmembrane protein.
  • the extracellular vesicle comprises a chimeric vesicle localization moiety which has been processed to a mature form lacking a signal peptide of a nascent or newly synthesized chimeric vesicle localization moiety, a transmembrane protein.
  • the chimeric vesicle localization moiety lacking a signal peptide or mature form may be any of the chimeric vesicle localization moiety as provided in FIGS. 10 - 12 or Table 5, corresponding to the chimeric vesicle localization moiety in vector #135, #140, #141, #142, #143 and #144.
  • nucleic acid sequences provided in Table 5 for chimeric vesicle localization moieties may be used to produce polypeptides comprising a chimeric vesicle localization moiety.
  • nucleic acid comprising a coding sequence for a targeting moiety of interest may be fused in-frame with a coding sequence for a chimeric vesicle localization moiety as provided in Table 5 to encode for a polypeptide comprising a targeting moiety and a chimeric vesicle localization moiety.
  • Examples of such nucleic acids encoding a polypeptide comprising an affinity peptide as a targeting moiety and a chimeric vesicle localization moiety can be seen in Table 3 for vector #135, #140, #141, #142, #143 and #144 as well as the amino acid sequence of said polypeptide in Table 3 and also in FIGS. 5 - 8 .
  • the cytosolic domain of one vesicle localization moiety is used to replace that of another so as to obtain a chimeric vesicle localization moiety with a surface-and-transmembrane domain of one vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety.
  • vesicle localization moieties having the arrangement of ABc, AbC, AbC, aBC, aBc and abC, where A, B and C correspond to the surface domain, transmembrane domain and cytosolic domain, respectively, of a first vesicle localization moiety and a, b, and c correspond to the surface domain, transmembrane domain and cytosolic domain, respectively, of a second vesicle localization moiety.
  • any chimeric vesicle localization moiety with surface domain, transmembrane domain and cytosolic domain, obtained by combining domains from about 3 or 4 distinct vesicle localization moieties the possible number of chimeric vesicle localization moieties contemplated are about 24 and 60, respectively.
  • chimeric vesicle localization moieties are ones with superior localization to EVs (over parental vesicle localization moieties contributing to the chimeric vesicle localization moiety), it is also contemplated that some of these chimeric vesicle localization moieties may have desirable qualities other than ability to associate with or be incorporated as part of an EV.
  • the chimeric vesicle localization moiety comprises a surface-and-transmembrane domain of a first (1 st ) vesicle localization moiety and a cytosolic domain of a second (2 nd ) vesicle localization moiety, which is a full-length surface-and-transmembrane domain of the 1 st vesicle localization moiety and a full-length cytosolic domain of a 2 nd vesicle localization moiety.
  • the surface domain and transmembrane domain are contiguous derived from a 1 st vesicle localization moiety and a cytosolic domain from a 2 nd vesicle localization moiety.
  • the chimeric vesicle localization moiety comprises a surface domain or portion thereof and a transmembrane domain or portion thereof of a 1 st vesicle localization moiety and a cytosolic domain or portion thereof of a 2 nd vesicle localization moiety.
  • the chimeric vesicle localization moiety comprises a surface domain or portion thereof, a transmembrane domain or portion thereof, and a cytosolic domain or portion thereof, where each domain is chosen from two or more vesicle localization moieties.
  • the chimeric vesicle localization moiety additionally comprises a signal peptide.
  • the nascent or newly synthesized polypeptide of the chimeric vesicle localization moiety comprises a signal peptide sequence at the N-terminus.
  • the nascent polypeptide or newly synthesized polypeptide is a polypeptide being produced or initially produced by ribosome translation of a mRNA encoding the chimeric vesicle localization moiety.
  • the nascent or newly synthesized polypeptide of the chimeric vesicle localization moiety comprises from amino-to-carboxyl terminus in the order: signal peptide, surface domain, transmembrane domain and cytosolic domain.
  • the nascent or newly synthesized polypeptide of the chimeric vesicle localization moiety may additionally comprise any one or more linkers, affinity peptides, epitope tags and/or glycosylation sites.
  • the signal peptide sequence may be a naturally occurring sequence or an engineered (not naturally occurring) sequence.
  • the engineered signal peptide sequence may be an artificial signal peptide sequence which directs strong protein secretion and expression in human cells.
  • the engineered signal peptide may be MWWRLWWLLLLLLLLWPMVWA.
  • affinity peptides include, but are not limited to, those shown in Table 3; THRPPMWSPVWP (SEQ ID NO.: 64), a targeting moiety(ies) or peptide for transferrin receptor (TfR), and THVSPNQGGLPS (SEQ ID NO.: 66), a targeting moiety(ies) or peptide for glypican-3 (GPC3).
  • suitable epitope tags include, but are not limited to, FLAG tags such as single or 3 ⁇ FLAG tags, Myc tags, V5 tags, S-
  • the chimeric vesicle localization moiety lacks a signal peptide.
  • the chimeric vesicle localization moiety is a mature or processed polypeptide.
  • the mature or processed polypeptide lacks the signal peptide sequence of the nascent polypeptide.
  • the mature or processed polypeptide comprises a glycosylation site.
  • the mature or processed polypeptide is a glycoprotein.
  • the glycoprotein comprises glycans.
  • the glycoprotein comprises N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan and/or GPI anchor.
  • the chimeric vesicle localization moiety is a mature or processed vesicle localizing polypeptide found in association or incorporated by an EV and lacks a signal peptide sequence present in the nascent polypeptide prior to maturation or processing.
  • “Surface domain” is a subset of the protein or polypeptide primary sequence that is exposed to the extra-EV environment.
  • the surface domain may be a loop between two transmembrane domains or it can contain one of the termini (amino or carboxy) of the protein.
  • Protein domain topology relative to the membrane bi-layer can be determined empirically by assessing what portions of the protein are digested by an external protease. More recently, characteristic amino acid patterns, such as basic or acidic residues in the juxta-membrane regions of the protein have been used to algorithmically assign probable topologies (extracellular versus cytosolic) to integral membrane proteins.
  • the surface domain of an EV localizing transmembrane protein may sometimes be referred to as an extracellular domain due to the same membrane topology of an EV and plasma membrane.
  • the “surface domain” may be a short peptide of approximately 10-15 amino acids.
  • the “surface domain” may be an unstructured polypeptide.
  • the “surface domain” is the entire surface domain of an integral membrane protein.
  • the “surface domain” is part or portion of the surface domain of an integral membrane protein.
  • the surface domain is amino terminal to the transmembrane domain and cytosolic domain. In an embodiment, the surface domain is at the N-terminus of the vesicle localization moiety or the chimeric vesicle localization moiety and is on the external surface of an extracellular vesicle, such as an exosome.
  • Transmembrane domain may be a span of about 18-40 aliphatic, apolar and hydrophobic amino acids that assembles into an alpha-helical secondary structure and spans from one face of a membrane bilayer to the other face, meaning that the N-terminus of the helix extends at least to and in many cases beyond the phospholipid headgroups of one membrane leaflet while the C-terminus extends to the phospholipid headgroups of the other leaflet.
  • the transmembrane domain connects an amino terminal surface domain with a carboxyl terminal cytosolic domain.
  • Cytosolic domain is a subset of the protein or polypeptide primary sequence that is exposed to the intra-EV or intracellular environment.
  • the cytosolic domain can be a loop between two transmembrane domains or it can contain one of the termini (amino or carboxy) of the protein. Its topology is distinct from that of the transmembrane and the surface domains.
  • the cytosolic domain is in the cytoplasmic side of a cell.
  • the cytosolic domain is in the lumen of a vesicle.
  • the cytosolic domain is at the C-terminus of the vesicle localization moiety or the chimeric vesicle localization moiety.
  • sequences corresponding to “surface domain,” “transmembrane domain” and “cytosolic domain” for the proteins disclosed herein may be found within the description under protein accession numbers provided herein.
  • Particularly useful examples are the proteins cataloged within UniProtKB (UniProt Release 2019_11 (11 Dec. 2019)) where under each accession number amino acid sequence along with features and functional domains are provided.
  • topological domains associated with each of the transmembrane vesicle localization moiety may be found in UniProKB accession number with the description of “extracellular” for the “surface domain,” “helical” for the “transmembrane domain” and “cytoplasmic” for the “cytosolic domain.” Amino acid sequences corresponding to “signal peptide” are also indicated as being processed out of the mature transmembrane protein.
  • Membranome membrane proteome of single-helix transiembrane proteins (membranome.org; Lomize, A. L. et al. (2017) Membranome: a database for proteome-wide analysis of single-pass membrane proteins. Nucleic Acids Res. 45:D250-D255 and Lomize, A. L. et al. (2016) Membranome 2.0: database for proteome-wide profiling of bitopic proteins and their dimers.
  • a “chimeric vesicle localization moiety” comprises the “surface-and-transmembrane domain” of one vesicle localization moiety and the “cytosolic domain” of a second vesicle localization moiety, wherein the two vesicle localization moieties are different and distinct proteins and are not isoforms.
  • the “chimeric vesicle localization moiety” comprises the “surface-and-transmembrane domain” of one vesicle localization moiety and the “cytosolic domain” of a second vesicle localization moiety, wherein the two vesicle localization moieties are different and distinct proteins and are not isoforms and wherein the “surface-and-transmembrane domain” may have a mutation.
  • the mutation may be a deletion, insertion or a substitution, so long as the resulting mutant retains at least 80% or at least about 90% of the EV association activity of the unmutated counterpart.
  • the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are not allelic or homologs. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are not orthologs. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are not paralogs. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two distinct genes which are paralogs.
  • the “chimeric vesicle localization moiety” is derived from combining domains of two proteins encoded by two nonhomologous genes. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two or more proteins encoded by two or more nonhomologous genes. In an embodiment, the “chimeric vesicle localization moiety” is derived from combining domains of two or more proteins encoded by two or more nonhomologous human genes. In an embodiment, the “chimeric vesicle localization moiety” is produced from combining domains of two or more human genes encoding transmembrane proteins. In a preferred embodiment, the “chimeric versicle localization moiety” is produced from combining two nonhomologous human genes or two human genes not placed within the same gene family, wherein the genes encode transmembrane proteins.
  • An “isoform” of a protein can be, e.g., a protein resulting from alternative splicing of a gene expressing the protein, a protein resulting from alternative promoter usage of a gene expressing the protein, or a degradation product of the protein.
  • “Surface-and-transmembrane domain” is a contiguous polypeptide containing both a domain that is exposed to extracellular or extra-EV solvent and a transmembrane domain as described above.
  • isolated means a state following one or more purifying steps but does not require absolute purity.
  • isolated extracellular vesicle e.g., exosome
  • Isolated extracellular vesicle or composition thereof means a extracellular vesicle, exosome or composition thereof passed through one or more purifying steps that separate the vesicle, extracellular vesicle, exosome or composition from other molecules, materials or cellular components found in a mixture or outside of the vesicle, extracellular vesicle or exosome or found as part of the composition prior to purification or separation. Isolation and purification may be achieved in accordance with conventional methods of recombinant synthesis or cell free protein synthesis. Separation procedures of interest include affinity chromatography.
  • Affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. For example, covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using natural biospecific binding of one molecular species to separate and purify a second species from a mixture.
  • Antibodies may be used in affinity chromatography. Preferably a microsphere or matrix is used as the support for affinity chromatography.
  • Such supports are known in the art and are commercially available, and include activated supports that can be combined to the linker molecules.
  • Affi-Gel supports based on agarose or polyacrylamide are low pressure gels suitable for most laboratory-scale purifications with a peristaltic pump or gravity flow elution.
  • Affi-Prep supports based on a pressure-stable macroporous polymer, may be suitable for preparative and process scale applications. Isolation may also be performed using methods involving centrifugation, filtration, size exclusion chromatography and vesicle flow cytometry.
  • a composition herein comprises an isolated or enriched set of vesicles that selectively target a tissue or cell of interest. Such vesicles can be loaded with a payload as described herein to be delivered to the cell or tissue of interest.
  • the chimeric vesicle localization moiety may comprise a surface-and-transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety.
  • the first and second vesicle localization moieties are distinct/different proteins and not isoforms.
  • the first and second vesicle localization moieties are distinct/different proteins and not an allelic variant.
  • the first and second vesicle localization moieties are distinct/different proteins and not a homolog.
  • first and second vesicle localization moieties are distinct/different proteins and not an ortholog. In an embodiment, the first and second vesicle localization moieties are distinct/different proteins but are paralogs. In a preferred embodiment, the first and second vesicle localization moieties are distinct/different proteins and are not paralogs.
  • the first and second vesicle localization moieties are distinct/different proteins from a eukaryote or of eukaryotic origin.
  • the eukaryote may include any an of animal, plant, fungi, and protist.
  • the first and second vesicle localization moieties are distinct/different proteins from a mammal or of mammalian origin.
  • the mammal may include, but is not limited to, a human, monkey, chimpanzee, ape, gorilla, cattle, pig, sheep, horse, donkey, kangaroo, rat, mouse, guinea pig, hamster, cat, dog, rabbit and squirrel.
  • the first and second vesicle localization moieties are distinct/different proteins from a human or of human origin.
  • the chimeric vesicle localization moiety is obtained using recombinant DNA methods.
  • the chimeric vesicle localization moiety can be produced from expression of a nucleic acid encoding amino acid sequence of the 1 st vesicle localization moiety and the 2 nd vesicle localization moiety.
  • the nucleic acid encoding the chimeric vesicle localization moiety can be introduced into an expression vector or system. Examples of nucleic acid sequences are provided in the Tables herein and the Sequence Listing provided herewith.
  • the expression vector or system may be introduced into a cell which expresses the chimeric localization moiety as a polypeptide.
  • the cell is a mammalian cell, more preferably a human cell.
  • the expression vector or system may be introduced into a producer cell, which produces extracellular vesicles, preferably exosomes.
  • the producer cell is a mammalian cell.
  • the producer cell is a human cell.
  • the expression vector or system may be used in an in vitro transcription and translation system to produce a chimeric vesicle localization moiety as a polypeptide.
  • the in vitro produced chimeric vesicle localization moiety may be isolated.
  • an isolated chimeric vesicle localization moiety may be introduced into an extracellular vesicle or exosome isolated from cells.
  • first vesicle localization moieties include, but are not limited to, ADAM10, ALCAM, CLSTN1, IGSF8, IL3RA, ITGA3, ITGB1, LAMP2, LILRB4, PTGFRN, and SELPLG.
  • Further examples of suitable vesicle localization moieties may include, but are not limited to, a growth factor receptor, Fc receptor, interleukin receptor, immunoglobulin, MHC-I or MHC-II component, CD antigen, and escort protein.
  • suitable second vesicle localization moieties include, but are not limited to, the same examples as described for the first vesicle localization moieties.
  • the vesicle-localization moiety may further comprise a peptide or protein with a modified amino acid.
  • the modified amino acid may result from an attachment of a hydrophobic group.
  • the attachment of a hydrophobic group may be myristoylation for attachment of myristate, palmitoylation for attachment of palmitate, prenylation for attachment of a prenyl group, farnesylation for attachment of a farnesyl group, geranylgeranylation for attachment of a geranylgeranyl group or glycosylphosphatidylinositol (GPI) anchor formation for attachment of a glycosylphosphatidylinositol comprising a phosphoethanolamine linker, glycan core and phospholipid tail.
  • the attachment of a hydrophobic group may be performed by chemical synthesis in vitro or is performed enzymatically in a post-translational modification reaction.
  • first and second vesicle localization moieties include, but are not limited to, any of ACE, ADAM10, ADAM15, ADAM9, AGRN, ALCAM, ANPEP, ANTXR2, ATP1A1, ATP1B3, BSG, BTN2A1, CALM1, CANX, CD151, CD19, CD1A, CD1B, CD1C, CD2, CD200, CD200R1, CD226, CD247, CD274, CD276, CD33, CD34, CD36, CD37, CD3E, CD40, CD40LG, CD44, CD47, CD53, CD58, CD63, CD81, CD82, CD84, CD86, CD9, CHMP1A, CHMP1B, CHMP2A, CHMP3, CHMP4A, CHMP4B, CHMP5, CHMP6, CLSTN1, COL6A1, CR1, CSF1R, CXCR4, DDOST, DLL1, DLL4, DSG1, EMB, ENG, EV12B, F11
  • Amino acid sequences and associated nucleic acid encoding sequences for the vesicle localization moieties may be obtained in Tables 1 and 2; where the sequences are not directly provided in the table, the sequences may be obtained from provided Accession Number and database referred to in the tables.
  • the first and second vesicle localization moieties from which a chimeric vesicle localization moiety is derived may be from any of the transmembrane proteins listed in Table 1 and 2 or a homologue thereof.
  • the chimeric vesicle localization moiety comprises a surface-and-transmembrane domain of a 1 st vesicle localization moiety selected from any of the transmembrane protein listed in Table 1 or a homologue thereof and a cytosolic domain of a 2 nd vesicle localization moiety selected from any of the transmembrane protein listed in Table 2 or a homologue thereof.
  • the chimeric vesicle localization moiety comprises a surface-and-transmembrane domain of a 1 st vesicle localization moiety selected from any of the transmembrane protein listed in Table 2 or a homologue thereof and a cytosolic domain of a 2 nd vesicle localization moiety selected from any of the transmembrane protein listed in Table 1 or a homologue thereof.
  • the chimeric vesicle localization moiety comprises a surface-and-transmembrane domain of a 1 st vesicle localization moiety selected from any of the transmembrane protein listed in Table 1 or a homologue thereof and a cytosolic domain of a 2 nd vesicle localization moiety from any of the transmembrane protein listed in Table 1 or a homologue thereof, but not selected for the 1 st vesicle localization domain.
  • nucleic acid sequences as provided in Tables 1 and 2 may be used to produce a chimeric vesicle localization moiety through recombinant DNA method.
  • the next adjacent amino acid of a surface domain is followed and joined to first amino acid of a transmembrane domain and the last amino acid of the transmembrane domain is joined to the first amino acid of a cytosolic domain.
  • a vesicle localization moiety in Tables 1 and 2 comprises a transmembrane protein in which from amino-to-carboxyl terminal direction, last amino acid of a surface domain is joined to first amino acid of a transmembrane domain, and further, last amino acid of the transmembrane domain is joined to first amino acid of a cytosolic domain.
  • a signal peptide sequence with its last amino acid joined to the first amino acid of the surface domain for the amino acid sequences in Table 1 and the nucleic acid sequences in Table 1 or the vesicle localization moiety coding sequences associated with each ENST number in Table 2.
  • the signal peptide is cleaved from the nascent protein to produce a mature vesicle localization moiety found associated with an EV.
  • Tables 1 and 2 provide full-length vesicle localization moieties with signal peptides and nucleic acid coding sequences.
  • Amino acid sequences of vesicle localization moieties and amino acid sequences for signal peptide, surface domain, transmembrane domain and cytosolic domain along with nucleic acid coding sequences may additionally be accessed through accession numbers associated with the UniProtKB and Ensembl ENSP and ENST identifiers.
  • the chimeric vesicle localization moiety comprises a surface-and-transmembrane domain of a 1 st vesicle localization moiety and a cytosolic domain of a 2 nd vesicle localization moiety.
  • the 1 st vesicle localization moiety may include any of ADAM10, ALCAM, CLSTN1, IGSF8, IL3RA, ITGA3, ITGB1, LAMP2, LILRB4, PTGFRN, and SELPLG or a homologue thereof.
  • the 2 nd vesicle localization moiety may be selected from the same group of transmembrane proteins so long as the first and second vesicle localization moieties are from different or non-homologous proteins.
  • Amino acid sequences and nucleic acid sequences encoding ADAM10, ALCAM, CLSTN1, IGSF8, IL3RA, ITGA3, ITGB1, LAMP2, LILRB4, PTGFRN, and SELPLG are provided in Table 1 along with Ensembl ENSP and ENST identifiers (Hunt, S. E. et al. (2016) Database, 2018, 1-12; doi: 10.1093/database/bay119; Yates, A. D. et al., (2019) Nucleic Acids Res. 48:D682-D688).
  • the chimeric vesicle localization moiety comprises a LAMP2 surface-and-transmembrane domain and has an amino sequence as provided in FIG. 9 or a LAMP2 protein with Accession Number ENSP00000360386 encoded by Transcript ID ENST00000371335 from Gene ID ENSG00000005893, based on assembled sequence in Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13; GenBank assembly accession GCA 000001405.28 and RefSeq assembly accession GCF_000001405.39).
  • LAMP2 protein with Accession Number ENSP00000360386 encoded by Transcript ID ENST00000371335 is LAMP2B.
  • the chimeric vesicle localization moiety comprising a LAMP2 surface-and-transmembrane domain additionally comprises a cytosolic domain of ADAM10, ALCAM, CLSTN1, IGSF8, IL3RA, ITGA3, ITGB1, LILRB4, PTGFRN, or SELPLG or a homologue or portion thereof.
  • the chimeric vesicle localization moiety comprising a LAMP2 surface-and-transmembrane domain additionally comprises a cytosolic domain of PTGFRN, ITGA3, IL3RA, SELPLG, ITGB1, or CLSTN1 or a homologue or portion thereof.
  • the cytosolic domain of PTGFRN has an amino acid sequence as provided in FIG. 5 or FIG. 10 or a homologue or portion thereof.
  • the homologue or portion may retain at least about 80% or at least about 90% of cytosolic domain activity of PTGFRN which may be determined by detecting its accumulation at an extracellular vesicle.
  • Accumulation may be assessed for a chimeric vesicle localization moiety on the basis of the percent of extracellular vesicle positive for the chimeric vesicle localization moiety, and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety and ignoring extracellular vesicles lacking the localization moiety, as measured by vesicle flow cytometry.
  • the mean abundance of localization moiety in an extracellular vesicle may be the mean concentration, density or amount of localization moiety in an extracellular vesicle positive for the localization moiety.
  • an alternative measure can also be used, including total number of extracellular vesicles positive for the localization moiety.
  • the cytosolic domain of ITGA3 has an amino acid sequence as provided in FIG. 5 or FIG. 10 or a homologue or portion thereof.
  • the homologue or portion may retain at least 80% or at least about 90% of cytosolic domain activity of ITGA3 which may be determined by detecting its accumulation at an extracellular vesicle.
  • the cytosolic domain of IL3RA has an amino acid sequence as provided in FIG. 6 or FIG. 10 or a homologue or portion thereof.
  • the homologue or portion may retain at least about 80% or at least about 90% of cytosolic domain activity of IL3RA which may be determined by detecting its accumulation at an extracellular vesicle.
  • the cytosolic domain of SELPLG has an amino acid sequence as provided in FIG. 6 or FIG. 11 or a homologue or portion thereof.
  • the homologue or portion retains at least about 80% or at least about 90% of cytosolic domain activity of SELPLG which may be determined by detecting its accumulation at an extracellular vesicle.
  • the cytosolic domain of ITGB1 may have an amino acid sequence as provided in FIG. 7 or FIG. 11 or a homologue or portion thereof, wherein the homologue or portion retains at least 80% or at least about 90% of cytosolic domain activity of ITGB1 which may be determined by detecting its accumulation at an extracellular vesicle.
  • the cytosolic domain of CLSTN1 may have an amino acid sequence as provided in FIG. 7 or FIG. 12 or a homologue or portion thereof, wherein the homologue or portion retains at least 80% or at least about 90% of cytosolic domain activity of CLSTN1 which may be determined by detecting its accumulation at an extracellular vesicle.
  • a homologue is an ortholog derived from a common ancestral gene and encodes a protein with the same function in different species.
  • a homologue is a paralog derived from a homologous gene that has evolved by gene duplication and encodes for a protein with similar but not identical function.
  • Homologous proteins, including orthologs and paralogs may be identified based on amino acid sequences, curated, grouped and aligned in publicly available databases, such as HomoloGene at the National Center for Biotechnology Information of the National Institutes of Health (NCBI Resource Coordinators (2016) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 44:D7-D9), OrthoDB (Waterhouse, R. M. et al.
  • an extracellular vesicle herein is engineered for enhanced targeting to a cell or tissue of interest.
  • Such engineered vesicles can be non-naturally occurring.
  • Such engineered vesicles can be ‘targeted’ or ‘guided’ via a functionalized moiety (a targeting moiety) for increased affinity to a cell, tissue, or organ of interest.
  • a vesicle can be engineered to include a heterologous expression of one or more targeting moieties.
  • Vesicle functionalization can occur by modification of vesicles such as exosomes, to display an exogenous protein or nucleic acid.
  • a “targeting moiety” can include, but is not limited to, a small molecule, glycoprotein, protein, peptide, lipid, carbohydrate, nucleic acid, or other molecules involved in EV trafficking and/or EV interaction with target cells.
  • the targeting moiety may be displayed inside or on the outside of a vesicle membrane or may span the inner membrane, outer membrane, or both inner and other membranes.
  • the targeting moiety is similarly displayed on the outside of a vesicle membrane, so as to be able to bind to the targeted cell surface receptor, ligand or moiety.
  • the targeting moiety may be expressed in exosomes that are “emptied” of natural cargo, “carry” a naturally occurring cargo or loaded with a payload for delivery to such as target cells or tissues.
  • an engineered vesicle is one that is functionalized or is engineered to express a targeting moiety (e.g., a protein, peptide or nucleic acid) that selectively targets a cell or tissue of interest.
  • a targeting moiety e.g., a protein, peptide or nucleic acid
  • Such engineered vesicle can be an exosome.
  • such engineered vesicle is an extracellular vesicle.
  • the engineered vesicle is an exosome.
  • an engineered vesicle comprises a chimeric vesicle localization moiety attached to a targeting moiety.
  • an engineered vesicle comprises a chimeric vesicle localization moiety attached to a targeting moiety, displayed outside the vesicle.
  • an engineered vesicle is an engineered extracellular vesicle comprising a chimeric vesicle localization moiety attached to a targeting moiety, displayed outside the EV.
  • an engineered vesicle is an engineered exosome comprising a chimeric vesicle localization moiety attached to a targeting moiety, displayed outside the exosome.
  • the vesicles described herein can be used to selectively target a cell, tissue, or organ of interest.
  • the target cell is an eukaryotic cell.
  • a target cell can be a cell from an animal such as a mouse, rat, rabbit, hamster, porcine, bovine, feline, or canine.
  • the target cells can be a cell of primates, including but not limited to, monkeys, chimpanzees, gorillas, and humans.
  • any of the extracellular vesicles disclosed herein may include one or more targeting moieties of interest. They can be embedded in or displayed on vesicle membranes.
  • the extracellular vesicle can be an exosome, and the targeting moiety can be displayed on the outer surface of the exosome.
  • the targeting moiety may be displayed/joined/attached to the surface domain of the chimeric localization moiety.
  • the invention provides an extracellular vesicle of the invention comprising a chimeric vesicle localization moiety comprising a surface and transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety, wherein a targeting moiety is attached or joined covalently or noncovalently to the surface domain of the first vesicle localization moiety.
  • the invention contemplates other types of domain swapping between different vesicle localization moieties including chimeric vesicle localization moieties having the arrangement of ABc, AbC, Abe, aBC, aBc and abC, where A, B and C correspond to the surface domain, transmembrane domain and cytosolic domain, respectively, of a first vesicle localization moiety and a, b, and c correspond to the surface domain, transmembrane domain and cytosolic domain, respectively, of a second vesicle localization moiety.
  • the targeting moiety can be displayed on a surface domain.
  • Targeting moieties can comprise a small molecule, glycoprotein, polypeptides, peptide, oligopeptide, protein, lipid, carbohydrate, nucleic acid polysaccharides, therapeutic drugs, imaging moieties or other molecules that facilitates the targeting of the vesicle to a cell or tissue of interest.
  • the term “polypeptide,” “peptide,” “oligopeptide,” and “protein,” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically, or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • a targeting moiety may be an antibody, a ligand or a functional epitope thereof that binds to a cell or tissue marker, for example, a cell surface receptor.
  • the term antibody can be a protein or polypeptide functionally defined as a binding protein and structurally defined as comprising an amino acid sequence that is recognized by one of skill in the art as being derived from a variable region of an immunoglobulin.
  • An antibody can comprise one or more polypeptides substantially encoded by immunoglobulin genes, fragments of immunoglobulin genes, hybrid immunoglobulin genes (made by combining the genetic information from different animals), or synthetic immunoglobulin genes.
  • the recognized, native, immunoglobulin genes can include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes and multiple D-segments and J-segments.
  • Light chains can be classified as either kappa or lambda.
  • Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • Antibodies may exist as intact immunoglobulins, as a number of well characterized fragments produced by digestion with various peptidases to produce, for example, antigen-binding fragments F(ab′) 2 , Fab and Fab′, or as a variety of fragments made by recombinant DNA technology, such as variable fragment (Fv), single chain variable fragment (scFv), diabodies, tascFv, bis-scFv, nanobody (e.g., V H H or V NAR fragment), and miniaturized “3G” fragment (Nelson, A. L. (2010) Antibody fragments. MAbs 2: 77-83; and Muyldermans, S. (2103) Nanobodies: natural single-domain antibodies. Ann.
  • Fv variable fragment
  • scFv single chain variable fragment
  • diabodies tascFv
  • tascFv single chain variable fragment
  • bis-scFv nanobody
  • miniaturized “3G” fragment
  • Antibodies can derive from many different species (e.g., rabbit, sheep, camel, human, or rodent, such as mouse or rat), or can be synthetic. Antibodies can be chimeric, humanized, or human. Antibodies can be monoclonal or polyclonal, multiple or single chained, fragments or intact immunoglobulins. In a preferred embodiment, the antibody is a scFv.
  • the targeting moiety is a peptide (e.g., an affinity peptide).
  • the targeting moiety may be an antibody fragment.
  • the antibody fragment may be any of F(ab′)2, Fab, Fab′, Fv, scFv, diabodies, tascFv, bis-scFv, nanobody and miniaturized “3G” fragment.
  • the antibody fragment is single chain Fv (scFv), wherein variable region of heavy chain (V H ) and variable region of light chain (V L ) are joined together by a flexible linker. The variable region of heavy chain fragment can precede the variable region of light chain fragment, or vice versa.
  • the flexible linker is often glycine-serine rich, such as a (GGGGS) 4 linker.
  • the scFv binds a target on the surface of a cell or tissue.
  • the scFv is attached to a chimeric vesicle localization moiety incorporated in an extracellular vesicle (such as an exosome) and displayed outside the extracellular vesicle (e.g., exosome).
  • the scFv is attached to a chimeric vesicle localization moiety and displayed outside an extracellular vesicle (e.g., exosome) preferentially or selectively targets a specific cell type or tissue.
  • the antibody fragment may be monospecific or bispecific. In an embodiment of the invention, the antibody fragment may be multivalent.
  • Suitable antibodies particularly single chain Fv antibodies; and fragments, include antibodies directed against any of Thy1, MHC class II, C3d-binding region of complement receptor type 2 (CR2), VCAM-1, E-selectin, alpha 8 integrin, integrin alpha-M (CD11b) and CD163.
  • Exemplary antibodies from which Fab and/or scFv antibodies may be prepared include OX7 antibody against Thy1 protein (Suana, A. J. et al., J. Pharmacol. Exp. Ther. 2011; 337:411-422; RT1 antibody against MHC class II protein (Hultman, K. L. et al., ACS Nano.
  • any of the targeting moieties described herein can enhance the selectivity of the vesicles towards the target cell of interest as compared to one or more other tissues or cells.
  • the one or more selective targeting moieties can be expressed on modified vesicles in a way that allows such modified vesicles to bind to intended targets.
  • the one or more targeting moieties can expose sufficient amount of amino acids to allow such binding.
  • the modified vesicles provided herein can comprise one or more targeting moieties that selectively target the vesicles to cells or tissue of interest by binding or physically interacting with markers expressed on such cells.
  • selective or “selectively” as used herein in the context of selective targeting or selective binding or selective interaction can refer to a preferential targeting, binding or interaction to a cell, tissue, or organ of interest as compared to at least one other type of cell, tissue or organ.
  • a “functional fragment” of a protein can mean a fragment of the protein which retains a function of a full-length protein from which it is derived, e.g., a targeting or binding function identical or similar to that of the full-length protein.
  • a “functional fragment” of an antibody can be its antigen binding portion or fragment, which confers binding specificity for the intact antibody.
  • a function can be similar to a function of a full-length protein if it retains at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of that function of the full-length protein. The function can be measured e.g., using an assay, e.g., an in vivo binding assay, a binding assay in a cell, or an in vitro binding assay.
  • sequence identity refers to a nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • sequence identity refers, in the context of two nucleic acid sequences or amino acid sequences, to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percent sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein (the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence which does not comprise additions or deletions comprises) can for optimal alignment of the two sequences.
  • the percentage can be calculated by determining the number of positions at which the identical nucleotide or amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to determine the percentage of sequence identity.
  • Sequence comparisons may be performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings; Needleman, S. B. and Wunsch, C. D. (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol.
  • the BLAST algorithm see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings; Altschul, S. F. et al. (1990) Basic local alignment search tool. J. Mol. Biol. 215:403-410; and Altschul, S. F. et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res.
  • the “percent identity” between two sequences may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health.
  • the BLAST program can be based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci.
  • the BLAST program can define identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences.
  • the program may be used to determine percent identity over the entire length of the sequences being compared. Default parameters can be provided to optimize searches with short query sequences, for example, with the BLASTP program.
  • the program can also allow use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton, J. C. and Federhen, S. (1993) Computers Chem. 17:149-163.
  • High sequence identity can include sequence identity in ranges of sequence identity of approximately 80% to 99% and integer values there between.
  • a “homolog” or “homologue” can refer to any sequence that has at least about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence homology to another sequence.
  • a homolog or homologue refers to any sequence that has at least about 98%, 99%, or 99.5% sequence homology to another sequence.
  • the homolog can have a functional or structural equivalence with the native or naturally occurring sequence.
  • the homolog can have a functional or structural equivalence with a domain, a motif or a part of the protein, that is encoded by the native sequence or naturally occurring sequence.
  • Homology comparisons may be conducted with sequence comparison programs.
  • Computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.
  • Sequence homologies may be generated by any of a number of computer programs, for example BLAST or FASTA, etc.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux, J. et al. (1984) Nucleic Acids Res. 12:387). Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel, F. M. et al.
  • Percent homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments can be performed over a relatively short number of residues.
  • one insertion or deletion may cause the following amino acid or nucleotide residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, the sequence comparison method can be designed to produce optimal alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology or identity score. This can be achieved by inserting “gaps” in the sequence alignment to try to maximize local homology or identity.
  • BLAST 2 Sequences is another tool that can be used for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health).
  • Homologous sequences can also have deletions, insertions or substitutions of amino acid residues which result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids may be grouped together based on the properties of their side chains alone.
  • Substantially homologous sequences of the present invention include variants of the disclosed sequences, e.g., those resulting from site-directed mutagenesis, as well as synthetically generated sequences.
  • the variants may be allelic variants due to different alleles.
  • the variants may be derived from the same gene or allele due to alternative transcription start site or alternative splicing, resulting in variants which are isoforms.
  • An extracellular vesicle of the present disclosure can be one that comprises (e.g., on its surface) one or more targeting moiety(ies) to a marker of interest.
  • a marker of interest may be a cell surface marker of a target cell of interest to which a vesicle of the present invention is intended to target or bind.
  • a vesicle of the present disclosure is one that comprises (e.g., on its surface) targeting moiety(ies) to a marker of interest or a homologue(s) of a marker of interest.
  • a vesicle comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different targeting moiety(ies).
  • a vesicle comprises a chimeric vesicle localization moiety attached to one or more targeting moiety(ies) to a marker of interest.
  • the marker of interest may be a cell surface marker.
  • a vesicle comprises two or more chimeric vesicle localization moieties, wherein each chimeric vesicle localization moiety comprises a different targeting moiety(ies) targeted to the same marker of interest.
  • a vesicle comprises two or more chimeric vesicle localization moieties, wherein each chimeric vesicle localization moiety comprises a different targeting moiety(ies) targeted to the different markers of interest.
  • a vesicle comprises two or more chimeric vesicle localization moieties, wherein each chimeric vesicle localization moiety comprises a different targeting moiety(ies) targeted to the different markers of interest present on the same cell.
  • a vesicle comprises two or more chimeric vesicle localization moieties, wherein each chimeric vesicle localization moiety comprises a different targeting moiety(ies) targeted to the different markers of interest present on different cell types.
  • a vesicle comprises two or more chimeric vesicle localization moieties, wherein each chimeric vesicle localization moiety comprises a different targeting moiety(ies) targeted to the different markers of interest present in a tissue.
  • a vesicle comprises two or more chimeric vesicle localization moieties, wherein each chimeric vesicle localization moiety comprises a different targeting moiety(ies) targeted to the different markers of interest present in different tissues.
  • a vesicle comprises a sufficient number of targeting moiety(ies) to selectively target cells of interest over other cells.
  • a vesicle comprises a sufficient number of targeting moiety(ies) to selectively target a tissue of interest over other tissues.
  • the vesicle comprises a concentration of a targeting moiety of interest that is 2, 3, 4, 5, 6, 8, 10, 12, 14, 17, 18, 20, 22, 25, 28, 30, 33, 35, 38, 40, 43, 44, 46, 48, 50, 52, 55, 57, 59, 62, 65, 68, 70, 72, 75, 78, 80, 82, 85, 89, 91, 92, 95, 100, 110, 120, 125, 130, 135, 145, 150, 155, 160, 170, 180, 185, 200, 210, 220, 230, 250, 270, 280, 290, 300, 310, 320, 330, 340, 350, 380, 400, 410, 430, 440, 450, 470, 490, 500, 510, 525, 540, 560, 580, 590, 600, 620, 650, 670, 680, 690, 700, 720, 740, 760, 780, 800, 820, 840, 8
  • the vesicle comprises a targeting moiety which is not naturally associated with a vesicle or an extracellular vesicle.
  • the vesicle comprises a targeting moiety of interest fused to a chimeric vesicle localization moiety.
  • the vesicle comprises two or more targeting moiety of interest fused to one or more chimeric vesicle localization moiety.
  • the “fusion protein” can be a single polypeptide derived from two separate polypeptides or portions of two separate polypeptides. As such, a chimeric vesicle localization may be considered a fusion protein.
  • the one or more targeting moieties of interest can be operably linked (directly or indirectly) to a chimeric vesicle localization moiety (e.g., as a fusion protein).
  • a targeting moiety may be linked non-covalently to a chimeric vesicle localization moiety mediated by interacting surfaces or partners in separate polypeptides comprising the targeting moiety and another polypeptide comprising the chimeric vesicle localization moiety.
  • Such interacting surfaces or partners may be inherently present on or be covalently attached to the targeting moiety and/or chimeric vesicle localization moiety.
  • Molecular forces maintaining non-covalent linkages or interactions include hydrogen bond, ionic bond, van der Waals interaction and hydrophobic bond.
  • a targeting moiety may be covalently linked to a chimeric vesicle localization moiety, and together the two can be referred to as a fusion protein comprising a targeting moiety and a chimeric vesicle localization moiety.
  • the chimeric vesicle localization moiety of the fusion protein can target the targeting moieties of interest (or other fused molecule) to a vesicle.
  • a chimeric vesicle localization moiety targets the targeting moieties of interest (or other fused molecule) to the membrane of a vesicle.
  • the chimeric vesicle localization moiety targets to the membrane of an exosome.
  • fusion proteins can be made with a chimeric vesicle localization moiety and a ligand (targeting moiety of interest) that binds a cell receptor of interest.
  • the ligand can be surface exposed and can selectively bind to a receptor or receptors on the surface of the target cell.
  • These fusion proteins of such targeting moieties can be loaded onto vesicles (e.g., exosomes and EVs) endogenously or exogenously.
  • nucleic acids encoding fusion proteins or such targeting moieties and chimeric vesicle localization moieties separately can be used to express the exosome localization moiety and targeting moieties.
  • vesicle localization moieties from which chimeric vesicle localization moieties may be produced by domain swapping include any of the following: ACE, ADAM10, ADAM15, ADAM9, AGRN, ALCAM, ANPEP, ANTXR2, ATP1A1, ATP1B3, BSG, BTN2A1, CALM1, CANX, CD151, CD19, CD1A, CD1B, CD1C, CD2, CD200, CD200R1, CD226, CD247, CD274, CD276, CD33, CD34, CD36, CD37, CD3E, CD40, CD40LG, CD44, CD47, CD53, CD58, CD63, CD81, CD82, CD84, CD86, CD9, CHMP1A, CHMP1B, CHMP2A, CHMP3, CHMP4A, CHMP4B, CHMP5, CHMP6, CLSTN1, COL6A1, CR1, CSF1R, CXCR4, DDOST, DLL1, DLL4, DSG
  • the chimeric vesicle localization moieties may be produced by domain swapping include any of the following: ADAM10, ALCAM, CLSTN1, IGSF8, IL3RA, ITGA3, ITGB1, LAMP2, L1LRB4, PTGFRN, and SELPLG or an isoform thereof, or a homologue thereof, or a functional fragment thereof.
  • Domain swapping is most easily achieved through recombinant DNA methods using coding sequence provided or referred to in Tables 1 and 2 to precisely dissect and fuse two different coding sequences in-frame with each other to obtain a single nucleic acid encoding a chimeric vesicle localization moiety.
  • Nucleic acid sequences encoding exemplary chimeric vesicle localization moieties may be obtained in Tables 3 and 5.
  • a chimeric vesicle localization moiety may be produced by domain swapping two non-homologous vesicle localization moieties. In an embodiment, a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are not orthologs. In an embodiment, a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are not paralogs. In an embodiment, a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are paralogs.
  • a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are not allelic variants. In an embodiment, a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are not isoforms. In an embodiment, a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are not related by an ancestral gene or gene duplication.
  • a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are related by gene duplication and have evolved to be paralogs encoded by homologous genes at a different genetic locus (not allelic).
  • a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties which are distinct and non-homologous proteins.
  • a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties, wherein the domains being swapped share less than about 95%, 90%, 70%, 50% or preferably less than about 30% amino acid sequence identity with gaps allowed in the sequence alignment to maximize sequence identity.
  • a chimeric vesicle localization moiety may be produced by domain swapping two vesicle localization moieties, wherein the domains being swapped differ in the length of the primary amino acid sequence by more than about 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2.3-fold, 2.7-fold or more preferably about 3-fold compared to the shorter domain.
  • the domains of a vesicle localization moiety may be determined in relation to membrane of a vesicle and may be described as surface domain (outside of the vesicle; also referred to sometimes as extracellular domain, which is topologically equivalent), transmembrane domain (spanning the lipid bilayer of the vesicle) and lumenal domain (in the interior of the vesicle; also referred to as a cytosolic domain prior to formation of a vesicle, which is topologically equivalent).
  • the three domains present in a vesicle localization moiety may be swapped with one or more domains from one or more other vesicle localization moiety.
  • the cytosolic domain or lumenal domain of a vesicle localization moiety is swapped with a cytosolic domain or lumenal domain of a second vesicle localization moiety so as to produce a chimeric vesicle localization moiety with a surface-and-transmembrane domain of a 11 vesicle localization moiety and a cytosolic domain of a 2 nd vesicle localization moiety.
  • engineered vesicles can involve generation of nucleic acids that encode, at least, in part, one or more of the cell-type specific or selective targeting moieties described herein, one or more of the targeting moiety(ies) described herein, one or more of the vesicle localization moieties including chimeric vesicle localization moieties described herein, one or more fusion proteins described herein, or a combination thereof.
  • expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the protein.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular expression system, e.g. mammalian cell, bacterial cell, cell-free synthesis, etc.
  • the control sequences that are suitable for prokaryote systems include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cell systems may utilize promoters, polyadenylation signals, and enhancers.
  • RNA capable of encoding the polypeptides of interest may be chemically synthesized.
  • codon usage tables and synthetic methods can readily utilize well-known codon usage tables and synthetic methods to provide a suitable coding sequence for any of the polypeptides of the invention.
  • a vector comprises nucleic acids encoding one or more cell-type specific or selective targeting moieties operably linked to nucleic acids that encode one or more vesicle localization moieties, preferably chimeric vesicle localization moieties.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate the initiation of translation.
  • operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation or through amplification reactions. Synthetic oligonucleotide adaptors or linkers may be used for linking sequences in accordance with conventional practice.
  • a vector comprises nucleic acids encoding the amino acid sequences set forth in Table 3 or the figures. In an embodiment, a vector comprises nucleic acids encoding the chimeric vesicle localization moiety produced from the vesicle localization moieties disclosed herein or in Table 3 or the figures. In one example, a vector comprises nucleic acids encoding a chimeric vesicle localization moiety operably linked to nucleic acids encoding any one or more of a targeting moiety(ies) of interest or cell-type specific or selective targeting moieties. In an embodiment, a cell-type specific or selective targeting moiety is a peptide.
  • a cell-type specific or selective targeting moiety is an antibody or an antibody fragment. In an embodiment, a cell-type specific or selective targeting moiety is a F(ab′)2, Fab or Fab′. In a preferred embodiment, a cell-type specific or selective targeting moiety is a scFv.
  • the nucleic acids may be natural, synthetic or a combination thereof.
  • the nucleic acids may be RNA, mRNA, DNA or cDNA.
  • Nucleic acid encoding the protein may be produced using known synthetic techniques, incorporated into a suitable expression vector using well established methods to form a protein-encoding expression vector which is introduced into a cell for protein expression using known techniques, such as transfection, lipofection, transduction and electroporation.
  • the nucleic acids may be isolated and obtained in substantial purity.
  • nucleic acids either as DNA or RNA
  • nucleic acids can be regulated by their own or by other regulatory sequences known in the art.
  • the nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art.
  • the expressed protein may localize or form an exosome or extracellular vesicle and released from the producing cell. Such exosomes or extracellular vesicles may be harvested from the culture medium.
  • the selected protein may be produced using recombinant techniques, or may be otherwise obtained, and then may be introduced directly into isolated exosomes by electroporation or transfection e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like.
  • the nucleic acids can also include expression vectors, such as plasmids, or viral vectors, or linear vectors, or vectors that integrate into chromosomal DNA.
  • Expression vectors can contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of cells. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria.
  • the expression vector can be integrated into the host cell chromosome and then replicate with the host chromosome or the expression vector may be an episome and replicate autonomously independent of the host chromosome.
  • Selection genes also can contain a selection gene, also termed a selectable marker.
  • the selection gene can encode a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the selective culture medium.
  • Selection genes can encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, G418, puromycin, hygromycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • An exemplary selection scheme can utilize a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene can produce a protein conferring drug resistance and thus survive the selection regimen.
  • Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well-known in the art.
  • a promoter that is capable of expressing a transgene in a mammalian nervous system cell is the EF1a promoter.
  • a promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • Other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus promoter (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, phosphoglycerate kinase (PGK) promoter, MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not
  • inducible or repressible promoters are also contemplated for use in this disclosure.
  • inducible promoters include a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, a tetracycline promoter, a c-fos promoter, the T-REx system of ThermoFisher which places expression from the human cytomegalovirus immediate-early promoter under the control of tetracycline operator(s), and RheoSwitch promoters of Intrexon.
  • Expression vectors typically have promoter elements, e.g., enhancers, to regulate the frequency of transcriptional initiation. These can be located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements can frequently be flexible, so that promoter function can be preserved when elements are inverted or moved relative to one another.
  • the expression vector may be a mono-cistronic construct, a bi-cistronic construct or multiple cistronic construct.
  • the two cistrons can be oriented in opposite directions with the control regions for the cistrons located in between the two cistrons.
  • the construct has more than two cistrons, the cistrons can be arranged in two groups with the two groups oriented in opposite directions for transcription.
  • polypeptides described herein can be desirable to modify the polypeptides described herein.
  • methods can include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other techniques (see, e.g., Gillam and Smith, Gene 8:81-97, 1979; Roberts et al., Nature 328:731-734, 1987, which is incorporated by reference in its entirety for all purposes).
  • the recombinant nucleic acids encoding the polypeptides described herein can be modified to provide preferred codons which can enhance translation of the nucleic acid in a selected organism or cell line.
  • the polynucleotides can also include nucleotide sequences that are substantially equivalent (homologues) to other polynucleotides described herein.
  • Polynucleotides can have at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to another polynucleotide.
  • a polynucleotide encoding a protein may be considered equivalent to a second polynucleotide encoding the same protein due to degeneracy of the genetic codon. Such polynucleotides are anticipated herein.
  • the nucleic acids can also provide the complement of the polynucleotides including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited herein.
  • the polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.
  • Nucleic acids which encode protein analogs or variants i.e., wherein one or more amino acids are designed to differ from the wild type polypeptide
  • Amino acid “substitutions” for creating variants can result from replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
  • positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the nucleic acid When the nucleic acid is introduced into a cell ex vivo, the nucleic acid may be combined with a substance that promotes transference of a nucleic acid into a cell, for example, a reagent for introducing a nucleic acid such as a liposome or a cationic lipid, in addition to any additional excipients. Electroporation applying voltages in the range of about 20-1000 V/cm may be used to introduce nucleic acid or protein into exosomes.
  • Transfection using cationic lipid-based transfection reagents such as, but not limited to, Lipofectamine® MessengerMAXTM Transfection Reagent, Lipofectamine® RNAiMAX Transfection Reagent, Lipofectamine® 3000 Transfection Reagent, or Lipofectamine® LTX Reagent with PLUSTM Reagent, may also be used.
  • the amount of transfection reagent used may vary with the reagent, the sample and the cargo to be introduced.
  • a vector carrying the nucleic acid of the present invention can also be used.
  • a composition in a form suitable for administration to a living body which contains the nucleic acid of the present invention carried by a suitable vector can be suitable for in vivo gene therapy.
  • the nucleic acid constructs can include linker peptides.
  • the linker peptides can adopt a helical, ⁇ -strand, coil-bend or turn conformations.
  • the linker motifs can be flexible linkers, rigid linkers or cleavable linkers.
  • the linker peptides can be used for increasing the stability or folding of the peptide, avoid steric clash, increase expression, improve biological activity, enable targeting to specific sites in vivo, or alter the pharmacokinetics of the resulting fusion peptide by increasing the binding affinity of the targeting domain for its receptor.
  • Folding refers to the process of forming the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure.
  • Non-covalent interactions are important in determining structure, and the effect of membrane contacts with the protein may be important for the correct structure.
  • the result of proper folding is typically the arrangement that results in optimal biological activity, and can conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc.
  • the linker peptides can generally be composed of small non-polar (Gly) or non-polar (Ser) amino acids.
  • the linker peptides can have sequences consisting primarily of stretches of glycine and/or serine residues. But can contain additional amino acids, such as Thr and Ala to maintain flexibility, as well as polar amino acids, such as Lys and Glu to improve solubility.
  • rigid linkers can have a Proline-rich sequence, such as (XP)n, with X designating any amino acid, preferably Ala, Lys or Glu.
  • cleavable linkers can be used susceptible to reductive or enzymatic cleavage, such as disulfide or protease sensitive sequences, respectively.
  • linker peptides can be linked to a reporter moiety, such as a fluorescent protein.
  • the nucleic acid sequence can also contain signal sequences that encode for signal peptides that function as recognition sequences for sorting of the resulting fusion protein to the vesicular surface.
  • the signal sequence can comprise a tyrosine-based sorting signal and can contain the NPXY where N stands for asparagine, P stands for proline, Y stands for tyrosine and X stands for any amino acid (alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine).
  • the signal sorting motif can comprise a YXXO consensus motif, where O stands for an amino acid residue with a bulky hydrophobic side chain.
  • the sorting signal can comprise a (DE)XXXL(LI) consensus motif where D stands for aspartic acid, E stands for glutamic acid, X stands for any amino acid, L stands for leucine and I stands for isoleucine.
  • the signal sequence can comprise a di-leucine-based signal sequence motif such as (DE)XXXL(LI) or DXXLL consensus motifs, where D stands for aspartic acid, E stands for glutamic acid, X stands for any amino acid, L stands for leucine and I stands for isoleucine.
  • the signal peptic can comprise an acidic cluster.
  • the signal peptide can comprise a FW-rich consensus motif, where F stands for phenylalanine and W stands for tryptophan.
  • the signal peptide can comprise a proline-rich domain.
  • the sorting signal comprises the consensus motif NPFX (1,2) D, where N stands for asparagine, P stands for proline, F stands for phenylalanine, D stands for aspartic acid and X stands for any amino acids.
  • the encoded signal peptides can be recognized by adaptor protein complexes AP-1, AP-2, AP-3 and AP-4.
  • the DXXLL signals are recognized by another family of adaptors known as GGAs.
  • the signal peptides can be ubiquitinated.
  • the signal peptide is an immunoglobulin i-chain signal peptide sequence, METDTLLLWVLLLWVPGSTGD.
  • the signal peptide is a human signal sequence.
  • the signal peptide is a computationally designed signal peptide.
  • the signal peptide sequence is MWWRLWWLLLLLLLLWPMVWA.
  • nucleic acids herein can be used for heterologous expression in a cell of a fusion protein comprising one or more chimeric vesicle localization moiety and one or more targeting moiety of interest, wherein the fusion protein localizes or is an integral part of an extracellular vesicle produced by the cell.
  • the vesicle is an extracellular vesicle or an exosome.
  • one or more nucleic acid encoding a chimeric vesicle localization moieties and one or more nucleic acid encoding a targeting moieties of interest can be used for heterologous expression in a cell to produce operably linked one or more chimeric vesicle localization moieties and one or more targeting moieties of interest wherein a targeting moiety associates with a chimeric vesicle localization moiety by non-covalent interaction through interacting partners or surfaces inherently present in or covalently attached to the targeting moiety of interest or chimeric vesicle localization moiety and wherein both the targeting moiety of interest and the chimeric vesicle localization are present at or associate with a vesicle, preferably an extracellular vesicle or exosome, produced by the cell.
  • Common GMP-grade cells used in such heterologous expression and from which vesicles may be isolated, including extracellular vesicles and exosomes include HEK293 (human embryonic kidneycell line), variants of HEK293, such as HEK293T, HEK 293-F, HEK 293T, and HEK 293-H, dendritic cells, mesenchymal stem cell (MSCs), HT-1080, PER.C6, HeLa, C127, BHK, Sp2/0, NS0 and any variants thereof, and any of the following types of allogeneic stem cell lines: Hematopoietic Stem Cells, such as bone marrow HSC, Mesenchymal Stem Cells, such as bone marrow MSC or placenta MSC, human Embryonic Stem Cells or its more differentiated progeny, such as hESC-derived dendritic cell or hESC-derived oligodendrocyte progenitor cell, Ne
  • any of the cells used for heterologous expression may serve as a source for vesicles, especially extracellular vesicles comprising one or more chimeric vesicle localization moiety(ies) operably linked to one or more targeting moiety(ies) of interest.
  • any of the cell used for heterologous expression may serve as a source for vesicles, especially extracellular vesicles comprising one or more chimeric vesicle localization moieties covalently linked to one or more targeting moieties of interest or a fusion protein comprising one or more chimeric vesicle localization moieties and to one or more targeting moieties of interest.
  • any of the polypeptides herein can be produced by a cell (or cell line) generating vesicles which contain the polypeptide.
  • the targeting moiety can be heterologously expressed by the cell producing the vesicle.
  • the cell producing the vesicle expresses a chimeric vesicle localization moiety and a targeting moiety wherein the targeting moiety associates with the chimeric vesicle localization moiety by a non-covalent linkage and wherein both the targeting moiety and the chimeric vesicle localization moiety associate with the vesicle.
  • the targeting moiety is displayed on the external surface or outside the vesicle.
  • the non-covalent linkage of a targeting moiety and a chimeric vesicle localization is mediated by interacting surfaces or partners between a polypeptide comprising the targeting moiety and a 2 nd polypeptide comprising the chimeric vesicle localization moiety.
  • interacting surfaces or partners may be inherently present on or is introduced to the polypeptide comprising the targeting moiety and the 2 nd polypeptide comprising the chimeric vesicle localization moiety.
  • Molecular forces maintaining non-covalent linkages or interactions include hydrogen bond, ionic bond, van der Waals interaction and hydrophobic bond.
  • the cell producing the vesicle also expresses a fusion protein comprising a chimeric vesicle localization moiety and a targeting moiety, which are covalently linked in a single polypeptide incorporated into a vesicle, preferably an extracellular vesicle or exosome, produced by the cell.
  • a fusion protein comprising a chimeric vesicle localization moiety and a targeting moiety, which are covalently linked in a single polypeptide incorporated into a vesicle, preferably an extracellular vesicle or exosome, produced by the cell.
  • an extracellular vesicle or exosome producing cell may be considered a producer cell (for an EV or exosome).
  • more than one targeting moieties may be attached to a single chimeric vesicle localization moiety.
  • more than one type of chimeric vesicle localization moiety covalently linked to one or more targeting moieties may be present at or are associated with a vesicle, wherein each type of chimeric vesicle localization moiety differs by at least one amino acid.
  • the targeting moiety is coupled to the vesicle by the producing cell, during vesicle biogenesis or prior to vesicle secretion or isolation.
  • the targeting moiety is coupled to the vesicle after the vesicles are produced and/or isolated.
  • Modified extracellular vesicles can be obtained from a subject, from primary cell culture cells obtained from a subject, from cell lines (e.g., immortalized cell lines), and other cell sources.
  • One such method includes engineering cells directly in culture to express targeting moieties that are then incorporated into the modified extracellular vesicles harvested as delivery vehicles from these engineered cells.
  • Cells which are used for modified extracellular vesicle production are not necessarily related to or derived from the cell targets of interest. Once derived, vesicles may be isolated based on their size, biochemical parameters, or a combination thereof.
  • Another method that can be used in conjunction with or independent of the direct cell engineering is physical isolation of particular subpopulations (subtypes) of modified vesicles with desired targeting moieties from the broad, general set of all vesicles produced by a subject.
  • Another method that can be used in conjunction with the previously described two methods or independently is direct incorporation of desired targeting moieties (e.g., proteins/polypeptides) on the vesicles surface.
  • desired targeting moieties e.g., proteins/polypeptides
  • a general population of extracellular vesicles or a specific population of extracellular vesicles are isolated from cell culture.
  • the isolated vesicles are then treated to incorporate desired targeting moieties into the vesicles (e.g., liposomal fusion) to generate modified vesicles.
  • desired targeting moieties e.g., proteins/polypeptides
  • the process can be direct engineering of cells for modified vesicles production followed by isolating target modified vesicles subpopulation.
  • the modified vesicles can be incorporated with the targeting moieties directly with or without cholesterol or other phospholipids.
  • the modified vesicle protein mixture can be created via gentle mixing and incubation or several cycles of freezing and thawing.
  • the modified vesicles can be derived from eukaryotic cells that can be obtained from a subject (autologous) or from allogeneic cell lines.
  • the subject may be any living organism. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Vesicles can be concentrated and separated from the circulatory cells using centrifugation, filtration, or affinity chromatography columns.
  • modified vesicle system described herein for example modified vesicles such as exosomes, can be used to deliver payloads to target cells.
  • the payload is embedded in the vesicle, e.g., the lipid bilayer.
  • the payload can be surrounded by the vesicle or lipid bilayer.
  • targeting moieties on the modified vesicles traffic the modified vesicles in the body to target cells, and the targeting moieties are also involved in target cell recognition and interaction.
  • Modified vesicles with these targeting moieties of interest can also be associated with or fused with other delivery vehicles, such as liposomes or adeno-associated viral vectors to enhance delivery to target cell. See György, Bence, et al. Biomaterials 35 (2014)26:7598-7609. Modified vesicles can carry a payload that is to be delivered to the target cell.
  • a payload can be, for example, a small molecule, polypeptide, nucleic acid, lipid, carbohydrate, ligand, receptor, reporter, drug, or combination of the foregoing (e.g., two or more drugs, or one or more drugs combined with a lipid, etc.).
  • payloads include, for example therapeutic biologics (e.g., antibodies, recombinant proteins, or monoclonal antibodies), RNA (siRNA, shRNA, miRNA, antisense RNA, mRNA, noncoding RNA, tRNA, rRNA, other RNAs), reporters, lipids, carbohydrates, nucleic acid constructs (e.g., viral vectors, plasmids, lentivirus, expression constructs, other constructs), oligonucleotides, aptamers, cytotoxic agents, anti-inflammatory agents, antigenic peptides, small molecules, and nucleic acids and polypeptides for gene therapy.
  • therapeutic biologics e.g., antibodies, recombinant proteins, or monoclonal antibodies
  • RNA siRNA, shRNA, miRNA, antisense RNA, mRNA, noncoding RNA, tRNA, rRNA, other RNAs
  • reporters lipids, carbohydrates, nucleic acid constructs (e.g., viral vectors
  • Payloads can also be complex molecular structures such as viral nucleic acid constructs (encoding transgenes) with accessory proteins for delivery to target cells where the nucleic acid construct can be (if needed) reverse transcribed, delivered to the nucleus, and integrated (or maintained extrachromosomally).
  • the construct with a desired transgene(s) can be specifically targeted to a site in the chromosome of the target cell using CRISPR/CAS and appropriate guide RNAs.
  • Payloads may be loaded into the extracellular vesicle internal membrane space, displayed on, or partially or fully embedded in the lipid bi-layer surface of the extracellular vesicle.
  • Examples of pharmaceutical and biologic payloads include drugs for treating organ diseases and syndromes, cytotoxic agents, and anti-inflammatory drugs.
  • the payloads can be fenretinide, Doxorubicin, Mertansine (i.e. DM1) or imatinib (i.e. Gleevec, STI-571) or any combination thereof.
  • RNA payloads examples include siRNAs, miRNAs, shRNA, antisense RNAs, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), long intergenic noncoding RNA (lincRNA), piwi interacting RNA (piRNA), ribosomal RNA (rRNA), tRNA, and rRNA.
  • noncoding RNA payloads include microRNA (miRNA), long non-coding RNA (lncRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), long intergenic non-coding RNA (lincRNA), piwi-interacting RNA (piRNA), ribosomal RNA (rRNA), yRNA and transfer RNA (tRNA). miRNAs and lncRNAs in particular are powerful regulators of homeostasis and cell signaling pathways, and delivery of such RNAs by an EV can impact the target cell.
  • Treatment payloads carried by the modified vesicles can include, for example nucleic acids such as miRNAs, mRNAs, siRNAs, anti-sense oligonucleotides (ASOs), DNA aptamers, CRISPR/Cas9 therapies that inhibit oncogenes, Cytotoxic transgene therapy to induce conditional toxicity, splice switching oligonucleotides or transgenes encoding toxic proteins.
  • the payload can be a nucleic acid payload listed in Table 4.
  • a payload can be a reporter moiety.
  • Reporters are moieties capable of being detected indirectly or directly. Reporters include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a luminescent protein, a receptor, a hapten, an enzyme, and a radioisotope.
  • Examples of reporters include one or more of a fluorescent reporter, a bioluminescent reporter, an enzyme, and an ion channel.
  • fluorescent reporters include, for example, green fluorescent protein from Aequorea victoria or Renilla reniformis , and active variants thereof (e.g., blue fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, etc.); fluorescent proteins from Hydroid jellyfishes, Copepod, Ctenophora, Anthrozoas, and Entacmaea quadricolor, and active variants thereof; and phycobiliproteins and active variants thereof.
  • Chemiluminescent reporters include, for example, placental alkaline phosphatase (PLAP) and secreted placental alkaline phosphatase (SEAP) based on small molecule substrates such as CPSD (Disodium 3-(4-methoxyspiro ⁇ 1,2-dioxetane-3,2′-(5′-chloro)tricyclo [3.3.1.13,7]decan ⁇ -4-yl)phenyl phosphate, p3-galactosidase based on 1,2-dioxetane substrates, neuraminidase based on NA-Star® substrate, all of which are commercially available from ThermoFisher Scientific.
  • CPSD Disodium 3-(4-methoxyspiro ⁇ 1,2-dioxetane-3,2′-(5′-chloro)tricyclo [3.3.1.13,7]decan ⁇ -4-yl)phenyl phosphate
  • Bioluminescent reporters include, for example, aequorin (and other Ca+2 regulated photoproteins), luciferase based on luciferin substrate, luciferase based on Coelenterazine substrate (e.g., Renilla, Gaussia , and Metridina), and luciferase from Cypridina, and active variants thereof.
  • the bioluminescent reporters include, for example, North American firefly luciferase, Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina luciferase, Metrida luciferase, OLuc, and red firefly luciferase, all of which are commercially available from ThermoFisher Scientific and/or Promega.
  • Enzyme reporters include, for example, ⁇ -galactosidase, chloramphenicol acetyltransferase, horseradish peroxidase, alkaline phosphatase, acetyleholinesterase, and catalase.
  • Ion channel reporters include, for example, cAMP activated cation channels.
  • the reporter or reporters may also include a Positron Emission Tunography (PET) reporter, a Single Photon Emission Computed Tomography (SPECT) reporter, a photoacoustic reporter, an X-ray reporter, and an ultrasound reporter.
  • PET Positron Emission Tunography
  • SPECT Single Photon Emission Computed Tomography
  • Nucleic acid payloads can be oligonucleotides, recombinant polynucleotides, DNA, RNA, or otherwise synthetic nucleic acids.
  • the nucleic acids can cause splice switching of RNAs in the target cell, turn off aberrant gene expression in the target cell, replace aberrant (mutated) genes in the chromosome of the target cell with genes encoding a desired sequence.
  • the replacement nucleic acids can be an entire transgene or can be short segments of the mutated/aberrant gene that replaces the mutated sequence with a desired sequence (e.g., a wild-type sequence).
  • the nucleic acid payloads can alter a wild-type gene sequence in the target cell to a desired sequence to produce a desired result.
  • the payload nucleic acids can also introduce a transgene into the target cell that is not normally expressed.
  • the payload nucleic acids can also cause desired deletions of nucleic acids from the genome of the target cell.
  • Appropriate genome editing systems can be used with the payload nucleic acids such as CRISPR, TALEN, or Zinc-Finger nucleases.
  • the efficiency of homologous and non-homologous recombination can be facilitated by genome editing technologies that introduce targeted double-stranded breaks (DSB).
  • DSB-generating technologies are CRISPR/Cas9, TALEN, Zinc-Finger Nuclease, or equivalent systems. See, Cong et al. Science 339.6121 (2013): 819-823, Li et al. Nucleic Acids Res. (2011); Gaj et al. Trends in Biotechnology 31.7 (2013): 397-405, all of which are incorporated by reference in their entirety for all purposes.
  • Payload nucleic acids can be integrated into desired sites in the genome (e.g., to repair or replace nucleic acids in the chromosome of the target cell), or transgenes can be integrated at desired sites in the genome including, for example, genomic safe harbor site, such as, for example, the CCR5, AAVS1, human ROSA26, or PSIP1 loci.
  • genomic safe harbor site such as, for example, the CCR5, AAVS1, human ROSA26, or PSIP1 loci.
  • Payloads can be incorporated into vesicles through several methods involving physical manipulation.
  • Physical manipulation methods include but are not limited to, electroporation, sonication, mechanical vibration, extrusion through porous membranes, electric current and combinations thereof, which cause disruption of vesicle membrane.
  • Loading of cargo to vesicles described herein may involve passive loading processes such as mixing, co-incubation, or active loading processes such as electroporation, sonication, mechanical vibration, extrusion through porous membranes, electric current and combinations thereof.
  • said loading can be done concomitantly with vesicle assembly.
  • Payloads of interest can be passively loaded into vesicles by incubation with payloads to allow diffusion into the vesicles along the concentration gradient.
  • the hydrophobicity of the drug molecules can affect the loading efficiency.
  • Hydrophobic drugs can interact with the lipid layers of the vesicle membrane and enable stable packaging of the drug in the vesicle's lipid bilayer.
  • purified exosome solution suspended in buffer solution can be incubated with payload.
  • the payload is dissolved in a solvent mixture that can include DMSO, to allow passive diffusion into exosomes. Following this, the payload-exosomes mixture is made free from un-encapsulated payload.
  • centrifugation or size-exclusion columns are used to remove precipitates from the supernatant.
  • LC/MS methods can be used for the measurement and characterization of payload in the exosome-payload formulation, following lysis and removal of the exosome fraction.
  • Nucleic acids of interest can be incubated with purified exosomes to allow transfection of purified exosomes in the presence of a suitable lipid-based transfection reagent. Centrifugation can be used to purify the suspension and isolate the transfected exosome population. Transfected exosomes can then be added to target cells or used in vivo.
  • Payload can be diffused into cells by incubation with cells that then produce exosomes that carry the payload.
  • cells treated with a drug can secrete exosomes loaded with the drug.
  • Pascucci et al. have treated SR4987 mesenchymal stroma cells with a low dose of paclitaxel for 24 h, then washed the cells and reseeded them in a new flask with fresh medium. After 48 h of culture, the cell conditioned medium was collected, and exosomes were isolated.
  • the paclitaxel-loaded exosomes from the treated cells had significant, strong anti-proliferative activities against CFPAC-1 human pancreatic cells in vitro, as compared with the exosomes from untreated cells (Pascucci, L. et al., Journal of Controlled Release, 192 (2014): 262-270.
  • Extracellular vesicles secreted from cells can be mixed with payloads and subsequently sonicated by using a homogenizer probe.
  • the mechanical shear force from the sonicator probe can compromise the membrane integrity of the exosomes and subsequently allow the drug to diffuse into the exosomes during this membrane deformation, especially, a hydrophilic drug.
  • extracellular vesicles from cells can be mixed with a payload, and the mixture can be loaded into a syringe-based lipid extruder with 100-400 nm porous membranes under a controlled temperature.
  • the exosome membrane can be disrupted during the extrusion process can allow vigorous mixing with the drug.
  • the number of effective extrusions can vary from 1-10 to effectively deliver drugs into exosomes.
  • Payload of interest can be incubated with exosomes at room temperature for a fixed amount of time. Repeated freeze-thaw cycles are then performed to ensure drug encapsulation.
  • the method can result in a broad distribution of size ranges for the resulting exosomes, and then, the mixture is rapidly frozen at ⁇ 80° C. or in liquid nitrogen and thawed at room temperature.
  • the number of effective freeze-thaw cycle may vary from 2-7 for effective encapsulation.
  • membrane fusion between exosomes and liposomes can be initiated through freeze-thaw cycles to create exosome-mimetic particles.
  • small pores can be created in exosomes membrane through application of an electrical field to exosomes suspended in a conductive solution.
  • the phospholipid bilayer of the exosomes can be disturbed by the electrical current. Payloads can subsequently diffuse into the interior of the exosomes via the pores. The integrity of the exosome membrane can then be recovered after the drug loading process.
  • nucleic acids e.g., mRNA, siRNA or miRNA can be loaded into exosomes using this method.
  • electroporation can be conducted in an optimized buffer such as trehalose disaccharide to aid in maintaining structural integrity and can inhibit the aggregation of exosomes.
  • an optimized buffer such as trehalose disaccharide to aid in maintaining structural integrity and can inhibit the aggregation of exosomes.
  • Membrane permeabilization can be initiated through incubation with surfactants, such as, saponin.
  • surfactants such as, saponin.
  • hydrophilic molecules can be assisted in exosome encapsulation by this process.
  • fluorophores and microbeads conjugated to highly specific antibodies can bind a particular antigen on the cell surface.
  • Specific antigen-conjugated microbeads can be used for exosome isolation and tracking in vivo.
  • compositions disclosed herein may comprise modified extracellular vesicles of the invention and/or liposomes with (or without) a payload, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • compositions are in one aspect formulated for intravenous administration or intracranial administration or intranasal administration to the central nervous system.
  • Compositions described herein may include lyophilized EVs (e.g., exosomes).
  • composition comprises an EV or exosome and a pharmaceutically acceptable excipient.
  • compositions may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • Suitable pharmaceutically acceptable excipients are well known to a person skilled in the art.
  • excipients include, but are not limited to, surfactants, lipophilic vehicles, hydrophobic vehicles, sodium citrate, calcium carbonate, and dicalcium phosphate.
  • the composition can be formulated into a known form suitable for parenteral administration, for example, injection or infusion.
  • the composition may comprise formulation additives such as a suspending agent, a preservative, a stabilizer and/or a dispersant, and a preservation agent for extending a validity term during storage.
  • compositions described herein may be administered to a patient trans arterially, subcutaneously, sublingually, intradermally, intranodally, intramedullary, intramuscularly, intranasally, intraarterially, into an afferent lymph vessel, by intravenous (i.v.) injection, or intracranially injection, or intraperitoneally.
  • the compositions of the present invention are administered to a patient by intradermal or subcutaneous injection.
  • the modified vesicles compositions described herein are administered by i.v. injection.
  • Compositions can be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
  • kits are provided.
  • Kits according to the invention include package(s) comprising any of the compositions of the invention (including the extracellular vesicles of the invention, chimerical vesicle localization moieties, fusion proteins, and nucleic acids).
  • packaging means any vessel containing compositions presented herein.
  • the package can be a box or wrapping.
  • Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including pre-filled syringes), bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • the kit can also contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.
  • Kits may optionally contain instructions for administering compositions of the present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of components of the composition herein by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for the present compositions. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies.
  • the kits can include compositions in the solid phase or in a liquid phase (such as buffers provided) in a package.
  • the kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
  • the kit may optionally also contain one or more other compositions for use in combination therapies as described herein.
  • the package(s) is a container for any of the means for administration such as intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, intra-arterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, or intradermal injection.
  • Additional vesicle localization moieties which may be used to produce a chimeric vesicle localization moiety
  • nucleic acid sequence coding a vesicle localization moiety may be found within sequence associated with an ENST number; note multiple ENST numbers associated with each vesicle localization moiety referred through its Gene Symbol or UniProtKB accession number potentially indicate multiple isoforms of a vesicle localization moiety.
  • Target anti-miRNA antimiR-494 Targets the “oncomiR”, miR-494 miRNA anti-miRNA antimiR-221/222 Targets the “oncomiR”, miR-221/222 miRNA anti-miRNA antimiR-132 Targets the “oncomiR”, miR-132 miRNA anti-miRNA antimiR-155 Targets the “oncomiR”, miR-155 miRNA Antisense ASO, OGX-011 Clusterin Oligonucleotide (ASO) Antisense EGFR antisense EGFR Oligonucleotide DNA (ASO) Antisense ASO, OGX-427 Hsp27 Oligonucleotide (ASO) Antisense ASO, ISIS- STAT3 Oligonucleotide STAT3Rx (ASO) Antisense ASO, AP 12009 TGFB2 Oligonucleodide (ASO) Antisense ASO,
  • Cytotoxic Herpes Simplex Converts the prodrug ganciclovir (or trans-genes Type 1 thymidine valacyclovir) into the highly toxic deoxyguanosine kinase (TK) triphosphate causing early chain termination of nascent DNA strands miRNA miRNA-34a Poorly understood tumor suppressor gene.
  • Targets include SIRT1, BCL2, YY1, MYC, CDK6, CCND1, FOXP1, HNF4a, CDKN2C, ACSL4, LEF1, ACSL1, MTA2, AXL, LDHA, HDAC1, CD44, BCL2, E2F3 miRNA miR-200 Poorly understood tumor suppressor gene.
  • Targets include ZEB1, CTNNB1, BAP1, GEMIN2, PTPRD, WDR37, KLF11, SEPT9, HOXB5, ERBB2IP, KLHL20, FOG2, RIN2, RASSF2, ELMO2, TCF7L1, VAC14, SHC1, SEPT7, FOG2 miRNA miR-15/16 Poorly understood tumor suppressor gene.
  • Targets include BACE1, DMTF1, C22orf5, BCL2, ARL2, CCNT2, TPPP3, VEGFA, RARS, FGF2, ZNF622, DNAJB4, PURA, SHOC2, LUZP1, FNDC3B, ITGA2, ATG9A, CA12, TMEM43, YIF1B, TMEM189, VTI1B, RTN4, TOMM34, NAA15, PNP, SRPR, IPO4, NAPg, PFAH1B2, SLC12A2, SEC24A, NOTCH2, PPP2R5C, KCNN4, UBE4A, KPNA3, RAB30, ACP2, SRPRB, EIF4E, ABCF2, TPM3, ARHGDIA, GALNT7, LYPLA2, CHORDC1, TMEM109, LAMC1, EGFR, GPAM, ADSS, PPIF, RFT1, TNFSF9, IGF2R, TXN2, GFPT1, SLC7A1, SQSTM
  • Targets include NIRF, NF2, CASP3, TRIM71 miRNA miR-26a Induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2 miRNA miR-143 MACC1 miRNA miR-145; miR-33a ERK5, c-Myc mRNA mRNAs encoding OX40L, IL-36 ⁇ , and IL-23 OX40L, IL-36 ⁇ , and IL-23 siRNA siRNA against targets Knockdown c-Myc/MDM2/VEGF siRNA siRNA against targets EphA2 oncoprotein siRNA siRNA against targets Oncogenic KRAS(G12D) siRNA siRNA against targets PLK1 (polo-like kinase-1) siRNA siRNA against targets protein kinase N3 (PKN3) gene expression in vascular endothelial cells siRNA siRNA against targets VEGF gene, kinesin spindle (KSP) protein gene Splice- SSO to Bcl-x A
  • SSOs switching oligonucleotides
  • HEK293F cells were maintained in serum-free media suspension cultures in shake flasks. Upon reaching a culture density of 2 ⁇ 10 6 cells/mL, each shake flask culture was transfected with individual plasmids corresponding to vector constructs provided in FIGS. 1 - 2 to produce the fusion proteins with the amino acid sequence provided in FIGS. 3 - 8 or Table 3, using PEI (MW: 25,000-1 mg/mL). Transfection reagent mixture was prepared in labeled tubes in 10% of the final desired culture volume of Opti-MEM® Reduced Serum Medium with 1 ⁇ g of DNA for every 1 mL of final culture volume and a 2:1 ratio of PEI to DNA, so 2 ⁇ g of PEI for every 1 mL of final culture volume.
  • Opti-MEM medium For example, for a 30 mL final culture volume, 3.0 mL of Opti-MEM medium is added to 15-mL tube with 30 ⁇ g DNA and vortexed gently. 60 ⁇ L PEI (at 1 mg/mL) is added to each 15-mL tube with DNA+medium. Mixture is immediately vortexed 3 ⁇ , 3s each after PEI addition. The mixture is incubated for 15 min at room temperature and then immediately added to the respective flasks. The incubator is set at 37° C. with 8% CO 2 on shaking platform at 125 RPM.
  • the transfection media was exchanged for fresh media, and the cells were grown for an additional 96 hours.
  • 96 hours following media exchange the cultures were transferred into 50-mL conical tubes and centrifuged at 3,220 ⁇ g for 30 min. The supernatant from these cultures were transferred to Amicon® Centrifugal Filter Units (100 kDa cut-off), concentrated and buffer exchanged into PBS.
  • EVs are then filtered using CaptoTM Core700 (Cytiva) Size Exclusion Chromatography (SEC) resin to remove non-EV-associated protein. Finally, the EVs are sterile filtered, using a 0.22 ⁇ M centrifugal filter column unit.
  • HEK293 or its variants include HEK293-F (Cellosaurus), FreeStyleTM 293-F (ThermoFisher) PER.C6, CHO-K1 or Hs 235.Sk (ATCC® CRL-7201TM).
  • stable cell lines may be created by co-transfecting an expression vector for a chimeric vesicle localization moiety or a fusion protein comprising a chimeric versicle localization moiety, such as a targeting moiety-chimeric vesicle localization moiety fusion protein, and a selectable marker, such as a drug selectable marker, and selecting for the selectable marker to obtain a double transfectant which expresses both the selectable marker as well as the chimeric vesicle localization moiety or fusion protein of interest.
  • EVs may be harvested from the culture media of such stable transfectants, wherein EVs may be modified with the chimeric vesicle localization moiety or fusion protein of interest.
  • a cassette is made for cloning nucleic acids encoding one or more targeting moieties of interest.
  • the cassette includes a polynucleotide encoding the LAMP2 or CLSTN1 vesicle localization moiety or a chimeric vesicle localization moiety (e.g., LAMP2 surface-and-transmembrane domains linked in frame to CLSTN1 cytosolic domain in the order as is naturally found for parental vesicle localization moieties, namely surface-transmembrane-cytosolic domains) for display on exosomes, a polynucleotide encoding a N-terminal signal sequence for membrane insertion (e.g., insertion into the endoplasmic reticulum), and optionally, a polynucleotide encoding an epitope tag, a glycosylation sequence and linker sequences, the latter to separate functional sequences when desired, such as, for example, a linker sequence between
  • a polynucleotide encoding a targeting moiety of interest is cloned into the cassette such that the open reading frame of the targeting moiety is linked in frame to the open reading frame of the signal sequence and the vesicle localization domain or chimeric vesicle localization domain, and optionally, an epitope tag, a glycosylation sequence and linker sequences, so as to produce a single open reading frame for a fusion protein comprising the signal sequence, targeting moiety of interest, vesicle localization domain or chimeric vesicle localization domain, and optionally, an epitope tag, a glycosylation sequence and linker sequences.
  • a targeting moiety of interest may be an affinity peptide or an scFv antibody and may be directed to a particular cell type or tissue through the affinity peptide or scFv antibody.
  • the nucleic acid for the fusion protein is inserted in an expression vector to permit expression of the fusion protein either constitutively or inducibly.
  • the expression vector preferably comprises a selectable or detectable marker, such as drug resistance gene (e.g., puromycine or G418) or a gene for a fluorescent protein (e.g., GFP and its variants).
  • drug resistance gene e.g., puromycine or G4108
  • a gene for a fluorescent protein e.g., GFP and its variants.
  • Exemplary fusion proteins comprising a targeting moiety of interest and a vesicle localization domain or chimeric vesicle localization domain may be seen in the figures.
  • a cell line such as HEK293 or its variants, HEK293-F (Cellosaurus), FreeStyleTM 293-F (ThermoFisher) PER.C6, CHO-K1 or Hs 235.Sk (ATCC® CRL-7201TM, is transfected with vectors including the cassette with a desired marker. Positive transfectants are obtained by flow cytometry or other cell sorting methods. In other cases, positive transfectants are enriched through antibiotic selection. Transfected cells are grown in exosome depleted or chemically defined media, suitable for exosome isolation. Following a period of culture, EVs are isolated from the conditioned media.
  • any cells in the conditioned media are cleared by centrifugation and filtration, and the EVs in the clarified media are concentrated using ultrafiltration. After concentration the exosomes are isolated using liquid chromatography using an appropriate column (e.g., Sephacryl S-300, Capto-Core 700, etc.)
  • an appropriate column e.g., Sephacryl S-300, Capto-Core 700, etc.
  • Extracellular vesicles labeled with a fluorescent dye or fluorescent protein permit tracking EVs in solution and cellular uptake of EVs, the latter resulting in transfer of the fluorescent dye or fluorescent protein to a cell indicating EV uptake by the cell.
  • Such labeled EVs are useful in assessing effectiveness of a targeting moiety on surface of an EV to preferentially target one cell type over another.
  • EVs may be labeled through the use of a fluorescent protein fusion, such as green fluorescent protein (GFP) and its variants, or protein reporters.
  • GFP green fluorescent protein
  • This alternative method often involves creation of fusion proteins to generate a vesicle localization moiety-protein reporter gene constructs and cellular expression of these fusion proteins to obtain exosomes.
  • targeting EVs are prepared in which the targeting EVs comprise a targeting moiety displayed on the external surface of fluorescently labeled EVs and exposed to a cell co-culture comprising two different cell types one of which is labeled with a 2 nd fluorescent dye and expresses a cell marker targeted by the targeting moiety.
  • Cell-based in vitro uptake assay is used to assess EV comprising a targeting moiety of interest (a targeting EV or TEV) to target a cell type or tissue.
  • a skeletal muscle cell line labeled with a fluorescent dye and containing a skeletal muscle target protein (i.e., a skeletal muscle marker protein) and a negative cell line not containing the skeletal muscle cell target are co-cultured.
  • the co-culture is then “dosed” with EVs for an indicated period.
  • the EVs have been engineered with a targeting motif that targets the nicotinic acetylcholine receptor found in skeletal muscle, such as an scFv for the nicotinic acetylcholine receptor or 3 ⁇ 5 ⁇ -conotoxin and derivatives targeting nicotinic acetylcholine receptor (see for example Tsetlin, V. and Kasheverov, I.
  • EVs are obtained from the conditioned media supernatant of cultured HEK293 cells.
  • the EVs are isolated using ultracentrifugation (size selection to enrich for a general EV population).
  • the EVs are loaded with a reporter (e.g., CPSD) or mRNA encoding a reporter (e.g., GFP), as described in Example 8.
  • a reporter e.g., CPSD
  • mRNA encoding a reporter e.g., GFP
  • Skeletal muscle cell line such as Hs 235.Sk (ATCC® CRL-7201TM) is grown to confluence and then the EVs with reporter are added to the skeletal muscle cell line. After incubating the skeletal muscle Hs235.Sk cells with the EVs, the excess EVs are washed away. The cells are then subjected to fluorescence microscopy to identify those cells that have obtained a reporter from the EVs. EV delivery to the cells is identified by reporter activity in cells.
  • EVs are obtained from the media of Hs 235.Sk (ATCC® CRL-7201TM). The EVs are isolated using ultracentrifugation (size selection to enrich for a general EV population). The EVs are loaded with a reporter (e.g., CPSD) or mRNA encoding a reporter (e.g., GFP), as described in Example 8.
  • a reporter e.g., CPSD
  • mRNA encoding a reporter e.g., GFP
  • mice B6.129X-Nfe212 tm1Ywk mice are injected with EVs containing the reporter CPSD. After 24 hours, the mice are sacrificed and the animal's skeletal myocytes are examined with fluorescence microscopy. EV delivery to skeletal muscle tissue is identified by reporter activity in the skeletal muscle cells.
  • Exosomes presenting targeting moieties of interest are engineered as described in herein.
  • the number of isolated exosomes are quantified using nanoparticle tracking analysis.
  • NTA measurements are obtained with a NanoSight NS300 instrument equipped with the Nanoparticle Tracking Analysis (NTA) 3.3 analytical software (Malvern Panalytical). Samples are diluted to achieve a particle count in the linear range of the instrument: between 20 and 150 particles on the screen at one time. Samples are loaded using the NanoSight Sample Assistant to automate the measurement of up to 96 samples in one run. Multiple 30 second videos of each sample flowing at a slow constant flow are obtained. These measurements are then analyzed using the batch process function.
  • NTA Nanoparticle Tracking Analysis
  • Example 8 Introducing Payloads into Engineered Exosomes Carrying Markers of Interest
  • An exosome is engineered to incorporate a targeting moiety(ies) of a skeletal muscle marker, such as a scFv or affinity peptide as a targeting moiety directed to a skeletal muscle marker, for example a subunit or multiple subunits of the nicotinic acetylcholine receptor found in skeletal muscle (e.g., ⁇ 1 ⁇ 1 ⁇ (adult) and ⁇ 1 ⁇ 1 ⁇ (fetal) nAChRs; see Tsetlin, V. and Kasheverov, I. (2014) Peptide and Protein Neurotoxin Toolbox in Research on Nicotinic Acetylcholine Receptors. In T.
  • a targeting moiety(ies) of a skeletal muscle marker such as a scFv or affinity peptide
  • a targeting moiety directed to a skeletal muscle marker for example a subunit or multiple subunits of the nicotinic acetylcholine receptor found in skeletal muscle (e.g., ⁇
  • an exosome is engineered to comprise any one of the following targeting moieties ENO2, JSRP1, VAPA, TMOD1 or a functional fragment thereof.
  • the engineered or isolated exosome or EV is loaded with a CRISPR gene editing system, a transgene or a miRNA.
  • miRNA may be selected from the group consisting of miR-133a, miR-1, miR-133, miR-133b, miR-181a-5p, miR-206, and miR-499.
  • the engineered or isolated exosome is loaded with fenretinide. The loaded exosome is then used to ameliorate insulin resistance of obese subject and/or reduce obesity in a subject.
  • Exosome protein input of ⁇ 300 ⁇ g (from about 1 ⁇ 10 ⁇ circumflex over ( ) ⁇ 7 exosomes) is suspended in 50 ⁇ l of sterile PBS.
  • a reaction mixture consisting of exosomes, 10 ⁇ l of Exo-Fect Reagent and nucleic acid of interest (20 pmol si/miRNA, 1 ⁇ g mRNA or 5 ⁇ g plasmid DNA) is put together and mixed by inversion.
  • the transfection solution is incubated in a shaker for 10 minutes at 37° C. and then placed on ice.
  • 30 ⁇ l of ExoQuick-TC reagent provided in the kit is added to the exosome sample suspension and mixed by inverting.
  • the transfected exosome sample is placed on ice for 30 minutes.
  • the sample is centrifuged at 13000-14000 rpm to pellet the exosomes.
  • the transfected exosomes are then resuspended in PBS and can be added to target cells or used in vivo for further applications.
  • exosomes may be isolated from HEK293 or engineered cells expressing fusion protein comprising a targeting moiety and vesicle localization moiety or a chimeric versicle localization moiety.
  • Sample isolated EVs are prepared in PBS buffer at a concentration of 2.10E+11 particles/mL.
  • Fenretinide (Selleck Chemicals: Cat No: S5233) was dissolved in 100% DMSO.
  • the fenretinide/DMSO stock solution can be 5 mM or 10 mM.
  • Protocol for introducing fenretinide into EV to obtain EV comprising fenretinide (fenretinide-EV, also called fen-EV, EV-fen or EV-fenretinide) and purifying the fenretinide-EV from extra-vesicular (not associated with an EV) fenretinide not incorporated into EVs:
  • Vector #91 construct when introduced into HEK293F cells produces a fusion protein comprising from amino-to-carboxyl terminus in the order: a signal sequence (for improved expression and endoplasmic reticulum association)-glycosylation site (for stabilization of fusion protein)-mature LAMP2 (Lysosome-associated membrane protein 2) protein with surface, transmembrane and cytosolic domains (for localization to exosomes).
  • the mature LAMP2 protein used lacks its natural signal sequence—the first 28 amino acids normally found at N-terminal of a nascent LAMP2 protein but is removed following association with a cell membrane, such as endoplasmic reticulum, such that the resulting processed, mature LAMP2 form is found associated with an exosome.
  • the fusion protein may additionally comprise peptide linkers. Such peptide linkers rich in glycine and serine amino acids may be found between the signal sequence, glycosylation site and LAMP2 protein.
  • epitope sequence such as that corresponding to 3x FLAG epitope tag
  • affinity peptide sequence may be found in between the signal sequence and the glycosylation site.
  • affinity peptides include, but are not limited to, THRPPMWSPVWP (SEQ ID NO.: 64), a targeting moiety or peptide for transferrin receptor (TfR), and THVSPNQGGLPS (SEQ ID NO.: 66), a targeting moiety or peptide for glypican-3 (GPC3).
  • This LAMP2 fusion protein serves as one parental vesicle localization moiety (see FIG. 1 , vector #91 for a schematic of a fusion protein comprising the mature LAMP2 protein (lacking its native LAMP2 signal peptide sequence); see FIG. 3 , vector #91 for the sequence of the LAMP2 fusion protein produced and FIG.
  • FIGS. 9 - 12 or Table 5 amino acid sequences corresponding to the surface, transmembrane and cytosolic domains are extracted from the sequences of the fusion proteins provided in FIGS. 3 - 8 or Table 3 so that only the vesicle localization moiety or chimeric vesicle localization moiety amino acid sequences are shown.
  • the surface domain (italic text) precedes the transmembrane domain (italic and bold) which is found between the surface and cytosolic domain (italic and underline) and connects these two domains.
  • a second parental vesicle localization moiety was constructed with mature CLSTN1 protein coding sequence, as schematically shown for vector #112 in FIG. 1 .
  • the mature CLSTN1 protein coding sequence lacks the first 28 codons of the nascent full length CLSTN1 protein coding sequence; the first 28 amino acids of the nascent CLSTN1 protein corresponds to its natural CLSTN1 signal sequence, which is normally removed upon association of the nascent protein with the endoplasmic reticulum and eventual incorporation of this processed mature CLSTN1 protein into an intraluminal vesicle before secretion from a multivesicular body (MVB) (Hanson, P. I. and Cashikar, A.
  • MVB multivesicular body
  • the primary amino acid sequence of the mature CLSTN1 protein differs from the full length nascent CLSTN1 protein (similarly to full length nascent LAMP2 protein) in lacking the native signal sequence, the first 28 amino acids at the N-terminus of the full-length nascent protein.
  • the CLSTN1 fusion protein has a similar arrangement of a non-native signal sequence at the amino terminus of the fusion protein along with epitope sequence and glycosylation site. Linkers are similarly present and in addition an affinity peptide is present in the CLSTN1 fusion protein (see FIG. 1 , vector #112 for a map of functional sequences encoding the fusion protein; FIG. 4 for the sequence of the parental CLSTN1 fusion protein produced from vector #112 and FIG. 9 for the sequence of the mature CLSTN1 protein).
  • Chimeric vesicle localization moieties were prepared primarily based on the surface domain and transmembrane domain of LAMP2 (surface-and-transmembrane domain of LAMP2) and cytosolic domain from other transmembrane proteins.
  • Type I transmembrane proteins were used, having the following characteristic after incorporation into an extracellular vesicle (e.g., an exosome): an amino-terminal surface domain, a single pass transmembrane domain and a carboxyl-terminal lumenal domain (also referred to its topological equivalence, as a cytosolic domain prior to the formation of an exosome but following insertion into the endoplasmic reticulum).
  • cytosolic domain (lumenal domain) of LAMP2 is replaced with the cytosolic domain of PTGFRN (vector #135), ITGA3 (vector #140), IL3RA (vector #141), SELPLG (vector #142), ITGB1 (vector #143) and CLSTN1 (vector #144), as schematically represented in FIGS. 1 and 2 .
  • Amino acid sequences for fusion proteins prepared from these chimeric vesicle localization moieties are shown in FIG. 3 - 8 or Table 3.
  • Sequences are shown in capital letter and bold for signal sequence, capital letter and underline for epitope sequence, shaded capital letter for affinity peptide, open boxed capital letter for peptide linker sequence, small letter for glycosylation site, capital letter and italic for surface domain, capital letter and bold italic for transmembrane domain and capital letter and underlined italic for cytosolic domain in FIGS. 3 - 8 .
  • the cytosolic domain of mature LAMP2 protein was removed and replaced with a highly charged tetrapeptide, KKPR, to stabilize truncated LAMP2 on surface of EVs (see FIG. 2 , vector #145 for a map of the fusion protein with the truncated LAMP2 lacking its natural cytosolic domain replaced by a tetrapeptide, KKPR, and FIG. 8 , #145 for the amino acid sequence of the fusion protein with the truncated LAMP2 lacking its native cytosolic domain).
  • isolated EVs are stained with fluorophore-conjugated anti-FLAG tag antibody and a membrane stain.
  • the stained vesicles are evaluated using vesicle flow cytometry (vFC) (Cytoflex—Beckman Coulter). EVs are identified as membrane stain-positive particles.
  • the amount of recombinant protein on each EV is detected using a fluorophore-conjugated antibody that binds specifically to the epitope sequence included in the primary sequence of the protein, and would only be available on the EV surface if the fusion protein is oriented in the intended topology (C-terminal domain in the lumen; N-terminal domain on the EV surface).
  • the amount of recombinant protein on each evaluated EV is determined by the antibody signal/membrane-stained particle.
  • EV populations were isolated from cells transfected with the indicated vector numbers. Isolated EVs were stained with a mouse monoclonal antibody specific to an epitope sequence encoded in the EV surface domain of each recombinant protein.
  • the Y-axis represents the relative amount (on average) of antibody bound to each EV ignoring EVs not labeled by the anti-FLAG epitope tag antibody, serving as an indirect measure of the amount of recombinant protein incorporated into each EV.
  • the background signal associated with EVs from mock transfected cells (Mock) has been subtracted from these values.
  • the fraction of the total EV population displaying a detectable amount of the recombinant protein is shown in FIG. 14 .
  • Example 11 Chimeric Vesicle Localization Moiety with a Surface-and-Transmembrane Domain of First Vesicle Localization Moiety and a Cytosolic Domain can Increase EV Localization
  • a fusion protein comprising a chimeric vesicle localization moiety having a LAMP2 surface-and-transmembrane domain and a non-native cytosolic domain from a number of different vesicle localization moieties PTGFRN (vector #135), ITGA3 (vector #140), IL3RA (vector #141), SELPLG (vector #142), ITGB1 (vector #143) and CLSTN1 (vector #144) showed dramatic improvement in the ability of the fusion protein to localize at an extracellular vesicle, increasing both the abundance of the fusion protein at an EV ( FIG. 13 ) as well as the fraction or percent of EVs positive for the fusion protein ( FIG. 14 ).
  • FIGS. 13 and 14 show that not only do chimeric vesicle localization moieties localize to EVs but localization of the fusion proteins is improved when a chimeric vesicle localization moiety is used in place of its non-chimeric counterpart (compare #135, 140, 141, 142, 143 and 144 with #91 or 112).
  • deleting the cytosolic domain of LAMP2 and replacing with a positively charged tetrapeptide, KKPR modestly improves localization of LAMP2 surface-and-transmembrane domain to EV (compare #145 with #91); however, the improvement in EV localization by transplanted cytosolic domains from a variety of vesicle localization moieties is much more robust—indicating that while a minimal cytosolic domain (i.e., KKPR) may be required for stable EV localization of a surface-transmembrane domain of a vesicle localization moiety (such as LAMP2), the cytosolic domain can modulate EV localization, affecting the efficiency of EV localization.
  • a minimal cytosolic domain i.e., KKPR
  • FIG. 15 Normalization of the data in FIG. 13 to illustrate fold increase in fusion protein abundance or concentration on EV surface relative to the fusion protein comprising a mature LAMP2 (nascent LAMP2 protein lacking its native signal sequence, the first 28 amino acids at the amino terminus of the nascent protein; vector #91 construct) is shown in FIG. 15 .
  • FIG. 14 normalization of the data in FIG. 14 to illustrate fold increase in percent or fraction of EVs positive for a fusion protein relative to the fusion protein comprising a mature LAMP2 protein produced by vector #99 construct is shown in FIG. 16 .
  • Replacing the cytosolic domain of the mature LAMP2 protein with the cytosolic domain of a variety of other vesicle localization moiety results in about a 4-fold increase in fusion protein abundance at an EV for a number of cytosolic domain examined obtained from PTGFRN (vector #135), ITGA3 (vector #140), IL3RA (vector #141), SELPLG (vector #142), and ITGB1 (vector #143), as seen in FIG. 15 .
  • fraction of total EVs positive for the various fusion proteins with a chimeric vesicle localization moiety increases 3-4 fold over the fusion protein comprising a non-chimeric vesicle localization moiety, namely the parental LAMP2 vesicle localization moiety (vector #91) which provided its LAMP2 surface-and-transmembrane domain to the various chimeric vesicle localization moieties (vector #135, 140, 141, 142, 143, and 144).
  • Example 12 Chimeric Vesicle Localization Moiety can Dramatically Improve EV Localization Over Parental Vesicle Localization Moieties
  • FIG. 15 shows fold increase in fusion protein abundance (or concentration) on EV surface relative to fusion protein produced by vector #91 construct (fusion protein with a mature LAMP2 protein having a contiguous surface-transmembrane-and-cytosolic domain but no native LAMP2 signal sequence), as detected by vesicle flow cytometry using a fluorophore-conjugated anti-FLAG epitope tag antibody.
  • the fusion protein produced by vector #112 concentrates at a much lower level, about 25% the abundance of the mature LAMP2-containing fusion protein (compare value of #91 and #112 in FIG. 15 ).
  • the new chimeric vesicle localization moiety increases by about 2-fold the abundance of the fusion protein over its parental LAMP2 (compare value of #91 and #144) or over 8-fold the abundance of the fusion protein over its parental CLSTN1 (compare value of #112 and #144), indicative of synergistic interaction between the surface-and-transmembrane domain of LAMP2 and the cytosolic domain of CLSTN1.
  • fusion protein comprising the parental LAMP2 vesicle localization moiety is better at associating with total EV population having a normalized value of 1.00 (#91) than the fusion protein comprising the parental CLSTN1 vesicle localization moiety with a normalized value 0.15 (#112).
  • a fusion protein comprising a chimeric vesicle domain produced from the two parental vesicle localization moieties has a normalized value of 3.79, reflecting over 3.5-fold increase over the parental LAMP2 vesicle localization moiety and over 25-fold over the parental CLSTN1 vesicle localization moiety.
  • Such a dramatic increase in association with total EV population which reaches about 55% (see FIG. 14 , #144) by a fusion protein comprising a chimeric vesicle localization moiety is unexpected.
  • the observed increase in EV localization is not unique to the use of CLSTN1 cytosolic domain to replace the LAMP2 cytosolic domain.
  • cytosolic domains also increase EV localization beyond that of the parental LAMP2 vesicle localization moiety, indicating that the cytosolic domain of PTGFRN, ITGA3, IL3RA, SELPLG, and ITGB1 may function in a similar manner as the cytosolic domain of CLSTN1 to synergistically increase EV localization, both concentrating at a single EV as well as associating with the total EV population.
  • a chimeric vesicle localization moiety comprising a surface-and-transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety can interact synergistically to increase accumulation at an extracellular vesicle.
  • Such a finding provides an approach not only to improve EV localization but potentially to change the composition of EVs as the chimeric vesicle localization moiety may interact with a different set of proteins or has altered affinity to the set of proteins recruited to an extracellular vesicle by the two native vesicle localization moieties.
  • FIGS. 13 - 16 are bar graphs showing abundance of a vesicle localization moiety or chimeric vesicle localization moiety at an EV having the localization moiety ( FIGS. 13 and 15 ) and fraction of total EVs positive for the vesicle localization moiety or chimeric vesicle localization moiety ( FIGS. 14 and 16 ).
  • EVs are isolated from culture media of cells transiently transfected with the expression construct (vector) indicated below each bar graph. The isolated EV population is labeled with a membrane-staining fluorescent dye with a spectral characteristic distinct a second fluorescent dye used to conjugate to an anti-FLAG antibody.
  • the fluorescent dye-labeled EV population is probed with the fluorophore-conjugated anti-FLAG antibody to detect presence of fusion protein comprising a vesicle localization moiety or a chimeric vesicle localization moiety, as all fusion proteins produced have 3 ⁇ FLAG epitope preceding the surface domain.
  • the resulting EVs are analyzed by vesicle flow cytometry (vFC) in a CytoFLEX benchtop flow cytometer (Beckman Coulter) to detect the EVs based on the membrane-staining fluorescent dye.
  • FIG. 13 is a plot of mean antibody fluorescence of an EV positively labeled with the anti-FLAG antibody for the fusion protein expressed by the indicated expression vector.
  • FIG. 14 is a plot of percent of total EV positively labeled by the anti-FLAG antibody.
  • FIGS. 15 and 16 show the fold changes in levels reported in FIGS. 13 and 14 , respectively, in relation to expression vector #91, which expresses a fusion protein comprising a mature LAMP2 protein.

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US11969504B2 (en) 2024-04-30
JP2023514975A (ja) 2023-04-12
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EP4097474A4 (fr) 2024-03-27
EP4097474A1 (fr) 2022-12-07
US20230218527A1 (en) 2023-07-13
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