WO2023133527A2 - Vésicules extracellulaires chargées avec des particules virales pour un apport de charge - Google Patents

Vésicules extracellulaires chargées avec des particules virales pour un apport de charge Download PDF

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Publication number
WO2023133527A2
WO2023133527A2 PCT/US2023/060261 US2023060261W WO2023133527A2 WO 2023133527 A2 WO2023133527 A2 WO 2023133527A2 US 2023060261 W US2023060261 W US 2023060261W WO 2023133527 A2 WO2023133527 A2 WO 2023133527A2
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modified
aav
viral
gvs
aav6
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PCT/US2023/060261
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English (en)
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WO2023133527A3 (fr
Inventor
Joshua A. HORWITZ
Joseph BOLEN
Raz C. BANOSIAN
Caroline K. HANLON
Michael P. Thomas
Jungyeon Hwang
Anne L. Burkhardt
Solly Weiler
Jacob T. MARTIN
Eyoung SHIN
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Puretech Lyt, Inc.
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Publication of WO2023133527A2 publication Critical patent/WO2023133527A2/fr
Publication of WO2023133527A3 publication Critical patent/WO2023133527A3/fr

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    • 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
    • A61K47/6905—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 the form being a colloid or an emulsion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • Viral particles such as adeno-associated viral (AAV) particles are promising gene delivery vehicles due to their effectiveness in directing transgene expression without accompanying immunogenic complications.
  • AAV adeno-associated viral
  • AAVs are single-stranded DNA (ssDNA), non-enveloped and replication-defective viruses capable of infecting human cells. More than ten distinct AAV serotypes have been identified, each having specificity for certain tissues and cell types. This makes AAVs an excellent platform for cell-specific gene delivery. Manipulation of their ssDNA genome, for example, to favor a self-complementary, transcription-ready configuration, leads to faster protein production than other common viral gene therapy vectors, such as lentiviral vectors, which require RNA to DNA conversion and DNA integration before transcription and protein production can occur.
  • ssDNA single-stranded DNA
  • lentiviral vectors which require RNA to DNA conversion and DNA integration before transcription and protein production can occur.
  • Viral particles are commonly administered to patients via an intravenous route or local injection. It is of interest to develop suitable methods for delivering viral particles via alternative routes, for example, oral delivery.
  • the present disclosure is based, at least in part, on the development of glycocalyx vesicles (GVs) such as extracellular vesicles (EVs) (c.g., whey-derived extracellular vesicles (WEVsj) loaded with viral particles such as adeno-associated viral (AA V) particles for delivering transgenes carried by the viral particle to a compartment of gastrointestinal tract via oral administration.
  • GVs glycocalyx vesicles
  • EVs extracellular vesicles
  • WEVsj whey-derived extracellular vesicles
  • viral particles such as adeno-associated viral (AA V) particles for delivering transgenes carried by the viral particle to a compartment of gastrointestinal tract via oral administration.
  • AA V adeno-associated viral particles
  • the present disclosure is based, at least in part, on the development of AAV serotypes that are capable of infecting intestinal epithelium cells with high efficiency.
  • AAV1 transduces the intestinal epithelium from both the basolateral and apical poles
  • AAV6 transduces such cells predominantly from the apical pole.
  • AAV6 variants e.g., AAV6.2FVFF
  • viral particle-loaded GVs such as EVs, which are optionally modified by surface lectins for binding to GI cells, methods of preparing such, and methods of using such for delivery of cargos carried by the viral particle via oral administration.
  • methods for delivering an agent to intestinal epithelium cells using with one or more AAV viral particles each of which comprises an AAV viral genome that expresses an agent.
  • the one or more AAV viral particles are AAV1 or AAV6, such as AAV6 variants disclosed herein, which are also within the scope of the present disclosures.
  • the present disclosure features a modified glycocalyx vesicle (GV), which is loaded with a viral particle and optionally modified on the surface one or more lectins that bind enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract.
  • the modified GV is an extracellular vesicle (EV), for example, whey extracellular vesicles (WE Vs).
  • the modified GV may comprise a lipid membrane and one or more proteins associated with the lipid membrane.
  • the one or more proteins comprise CD9, CD81 , BSG, SLC3A2, or a combination thereof. Alternatively or in addition, the one or more proteins do not include CD63 and/or LAMP1.
  • the modified extracellular vesicle may comprise sialic acid associated with the lipid membrane of the extracellular vesicles.
  • the viral particle loaded to the modified extracellular vesicle is an adeno-associated virus, e.g., serotype 1 (AAV1) or AAV serotype 6 (AAV6) particles.
  • the AAV viral particle is an AAV6 viral particle, which comprises a modified AAV6 capsid protein and the AAV viral genome.
  • the modified AAV6 capsid protein may comprise mutations at: (a) position 129 of SEQ ID NO: 2; and (b) position 492 and/or position 705 of SEQ ID NO: 2; and wherein the AAV particle infects human intestinal epithelium cells.
  • the modified AAV6 capsid protein further comprises mutations (e.g., amino acid substitutions) at position 445, position 502, position 663, position 731 in SEQ ID NO:2, or a combination thereof.
  • the amino acid substitutions may comprise F129L, Y445F, T492V, T502S, S663L, Y705F, ⁇ 73 IF, or a combination thereof.
  • the modified AAV6 capsid protein comprises amino acid substitutions of F129L, Y445F, T492V, Y705F, and Y731F.
  • the one or more lectins, the viral particle or both are embedded in the lipid membrane, associated to one or more of the proteins associated with the lipid membrane, and/or associated to the sialic acid associated with the lipid membrane.
  • the viral particle is encapsulated by the lipid membrane, e.g., fully or partially encapsulated by the lipid membrane.
  • the viral particle can be embedded in the lipid membrane, associated to one or more of the proteins associated with the lipid membrane, associated to the sialic acid associated with the lipid membrane and/or encapsulated by the lipid membrane, e.g., fully or partially by the lipid membrane.
  • the modified GV such as modified EV displays one or more lectins that bind enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract (e.g., duodenum, upper jejunum, lower jejunum, ileum, cecum, colon, or rectum).
  • GI gastrointestinal
  • the one or more lectins comprise ECL, SBA, GSL2, UEA, PNA, GSL1, WGA, PHAL and DBA.
  • the lectin is ECL, UEA1, or a combination thereof.
  • the one or more lectins are attached to the modified extracellular vesicle via a receptor-ligand pair, for example, biotin-streptavidin, or nitrilotriacetic acid-His tag.
  • the lipid membrane of the modified GV e.g., EV
  • the lipid membrane of the modified GV comprises phospholipids, cholesterol, and/or tocopherol, which is conjugated to polyethylene glycol (PEG) chains, and wherein the one or more lectins form a covalent bond to a functional moiety linked to the PEG chain.
  • Exemplary functional moieties include, but are not limited to, a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amine group, a phosphate group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain-promoted alkyne-nitrone cycloaddition (SPANG).
  • CuAAC Copper(I)-catalyzed azide-alkyne cycloaddition
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • SPANG strain-promoted alkyne-nitrone cycloaddition
  • the PEG chains have a molecular weight ranging from about 1-10 kDa, optionally ranging from about 2-5 kDa.
  • the lectin is linked to a lipid and a PEG chain to form a lectin- lipid- PEG conjugate, which is integrated into the lipid membrane of the extracellular vesicle via hydrophobic insersion.
  • the AAV1 or AAV6 particles loaded therein may comprise an AAV viral genome, which comprises an expression cassette comprising a gene of interest.
  • the AAV viral genome may further comprise a promoter, an intron, one or more polyadenylation signaling sequences, or a combination thereof.
  • the expression cassette comprises a promoter, which optionally is a short CMV promoter.
  • the expression cassette further comprises a Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE).
  • WP Woodchuck Hepatitis Virus
  • WPRE Woodchuck Hepatitis Virus
  • the AAV viral genome is a self-complementary AAV viral genome.
  • the gene of interest carried by the viral particle may encodes a therapeutic protein or a therapeutic nucleic acid.
  • the gene of interest may encode a therapeutic protein. Examples include an antibody, an enzyme, a growth factor, a hormone, a vaccine antigen, an antithrombotic, an antithrombolytic, a toxin, a chemokine, a cytokine, an anti-viral peptide, or an immunogen.
  • a pharmaceutical composition comprising one or more of the modified GVs such as EVs provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for oral administration.
  • the pharmaceutical composition may comprise two or more AAV1 and/or AAV6 viral particles (e.g., those disclosed herein), wherein the AAV viral genomes in the two or more AAV1 and/or AAV6 viral particles express two or more polypeptides, which form a multi-chain protein.
  • the multi-chain protein is a full-length antibody, a hetero-muhimeric immunogen, or a hetero-multimeric enzyme.
  • the present disclosure features a method for delivering a viral particle to a compartment of a gastrointestinal (GI) tract, the method comprising administering orally to a subject in need thereof an effective amount of any of the pharmaceutical compositions set forth herein comprising any of the modified GVs such as modified EVs (e.g., WEVs) as also disclosed herein.
  • modified GVs such as modified EVs (e.g., WEVs) as also disclosed herein.
  • the present disclosure provides any of the pharmaceutical compositions disclosed herein for use in delivering the cargo carried by the viral particles loaded to the modified glycocalyx vesicles (e.g., extracellular vesicles) in the pharmaceutical composition and for treating a disease targeted by such a therapeutic cargo.
  • the modified GVs such as EVs (e.g., WEVs) disclosed herein or pharmaceutical comprising such for manufacturing a medicament for treatment of intended diseases based on the therapeutic cargo carried by die viral particles loaded in the GVs.
  • a method for preparing GVs may comprise: (i) incubating a formulation comprising a population of GVs, a viral particle and one or more lectins under conditions allowing for tethering of the viral particle and the optional one or more lectins to the GVs; wherein the one or more lectins are lectins that bind enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract; and (ii) collecting modified GVs produced in step (i), which are loaded with the viral particle and surface modified with the one or more lectins.
  • GI gastrointestinal
  • the formulation further comprises PEG.
  • the method may comprise: (i) applying a high shear force to a formulation comprising the GVs such as EVs and the viral particles; and (ii) collecting the formulation comprising GVs loaded with the viral particles formed in step (i).
  • step (i) may be performed using a microfluidic device.
  • the microfluidic device comprises one or more channels having a diameter of about 75 pin to about 1,100 pm.
  • the formulation runs through the microfluidic device for one or more passes in step (i).
  • Each pass may comprise a pressure of about 10,000 to about 40,000 psi. In one example, the pressure is about 20,000 psi.
  • the formulation runs through the microfluidic device for up to three passes. In one specific example, the formulation runs through the microfluidic device for one pass under a pressure of about 20,000 psi .
  • step (i) comprises probe sonication.
  • the probe sonication comprises about 5-30 seconds pulse on.
  • the probe sonication is performed under a controlled temperature.
  • the formulation further comprises polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG may have a molecular weight ranging from about 300 Da to about 10,000 Da.
  • the PEG may have a molecular weight ranging from about 400 Da to about 8,000 Da, e.g., about 400 Da to about 3,000 Da.
  • PEG having a molecular weight of about 400 can be used.
  • the PEG in the formulation is about 10% (w/v) to about 40% (w/v). In some examples, the PEG in the formulation is about 30% (w/v).
  • any of the method disclosed herein may further comprise removing PEG from the formulation after the loading step ( ⁇ ?.g. , by a high shear force or by electroporation).
  • the PEG may be removed by tangential flow filtration (TFF).
  • the formulation in step (i) comprises the GVs ⁇ e.g., EVs) and the viral particles at a ratio of 1:1.
  • the glycocalyx vesicles are EVs such as whey extracellular vesicles (WEVs),
  • the GVs comprise a lipid membrane and one or more proteins associated to the lipid membrane.
  • the one or more proteins comprise CD9, CD81, BSG, SLC3A2, or a combination thereof. In some embodiments, the one or more proteins do not include CD63 and/or LAMPE In some embodiments, the GVs comprise sialic acid associated with the lipid membrane.
  • the viral particle can be an adeno-associated virus, e.g., serotype 1 (AAV1) or AAV serotype 6 (AAV6) particles as disclosed herein (e.g., the AAV6 variants disclosed herein).
  • AAV1 or AAV6 particles may comprise an AAV viral genome, which comprises an expression cassette comprising a gene of interest.
  • the AAV viral genome further comprises a promoter, an intron, one or more polyadenylation signaling sequences, or a combination thereof.
  • the expression cassette comprises a promoter, which optionally is a short CM V promoter.
  • the expression cassette further comprises a Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE).
  • WP Woodchuck Hepatitis Virus
  • WPRE Woodchuck Hepatitis Virus
  • the AAV viral genome is a self-complementary AAV viral genome.
  • any of the viral particles disclosed herein, for example, AAV1 or AW6, may carry a gene of interest in the viral genome.
  • the gene of interest encodes a therapeutic protein or a therapeutic nucleic acid.
  • the gene of interest encodes a therapeutic protein, which optionally is an antibody, an enzyme, a growth factor, a hormone, a vaccine antigen, an antithrombotic, an antithrombolytic, a toxin, a chemokine, a cytokine, an anti-viral peptide, or an immunogen.
  • the present disclosure provides a method tor delivering an agent to intestinal epithelium cells, the method comprising contacting intestinal epithelium cells with one or more AAV viral particles, each of which comprises an AAV viral genome that expresses an agent.
  • the one or more AAV viral particles are A AVI or AAV6 as disclosed herein, for example, any of the AAV6 variants disclosed herein.
  • the contacting step comprises administering (e.g., orally) to a subject in need thereof a pharmaceutical composition disclosed herein that comprises the one or more AAV viral particles.
  • the AAV viral genome comprises an expression cassette, which comprises a gene of interest encoding the agent, which is protein-based or nucleic acid-based.
  • the expression cassette further comprises a promoter, an intron, one or more polyadenylation signaling sequences, or a combination thereof.
  • the expression cassette further comprises a Woodchuck Hepatitis Virus (WHP) post transcriptional regulatory element (WPRE),
  • WPRE Woodchuck Hepatitis Virus
  • the expression cassette comprises a promoter, which optionally is a short CMV promoter.
  • the viral genome is a self-complementary AAV viral genome.
  • the gene of interest encodes a therapeutic protein or a therapeutic nucleic acid.
  • the gene of interest encodes a therapeutic protein, which optionally is an antibody, an enzyme, a growth factor, a hormone, a vaccine antigen, an antithrombotic, an antithrombolytic, a toxin, a chemokine, a cytokine, an anti-viral peptide, or an immunogen.
  • the intestinal epithelium cells are in contact with two AAV viral particles, and wherein the AAV viral genomes in the two AAV viral particles collectively express two polypeptides, which form a multi-chain protein.
  • the multi-chain protein is a full-length antibody, a hetero-multimeric immunogen, or a hetero-multimeric enzyme.
  • the present disclosure also features an adeno-associated viral (AAV) particle, comprising a modified AAV6 capsid protein as disclosed herein and the AAV viral genome.
  • AAV adeno-associated viral
  • a nucleic acid encoding the modified AAV6 capsid protein.
  • the nucleic acid can be a vector, for example, an expression vector or an AAV vector.
  • a host cell comprising any of the nucleic acids disclosed herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more A AV 6 viral particles as those disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises two or more AAV6 viral particles.
  • the AAV viral genomes in the two or more AAV6 viral particles express two or more polypeptides, which form a multi-chain protein, for example, a full-length antibody, a hetero-multimeric immunogen, or a hetero-multimeric enzyme.
  • any of the modified GVs such as EVs disclosed herein, the AAV1 or AAV6 viral particles disclosed herein, or pharmaceutical compositions comprising such for use in delivering a gene of interest to intestinal epithelium cells in a subject, e.g., via oral administration.
  • any of the modified E Vs disclosed herein, the AAV 1 or AAV6 viral particles disclosed herein, or pharmaceutical compositions comprising such for use in manufacturing a medicament for treating a disease that is targeted by the therapeutic agent expressed from the gene of interest are provided herein.
  • FIGs. 1A-1D include diagrams showing the stability of WE V particles under microfluidization.
  • FIG. 1A Graph showing concentration WEV particles by microfluidizer under 10,000 psi and 30,000 psi, single and triple pass.
  • FIG. IB Graph showing mean particle size of WEV particles by microfluidizer under 10,000 psi and 30,000 psi, single and triple pass.
  • FIG. 1C Transmission electron microscopy images of WEV particles by microfluidizer under 10,000 psi, triple pass.
  • FIG. ID Transmission electron microscopy images of WEV particles by microfluidizer under 30,000 psi, triple pass.
  • FIGs. 2A-2B include diagrams showing the stability of adeno-associated virus (AAV) particles by microfluidizer.
  • FIG. 2A Graph showing concentration AAV particles by microfluidizer under 10,000 psi and 30,000 psi, single and triple pass as determined by Zetasizer (left bar in each microfluidization condition) and ELISA (right bar in ach microfluidization condition).
  • FIG. 2B Graph showing mean particle size of A AV particles by microfluidizer under 10,000 psi and 30,000 psi, single and triple pass.
  • FIGs. 3A-3B include diagrams showing the stability of WEV particles under probe sonication.
  • FIG. 1A Graph showing concentration AAV particles by microfluidizer under 10,000 psi and 30,000 psi, single and triple pass as determined by Zetasizer (left bar in each microfluidization condition) and ELISA (right bar in ach microfluidization condition).
  • FIG. 3A Graph showing concentration WEV particles by probe sonication under mild and harsh conditions.
  • FIG. 3B Graph showing mean particle size of WEV particles by byprobe sonication under mild and harsh conditions. Mild condition: temperature controlled; 5 sec pulse on. Harsh condition: temperature uncontrolled; ,30sec pulse on.
  • FIGs. 4A and 4B include diagrams showing the stability of AAV particles under probe sonication.
  • FIG. 4A Graph showing concentration WEV particles by probe sonication under mild and harsh conditions.
  • FIG. 4B Graph showing mean particle size of AAV particles by by probe sonication under mild and harsh conditions. Mild condition: temperature controlled; 5sec pulse on. Harsh condition: temperature uncontrolled; 30sec pulse on.
  • FIG.5 is a diagram showing the association of A AV- WEV efficiencies by various methods (microfluidizer, probe sonication and electroporation) in the presence of various fusogenic agents as indicated.
  • fusogenic agent indicated at the x-axis from left to right: microfluidizer, sonication, and electroporation.
  • FIGs. 6A and 6B include a schematic illustration of exemplary self-complementary adeno-associated virus (sc AAV) genomes carrying genes of interest (GOT).
  • FIG. 1A Schematic illustration of a scAAV genome resulting from AAV production using plasmid pTRS-KS-EGFP.
  • FIG. 1A Schematic illustration of a scAAV genome resulting from AAV production using plasmid pTRS-KS-EGFP.
  • IB Schematic illustration of exemplary scAAV genome carrying a GOI.
  • Upper panel Design of the exemplary scAAV genome.
  • Bottom panel Expression cassette for two GOIs, EPO and NLuc linked by a 2A self-cleaving peptide.
  • FIGs. 7A-7F include a schematic illustration of intestinal organoid architecture and culture formats used herein.
  • FIG. 7A Schematic illustration of a representative structure of an intestinal organoid.
  • FIG. 7B Schematic showing a 3D culture format with basolateral pole exposed.
  • FIG. 7C Schematic showing a 2D culture format with apical pole exposed.
  • FIG. 7D Schematic showing a 2D trans- well culture format with bi-directional secretion of the GOI gene product.
  • FIG. 7E a photo showing transduction efficiency of various AAV-GFP serotype particles as indicated observed in a 3D organoid culture format (see FIG. 7B), in which the basolateral pole is exposed.
  • FIG. 7F a photo showing transduction efficiency of various scAAV-GFP serotype vectors as indicated observed in a 2D organoid culture format (see FIG. 7C), in which the apical pole is exposed.
  • FIGs. 8A and 8B show's AAV transduction efficiency using AAV1 and AAV6 in the 2D trans-well organoid culture format disclosed herein (see FIG. 7D above).
  • FIG. 8A Diagram showing luminescence of apical and basolateral NanoLuc® Luciferase (NLuc) secretion following AAV transduction using AAV1, AAV6, and variants of AAV6 as indicated.
  • FIG. 8B Diagram showing electrochemiluminescence (ECL) signal of apical and basolateral human erythropoietin (EPO) secretion following AAV transduction using A AVI, AAV6, and variants of AAV6 as indicated. The results show bi-directional (apical and basolateral) secretion of NLuc following AAV-EPO/Nluc transduction.
  • FIG. 9 is a schematic showing mutations in AAV6 capsid protein as relative to the
  • AAV1 capsid protein AAV1 capsid protein.
  • FIGs. 10A and 10B include diagrams showing bi-directional secretion of encoded proteins by AAV transduced cells.
  • FIG. 10A Diagram showing AAV transduction efficiency obtained from an NLuc luminescence assay, using AAV1, AAV6, and variants of AAV6 as indicated.
  • FIG. 10B Diagram showing AAV transduction efficiency indicated by EPO secretion using A AVI, AAV6, and variants of AAV6 as indicated.
  • FIGs. 11A and 11B include diagrams showing expression of nanoluciferase (nLuc) and erythropoietin (EPO) mediated by adeno-associated virus serotype 1 (AAV1) and serotype 6 (AAV6) infection of luminal cells of the duodenum in fasted female C57B1/6 mice after direct administration of the AAV viral particles to the duodenum.
  • FIG. 11A AAV transduction efficiency obtained from an NLuc luminescence assay from mouse plasma, using AAV1 and AAV6 as indicated.
  • FIG. 11B spleen sizes and weights of female C57B1/6 indicating EPO-mediated splenomegaly in the treated animals using AAV1 and AAV6 as indicated.
  • FIG. 12 is a chart showing detection of WGA binding to mouse duodenal by an in vivo imaging system (IVIS).
  • IVIS in vivo imaging system
  • FIGs. 13A-13C include diagrams showing delivery of reporter proteins via WEV-AAV- lectin particles using the Plasma NanoGio® assay.
  • FIG. 13A total flux levels in plasma samples of treated mice on Day 2 post administration of the WEV-A AV-1 ectin particles.
  • FIG. 13A total flux levels in plasma samples of treated mice on Day 2 post administration of the WEV-A AV-1 ectin particles.
  • FIG. 13B total flux levels in plasma samples of treated mice on Day 4 post administration of the WE V- AAV- lectin particles.
  • FIG. 13C total flux levels in plasma samples of treated mice on Day 10 post administration of the WEV-A AV-1 ectin particles.
  • FIGs. 14A and 14B include diagrams showing delivery levels of reporter proteins in mice treated by the WEV-AAV-lectin particles as indicated.
  • FIG. 14A total flux levels in animals treated by the particles at different time points as indicated.
  • FIG. 14B weight of spleens from the various treatment groups at Day 10 post treatment.
  • FIG. 15 is a schematic illustration of surface functionalization strategies to conjugate lectin molecules to particle surfaces.
  • FIG. 16 is a schematic showing exemplary approaches for surface engineering of lectins, including hydrophobic insertion and covalent modifications.
  • FIG. 17 is a schematic depicting assembly of modified WE Vs carrying AAV payloads and surface lectin molecules.
  • FIGs. 18A-18H include diagrams showing AAV transduction-mediated expression of transgenes in the intestinal epithelial monolayer of Balb/c mouse, non-human primates, and human donors
  • FIG. 18A Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in Balb/c mouse obtained from an NLuc luminescence assay, using AAV1, AAV6, and AAV6.2FVFF as indicated in the respective viral loads.
  • FIG. 18B Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in Balb/c mouse indicated by EPO secretion using A AVI , AAV6, and AAV6.2FVFF as indicated in the respective viral loads.
  • FIG. 18A Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in Balb/c mouse obtained from an NLuc luminescence assay, using AAV1, AAV6, and AAV6.2FVFF as indicated in the respective viral loads.
  • FIG. 18B Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in Balb/c mouse indicated
  • FIG. 18C Image of A AV 1 -transduction of intestinal epithelial monolayer in Balb/c mouse for expression of GFP by epifluorescence microscopy at viral dose of 1E10.
  • FIG. 18D Image of AAV6-transduction of intestinal epithelial monolayer in Balb/c mouse for expression of GFP by epifluorescence microscopy at viral dose of 1E10.
  • FIG. 18E Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in NHP obtained from an NLuc luminescence assay, using AAV1, AAV6, and AAV6.2FVFF as indicated in the respective viral loads.
  • FIG. 18C Image of A AV 1 -transduction of intestinal epithelial monolayer in Balb/c mouse for expression of GFP by epifluorescence microscopy at viral dose of 1E10.
  • FIG. 18E Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in NHP obtained from an NLuc luminescence assay, using AAV1, AAV6, and AAV6.2FVFF as indicated
  • FIG. 18F Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in NHP indicated by EPO secretion using AAV1, AAV6, and AAV6.2FVFF as indicated in the respective viral loads.
  • FIG. 18G Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in human donor 1 (duodenal and jejunal) obtained from an NLuc luminescence assay, using AAV1, AAV 6, and AAV6.2FVFF as indicated in the respective viral loads.
  • FIG. 18G Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in human donor 1 (duodenal and jejunal) obtained from an NLuc luminescence assay, using AAV1, AAV 6, and AAV6.2FVFF as indicated in the respective viral loads.
  • 18H Diagram showing the species screen of A AV transduction efficiency of intestinal epithelial monolayer in C57BI/6 mouse, Balb/c mouse, day 7 human donor 1 (jejunal and duodenal) obtained from an NLuc luminescence assay, using AAV1, AAV6, and AAV6.2FVFF as indicated in the respective viral loads.
  • FIG. 19 Diagram showing AAV transduction efficiency of intestinal epithelial monolayer in rat obtained from an NLuc luminescence assay-day 3, using AAV1 , AAV6, and AAV6.2FVFF at IE 10 viral load in matrigel and gelatin basement membrane matrix.
  • FIG. 20 is a diagram showing expression of the NLuc transgene in mouse via delivery of GV-AAV-NLuc-EPO complex (ORA(WAV) to mouse intestinal tissues.
  • Glycocalyx vesicles (a.k.a., glycocalyx stabilized vesicles) are vesicles carrying a glycocalyx.
  • the glycocalyx known as the precellular matrix, is a glycoprotein and glycolipid covering that surrounding the cell membranes of bacteria, epithelial cells, or other cells.
  • the GVs disclosed herein are extracellular vesicles (EVs), which are lipid membrane-containing vesicles naturally released by many types of cells.
  • EVs can carry various types of cargos such as protein, nucleic acids, lipids, metabolites, etc.
  • EVs include various subtypes based mostly on biogenesis, for example, cell pathway, cell or tissue identity, condition of origin, etc.. Examples include ectosomes, microvesicles, microparticles, exosomes, oncosomes, apoptotic bodies, exomeres, etc.
  • Ectosome,” “microvesicle” (MV), and “'microparticle” (MP) are particles released from the surface of cells.
  • exosome biogenesis begins with pinching off of endosomal invaginations into the multi vesicular body (MVB), forming intraluminal vesicles (ILVs).
  • EVs are an optimal vehicle for oral delivery of therapeutic agents because of their stability profile at acidic pH and other high-stress or degradative conditions.
  • Viral particles such as adeno-associated viral (AAV) particles, are a commonly used vehicle for delivering therapeutic agents (e.g. , for delivering transgenes in gene therapy).
  • AAVs when administered to the systemic circulation (via any route), generate immune responses that render subsequent treatments ineffective at delivering genetic cargo to the target cell types.
  • a significant part of the population has preexisting neutralizing antibodies for the major AAV serotypes, making even the first treatment inefficient and leading to elimination of many patients from the trials.
  • Oral delivery of AAVs to the gut holds the distinct advantage of bypassing humoral immune responses that may be directed against the viral particles, due to a dearth of antibodies and a generally immune-tolerizing environment in the small intestine. Delivery of the genetic payload therefore proceeds unimpeded and should allow for repeated dosing without loss of effectiveness. Safety is a primary concern with any gene therapy approach.
  • Glycocalyx Vesicles e.g., extracellular vesicles (EVs)
  • EVs extracellular vesicles
  • GVs purified from a suitable source typically have a high sialic acid content, providing a negatively charged anti-adhesive coating.
  • mucus layers in portions of the Gl tract may have heavily sialylated glycocalyx.
  • the heavily negatively charged GVs can be repelled by the also negatively charged mucosa glycocalyx, promoting proximal to distal transit of GVs through the GI lumen.
  • the identification of neutral lectins that are capable of binding to GI compartments/cells, e.g., enterocytes with low binding affinity to GVs are useful to create an adhesive surface for enterocytes so as to facilitate delivery of the viral particle payloads to GI cells.
  • Such lectins can be tethered (e.g., chemically) to the GV surface, as di sclosed herein.
  • the present disclosure provides the Orasome platform strategy involving glycacalyx vesicles such as extracellular vesicles (e.g., WEVs) carrying viral particle payloads and surface neutral lectins that bind to GI cells for delivery of cargos of interest via oral administration.
  • glycacalyx vesicles such as extracellular vesicles (e.g., WEVs) carrying viral particle payloads and surface neutral lectins that bind to GI cells for delivery of cargos of interest via oral administration.
  • WEVs extracellular vesicles
  • the Orasome technology relies on extracellular vesicles which are loaded with one or more payloads of interest. See, e.g., FIG.
  • the GVs may optionally be modified on its surface with one or more lectin(s) that binds GI cells, such as those disclosed herein.
  • the payload may be viral particles such as adeno-associated viral particles (e.g., enterocyte-tropic AAV serotypes such as A A VI or AAV 6), which may be loaded on the surface of EVs (or encapsulated or partially encapsulated).
  • the extracellular’ vesicles may also have one or more lectins as disclosed herein tethered on the surface. The one or more lectins are able to target GI cells but do not promote the binding or association of GVs, e.g., EVs such as WEVs to one another.
  • the modified GVs Upon oral administration, the modified GVs are delivered to GI compartments and release the viral particle payload via membrane metabolism.
  • the released viral particles enter GI cells (e.g., enterocytes) and express the cargo (e.g., therapeutic protein or therapeutic nucleic acid) encoded by the transgene carried by the viral genome.
  • the cargo thus expressed is secreted by the enterocytes, leading to systemic delivery of the cargo for, e.g., treatment purposes.
  • G Vs Glycocalyx Vesicles
  • the GVs for use in the present disclosure may be ectosomes, microvesicles (MVs), exosomes, and/or apoptotic bodies, which are subtypes of EVs differentiated based on their biogenesis, release pathways, size, content, and function.
  • the GVs for use in the present disclosures are ectosomes.
  • Glycocalyx vesicles for example, extracellular vesicles such as WEVs, can encapsulate or otherwise carry therapeutic cargos such as miRNA species, and can enable oral delivery of a variety of therapeutic agents.
  • the present disclosure harnesses lectin-modified GVs such as lectin-modified EVs to meet the urgent need for suitable delivery vehicles for therapeutics that were previously not orally administrable or suffered from other delivery challenges such as poor bioavailability, storage instability, metabolism, off-target toxicity, or decomposition in vivo.
  • the glycocalyx vesicles such as extracellular vesicle is approximately round or spherical in shape.
  • the GV is approximately ovoid, cylindrical, tubular, cube, cuboid, ellipsoid, or polyhedron in shape.
  • the GV may be part of a cluster, collection, or formation of extracellular vesicles.
  • compositions comprising GVs such as EVs for use in delivering cargos such as those disclosed herein may have a relative abundance of proteins with a molecular weight of about 25-30 kDa (e.g., casein) no greater than about 40% and/or a relative abundance of proteins with a molecular weight of about 10-20 kDa (e.g., lactoglobulin) no greater than 25 % .
  • the GVs such as EVs (e.g., WEVs) can be about 20 nm - 1000 nm in diameter or size. In some embodiments, the GV is about 20 nm to about 200 nm in size. In some embodiments, the GV is about 20 nm to about 190 nm or about 25 nm to about 190 nm in size. In some embodiments, the GV is about 30 nm to about 180 nm in size. In some embodiments, the GV is about 35 nm to about 170 nm in size. In some embodiments, the GV is about 40 nm to about 160 nm in size. In some embodiments, the GV is about 50 nm to about
  • the GV is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 1 15 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 .
  • an average vesicle size in a vesicle composition or plurality of vesicles isolated or derived from whey is about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nrn, about 55 nm, about 60 nm, about 65 nrn, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm
  • an average vesicle size in a vesicle composition or plurality of vesicles isolated or derived from whey is about 20 nm to about 200 nm, about 20 nm to about 190 nm, about 25 nrn to about 190 nm, about 30 nm to about 180 nrn, about 35 nm to about 170 nm, about 40 nrn to about 160 nm, about 50 nm to about 150, about 60 to about 140 nm, about 70 to about 130, about 80 to about 120, or about 90 to about 110 nm in average size.
  • the size of the GVs disclosed herein is determined by Dynamic Light Scattering (DLS) or nanoparticle tracking analysis (NTA).
  • DLS Dynamic Light Scattering
  • NTA nanoparticle tracking analysis
  • the glycocalyx vehicles (GVs) described herein can be derived from any suitable source, for example, cultured cells capable of producing GVs (e.g., EVs), biological samples such as tissue samples or body fluid samples.
  • GVs e.g., EVs
  • biological samples such as tissue samples or body fluid samples.
  • the GVs may be purified from whey, which refers to the liquid remaining after milk has been curdled and strained.
  • WE Vs Whey-derived vesicles
  • the WE Vs are purified from sweet whey, which can be a byproduct resulting from the manufacture of rennet types of hard cheese.
  • the WEVs may be purified from acid whey, which can be a byproduct during the manufacturing of acid types of dairy products (e.g., cottage cheese or strained yogurt).
  • Whey may be from any suitable mammal species, for example, primates (e.g., human, ape, monkey, lemur), rodentia (e.g., mouse, rat, etc), carnivora (e.g., cat, dog, etc.), lagomorpha (e.g. , rabbit, etc), cetartiodactyla (e.g., pig, cow, deer, sheep, camel, goat, bufflo, yak, etc.), perissodactyla (e.g., horse, donkey, etc.).
  • primates e.g., human, ape, monkey, lemur
  • rodentia e.g., mouse, rat, etc
  • carnivora e.g., cat, dog, etc.
  • lagomorpha e.g. , rabbit, etc
  • cetartiodactyla e.g., pig, cow, deer, sheep, camel, goat, bufflo,
  • the whey product or vesicles derived therefrom is from human, cow, buffalo, pig, goat, rat, mouse, sheep, camel, donkey, horse, llama, alpaca, vicuna, reindeer, moose, or yak milk or colostrum.
  • the whey product is from cow. Methods for preparing whey products are known in the art.
  • a whey product for use in purifying the WEVs can be lyophilized.
  • Lyophilized whey products can be reconstituted using standard procedures as recommended by manufacturer’s instruction and/or as known in the art, for example, by mixing distilled water with lyophilized whey or the GVs derived therefrom at room temperature such that the whey or the GVs derived therefrom is present at a suitable final concentration by weight relative to water.
  • the GVs described herein can be any types of particles found in a suitable source as disclosed herein.
  • the GVs used in the methods described herein may comprise one or more of the following molecules: lipid, protein, glycoprotein, glycolipid, lipoprotein, phospholipid, phosphoprotein, peptide, glycan, fatty acid, sterol, steroid, and combinations thereof.
  • the GVs described herein comprise a lipid-based membrane to which one or more proteins are associated.
  • the proteins may be attached to the surface of the lipid membrane or embedded in the lipid membrane. Alternatively or in addition, the proteins may be encapsulated by the lipid membrane.
  • the GVs may contain endogenous RNA, such as miRNA.
  • the GVs disclosed herein may comprise one or more lipids selected from faty acid, sterol, steroid, cholesterol, and phospholipid.
  • the lipid membrane of the extracellular vesicles described herein may comprise ceramides or derivatives thereof, gangliosides, phosphatidylinositols (PI) such as alpha-lysophosphatidylinositol (LPI), phosphatidyl serine (PS), cholesterol (CHOL), phosphatidic acids (PA), glycerol or derivatives thereof, such as diacylglycerol (DAG) or phosphatidylglycerol (PG), sphingolipids, or combinations thereof.
  • PI phosphatidylinositols
  • LPI alpha-lysophosphatidylinositol
  • PS phosphatidyl serine
  • PA phosphatidic acids
  • glycerol or derivatives thereof such as diacylgly
  • Ceramides are a family of lipid molecules composed of sphingosine and a fatty acid. Examples include, but are not limited to, ceramide (Cer), lactosylceramide (LacCer), hexosylceramide (HexCer), and globotriaosylceramide (Gb3).
  • Gangliosides are a family of molecules composed of a glycosphigolipid with one or more sialic acids, for example, n-acetylneuraminic acid (NANA). Examples include, but are not limited to, GM1, GM2, GM3, GDla, GDlb, GD2, GTlb, GT3, and GQ1.
  • Sphingolipids are a class of lipids containing a backbone of sphingoid bases and a set of aliphatic amino alcohols that includes sphingosine. Examples include sphingomyelin (SM).
  • the extracellular vesicles may contain lipids such as phosphatidylcholines (PC), cholesteryl ester (CE), phosphatidylethanolamine (PE), and/or lysophosphatidylethanolamine (LPE).
  • PC phosphatidylcholines
  • CE cholesteryl ester
  • PE phosphatidylethanolamine
  • LPE lysophosphatidylethanolamine
  • the GVs may comprise one or more proteins, which may be associated with the lipid membranes also described herein.
  • a “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long.
  • a protein may refer to an individual protein or a collection of proteins.
  • a peptide may contain ten or more amino acids but less than 50.
  • a polypeptide or a protein may contain 50 or more amino acids.
  • a peptide, polypeptide, or protein may have a mass from about 10 kDa to about 30 kDa, or about 30 kDa to about 150 or to about 300 kDa.
  • Exemplary proteins may contain only natural amino acids, although non-natural amino acids (?. ⁇ ?., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification.
  • a protein may also be a single molecule or may be a multi-molecular complex.
  • a protein may be a fragment of a naturally occurring protein or peptide.
  • a protein may be naturally occurring, recombinant, synthetic, or any combination of these.
  • the EVs disclosed herein are WEVs purified from whey. Such WEVs may comprise ectosomes.
  • the WEVs disclosed herein comprise one or more proteins of CD9, CD81, BSG, and SLC3A2.
  • the WEVs disclosed herein are free of CD63 and/or LAMP1, e.g., detection of the involved proteins (e.g., CD63 and LAMP!) by a conventional method or only marginal signal is detected such that presence or absence of the involved proteins cannot be determined.
  • Any of the protein moieties in the GVs may be glycosylated, i.e., linked to one or more glycans at one or more glycosylation sites.
  • a glycan is a compound consisting of one or more monosaccharides linked glycosidically, including for example, the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.
  • Glycans can be homo- or heteropolymere of monosaccharide residues and can be linear or branched.
  • Glycans can have O-glycosidic linkages ( li nked to oxygen in a serine or threonine residue of a peptide chain) or N-Linked linkages (linked to nitrogen in the side chain of asparagine in the sequence Asn-X- Ser or Asn-X-Thr, where X is any amino acid except proline). Glycans bind lectins and have many specific biological roles in cell-cell recognition and cell-matrix interactions.
  • glycosylated proteins that can be present in the biological membrane of a GV as described herein can include any appropriate glycan.
  • glycans include, without limitation, N-glycans (e.g., N-acetyl-glucosamines and N-glycan chains), O-glycans, C- glycans, sialic acid, galactose or mannose residues, and combinations thereof.
  • the glycan is selected from an alpha-linked mannose, Gal p 1-3 GalNAc 1 Ser/Thr, GalNAc, or sialic acid.
  • the GV comprises one or more glycoproteins or glycopolypeptides having a glycan selected from: galactose, mannose, O- glycans, N-acetyl- glucosamines, and/or N-glycan chains or any combination thereof.
  • a glycan selected from: galactose, mannose, O- glycans, N-acetyl- glucosamines, and/or N-glycan chains or any combination thereof.
  • the GV comprises one or more glycoproteins or glycopolypeptides having a glycan selected from: D- or L- glucose, erythrose, fucose, galactose, mannose, lyxose, gulose, xylose, arabinose, ribose, 2'-deoxyribose, glucosamine, lactosamine, polylactosamine, glucuronic acid, sialic acid, sialyl-Lewis X (SLex), N-acetyl-glucosamine, N- acetylgalactosamine, neuraminic acid, N-glycolylneuraminic acid (NeuSGc), N- acetylneuraminic acid (NeuSAc), an N-glycan chain, an O-glycan chain, a Core 1 , Core 2, Core 3, or Core 4 structure, or a phosphate- or acetate-modified analog thereof or a combination thereof.
  • the GV comprises a glycoprotein having one or more of the following glycans: terminal b-galactose, terminal a-galactose, N-acetyl-D-galactosamme, N-acetyl-D- galactosamine, and N-acetyl-D-glucosamine,
  • any of the glycans described herein may exist in free form in the GVs, which are also within the scope of the present disclosure.
  • the GVs may be treated by a suitable approach (e.g., enzyme digestion) to reduce the amount, of surface sialic acid residues or remove substantially surface sialic acid residues.
  • the GVs or a composition comprising such contain proteins having a molecular weight of about 25-30 kDa at a relative abundance of no greater than 40% (e.g., less than about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or less).
  • relative abundance of protein X in the composition refers to the percentage of protein X in the total protein content in the composition.
  • the proteins having a molecular weight of about 25-30 kDa are caseins.
  • the GVs or the composition comprising such may be substantially free of casein, e.g., cannot be detected by a conventional method or only a trace amount can be detected by the conventional method.
  • the GVs or a composition comprising such contain proteins having a molecular weight of about 10-20 kDa at a relative abundance of no greater than 25% (e.g., less than about 25%, about 20%, about 15%, about
  • the proteins having a molecular weight of about 10- 20 kDa are lactoglobulins.
  • the GVs or the composition comprising such may be substantially free of lactoglobulins.
  • casein refers to a family of related phosphoprotein commonly found in mammalian whey having a molecular weight of about 25-30 kDa.
  • Exemplary species include alpha-Sl -casein (aSl), alpha-S2-casein (aS2), p-casein, K-casein.
  • casein protein may refer to a specific species as known in the art, for example, those noted above. Alternatively, it may refer to a mixture of at least two different species. In some instances, casein can be the population of all casein proteins found in the whey of a mammal, for example, any of those described herein (e.g., cow, goat, sheep, yak, buffalo, camel, or human).
  • Lactoglobulin including a-lactoglobulin and P-lactoglobulin, is a family of whey proteins found in mammalian whey having a molecular weight of about 10-20 kDa.
  • p- lactoglobulin typically has a molecular weight of about 1 8 kDa and a-lactoglobulin typically has a molecular weight of about 15 kDa.
  • lactoglobulin may refer to one particular species, e.g., a-lactoglobulin or p-lactoglobulin. Alternatively, it may refer to a mixture of different species, for example, a mixture of a-lactoglobulin and P-lactoglobulin.
  • casein and/or lactoglobulin- depleted GVs such as EVs or compositions comprising such have a higher cargo loading capacity, e.g., oligonucleotide loading capacity, as compared with extracellular vesicles prepared by the conventional ultracentrifugation method.
  • cargo loading capacity e.g., oligonucleotide loading capacity
  • the GVs such as EVs described herein are stable under, for example, harsh conditions, e.g., low or high pH, sonication, enzyme digestion, freeze-thaw cycles, temperature treatment, etc.
  • Stable or stability means that the GVs maintain substantially the same intact physical structures and substantially the same functionality as relative to the GVs under normal conditions.
  • a substantial portion of the GVs e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above
  • the GVs may be resistant to enzymatic digestion such that a substantial portion of the GVs (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above) would have no substantial structural changes in the presence of enzymes such as digestive enzymes. Further, the GVs that are stable after multiple rounds of freeze-thaw cycles (e.g., up to 6 cycles) would have a substantial portion (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above) that has no substantial structural changes and/or functionality changes after the multiple freezethaw cycles.
  • a substantial portion of the GVs e.g., at least 60%, at least 70%, at least 80%, at least 90%, or above
  • the GVs described herein are able to deliver their cargo while withstanding stressed conditions or conditions under which the therapeutic agent would become deactivated, metabolized, or decomposed, e.g., saliva, digestive enzymes, acidic conditions in the stomach, peristaltic motions, and/or exposure to the various digestive enzymes, for example, proteases, peptidases, lipases, amylases, and nucleases that break down ingested components in the gastrointestinal tract.
  • the GVs such as EVs is stable in the gut or gastrointestinal tract of a mammalian species.
  • the GV is stable in the esophagus of a mammalian species. In some embodiments, the GVis stable in the stomach of a mammalian species. In some embodiments, the GVis stable in the small intestine of a mammalian species. In some embodiments, the GVis stable in the large intestine of a mammalian species. In some embodiments, the GVis stable at a pH range of about pH 1.5 to about pH 7.5. In some embodiments, the GVis stable at a pH range of about pH 2.5 to about pH 7.5. In some embodiments, the GVis stable at a pH range of about pH 4.0 to about pH 7.5.
  • the GVis stable in the. presence, of digestive enzymes, such as, for example, proteases, peptidases, nucleases, pepsin, pepsinogen, lipase, trypsin, chymotrypsin, amylase, bile and pancreatin (digestive enzymes in pancreas).
  • digestive enzymes such as, for example, proteases, peptidases, nucleases, pepsin, pepsinogen, lipase, trypsin, chymotrypsin, amylase, bile and pancreatin (digestive enzymes in pancreas).
  • the GVsdisclosed herein can protect cargo loaded therein (e.g., oligonucleotides) from enzyme digestion (e.g., nuclease digestion).
  • the GVs disclosed herein are stable after multiple rounds of freeze-thaw cycles.
  • the. GVsare stable after at least two freeze-thaw cycles e.g., at least 3 cycles, at least 4 cycles, at least 5 cycles, or at least 6 cycles.
  • the GVsare stable up to 10 freeze-thaw cycles e.g., up to 9 cycles, up to 8 cycles, up to 7 cycles, or up to 6 cycles.
  • the GVsdisclosed herein are stable after temperature treatment, e.g., incubated at a low temperature (e.g., at 4 °C) for a period (e.g., 1-3 days) or at a high temperature for period, e.g., at 60-80 °C for 30 minutes to 2 hours or at. 100-120 C C for 5-20 minutes.
  • a low temperature e.g., at 4 °C
  • a high temperature e.g., at 60-80 °C for 30 minutes to 2 hours or at. 100-120 C C for 5-20 minutes.
  • colloidal stability refers to the long-term integrity of dispersion and its ability to resist phenomena such as sedimentation or particle aggregation. This is typically defined by the time that dispersed phase particles can remain suspended without producing precipitates.
  • the GVs may be stable under physical processes, for example, sonication, centrifugation, and filtration. e. Glycocalyx Vesicle Modification
  • any of the GVs such as EVs disclosed herein may be further modified to alter one or more lipids, proteins, glycoproteins, glycolipids, lipoproteins, phospholipids, phosphoproteins, peptides, glycans, fatty acids, and/or sterols present in the natural GV.
  • the GVis modified by altering the quantity, concentration, or amount of a biomolecule naturally present e.g., the addition or complete or partial removal of a biomolecule naturally present (e.g., carbohydrate, such as a glycan; fatty acid, lipid).
  • the GVis modified by the addition of a biomolectde not. naturally present e.g., carbohydrate, such as a glycan; fatty acid; lipid, or protein, e.g., a glycoprotein).
  • the GVis modified to alter one or more lipids in the GV.
  • the lipid component of the extracellular vesicle is modified or altered, e.g., via the addition of one or more lipids not naturally present in the GVor by altering the amount (increasing or decreasing) of one or more lipids naturally present in the GV.
  • the GVis modified to reduce or remove one or more lipids For example, methyl-beta-cyclodextrin can be used to extract cholesterol from GVs.
  • the lipid component of the GVcan be altered or modified by known methods, including, for example, fusion with another vesicle having a lipid bilayer, e.g., liposome and/or lipid nanoparticle.
  • the altering the amount or content of the lipids on the GVs such as EVs affects the ability of the GVto interact, bind and/or fuse with another vesicle, e.g., a lipid particle encapsulating a particle to which a nucleic acid is attached as those disclosed herein.
  • altering the amount or content of lipids in the GV alters the overall charge of the GV.
  • the lipid contents of the GV may be altered such that it is negatively charged, which would facilitate its fusion with positively charged lipid particles comprising viral particles.
  • altering the charge of the vesicle makes the vesicle more attractive for interactions, binding and/or fusion with another vesicle, e.g., a nanoparticle, e.g., a lipid nanoparticle.
  • lipid nanoparticles and GVshaving lipid contents with opposite electrostatic charges are used to promote or improve interactions, binding and/or fission between the two types of particles.
  • interactions, binding and/or fusion is achieved between cargo-carrying lipid nanoparticles comprising negatively charged lipids and GVscomprising positively charged lipids.
  • fusion is carried out between cargo-carrying lipid nanoparticles comprising positively charged lipids and GVs comprising negatively charged lipids.
  • a GVhaving a decreased number of one or more of its native glycoprotein(s) is produced using an enzyme selected from a serine protease, cysteine protease or metalloprotease.
  • the enzyme is selected from trypsin, AspN, GluC, ArgC, chymotrypsin, proteinase K, and Lys-C.
  • the biological membrane is modified such that one or more of its native glycoprotein(s) is eliminated or not present. In some embodiments, the biological membrane is modified such that one or more of its native glycoprotein(s) is reduced.
  • the GVis modified to alter the amount or content of carbohydrate moieties present on a glycopolypeptide present in or associated with the GV. In some embodiments, the GVis modified to increase, decrease, or otherwise alter the glycan content of the GV, e.g., via the addition of one or more glycans not naturally present in the GVor by altering the amount (increasing or decreasing) of one or more glycans naturally present in the GV. In some embodiments, the biological membrane of the GVis modified such that one or more of its native glycoprotein(s) is altered.
  • the GVis modified to decrease or remove one or more glycoprotem(s) having one or more of the sugar moieties, to which the surface displayed lectin binds.
  • the GVcan be treated by an enzyme capable of removing glycans or sugar residues, e.g., glycosidase, exoglycosidase, endoglycosidase, glycoamidase, neuraminidase, galactosidase, peptide:N- glycosidase (PNGase), glycohydrolase, and any combination thereof.
  • the enzyme is selected from a p-N-acetylglucosaminidase, PNGase F, p (1-4) Galactosidase, O-Glycosidase, N- Glycosidase, N-glycohydrolase, Endo H, Endo D, Endo F2, EndoF3, and any combination thereof.
  • the GVsdisclosed herein has a modified glycocalyx as compared with the naturally-occurring counterpart.
  • Glycocalyx refers to the precellular matrix composed of glycoproteins and/or glycolipids that surrounds naturally-occurring GVs.
  • the glycocalyx of the GVscan be modified by removing surface sialic acid residues, e.g., by sialidase treatment.
  • the sugar content of glycocalyx of the GVs may be altered via, e.g., treatment of a glycosylation enzyme, a glycosyltransferase enzyme, or a combination.
  • two or more native glycoprotein(s) are altered such that at least one glycoprotein has an increased number of glycan residues and at least one other glycoprotein has a decreased number of glycan residues or is missing its glycan residue(s), wherein the glycoprotein(s) having an increased number of glycan residues is different from the glycoprotein(s) having a decreased number of glycan residues or missing glycan residues.
  • the one or more native glycoprotein(s) is altered such that it comprises a modified glycan.
  • the modified glycan comprises at least one carbohydrate moiety that differs from that of the glycan in the native glycoprotein(s).
  • the modified glycan comprises one or more galactose, mannose, O-glycans, N- acetyl- glucosamines, and/or N-glycan chains or any combination thereof.
  • the glycan is selected from comprises one or more D- or L- glucose, erythrose, fucose, galactose, mannose, lyxose, gulose, xylose, arabinose, ribose, 2'-deoxyribose, glucosamine, lactosamine, polylactosamine, glucuronic acid, sialic acid, sialyl-Lewis X (SLex), N-acetyl-glucosamine, N- acetyl-galactosamine, neuraminic acid, N- glycolylneuraminic acid (NeuSGc), N- acetylneuraminic acid (NeuSAc), an N-glycan chain, an O
  • the modified glycan lacks a portion of one or more of its carbohydrate chain(s). In some embodiments, the modified glycan is missing one or more of its carbohydrate chain(s). In some embodiments, the modified glycan comprises one or more altered carbohydrate chain(s). In some embodiments, the one or more native glycoprotein(s) is altered such that at least one glycan present on the glycoprotein(s) is substituted with a glycan that is not naturally present in the native glycoprotein(s). See also WO2018170332, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.
  • the GVs such as EVs disclosed herein can be treated by neuraminidase and/or sialidase to remove surface sialic acid residues.
  • the GVsused herein may be substantially free of surface sialic acid residues (e.g., not detectable by a conventional method.)
  • altering the number or content of the glycan residues on the GVs affects the colloidal stability of the GV. In some embodiments, altering the number or content of the glycan residues on the GVmodulates the interaction between extracellular vesicles and GI cells, e.g., enhances the uptake of GVsin G1 cells. In some embodiments, the altering the number or content of the glycan residues on the GVaffects the ability of the GVto interact, bind and/or fuse with another vesicle, e.g., a lipid particle encapsulating a nucleic acid-attaching particle (e.g., a viral particle) as those disclosed herein.
  • another vesicle e.g., a lipid particle encapsulating a nucleic acid-attaching particle (e.g., a viral particle) as those disclosed herein.
  • altering the number or content of the glycan residues alters the overall charge of the GV. In some embodiments, altering the number or content of the glycan residues in the GVsresults in a GVwith greater positive charge as compared to the unaltered vesicle. In some embodiments, altering the number or content of the glycan residues in the GVsresults in a GVwith greater negative charge as compared to the unaltered vesicle. In some embodiments, altering the charge of the vesicle makes the vesicle more attractive for interactions, binding and/or fusion with another vesicle, e.g., the lipid particle as disclosed herein.
  • lipid nanoparticles having lipid contents and GVshaving lipid and/or glycan or glycoprotein contents with opposite electrostatic charges are used to promote or improve interactions, binding and/or fusion between the two types of particles.
  • interactions, binding and/or fusion is achieved between cargo- carrying lipid nanoparticles comprising negatively charged lipids and GVscomprising positively charged lipids and/or glycoprotein or glycan contents.
  • fusion is carried out between cargo-carrying lipid nanoparticles comprising positively charged lipids and GVscomprising negatively charged lipids and/or glycoprotein or glycan contents.
  • altering the number or content of the glycan residues on the GVs improves the ability of the GVand/or the fused vesicle as described herein to be enriched and/or purified.
  • altering the number or content of the glycan residues on the GV improves the ability of the GVand/or the fused vesicle as described herein to be detected in vitro or in vivo.
  • anti-glycan antibodies or lectins are used to enrich and/or purify GVsand/or fused vesicles as described herein.
  • anti-glycan antibodies or lectins are used to detect and/or purify GVsand/or fused vesicles as described herein. Accordingly, methods to enrich and/or purify these GVsor fused vesicles are contemplated which comprise contacting anti-glycan antibodies or lectins with GVsand/or fused vesicles. In some embodiments, methods to detect GVsor fused vesicles using anti-glycan antibodies or lectins are contemplated. In some embodiments, the GVs such as EVsare modified to alter one or more proteins in the GV. In some embodiments, levels of existing GVproteins are reduced. In some embodiments, proteins which do not naturally occur in the GVare added.
  • the GVcan be modified to display a functional agent on the surface.
  • the functional agent may be any molecule having a desired bioactivity
  • Functional agent any molecule having a desired bioactivity, for example, targeting a particular tissue, reacting with a cognate ligand for surface modification, or facilitating purification of the GVsdisclosed herein.
  • a functional agent may facilitate interaction and/or fusion between the GVand a lipid particle comprising a particle to which a nucleic acid is attached as those disclosed herein so as to load the nucleic acid-containing particle into the GVs.
  • the functional agent may be a member of a receptor-ligand pair (e.g., biotin-streptavidin pair omilrilotriacetic acid (NTA)-His-tag pair), which can facilitate fusion of the GV to a lipid particle that displays the other member of the receptor- ligand pair.
  • the functional agent may be a tag commonly used for purification purposes, for example, a protein tag (e.g., His-tag, FLAG, etc.).
  • GVsisolated from a natural source may be subject to extrusion (e.g., once or multiple times) through a filter having a suitable size, e.g., 50 nM, 75 nM, or 100 nM, to change size distribution.
  • GVsisolated from one or more natural sources may be subject to homogenization (e.g., under high pressure in some instances) to cause fusion of particles.
  • extrusion or homogenization may be performed to GVsisolated from a natural source in the presence of other natural or artificial lipid membrane vesicles or protein micelles or aggregates to produce fused particles.
  • Such fusion may lead to change of protein and/or lipid content of the resultant particles, for example, incorporating non- naturally occurring lipids, which may present in the artificial lipid membrane particles.
  • additional lipids may be incorporated into GVsisolated from a natural source via saturation of the GVswith specific lipids of interest or incubating the GVswith lipid films, which may contain lipids of interest (e.g., cholesterol, phospholipids, ceramides, and/or sphingomy el ins .) .
  • any of the functional agents disclosed herein can be conjugated to a
  • the GV such as an EVusing a conventional method directly.
  • the GVcan be first modified by one or more polyethylene glycol (PEG) chains on the surface.
  • PEG chains may have a molecular weight ranging from about 1 kDa to about 10 kDa.
  • a functional chemical moiety may be added to the PEG chain and one or more of the functional agents may be linked to the PEG chain via the functional moiety, either directly or via a linker.
  • the functional moiety may be a chemical group capable of reacting with another group to form a covalent fond. Examples include, but are not limited to, a thiol group, an amine group, or an azide group. f . Glycocalyx Vesicle Preparation
  • glycocalyx vesicles such as extracellular vesicles (EVs) may be harvested from a suitable source as disclosed herein, e.g., whey.
  • a suitable source e.g., whey.
  • the GVdare produced and subsequently isolated from mammary epithelial cells lines adapted to recapitulate the GVsarchitecture of that naturally occurring in whey.
  • the GVsare provided using a cell line one in a batch-like process, wherein the GVsmay be harvested periodically from the cell line media.
  • the challenge with a cell line-based production methods is the potential for contamination from exosomes present in fetal bovine serum (media used to grow cells).
  • this challenge can be overcome with the use of suitable serum free media conditions so that GVspurified from the cell line of interest are harvested from the culture medium.
  • a filter such as a 0.2 micron filter is used to remove larger debris from a whey solution.
  • the method for separation of GV s includes separation based on specific GV properties such as size, charge, density, morphology, protein content, lipid content, or epitopes recognized by antibodies on an immobilized surface (immuno-isolation).
  • antibodies directed against epitopes located on a polypeptide selected from one or more of CD9, CD81, BSG, and/or SLC3A2 may be used to enrich WEV particles.
  • antibodies specific to CD63 or LAMP1 may be used for negative selection.
  • the separation method comprises a centrifugation step. In some embodiments, the separation method comprises PEG based volume excluding polymers.
  • the separation method comprises ultra-centrifugation to separate the desired GVsfrom bulk solution.
  • sequential steps involving initial spins at 20,000 x g for up to 30 minutes followed by multiple spins at ranges of about 100,000 x g to about 120,000 x g for about 1 to about 2 hours provides a pellet or isolate rich in GVssuch as EVs (e.g., WEVs).
  • EVs e.g., WEVs
  • ultracentrifugation provides EVs such as WEVs that can be resuspended, for example, in phosphate buffered saline or a solution of choice.
  • the vesicles are further assessed for desired properties by assessing their attributes when exposed to a sucrose density gradient and picking the fraction in 1.13-1.19 g/mL range.
  • isolation of vesicles of the present disclosure includes using combinations of filters that exclude different sizes of particles, for example 0.45 uM or 0.22 uM filters can be used to eliminate vesicles or particles bigger than those of interest.
  • GVs such as EVs may be purified by several means, including antibodies, lectins, or other molecules that specifically bind vesicles of interest, eventually in combination with beads ( ⁇ ?.g., agarose/sepharose beads, magnetic beads, or other beads that facilitate purification) to enrich for the desired vesicles.
  • a marker derived from the vesicle type of interest may also be used for purifying vesicles.
  • vesicles expressing a given biomarker such as a surface-bound protein may be purified from cell-free fluids to distinguish the desired vesicle from other types.
  • vesicles include density gradient centrifugation (e.g. sucrose or optiprep gradients), and electric charge separation. All these enrichment and purification techniques may be combined with other methods or used by themselves.
  • a further way to purify vesicles is by selective precipitation using commercially available reagents such as ExoQuickTM (System Biosciences, Inc.) or Total Exosome Isolation kit (InvitrogenTM Life).
  • isolation of the GVs such as EVs is achieved by centrifuging raw a whey product at high speeds to isolate the vesicle.
  • EVs such as WE Vs can be isolated in a manner that provides amounts greater than about 50 mg (e.g., greater than about 300 mg) of vesicles per 100 mL of whey.
  • the WEVs may be prepared by a method comprising the steps of: providing a quantity of whey product: and performing a centrifugation, e.g., sequential centrifugations, on the whey to yield greater than about 50 mg of WEVs per 100 mL of whey.
  • the sequential centrifugations yield greater than 300 mg of WEVs per 100 mL of milk.
  • the series of sequential centrifugations comprises a first centrifugation at 20,000 x g at 4 °C for 30 min, a second centrifugation at 100,000 x g at 4 °C for 60 min, and a third centrifugation at 120,000 x g at 4 °C for 90 min.
  • the isolated vesicles can then be stored at a concentration of about 5 mg/mL to about 10 mg/mL to minimize coagulation and allow the isolated vesicles to effectively be used for the encapsulation or loading of one or more therapeutic agents.
  • the isolated vesicles are passed through a 0.22 pm filter to remove any coagulated particles as well as microorganisms, such as bacteria.
  • GVs such as EVs
  • the methods involve one or more steps to reduce or eliminate caseins and/or lactoglobulins from the input whey materials.
  • Caseins are proteins found in whey that have a molecular weight or about 25-30 kDa. Lactoglobulins are also found in whey that have a molecular weight of about 10-20 kDa, Briefly, such a method may involve one or more defatting steps to remove abundant, whey proteins following conventional methods or those disclosed herein.
  • the whey samples can be subject to one or more steps to disrupt casein micelles, coagulate casein and remove casein from the whey sample.
  • the casein-depleted whey sample can thus be subject to steps to enrich GVs, for example, those approached known in the art or disclosed herein, e.g., chromatography-based methods (e.g,, for scalable preparation) and ultracentrifugation-based methods.
  • steps to enrich GVs for example, those approached known in the art or disclosed herein, e.g., chromatography-based methods (e.g,, for scalable preparation) and ultracentrifugation-based methods.
  • casein removal may be achieved chemically, e.g., by acidification.
  • a suitable acid solution e.g., acetic acid, hydrochloric acid, citric acid, etc.
  • powder of a suitable acid e.g., citric acid powder
  • acidification of whey may be achieved by saturation of the whey with CO2 gas.
  • casein removal may be achieved using enzymes capable of coagulating or digesting casein, for example, using rennet.
  • rennet refers to a mixture of enzymes capable of curdling caseins in whey.
  • the rennet used in the methods disclosed herein is derived from an animal, e.g. , a complex set of enzymes produced in the stomachs of a ruminant mammal such as calf.
  • a rennet may comprise chymosin, which is a protease enzyme that curdles casein in whey, and optionally other enzymes such as pepsin and lipase.
  • the rennet used in the methods disclosed herein is derived from a plant, e.g., a vegetable rennet.
  • Vegetable rennet can be an enzyme or a mixture of enzymes that coagulates milk and separates the curds and whey from milk.
  • the vegetable rennet used herein can be a commercially available vegetable rennet extracted from a mold such as mucor miehei.
  • one or more recombinant casein coagulation enzymes may be used for casein removal.
  • Such recombinant enzymes may be produced using a suitable host (e.g., bacterium, yeast, insect cell, or mammalian cell) by the conventional recombinant technology.
  • the method disclosed herein may involve the use of a Ca2+ chelating agent such as EDTA or EGTA to disrupt casein micelles, which can be then removed.
  • the resultant whey sample can be subject to one or more steps to enrich the GVs contained therein, e.g., ultracentrifugation, size exclusion chromatography, affinity purification, tangential flow filtration, or a combination thereof.
  • the method disclosed herein may comprise a tangential flow filtration (TFF) step for GVenrichment.
  • the method may further comprise a size exclusion chromatography following the TFF step.
  • the enrichment may be achieved by a conventional approach such as ultracentrifugation.
  • Suitable GVs may also be derived by artificial production means, such as from exosome-secreting cells and/or engineered as is known in the art.
  • GVscan be further characterized by one or more of nanoparticle tracking analysis to assess particle size, transmission electron microscopy to assess size and architecture, immunogold labeling of vesicles or their contents prior to electron microscopy to track species of interest associated with exosonies, immunoblotting, or protein content assessment using the Bradford Assay.
  • viral particles disclosed herein refer to small virus-like particles (e.g., artificial) comprising a protein shell encapsulating a viral genome, which may comprise viral elements essential for assembly of the viral particles and a transgene, which may encode an agent of interest (e.g., a therapeutic or diagnostic agent).
  • the viral particles may infect target host cells (e.g., human cells such as human intestinal epithelium cells) but lacks the capacitiy to proliferate in the host cells.
  • the viral particles disclosed herein may be those that are commonly used in drug/gene delivery. Examples include, but are not limited to, adeno viral particles, retroviral particuels such as lentiviral particles, and adeno-associated viral (AAV) particles.
  • adeno viral particles retroviral particuels such as lentiviral particles
  • AAV adeno-associated viral
  • the viral particles disclosed herein are AAV particles.
  • Adeno- associated virus (AAV) is a member of the Parvovirus family.
  • AAV is a small non -enveloped virus having a single-stranded linear DNA genome. Recombinant AAVs have emerged as one of the most effective vehicles for gene delivery due to their ability to transduce a variety of mammalian cells and their non-pathogenicity and low immunogenicity in humans.
  • AAV viral particles or AAV virus used interchangeably, refer to a viral particle composed of at least one AAV capsid protein (e.g. , all of the capsid proteins) and an encapsidated AAV viral genome.
  • An AAV viral particle is capable of transducing mammalian target cells and the host specificity depends on the serotype of the AAV viral particle.
  • the genome of an AAV is a single-stranded DNA chain (ssDNA), which can be either positive- or negative-sensed.
  • the AAV genome contains inverted terminal report (ITR) sequences at both ends of the DNA strand and two open reading frames, rep and cap, encoding the Rep protein and the capsid proteins, respectively.
  • ITR sequence in a naturally- occurring AAV genome is about 145 bp long and capable of forming a hairpin, allowing for self-priming and synthesis of the second DNA strand.
  • the ITR sequence also plays a role in efficient packaging of the AAV genome into capsid to form a fully assembled AAV virus.
  • the rep gene encodes Rep proteins, which enable viral DNA replication and integration into the host genome.
  • the cap gene produces three overlapping messenger RNA sequences coding for three capsid proteins, VP1 , VP2, and VP3.
  • an AAV capsid is composed of a mixture of VPl , VP2, and VP3 arranged in icosahedral symmetry.
  • AAV virus of different serotypes contain different capsid proteins.
  • AAV viral particles of specific serotypes for use in delivering genes of interest to intestinal epithelium cells.
  • Use of such AAV viral particles in gene delivery can restrict both infection and gene expression to target cells of interest, for example, intestinal epithelium cells (e.g., gut enterocytes).
  • Gut enterocytes are terminally differentiated intestinal epithelial cells of the villi. Such cells typically have a halflife of only several days, naturally becoming replaced by fresh cells after undergoing apoptosis. Any genotoxicity resulting from treatment is therefore naturally corrected within days upon apoptosis.
  • the present disclosure provides AAV viral particles of a specific serotype, which is capable of transducing intestinal epithelium cells such as enterocytes.
  • Specific AAV serotypes capable of transducing intestinal epithelium cells are identified in the screening assays disclosed herein. See Examples below.
  • Such AAV viral particles e.g., AAV1 or AAV6 can be used as vehicles to deliver genes of interest to intestinal epithelium cells.
  • the present disclosure features AAV vectors (e.g., plasmids) designed for delivery and expression of one or more genes of interest.
  • An AAV vector comprises a polynucleotide, which comprises naturally-occurring AAV genome elements (e.g., ITR sequences) or variants thereof and one or more non-AAV elements (heterologous elements), for example, an expression cassette for one or more genes of interest.
  • This polynucleotide corresponds to the AAV genome to be packed into the AAV viral particles disclosed herein.
  • the heterologous elements can be integrated by recombinant techniques into or in place of the AAV genomic coding region (e.g., replacing the original AAV rep and cap genes in an AAV genome), and is typically flanked on either side by ITR regions. This means that an ITR may appear both upstream and downstream from the heterologous elements. In some instances, a single ITR may be sufficient to carry out the functions normally associated with configurations comprising two ITRs (see, for example, WO 94/13788).
  • the AAV vector comprises a regular AAV genome sequence, in which two ITR sequences are flanking the gene of interest and the regulatory elements, if any.
  • the AAV vector comprises a self-complementary AAV genome sequence, in which the coding region form an intra-molecular double-stranded DN A template.
  • the two complementary halves of the self- complementary vector can associate to form one self-annealing, partially double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription, thereby leading to fast expression of the encoded agents of interest in most of the infected cells.
  • dsDNA partially double stranded DNA
  • the ITR sequences contained in the AAV vectors may be derived from a naturally- occurring AAV genome. In some embodiments, the ITR sequences can be naturally-occurring. In other embodiments, the ITR sequences can be modified relative to the naturally-occurring counterpart, for example, an internal ITR lacking a terminal resolution site.
  • the ITR sequences for use in the AAV vectors disclosed herein may be derived from any serotype of AAV virus. In some examples, the ITRs can be derived from AAV2.
  • the expression cassette of one or more genes of interest in the AAV vectors disclosed herein typically comprises one or more coding regions in operable linkage to one or more regulatory elements, e.g., promoter, enhancer, polyA signal region, etc.
  • the expression cassette in the AAV vector comprises one coding region. In other embodiments, the expression cassette in the AAV vector comprises two or more coding regions.
  • the expression cassette may be in multici stronic format (c.g., bicistronic), in which the coding regions are linked by a region such as a segment encoding a 2A self-cleaving peptide, a ribosome binding site, an internal ribosome entry site, or the like to allow for simultaneous expression of two or more polypeptides.
  • the genets) of interest carried by any of the AAV vector disclosed herein may encode a therapeutic agent, e.g., a therapeutic protein or a therapeutic nucleic acid. Details of such therapeutic agents are provided below.
  • an AAV vector disclosed herein may comprise multiple GOIs, each encoding a subdomain or a subunit of a functional protein or protein complex.
  • the multiple GOIs may be located on multiple AAV vectors. The multiple GOIs collectively can produce the functional protein or protein complex.
  • an AAV vector disclosed herein may comprise two coding sequences, one for the heavy chain (e.g., heavy chain variable domain) of an antibody and the other for the light chain (e.g., light chain variable domain) of the antibody.
  • An AAV viral particle e.g., any of the AAV6 variants disclosed herein
  • encapsulating the AAV genome polynucleotide carried by such an AAV vector could produce both the heavy chain and the light chain of the antibody in the target cells (e.g., intestinal epithelium cells), to which the AAV viral particle infects.
  • This approach can also be applied to production of multiple subunits or subdomains of other proteins or protein complex using one AAV vector.
  • a first AAV vector as disclosed herein may comprise a coding sequence for the heavy chain (e.g., a full-length heavy chain) of an antibody and a second AAV vector may comprise a coding sequence for the light chain (e.g., a full-length light chain) of the antibody.
  • AAV viral particles such as any of the AAV6 variants disclosed herein may be generated using the first AAV vector and the second AAV vector. Delivering the AAV viral particles (a heterogeneous population) to desired target cells such as intestinal epithelium cells would lead to production of the multi-chain antibody in the target cells. This approach can also be applied to production of multiple subunits or subdomains of other proteins or protein complex using two or more AAV vectors.
  • the expression cassette in the AAV vector may also comprise one or more regulatory elements, which are operably linked to the coding region in a manner allowing for transcription, translation and/or expression in a cell transduced with the AAV viral particle the product (e.g., the therapeutic agent) encoded by the genes of interest.
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the expression cassette comprises a promoter in operable linkage with the gene of interest to control its expression.
  • the promoter is an AAV promoter.
  • the promoter Is a heterologous promoter (non- AAV promoter).
  • the promoter may be a constitutive promoter.
  • the promoter may be an inducible promoter.
  • the promoter is a tissuespecific promoter (e.g., a promoter specific for gene expression in intestinal epithelium cells).
  • constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41:521 - 530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the p-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl promoter.
  • the promoter for use in constructing the expression cassette in the A AV vector disclosed herein may be a CMV promoter.
  • the CMV promoter is a regular CMV promoter. In other instances, the CMV promoter is a short CMV promoter (also known as minimal CMV promoter). See, e.g., Schlabach et al., PNAS 2010 107(6):2538-2543.
  • Inducible promoters allow regulation of gene expression by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state ⁇ e.g., acute phase), a particular differentiation state of the cell, or in replicating cells only.
  • inducible promoters regulated by exogenously supplied compounds include, but are not limited to, the zinc-inducible sheep metallothionine (MI') promoter, the dexamethasone (Dex) -inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline- repressible promoter, the tetracycline-inducible promoter, the RU486-inducible promoter, and the rapamycin-inducible promoter.
  • Other types of inducible promoters include those that can be regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or
  • the expression cassette disclosed here may further comprise one or more additional regulatory elements, for example, a control sequences such as appropriate transcription initiation, termination, and/or enhancer sequences; efficient RNA processing signals (e.g., splicing and polyadenylation (poly A) signals such as the SV40 poly A signal sequence, or the human growth factor poly A signal sequence); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence): sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • the regulatory elements may comprise one or more intron sequences, for example, a S V40 intron sequence, or an MVM intron sequence.
  • the regulatory elements may comprise a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) such as WPRE3. Exemplary regulatory elements are also provided in FIGs. 6A and 6B.
  • WPRE Woodchuck hepatitis virus posttranscriptional regulatory element
  • the AAV particles provided herein can be AAV1 or AAV6 particles, which can transduce intestinal epithelium cells, particularly via the apical pole.
  • the AAV1 or AAV6 particles can contain wild-type capsid proteins.
  • the AAV1 or AAV6 particles may contain at least one mutated capsid proteins as relative to the wild-type counterpart.
  • the AAV1 or AAV6 variant disclosed herein has a higher transduction efficiency to mammalian intestinal epithelium cells ( ⁇ ?.g., mouse intestinal epithelium cells and/or human intestinal epithelium cells) relative to its wild-type counterpart.
  • An A AV genome comprises a cap gene encoding various capsid proteins, including VP1, VP2, and VP3, which have overlapping sequences.
  • the positions of mutations in AAV 6 variant capsid proteins disclosed herein refer to the positions in the polypeptide sequence encoded by a cap ORF. Accordingly, one would understand that these mutations would present in the corresponding positions in VP1, VP2, and/or VP3 capsid proteins in an AAV1 or AAV6 variant serotype as disclosed herein.
  • amino acid sequences of the polypeptides encoded by the wild-type AAV1 cap ORF and the AAV6 ORF are provided below':
  • AAV1 Capsid Protein Sequence (SEQ ID NO:1):
  • AAV6 Capsid Protein Sequence (SEQ ID NO:2):
  • AAV1 and AAV6 serotypes disclosed herein refer to AAV particles containing AAV1 and AAV6 capsid proteins, respectively (wild-type or variants such as those disclosed herein).
  • the AAV particles disclosed herein may contain a matched viral genome (e.g., derived from AAV1 or AAV 6 viral genome).
  • the AAV particles disclosed herein may contain AAV1 or AAV6 capsid proteins and a viral genome derived from a different viral serotype (e.g., from AAV2).
  • the AAV 1 or AAV6 variant serotype disclosed herein comprise modified capsid proteins that comprise mutations at position 129 and position 492 of SEQ ID NO:1 or SEQ ID NO:2, for example, amino acid substitutions such as F129L and/or T492V.
  • modified capsid proteins may further comprise mutations at one or more of positions 445, 502, 663, 705, and 731 in SEQ ID NO:1 or SEQ ID NO:2, for example, amino acid substitutions such as Y445F, T502S, S663L, Y705F, and/or Y73 IF
  • the AAV1 or AAV 6 variant serotype disclosed herein comprise modified capsid proteins that comprises mutations at position 129 and position 705 of SEQ ID NO: 1 or SEQ ID NO:2, for example, amino acid substitutions such as F129L and/or Y705F.
  • modified capsid proteins may further comprise mutations at one or more of positions 445, 492, 502, 663, and 731 in of SEQ ID NO:1 or SEQ ID NO:2, for example, amino acid substitutions such as Y445F, T492V, T502S, S663L, and/or Y73 IF.
  • the mutant AAV capsid protein may comprise mutations at positions 129 (e.g. , F129L), 445 (e.g., Y445F), 492 (e.g., T492V), 705 (e.g., Y705F), and 731 (e.g., Y731F) of of SEQ ID NO: I or SEQ ID NO:2.
  • Such a mutant may further comprise mutations at position 502 and/or position 663 of SEQ ID NO:2, for example, T502S and/or S663L.
  • any of the A.AV1 or AAV 6 variant serotype particals disclosed herein may comprise a mutated VP1 capsid protein comprising mutations as position 129 and position 492 of SEQ ID NO: 1 or SEQ ID NO:2 or mutations at position 129 and position 705 of SEQ of SEQ ID NO: 1 or SEQ ID NO:2.
  • the mutated VP1 capsid protein may further comprise one or more mutations at positions 445, 502, 663, and 731 in of SEQ ID NO:1 or SEQ ID NO:2.
  • the AAV6 variant serotype partical may comprise a mutated VP2 capsid protein and/or a mutated VP3 capsid protein comprising mutations at position 492 and/or 705 of of SEQ ID NO: 1 or SEQ ID NO:2, and optionally one or more mutations at positions 445, 502, 663, and 731 in of SEQ ID NO: 1 or SEQ ID NO:2.
  • the mutations can be amino acid residue substitutions such as those described herein.
  • the AAV6 variant capsid proteins are produced by a cap ORF encoding the following variant polypeptide: (mutations underlined and in boldface):
  • AAV6.2 (SEQ ID NO:3)
  • AAV6TM (SEQ ID NO:4)
  • AAV6.2FVFF (SEQ ID NO:5)
  • an AAV1 or AAV6 variant capsid protein disclosed herein e.g. ,
  • VP1, VP2, and/or VP3 may be produced from a cap ORF encoding an amino acid sequence at least 80% (e.g., at least 85%, at, least 90%, at least, 95%, at least 98% or above) identical to of SEQ ID NO:1 or SEQ ID NO:2 and comprise the mutations disclosed herein.
  • the “percent identity’’ of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 8'7:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993.
  • any of the AAV variant capsid proteins disclosed herein may comprise conservative amino acid residue substitutions, for example, at positions other than those noted above (e.g., position 129, 445, 492, 502, 663, 705, and/or 731 of of SEQ ID NO:1 or SEQ ID NO:2), relative to the wild-type counterpart.
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J.
  • Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • the AAV1 or AAV6 variant serotype comprising any of the AAV1/AAV6 variant capsid proteins disclosed herein may infect (e.g., infect specifically) intestinal epithelium cells
  • AAV particle can infect the target cell or tissue in a much greater level compared to other types of cells or tissue (e.g., at least 1 fold greater, at least 2 fold greater, at least 5 folder greater, or at least 10 fold greater).
  • the AAV6 variant serotype comprising any of the AAV6 variant capsid proteins disclosed herein may have a transduction efficiency to intestinal epithelium cells at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or above) higher than the wild- type counterpart.
  • Transduction efficiency of a specific type of cells can be determined via a conventional method or a method disclosed herein (see Examples below).
  • any of the AAV vectors disclosed herein may be delivered to a suitable host cell, such as a packaging host cell, for producing AAV viral particles following routine practice.
  • the host cell for use in producing AAV viral particles may be prokaryotic (e.g., bacterial) cells, and or eukaryotic cells (e.g., insect cells, yeast cells and mammalian cells).
  • the host cells are mammalian cells. Examples include, but are not limited to, A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO,
  • WI38 HeLa, 293 cells (which express functional adenoviral El), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells.
  • one or more vectors carrying AAV rep gene and/or AAV cap gene may be introduced into the host cell together with the AAV vector.
  • the cap gene may encode any of the A AV6 capsid variant polypeptides disclosed above (e.g. , SEQ ID NO:5).
  • the rep gene may be of the same serotype as the serotype of the ITRs in the AAV vector. Alternatively, the rep gene may be from a cross-complementing serotype relative to the serotype of the ITR sequences.
  • the AAV vector and the one or more vectors carrying the rep gene and the cap gene may be introduced into the host cell by a conventional method.
  • the host cell may stably express capsid proteins such as the AAV6 variant capsid proteins disclosed herein under the control of a suitable promoter.
  • the capsid proteins may be under the control of an inducible promoter.
  • the host cell may stably express the Rep protein under the control of a suitable promoter.
  • expression of the Rep protein may be under the control of an inducible promoter.
  • an AAV vector can be introduced into a packaging host cell, which may stably express both a Rep protein and capsid proteins such as the A.AV6 variants disclosed herein.
  • the packaging host cell may also have helper functions in order to package the recombinant AAV viral particles as disclosed herein. In some examples, these functions may be supplied by a herpesvirus.
  • the necessary helper functions can be provided by a human or non-human primate adenovirus, such as those known in the art, e.g., those available from the American Type Culture Collection (ATCC), Manassas, Va. (US).
  • the host cell may be provided with and/or contains an Ela gene product, an Elb gene product, an E2a gene product, and/or an E4 ORF6 gene product.
  • the adenovirus Ela, Elb, E2a, and/or E4ORF6 gene products, as well as any other desired helper functions, can be provided using a conventional method. Coding sequences of these gene products may be located on separate vectors. Alternatively, two or more coding sequences may be located on the same vector.
  • Such vectors may be introduced into the host cells via conventional approaches, for example, transfection, infection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion, among others.
  • One or more of the adenoviral genes may be stably integrated into the genome of the host cell, stably expressed as episomes, or expressed transiently.
  • the gene products may all be expressed transiently, on an episorne or stably integrated, or some of the gene products may be expressed stably while others are expressed transiently.
  • the promoters for each of the adenoviral genes may be selected independently from a constitutive promoter, an inducible promoter or a native adenoviral promoter.
  • the promoters may be regulated by a specific physiological state of the organism or cell (?'. ⁇ ?., by the differentiation state or in replicating or quiescent cells) or by exogenously added factors, for example.
  • any of the AAV vectors or AAV genome disclosed herein comprise one or more genes of interest (GOIs).
  • the GOIs encode or produce therapeutic agents, which may be nucleic-acid based or protein-based.
  • the GOIs encode or produce diagnostic agents, which may be nucleic-acid based or protein-based.
  • Any of the AAV viral particles disclosed herein e.g.
  • the AAV6 variant can be used to deliver the GOIs to desired target cells, for example, intestinal epithelium cells, thus producing the encoded therapeutic agents or diagnostic agents in the target cells, which could then secrete the encoded therapeutic agents to release those to the circulation system (e.g., via the basolateral pole) or to the lumen of the gut (e.g. , via the apical pole).
  • desired target cells for example, intestinal epithelium cells
  • Exemplary agents encoded by the GOIs can be a peptide, a polypeptide, or protein.
  • the GOIs may encode and produce a nucleic acid-based therapeutic agent, for example, an interfering RNA, an antisense oligonucleotide, or an aptamer.
  • a nucleic acid-based therapeutic agent for example, an interfering RNA, an antisense oligonucleotide, or an aptamer.
  • the GOI(s) carried by any of the AAV vectors or AAV viral particles may encode a protein-based agent (e.g., a peptide, a polypeptide, or a protein), for example, a protein-based therapeutic agent or a protein-based diagnostic agent.
  • a protein-based agent e.g., a peptide, a polypeptide, or a protein
  • the protein-based agent may be a naturally occurring polypeptide. Alternatively, it may be a modified version of a naturally occurring polypeptide or a non-naturally (synthetic) polypeptide.
  • Non-limiting examples of suitable protein-based agents include antibodies (e.g., directed against a cellular or pathogenic target), hormones, growth factors, cofactors, enzymes (e.g., metabolic enzymes, immunoregulatory enzymes, gastrointestinal enzymes, growth regulatory enzymes, coagulation cascade enzymes), cytokines, chemokines, vaccine antigens, antithrombotics, antithrombolytics, toxins, or an antitoxin.
  • a GOI may comprise a coding sequence for a subunit of a multichain protein complex or a subdomain of a single-chain protein. Multiple GOIs, which may be located on one AAV vector or AAV genome, or located on multiple AAV vectors or AAV gemones, collectively can produce a functional protein or protein complex.
  • the protein-based agent is an antibody (e.g., a therapeutic antibody), which may be directed against a cellular target.
  • An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • antibody encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen- binding fragments thereof (such as Fab, Fab', F(ab‘)2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g. , nanobody), single domain antibodies (e.g., a VH only antibody), multispecific antibodies (eg., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • antigen- binding fragments thereof such as Fab, Fab', F(ab‘)2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g. , nanobody),
  • An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG I, IgG2, IgG3, IgG4, IgAl and IgA'2, Tire heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the antibodies may target checkpoint molecules (e.g., PD-1 or PD- LI). See examples in Table 1 below.
  • the antibodies may target cytokines, e.g., inflammatory cytokines such as TNF-alpha or IL-6 or receptors thereof such as IL-6R.
  • the antibodies may target pathogenic antigens, for example, antibodies capable of neutralizing a pathogen such as a virus, a bacterium, a fungus, a helminth, or a parasite.
  • a neutralizing antibody may be a broadly neutralizing antibody or non-broadly neutralizing antibodies.
  • a broadly neutralizing antibody can recognize, bind to, and block many strains of a particular pathogen, such as a virus.
  • Broadly neutralizing antibodies generally target certain conserved epitopes of the pathogen, e.g., a viral pathogen.
  • non-broadly neutralizing antibodies are specific for individual viral strains with unique epitopes.
  • a type of neutralizing antibody may recognize and block one or more types of a pathogen from entering its target cells.
  • Broadly neutralizing antibodies may also activate other immune cells to help destroy pathogen-infected cells.
  • such antibodies are isolated from patients recovered from an infection. These antibodies from recovered patients can be isolated and either be used directly as a therapeutic agent or are sequenced and subsequently produced using recombinant techniques known in the art. Alternatively, antibodies capable of binding to the pathogenic target antigens can be isolated from a suitable antibody library following routine selection processes as known in the art.
  • Such antibodies can be made fully human (humanized) and recombinantly produced from cell lines according to methods known in the art.
  • two, three or more neutralizing, e.g., broadly neutralizing, non-broadly neutralizing antibodies, or a combination thereof, can be combined in order to achieve virus control.
  • a combination of AAV vectors or A AV viral particles, which collectively encode a combination of such antibodies may be used for delivering and producing the combination of antibodies to desired target cells such as intestinal epithelium cells.
  • the neutralizing antibodies disclosed herein may target a corona vims such as SARS (e.g., SARS-CoV-2) and thus be effective in treating diseases caused by SARS infection such as COVID-19.
  • SARS e.g., SARS-CoV-2
  • the neutralizing antibodies can be isolated from patients recovered from an infection, e.g., a coronavirus infection.
  • the antibodies can be isolated from a human patient recovered from COVID-19.
  • Such antibodies may be sequenced and subsequently produced using recombinant techniques known in the art.
  • such neutralizing antibodies may be isolated from a suitable antibody library following routine selection processes as known in the art, using a suitable antigen from the vims, for example, the Spike protein of SARS-CoV-2.
  • the neutralizing antibodies are fully human (humanized) and recombinantly produced from cell lines.
  • Non-limiting examples of neutralizing antibodies targeting SARS- CoV-2 include REGN3048 and REGN 3051 (Regeneron Pharmaceuticals).
  • the protein-based agent encoded by a GOT may be a therapeutic peptide, e.g. , hormone.
  • a non-limiting example of such biologic agents include Glucagon-like peptide 1 (GLP-1) and derivatives thereof or other GLP-1 receptor agonists, including but not limited to exenatide, liraglutide, taspoglutide, lixisenatide, semaglutide, albiglutide, dulaglutide, and langlenatide. See Table 2 below.
  • the protein-based agent may be a growth factor, for example, erythropoietin.
  • the protein-based agent may be a factor involved in the coagulation cascade, for example.
  • the protein-based agent can be an enzyme (e.g., metabolic enzymes, immunoregulatory enzymes, gastrointestinal enzymes, growth regulatory enzymes, or coagulation cascade enzymes).
  • Other exemplary protein-based agents include, but are not limited to, cytokines, vaccine antigens, antithrombotics, anti thromboly tics, toxins, or an antitoxin. Table 2 provides additional examples of proteinbased agents.
  • a GOI disclosed herein may encode or produce a nucleic acid, for example, a nucleic acid-based therapeutic agent.
  • the oligonucleotide therapeutic agent can be a single- stranded or double -stranded DNA, 1RNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA, morpholino oligonucleotide, or ssDNA.
  • the nucleic acid-based agent is a ncRNA of about 30 to about 200 nucleotides (nt) in length or a long non-coding RNA (IncRNA) of about 200 to about 800 nt in length.
  • the IncRNA is a long intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRN A), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA.
  • the ncRN A is selected from a piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA).
  • IncRN As Human and other mammalian genomes pervasively transcribe tens of thousands of long non-coding RNAs (IncRN As).
  • GenCode version #2-7 catalogs just under 16,000 IncRNAs in the human genome, producing nearly 28,000 transcripts; when other databases are included, more than 40,000 IncRNAs are known.
  • IncRNAs are a group that is commonly defined as transcripts of more than 200 nucleotides ( ⁇ ?.g., about 200 to about 1200 nt, about 2500 nt, or more) that lack an extended open reading frame (ORF).
  • the term “non-coding RNA” (ncRNA) includes IncRNA as well as shorter transcripts of, e.g., less than about 200 nt, such as about 30 to 200 nt.
  • IncRNAs modulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors and p53 and thus provide an additional layer of flexibility and robustness to cell cycle progression.
  • some IncRNAs are linked to mitotic processes such as centromeric satellite RNA, which is essential for kinetochore formation and thus crucial for chromosome segregation during mitosis in humans and flies.
  • Another nuclear IncRN A, MA- lincl regulates M phase exit by functioning in cis to repress the expression of its neighboring gene Pura, a regulator of cell proliferation. Since deregulation of the cell cycle is closely associated with cancer development and growth, cell cycle regulatory IncRNAs may have oncogenic properties.
  • the nucleic acid-based agent encoded by a GOI can be a non-coding RNA (ncRNA).
  • ncRNA non-coding RNA
  • the ncRNA is a long non-coding RNA (IncRNA) of about 200 nucleotides (nt) in length or greater.
  • the IncRNA can be about 200 nt to about 1 ,200 nt in length.
  • the IncRNA is about 200 nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400, or about 300 nt in length.
  • the ncRNA can be of about 25 nt or about 30 nt to about 200 nt in length.
  • the nucleic acid -based agent is a miRNA.
  • miRNAs are small non-coding RNAs that are about 17 to about 25 nucleotide bases (nt) in length in their biologically active form.
  • the miRNA is about 17 to about 25, about 17 to about 24, about 17 to about 23, about 17 to about 22, about 17 to about 21, about 17 to about 20, about 17 to about 19, about 18 to about 25, about 18 to about 24, about 18 to about 23, about 18 to about 22, about 18 to about 21, about 18 to about 20, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 19 to about 22, about 19 to about 21, about 20 to about 25, about 20 to about 24, about 20 to about 23, about 20 to about 22, about 21 to about 25, about 21 to about 24, about 21 to about 23, about 22 to about 25, about 22 to about 24, or about 22 nt in length. miRNAs regulate gene expression post- transcriptionally by decreasing target mRNA translation. In some instances, miRNAs function as negative regulators.
  • miRNAs There are generally three forms of miRNAs: primary miRNAs (pri- miRNAs), premature miRNAs (pre-rniRNAs), and mature miRNAs, all of which are within the scope of the present disclosure.
  • Primary miRNAs are expressed as stem-loop structured transcripts of about a few hundred bases to over 1 kb.
  • the pri- miRNA transcripts are cleaved in the nucleus by Drosha, an RNase II endonuclease that cleaves both strands of the stem near the base of the stem loop. Drosha cleaves the RNA duplex with staggered cuts, leaving a 5' phosphate and 2 nt overhang at the 3' end.
  • the cleaved product, the premature miRNA (pre-miRNA) is about 60 to about 110 nt long with a hairpin structure formed in a fold-back manner.
  • Pre-miRNA is transported from the nucleus to the cytoplasm by Ran- GTP and Exportin-5.
  • Pre-rniRNAs are processed further in the cytoplasm by another RNase II endonuclease called Dicer. Dicer recognizes the 5' phosphate and 3' overhang and cleaves the loop off at the stem-loop junction to form miRNA duplexes.
  • the miRNA duplex binds to the RNA-induced silencing complex (RISC), where the antisense strand is preferentially degraded and the sense strand mature miRNA directs RISC to its target site. It is the mature miRNA that is the biologically active form of the miRNA and is about 17 to about 25 nt in length.
  • the miRNAs encapsulated by the microvesicles of the presently-disclosed subject matter are selected from miR-155, which is known to act as regulator of T- and B-cell maturation and the innate immune response, or miR-223, which is known as a regulator of neutrophil proliferation and activation.
  • Other non -natural miRNAs such as iRNAs (c.g. siRNA) or natural or non- natural oligonucleotides may be present in the whey-purified vesicles and represent an encapsulated therapeutic agent, as the term is used herein.
  • the nucleic acid-based agent disclosed herein is a siRNA.
  • siRNA Small interfering RNA
  • siRN As generally exert their biological effects through the RN A interference (RNAi) pathway.
  • siRNAs generally have 2. nucleotide overhangs that are produced through the enzymatic cleavage of longer precursor RNAs by the ribonuclease Dicer. siRNAs can limit the expression of specific genes by targeting their RNA for destruction through the RNA interference (RNAi) pathway.
  • the RNA is an siRNA molecule comprising a modified ribonucleotide, wherein said siRNA (a) comprises a two base deoxynucleotide “IT’ sequence at its 3' end, (b) is resistant to RNase, and (c) is capable of inhibiting viral replication.
  • the siRN A molecule comprises a nucleotide sequence at least 80% identical to the nucleotide sequence of siRNAS, siRNACl, siRNAC2, siRNASBl , siRNA5B2 or siRNA5B4.
  • the nucleic acid-based agent can be a doublestranded (dsRNA) molecule that mediates RNA interference in target cells.
  • nucleic acid-based agents include antisense RNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, tRNA or other nucleic acids and analogs thereof described herein.
  • ceRNA competing endogenous RNA
  • snRNA small nuclear RNA
  • snoRNA small nucleolar RNA
  • pseudo-gene rRNA
  • tRNA tRNA or other nucleic acids and analogs thereof described herein.
  • the nucleic acid-based agent described herein target RNAs encoding the following polypeptides: vascular endothelial growth factor (VEGF); Apolipoprotein B (ApoB); luciferase flue); Androgen Receptor (AR); coagulation factor VII (FVII); factor VIII (FVIII, also known as anti -hemophilic factor (AHF)); factor IX (FIX, also known as Christmas factor); Factor XI (FXI, also known as plasma thromboplastin antecedent): factor I (FI, also known as fibrinogen): factor II (FII, also known as protheombin); factor V (FV, also known as proaccelerin); factor X (FX, also known as Stuart-Power factor); factor XII (FXII, also known as Hageman Factor); factor XIII (FXIII, also known as fibrin stabilizing factor): hypoxia-inducible factor I, alpha subunit (Hif-
  • Exemplary single stranded oligonucleotide agents are shown in Table 1 below. Additional suitable miRNA targets are described, e.g., in John et al., PLoS Biology 2:1862-1879, 2004 (correction in PLoS Biology 3:1328, 2005), and The microRNA Registry (Griffiths- Jones S., NAR 32:D1O9-D111, 2004).
  • the therapeutic agent encoded by the GO1 disclosed herein is an allergen, adjuvant, antigen, or immunogen.
  • the allergen, antigen, or immunogen elicits a desired immune response to increase allergen tolerance or reduce the likelihood of an allergic or immune response such as anaphylaxis, bronchial inflammation, airway constriction, or asthma.
  • the allergen, antigen, or immunogen elicits a desired immune response to increase viral or pathogenic resistance or elicit an anticancer immune response.
  • the allergen or antigen elicits a desired immune response to treat an allergic or autoimmune disease.
  • an autoantigen may be used to increase immunological tolerance, thereby benefiting treatment of the corresponding autoimmune disease or decreasing an autoimmune response.
  • adjuvant refers to any substance which enhances an immune response (e.g.
  • a mechanism such as: recruiting of professional antigen-presenting cells (APCs) to the site of antigen exposure; increasing the delivery of antigens by delay ed/slow release (depot generation); immunomodulation by cytokine production (selection of Thl or Th2 response); inducing T-cell response (prolonged exposure of peptide-MHC complexes (signal 1) and stimulation of expression of T-cell- activating co- stimulators (signal 2) on an APC surface) and targeting (e.g., carbohydrate adjuvants which target lectin receptors on APCs), and the like.
  • APCs professional antigen-presenting cells
  • the allergen can be a food allergen, an animal allergen (e.g., pet such as dog, cat, or rabbit), or an environmental allergen (such as dust, pollen, or mildew).
  • the allergen is derived from abalone, perlenioen, acerola, Alaska pollock, almond, aniseed, apple, apricot, avocado, banana, barley, bell pepper, brazil nut, buckwheat, cabbage, chamomile, carp, carrot, casein, cashew, castor bean, celery, celeriac, cherry, chestnut, chickpea, garbanzo, bengal gram, cocoa, coconut, cod, cotton seed, courgetti, zucchini, crab, date, egg (e.g.
  • hen’s egg fig, fish, flax seed, linseed, frog, garden plum, garlic, gluten, grape, hazelnut, kiwi fruit (Chinese gooseberry), legumes, lentil, lettuce, lobster, lupin or lupine, lychee, mackerel, maize (corn), mango, melon, milk (e.g., cow), mollusks, mustard, oat, oyster, peach, peanut (or other ground nuts or monkey nuts), pear, pecan, persimmon, pistachio, pine nuts, pineapple, pomegranate, poppy seed, potato, pumpkin, rice, rye, salmon, sesame, shellfish (e.g., crustaceans, black tiger shrimp, brown shrimp, greasyback shrimp, Indian prawn, neptune rose shrimp, white shrimp), snail, soy, soybean (soya), squid, strawberry, sulfur dioxide (sulfites), sunflower seed, tomato, tree nuts, tuna,
  • the allergen can be an allergenic protein, peptide, toxin, venom, nucleic acid, or other allergen, such as those listed at allergenonline.org.
  • the allergen can be derived from an airborne fungus, mite or insect allergen, plant allergen, venom or salivary allergen, animal allergen, contact allergen, parasitic allergen, or bacterial airway allergen.
  • the agent encoded by the GOI may be an autoimmune antigen.
  • exemplary autoantigens and the corresponding autoimmune disorders are provided in Table 4 below.
  • the GOIs disclosed herein may encode or capable of producing one or more therapeutic agents (e.g., nucleic acid-based or protein-based) targeting an infection, for example, infection caused by a virus such as a coronavirus (e.g., SARS such as SARS-CoV-2).
  • therapeutic agents e.g., nucleic acid-based or protein-based
  • examples include a vaccine antigen or a neutralizing antibody, a small molecule, a polypeptide therapeutic agent, or a nucleic acid (e.g., those designed for producing such protein-based therapeutic agents).
  • the one or more therapeutic agents ⁇ e.g., nucleic acid-based or protein-based) encoded or to be produced by the GOI(s) may target a metabolic disease.
  • a metabolic disease examples include a therapeutic antibody, a polypeptide anti-pathogenic agent, or a nucleic acid (e.g., those designed for producing such protein-based therapeutic agents).
  • Exemplary agents for treating a metabolic disease are provided in Tables 1-4 herein.
  • the one or more therapeutic agents e.g., nucleic acid-based or protein-based
  • the one or more therapeutic agents may target an immune disorder.
  • examples include a therapeutic antibody, an immunomodulator, a polypeptide (e.g., an autoantigen), or a nucleic acid (e.g., those designed for producing such protein-based therapeutic agents).
  • Exemplary anti-immune disorder agents are provided in Tables 1-4 herein.
  • the GOIs may encode or capable of producing one or more anti- infectious agents, including, but not limited to, antiviral agents, anti-malarial, antiinflammatory, anti-bacterial, anti-fungal, anti-protozoal, IL-6 inhibitors, Jak Inhibitors (e.g., baricitinib, fedratinib, ruxolitimb, tofacitinib, oclacitinib, peficitinib, upadacitinib, filgotinib, cerdulatatinib, gandotinib, lestaurtinib, momelotinib, pacritinib, abrocitinib, cucurbitacini, and CHZ868), interferon, kinase inhibitor, protease inhibitor, antibodies, (such as anti-Jak or anti-
  • IL-6 antibodies IL-6 receptor antagonists, or anti-T cell antibodies
  • antibodies directed against pathogenic targets e.g., broadly neutralizing antibodies
  • other polypeptides such as decoy receptors, growth factors or cytokines (e.g., anti-inflammatory cytokines), and viral antigens).
  • the antiviral agents disclosed herein refer to agents capable of inhibiting viral infection by any mechanism of action.
  • an antiviral agent may suppress the activity of one or more viral proteases, leading to blockade of viral protein synthesis and/or viral replication.
  • an antiviral agent may block virus entry into the host cells, for example, via inhibition of binding of virus to cell receptor or inhibits membrane fusion.
  • an antiviral agent may target viral nucleic acid synthesis, for example, inhibiting RNA-dependent RNA polymerase activity.
  • Such antiviral agent may be nucleoside analogs.
  • an antiviral agent may impair endosome trafficking within the host cells and/or limit viral assembly and release.
  • the GOIs disclosed herein may encode one or more checkpoint blockade inhibitors, for example, an anti-CTLA4 antibody, or an anti-PDl/PD-Ll antibody.
  • Exemplary anti-CTLA-4 antibodies include Yervoy (ipilimumab), tremelimumab, AK-104 (PD-1 bispecific), KN-046 (PD-1 bispecific), BMS-986218, CG-0070, MK-1308, zalifrelimab, ATOR-1015, MEDI-5752, MGD-019, XmAb-20717, and XmAb-22841.
  • Exemplary anti-PD- l/'PD-Ll antibodies include Pembrolizumab, Nivolumab, Atezolizumab, Avelumab, Durvalumab, Sintilimab, Toripalimab, Tislelizumab, Camrelizumab, Cemiplimab, HLXIO, Balstilimab, Dostarlimab, Budigalimab, Penpulimab, MEDI0680/ AMP-514, Pidilizumab, Cosibelimab, CS1001, and FAZ053. See also Table 3 for additional examples.
  • Lectins fire a family of proteins capable of binding to carbohydrate molecules or carbohydrate moieties that is a part of other molecules, for example glycoproteins or glycolipids.
  • the sugar binding specificities of exemplary lectins are provided in Tables 8-11 in Example 10 below. Lectins typically do not have enzymatic activity.
  • the lectins for use in the modified GVs such as EVs disclosed herein have binding specificity to specific sites or cells in the GI tract, for example, small intestine and large intestine in the GI tract.
  • the lectins disclosed herein binds specific sites or cells in the human GI tract, for example, the small intestine or the large intestine in the human tract.
  • the lectins disclosed herein may bind to enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract, e.g., human GI tract.
  • Exemplary GI tract compartment to which the lectins bind may include duodenum, upper jejunum, lower jejunum, ileum, cecum, colon, or rectum.
  • the lectin disclosed herein is erytlirina cristagalli lectin (ECL), also known as EGA.
  • ECL erytlirina cristagalli lectin
  • EGA erytlirina cristagalli lectin
  • the lectin disclosed herein is a soybean agglutinin (SB A), also known as SBL, which is a family of lec tins found in soybean.
  • SB soybean agglutinin
  • SB have a molecular weight of 120 kDa and an isoelectric point near pH 6.0.
  • SB preferentially bind to oligosaccharide structures with terminal a- or p-linked N ⁇ acetylgalactosamine, and to a lesser extent, galactose residues.
  • the lectin disclosed herein is Griffonia (Bandeiraea) Simplicifolia Lectin, including GSL-I (GSL-1), GSL-II (GSL-2), or a combination thereof.
  • GSL I is a family of glycoproteins with molecular weights of approximately 114 kDa. There are two types of subunits, termed A and B, with slightly different molecular weights. These subunits combine to form tetrameric structures, resulting in five isolectins.
  • the A-rich lectin preferentially agglutinates blood group A erythrocytes and thus appeal’s to be specific for a-N- acetylgalactos amine residues
  • the B-rich lectin preferentially agglutinates blood group B cells and is specific for a-galactose residues.
  • GSL-2 is a dimeric glycoprotein composed of two subunits of nearly identical size with each subunit having disulfide-linked chains and a binding site for a- or 0-linkedN-acetylglucosamine residues.
  • the lectin disclosed herein is Ulex Europaeus Agglutinin (UEA), for example, UEA-I, UEA II, or a combination thereof.
  • UEA-I consists of two subunits and reacts strongly with a(l,2) linked fucose residues but poorly or not at all with a(l ,3) or a(l,6)-linked fucose.
  • UEA-II is a glycoprotein that consists of four 24,000 Da monomer subunits, which require Ca2+ for binding to its ligands through carbohydrate recognition domain. It is specific for di-N-acetylchitobiose, an oligomer of GlcNAc.
  • the lectin disclosed herein is peanut agglutinin (PNA), which binds preferentially to the T-antigen, a galactosyl (p-1 ,3) N-acetylgalactosamine structure present in many glycoconjugates such as M and N blood groups, gangliosides, and many other soluble and membrane-associated glycoproteins and glycolipids.
  • PNA peanut agglutinin
  • p-1 ,3 galactosyl
  • the protein is 273 amino acids in length with the first 23 residues acting and a signal peptide, which is subsequently cleaved.
  • the lectin disclosed herein may be wheat germ agglutinin (WGA), which binds N-acetyl-D-glucosamine and Sialic acid.
  • WGA wheat germ agglutinin
  • CBM18 Carbohydrate- binding module
  • the lectin disclosed herein may be Phaseolus Vulgaris Leucoagglutinin (PHA), which is a family of lectin each consisting of four subunits. There are two different types of subunits. One appears to be involved primarily in red cell agglutination and has been designated the “E” subunit (PHA-E for erythroagglu tinin). The other type is involved in lymphocyte agglutination and mitogenic activity and has been termed the “L” subunit (PHA-L for leucoagglutinin). These subunits combine to produce five isolectins. PHA has carbohydrate-binding specificity for a complex oligosaccharide containing galactose, N- acetylglucosamine, and mannose.
  • PHA Phaseolus Vulgaris Leucoagglutinin
  • the lectin disclosed herein may be Dolichos Biflorus (Horse Gram) agglutinin (DBA), which is a glycoprotein having a molecular weight of about 111 kDa and consists of 4 subunits of approximately the same size.
  • DBA has a carbohydrate specificity toward a-linked N-acetylgalactosamine. It is commonly used to examine secretor status in blood group A individuals by hemagglutination inhibition assays and in blood typing.
  • any of the lectins for use in the modified GVs disclosed herein may be prepared by a conventional method.
  • the lectin may be ECL.
  • the lectin may be LEA, such as UEA1.
  • the lectin may be isolated from a suitable natural source.
  • the lectin may be produced by the conventional recombman t technol ogy .
  • any of the lectins disclosed herein can be attached to the surface of the modified GV sin any suitable means.
  • the lectin can be displayed directly on the surface of the GVsallowing for its binding to the corresponding sugar moiety.
  • at least a portion of the lectin can be embedded in the bilayer of the lipid membrane of the GVs.
  • the lectin may be associated with lipids in the lipid membrane of the GVsvia, e.g., covalent linkage or non-covalent interaction.
  • the lectin can be attached to one or more proteins in the lipid membrane of the GVs.
  • the lectin may be part of a fusion protein with a protein of the GVs.
  • the lectin may be associated to a protein of the GVvia covalent linkage or non-covalent interaction.
  • the lectin may be linked to the protein of the GV via a linker, e.g., a peptide linker or a chemical linker.
  • Another aspect of the present disclosure provides methods for preparing GVshaving surface modification of one or more lectins, and optionally loaded with a suitable cargo such as those disclosed herein.
  • Various surface functionalization strategies are provided in FIG. 15. Such functionalization strategies may be applied individually, or in combination.
  • surface of GVs may be functionalized by attaching a functional moiety, for example a member of a receptor/ligand pair, or a PEG chain conjugated to a functional group.
  • a functional moiety for example a member of a receptor/ligand pair, or a PEG chain conjugated to a functional group.
  • one or more lectins may be conjugate to the EVs via interaction with the functional moiety, either directly or indirectly.
  • the one or more of the lectins can be conjugated to the PEG chain via covalent bond with the functional group, directly or via a second functional group attached to the lectins.
  • the lectins are conjugated to the other member of the receptor/ligand pair and attach to the EVs via receptor-ligand binding. See examples provided in FIG. 14.
  • the one or more lectins disclosed herein may be conjugated to the surface of the EVs such as WEVs (e.g., cargo-loaded).
  • the one or more lectins can be conjugated to a lipid via a PEG linker.
  • the lipid can be integrated into the lipid membrane of the EVs such as WEVs, the lipid particles or the hybrid EVs. See, e.g., FIG. 16, top panel.
  • the PEG linker may have a molecular weight of about IkDa to about 10 kDa, for example, about 3kDa to about 8 kDa. In some examples, the PEG linker may have a molecular weight of about 5 kDa.
  • the one or more lectins may be attached to EVs via covalent bonding. See, e.g., FIG. 16, bottom panel.
  • the one or more lectins can be conjugated to a first functional moiety, which can react with a second functional moiety attached to the surface of the EVs.
  • Exemplary functional moieties are provided elsewhere in the present disclosure. See, e.g., FIG. 15.
  • the one or more lectins can be incubated with the EVs under suitable conditions allowing for reactions between the two functional moieties to form a covalent bond, thereby attaching the one or more lectins on the surface of the EVs, the lipid particles, or the hybrid GVs.
  • the one or more lectins can be conjugated to a member of a receptor-ligand pair.
  • the other member of the receptor-ligand pair can be conjugated to the surface of the EVs.
  • the two members of the receptor- ligand pair bind to each other, thereby producing EVs, lipid particles, or hybrid GVs having surface modification of the one or more lectins.
  • the receptor-ligand pair is biotin-strepta vidin.
  • the receptor-ligand pair is nitrilotriacetic acid-His tag.
  • the one or more lectins may be tethered to the GVs via a fusion approach, in which lectin-carrying nanoparticles may be fused with GVs, such that the lectins would be displayed by the hybrid GVs or fused GVs thus formed.
  • Exemplary methods for making GVs displaying one or more lectins disclosed herein are provided below.
  • a method for making lectin-displaying extracellular vesicles such as WEVs, the method comprising: (i) incubating extracellular vesicles with one or more lectins to allow for attachment of the one or more lectins onto the extracellular vesicles, thereby producing lectin-displaying extracellular vesicles; and(ii) collecting the lectin-displaying extracellular vesicles produced in step (i).
  • the one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract, e.g., those disclosed herein.
  • GI gastrointestinal
  • a method for making lectin-displaying extracellular vesicles comprising: (i) contacting GV s(e.g., EVs such as WEVs) with a lipid nanoparticle carrying one or more lectins to allow for fusion of the GVand the lipid nanoparticle, thereby forming a hybrid GV displaying the one or more lectins, and (ii) collecting the fused GVs.
  • GV s e.g., EVs such as WEVs
  • a lipid nanoparticle carrying one or more lectins to allow for fusion of the GVand the lipid nanoparticle, thereby forming a hybrid GV displaying the one or more lectins
  • collecting the fused GVs bind enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract, e.g., those disclosed herein.
  • GI gastrointestinal
  • a method for making lectin -displaying GVs comprising: (i) incubating a hybrid GVswith one or more lectins to allow for attachment of the one or more lectins onto the hybrid GVs, thereby producing lectin-displaying GVs; and (ii) collecting the lectin-displaying GVs.
  • the method may further comprise fusing a lipid nanoparticle with an GV to form the hybrid GV.
  • the one or more lectins bind enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract, e.g. , those disclosed herein.
  • GI gastrointestinal
  • the lipid nanoparticles may carry any of the cargos as disclosed herein to produce cargo-loaded and lectin-modified GVs.
  • the GVs either before cargo-loading and/or lectin-modification or after cargoloading and/or lectin-modification, may be treated by one or more glycosidases (e.g., sialidase) to reduce surface sugar contents, for example, to remove sialic acid. Doing so could reduce or eliminate binding of the GVs to lectins, e.g., those attached to other GVs.
  • glycosidases e.g., sialidase
  • any of the lipid particles may comprise a functional moiety on the surface (surface functionalization) for association with the one or more lectins disclosed herein.
  • the functional moiety may be linked to a lectin (e.g., those disclosed herein) directly via a covalent bond formed between the functional moiety and a second functional moiety on the lectin.
  • the functional moiety may be conjugated to a functional agent, which may interact with a lectin, either directly or indirectly (via a second functional agent).
  • a functional moiety refers to any functional group capable of interacting with another moiety by a covalent fond or non-covalent interaction.
  • the functional moiety used herein can be a chemically functional group, which refers to an atom or a group of atoms responsible for the characteristic chemical reactions of molecules carrying the functional group.
  • exemplary functional groups include a hydroxyl group, a carbonyl group, a carboxyl group, thiol group, an amino group, a sulfhydryl group, and a phosphate group.
  • the functional moiety may be a functional group reactive in a click chemistry reaction, for example, an azide group, a Dibenzocyclooctyne (DBCO) group, or a functional group reactive in a Copper(I)-catalyzed azide-alkyne cycloaddition (CuA AC), in strain-promoted azide-alkyne cycloaddition (SPAAC), or in strain- promoted alkyne -nitrone cycloaddition (SPANC).
  • the functional moiety may be a peptide substrate of a sortase, for example, a peptide comprising a motif of LPETG or a polyG or poly A tail.
  • Peptide substrates of sortases are known in the art. See, e.g., 20160122707, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.
  • the functional moiety is conjugated to a PEG chain connected to one or more lipids in the lipid layer of the lipid particles.
  • the PEG chain may have a molecular weight of about 1 kDa to 10 kDa. In some examples, the PEG chain has a molecular weight of about 2 kDa to about 5 kDa.
  • the functional moiety (e.g., conjugated to the PEG chain) may react with a second functional moiety conjugated to the lectin to form a covalent bond, thereby- linking the lectin to the surface of the lipid particles.
  • the reaction may occur between an azide group on the surface of the lipid particle and a DBCO group on the lectin. See, e.g., FIG. 14.
  • the functional moiety (e.g., conjugated to the PEG chain) may react with a functional agent, e.g., via a covalent bond, to attach the functional agent to the surface of the lipid particle.
  • the functional agent may be a member of a receptor-ligand pair.
  • the other member of the receptor-ligand pair can be conjugated to the lectin.
  • the lectin Via interaction between members of the receptor-ligand pair, the lectin can be conjugated on the surface of the lipid particles.
  • Exemplary receptor-ligand pairs include biotin- streptavidin or nitrilotriacetic acid- His tag.
  • the resultant GVs or fused vesicles having surface modification of lectins may be enriched by conventional methods or approached disclosed herein, eg., ion-exchange chromatography, affinity chromatography, or a combination thereof.
  • the lectin-modified vesicles may be selectively collected by negative selection (e.g., excluding lipid nanoparticles) or positive selection (e.g., collecting specifically the fused vesicles).
  • the lectin-modified vesicles may be enriched by fractionation based on particle size, for example, SEC.
  • the fused vesicles may be enriched via an affinity binding approach, using a target molecule that specifically binds lectin-modified, cargo-carrying vesicles.
  • target molecule may be a lectin, for example, Con A, RCA, WGA, DSL, J acalin, and any combination thereof.
  • the lectin-modified vesicles may be enriched using one or more columns (e.g., an ion-exchange column and/or an affinity column) that selectively bind unfused lipid nanoparticles and/or GVs.
  • the lectin-modified vesicles may be enriched using one or more columns (e.g., an ion-exchange column and/or an affinity column) that selectively bind the lectin-modified, cargo-carrying vesicles.
  • columns e.g., an ion-exchange column and/or an affinity column
  • the lectin-modified, optionally cargo-carrying, GVs may be treated by a suitable approach (e.g., enzymatic digestion) to reduce or remove surface sialic acid residues.
  • a suitable approach e.g., enzymatic digestion
  • one or more of neuraminidase and/or sialidase may be used for removing surface sialic acid residues.
  • the present disclosure provides methods for loading any of the viral particles such as AAV particles onto GVs disclosed herein such as EVs (e.g., WEVs).
  • the loading methods may involve incubation of the viral particles (e.g., AAV1 or AAV6) and the GVs (e.g., EVs) under suitable conditions allowing for tethering of the viral particles to the GVs.
  • Such an incubation step may also contain the one or more lectins disclosed here such that viral particle loading and surface lectin modification could occur in one step.
  • the loading methods disclosed herein may involve a high shear force such as microfludization or sonication.
  • the loading methods disclosed herein may involve electroporation. Any of the loading methods disclosed herein may be performed in the presence of a fusogenic agent such as PEG.
  • a method for preparing GVs loaded with a viral particle via incubation may comprise incubating a formulation comprising a population of GV s(e.g. leave EVs) and a viral particle (e.g., AAV1 or AAV6, including any AAV6 variant disclosed herein) under conditions (e.g., suitable temperature and/or time period) allowing for tethering of the viral particle to the GVs.
  • a formulation comprising a population of GV s(e.g. continue EVs) and a viral particle (e.g., AAV1 or AAV6, including any AAV6 variant disclosed herein) under conditions (e.g., suitable temperature and/or time period) allowing for tethering of the viral particle to the GVs.
  • the formulation may further comprise one or more lectins that bind enterocytes, Tuft cells, Goblet cells, and/or Peyer’s patches at a compartment of a gastrointestinal (GI) tract such that the lectins can alto attach to the EVs during the incubation.
  • GI gastrointestinal
  • the formulation may comprise one or more agents that can facilitate attachment of the viral particles and/or the lectins to the GVs.
  • PEG may be used for this purpose.
  • suitable cationic lipids may be used as well.
  • provided herein is a method for preparing viral particle-loaded EVs such as WE Vs under a high shear force, for example, using a microfluidic device or a sonication device.
  • the loading method disclosed herein involves microfluidics (e.g., continuous -flow microfluidic or droplet-based microfluidic methods).
  • Microfluidics involve manipulating and controlling fluids, usually in the range of microliters (1 O’ 6 ) to picoliters (IO - 12 ), in networks of channels with dimensions from tens to hundreds of micrometers.
  • Fluid handling can be manipulated by components such as microfluidic pumps or niicrofluidic valves.
  • Microfluidic pumps can supply fluids in a continuous way or can be used for dosing.
  • Microfluidic valves can inject precise volumes of sample or buffer.
  • any of the EVs such as WEVs disclosed herein can be mixed with viral particles as also disclosed herein (e.g., AAV particles) at a suitable ratio, for example, 1:10 to 10:1 (by particle numbers) to form a suspension.
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1:8 to 8:1.
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1:5 to 5:1,
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1:3 to 3:1.
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1 :2 to 2:1.
  • the suspension contains the EVs and the viral particles at a ratio of 1:10, 1:9, 1:8, 1:7, 1 :6, 1:5, 1:4, 1:3, or 1:2. In some instances, the suspension contains the EVs and the viral particles at a ratio of 10:1, 9;1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. In one specific example, the suspension contains the EVs and the viral particles at a ratio of 1:1.
  • the suspension containing the GV's and the viral particles can then be loaded onto a niicrofluidic device, which comprises one or more channels (e.g., of glass and/or polymer materials).
  • the one or more channels of the microfluidic device may have a diameter of about 75 pm to about 1, 100 pm.
  • the suspension may pass the channels once or multiple times (e.g., up to 3 times). In each pass, a constant pressure railing from about 10,000 to about 40,000 psi is applied to the suspension, resulting in loading of the viral particles onto the EVs in the suspension.
  • the suspension may pass the channel once under the pressure of 10,000-40,000 psi (e.g., 10,000, 20,000, 30,000, or 40,000 psi).
  • the suspension may pass the channel twice under the pressure of 10,000-40,000 psi (e.g., 10,000, 20,000, 30,000, or 40,000 psi). In some examples, the suspension may pass the channel three times under the pressure of 10,000-40,000 psi (e.g., 10,000, 20,000, 30,000, or 40,000 psi). In one specific example, the suspension may pass the channel once under the pressure of 20,000 psi. In another specific example, the suspension may pass the channel multiple times (e.g., twice or three times) under the pressure of 20,000 psi.
  • the loading method disclosed herein involves sonication, the act of applying sound energy to agitate particles in a sample.
  • ultrasonic frequencies >20 kHz
  • a probe sonicator may be used in the loading method disclosed herein.
  • any of the GVs such as E Vs disclosed herein can be mixed with viral particles as also disclosed herein (e.g. , AAV particles such as AAV1 or AAV 6) at a suitable ratio, for example, 1 :10 to 10:1 (by particle numbers) to form a suspension.
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1:8 to 8:1.
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1 :5 to 5:1.
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1:3 to 3:1 .
  • the suspension contains the EVs and the viral particles at a ratio ranging from 1:2 to 2:1. In some instances, the suspension contains the GVs and the viral particles at a ratio of 1: 10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, or 1:2. In some instances, the suspension contains the EVs and the viral particles at a ratio of 10:1, 9: 1, 8:1, 7: 1 , 6:1, 5: 1 , 4:1, 3: 1 , or 2:1. In one specific example, the suspension contains the EVs and the viral particles at a ratio of 1:1 .
  • the suspension can then be placed in a sonicator such as a probe sonicator and sonication can be applied to the suspension, allowing for loading of the viral particles onto the GVs.
  • a sonicator such as a probe sonicator
  • sonication may be performed under mild conditions, for example, at a controlled temperature.
  • the viral particle-loaded GVs produced by any of the methods disclosed herein can be isolated, for example, via size selection, positive selection, negative selection, or a combination thereof.
  • the one or more lectins disclosed herein may be included in the process disclosed herein such that the lectins can also be tethered to the GVs to produce modified EVs loaded with the viral particles and surface modified by the lectins.
  • a method for preparing viral particle-loaded GVs such as EVs via electroporation which uses a pulse of electricity to briefly open pores in the GVs and/or viral particles, thereby allowing for loading of the viral particles onto the GVs.
  • any of the GVs such as EVs disclosed herein can be mixed with viral particles as also disclosed herein (e.g., AAV particles) at a suitable ratio, for example, 1:10 to 10:1 (by particle numbers) to form a suspension.
  • the suspension contains the GVs and the viral particles at a ratio ranging from 1:8 to 8:1.
  • the suspension contains the GVs and the viral particles at a ratio ranging from 1 :5 to 5:1. In some instances, the suspension contains the GVs and the viral particles at a ratio ranging from 1:3 to 3:1. In some instances, the suspension contains the GVs and the viral particles at a ratio ranging from 1:2 to 2:1. In some instances, the suspension contains the GVs and the viral particles at a ratio of 1 : 10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, or 1 :2. In some instances, the suspension contains the GVs and the viral particles at a ratio of 10:1, 9;1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1. In one specific example, the suspension contains the GVs and the viral particles at a ratio of 1:1.
  • the suspension can be placed in vial suitable for electroporation, which can be placed into an electroporation device. Electroporation can be performed following routine practice or nianufactuer’ s protocol based on the electroporation device used to produce viral particle loaded GVs.
  • the viral particle-loaded GVs produced by any of the methods disclosed herein can be isolated, for example, via size selection, positive selection, negative selection, or a combination thereof.
  • the one or more lectins disclosed herein may be included in the process disclosed herein such that the lectins can also be tethered to the GVs to produce modified EVs loaded with the viral particles and surface modified by the lectins.
  • any of the loading methods disclosed herein may be performed in the presence of one or more fusogenic agents to enhance loading efficiency of the viral particles onto the GVs.
  • a suitable fusogenic agent may be placed in any of the suspensions containing the viral particles and the GVs as disclosed herein at a suitable concentration.
  • the fusoenic agent-containing suspension can be subject to a high shear force (e.g., microfluidization or sonication) or electroporation as disclosed herein to produce viral particle-loaded GVs.
  • the fusogenic agent used in any of the loading methods disclosed herein is polyethylene glycol (PEG).
  • the PEG used in a loading method may have a molecule weight ranging from about 400 Da to about 8,000 Da.
  • the PEG may have a molecule weight ranging from about 400 Da to about 1,000 Da (e.g., 400, 500, 600, 700, 800, 900, or 1 ,000 Da). In some examples, the PEG may have a molecule weight ranging from about 1,000 Da to about 3,000 Da (e.g., 1,500, 2,000, 2,500, or 3,000 Da). In some examples, the PEG may have a molecule weight ranging from about 3,000 Da to about 8,000 Da (e.g., 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, or 8,000 Da).
  • the PEG used in any of the loading methods disclosed herein may have a concentration (w/v) ranging from about 10% to about 40%.
  • the PEG may have a concentration (w/v) ranging from about 10% to about 20% (e.g., 10%, 15% or 20%).
  • the PEG may have a concentration (w/v) ranging from about 20% to about 30% (e.g., 20%, 25% or 30%).
  • the PEG may have a concentration (w/v) ranging from about 30% to about 40% (e.g,, 30%, 35% or 40%).
  • PEG having a molecule weight of about 400 Da at a concentration of about 25%-35% may be used in a loading method disclosed herein (e.g., via microfludization or sonication).
  • PEG having a molecule weight of about 1,000 Da at a concentration of about 25%-35% may be used in a loading method disclosed herein (e.g., via microfludization or sonication).
  • PEG having a molecule weight of about 3,000 Da at a concentration of about 25%-35% (e.g., 30% w/v) may be used in a loading method disclosed herein (e.g., via microfludization or sonication).
  • PEG having a molecule weight of about 8,000 Da at a concentration of about 25%-35% (e.g., 30% w/v) may be used in a loading method disclosed herein (e.g., via microfludization or sonication).
  • the fusogenic agent such as PEG may be removed from the resultant composition containing the viral particle-loaded the GVs. Conventional methods can be used for removal of the fusogenic agent such as PEG. For example, PEG may be removed by tangenical flow filtration (TFF).
  • modified GVs such as modified extracellular’ vesicles (e.g., modified WEVs), which are loaded with any of the viral particles disclosed herein such as the AAV particles (AAV1 or A AV6) and optionally display one or more lectins targeting GI cells as also disclosed herein.
  • modified EV particles may be produced by any of the loading and tethering methods disclosed herein.
  • the modified EVs disclosed herein can be used as suitable delivery vehicles for therapeutics or diagnostics that were previously not orally administrable or suffered from other delivery challenges such as poor bioavailability, storage instability, metabolism, off-target toxicity, or decomposition in vivo, thereby satisfying a long-felt but unmet need.
  • the modified EVs may comprise any of the GVs such as EVs as disclosed herein and one or more viral particles.
  • the modified GVs carry surface lectins that target GI cells such as those disclosed herein (e.g., ECL and/or UEAl).
  • the GVs such as EVs comprise a lipid membrane, to which the one or more viral particles are associated (incorporated or loaded, which are used interchangeably).
  • the viral particle- loaded GVs contain a naturally-occurring lipid membrane, to which one or more proteins (e.g., those disclosed herein) are associated.
  • the viral particle-loaded GVs disclosed herein are not derived from fusion of GVs with any artificial lipid nanoparticles and thus do not contain synthetic lipids or others from any artificial lipid nanoparticles.
  • the protein content may be the same as in naturally-occurring GVs. Alternatively, the protein content may be modified, for example, with addition or removal of certain proteins.
  • the viral particle-loaded GVs such as viral particle-loaded EVs disclosed herein comprise one or more proteins of CD9, CD81, BSG, and SLC3A2.
  • the EVs disclosed herein are free of CD63 and/or LAMP1, e.g., detection of the involved proteins (e.g., CD63 and LAMP1 ) by a conventional method or only marginal signal is detected such that presence or absence of the involved proteins cannot be determined.
  • the GV portion in the modified GVs is naturally -occurring and nonmodified.
  • the GV portion may be modified, for example, to incorporate a targeting moiety that recognizes specific tissues/cells or to incorporate one or more protecting moieites to enhance stability.
  • any of the modified GVs disclosed herein may carry (via the viral particle) one or more agents of interest, such as any of the therapeutic agents disclosed herein or a diagnostic agent.
  • agents of interest such as any of the therapeutic agents disclosed herein or a diagnostic agent.
  • Such GVs can be used to deliver the viral particles and thus the agents of interest to target host cells via, e.g. , oral administration.
  • the GOT carried in the viral genome of the viral particles may produce the agents of interest (e.g., therapeutic or diagnostic agents such as those discloed herein) in the target cells, for example, in intestinal epithelium cells.
  • the target cells such as intestinal epithelium cells secrete the encoded therapeutic agents or diagnostic agents to release those to the circulation system (e.g., via the basolateral pole) or to the lumen of the gut (e.g., via the apical pole).
  • modified GVs such as modified EVs disclosed herein may be mixed with one or more pharmaceutically acceptable carrier, adjuvant, or vehicle to form a pharmaceutical composition, which is also within the scope of the present disclosure.
  • the amount of the viral particles such as AAV viral particles in the modified GVs is an amount effective for delivering the agents of interest that can produced by the GOIs in the viral genome (e.g., the AAV genome), which can be used for treating relevant disease, disorder, or condition in a patient in need thereof.
  • the pharmaceutical composition as disclosed herein is formulated for oral administration to a patient.
  • patient means an animal, for example a mammal, such as a human.
  • compositions of this invention refers to a nontoxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the therapeutic -loaded vesicle with which it is formulated.
  • Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, poly acrylates, waxes, polyethylene
  • compositions of the present disclosure may be administered via a suitable route, for example, by oral administration.
  • the compositon may be administered via a parenteral route.
  • parenteral includes subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the modified GVs such as modified EVs or pharmaceutical compositions thereof are administered by an oral, intravenous, subcutaneous, intranasal, inhalation, intramuscular, intraocular, intraperitoneal, intratracheal, transdermal, buccal, sublingual, rectal, topical, local injection, or surgical implantation route.
  • the administration route is oral.
  • compositions of this disclosure may be orally- administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions, hi the case of tablets for oral use, carriers commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • the pharmaceutical compositions for oral administration as described herein may be administered to a subject with or without food. In some embodiments, pharmaceutically acceptable compositions disclosed herein are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.
  • the therapeutic or diagnostic attributes of the viral particles e.g.,
  • AAV particles are achieved via non-oral means. Achieving systemic distribution of the encapsulated therapeutic agent using whey-derived vesicles following delivery would be the major objective of this approach but it is also possible to achieve selective delivery to sites of interest through the use of targeting ligands (e.g., antibodies, peptides, aptamers, or others: see, e.g., Friedman, A. D. et al., Carr Pharm Des 2013; 19(35): 6315—6329).
  • targeting ligands e.g., antibodies, peptides, aptamers, or others: see, e.g., Friedman, A. D. et al., Carr Pharm Des 2013; 19(35): 6315—6329.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxy ethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • surfactants such as polysorbates (Tween® compounds), sorbitan esters (Span® compounds) and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions of this disclosure may be administered in the form of suppositories for rectal administration.
  • suppositories for rectal administration.
  • suppositories can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter, beeswax and polyethylene glycols.
  • compositions of this disclosure may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific therapeutic-loaded GV such as EV employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated.
  • the amount of a therapeutic- loaded GVs such as EVs of the present disclosure in the composition may also be depend upon the particular therapeutic-loaded vesicle in the composition.
  • modified GVs e.g., modified EVs such as modified WEVs
  • compositions comprising such may be used to deliver the GOIs contained in the viral particles to a subject, e.g., to an intestinal site via oral administration, where the viral particles such as AAV particles can enter into host cells and produce therein the encoded agents of interest, such as therapeutic nucleic acids or therapeutic proteins.
  • the host cells e.g., intestinal epithelium cells such as enterocytes
  • the agents of interest can release the agents of interest into the circulation system of the body, thereby achieving systemic delivery of the agents of interest.
  • GVs as a delivery vehicle for viral particles can enhance desirable properties of the biological molecule such as improving oral bioavailability, for example by minimizing destruction of the nucleic acid capable of producing the agents of interest in the gut or minimizing liver first-pass effect; or improving delivery of the nucleic acid to a target tissue; or increasing the solubility and stability of the nucleic acid in vivo.
  • an effective amount of any of the modified GVs can be administered to a subject in need of the treatment via a suitable route, e.g., those described herein.
  • the modified GVs may be administered orally.
  • the the modified GVs would be effective in treating or diagnosing target diseases of interest, depending upon the agents of interest that can be produced by the GOIs carried by the viral particles loaded therein.
  • the GOIs carried by the viral particles can produce an autoimmue antigen.
  • GVs loaded with such viral particles can be used to treat, prevent, or ameliorate an autoimmune disease, such as Rheumatoid Arthritis, Diabetes Mellitus, Insulin-DependentLupus Erythematosus (Systemic), Multiple Sclerosis, Psoriasis, Pancreatitis, Inflammatory Bowel Diseases, Crohn’s disease, ulcerative colitis, Sjogren’s Syndrome, autoimmune encephalomyelitis, experimental Graves’ Disease, Sarcoidosis, Scleroderma, primary biliary cirrhosis, Chronic lymphocytic thyroiditis, Lymphopenia, Celiac Disease, Myocarditis, Chagas Disease, Myasthenia Gravis, Glomerulonephritis, IGA, Aplastic Anemia, Lupus Nephritis, Hamman-Rich syndrome, Hepatitis, Chronic Active Dermatitis, a autoimmune
  • Addison’s disease Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Axonal & neuronal neuropathy (AMAN), Behcet’s disease, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Cicatricial pemphigoid/benign mucosal pemphigoid, Cogan’s syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn’s disease.
  • Pars pianitis peripheral uveitis
  • Parsonnage- Turner syndrome Pemphigus
  • Peripheral neuropathy Perivenous encephalomyelitis
  • Pernicious anemia PA
  • POEMS syndrome polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes
  • Polyarteritis nodosa Polymyalgia rheumatica
  • Polymyositis Postmyocardial infarction syndrome
  • Postpericardiotomy syndrome Postpericardiotomy syndrome.
  • RLS Restless legs syndrome
  • RA Rheumatoid arthritis
  • SPS Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocarditis
  • SBE Subacute bacterial endocardi
  • the GOIs carried by the viral particles loaded onto the GVs may produce a therapeutic agent (nucleic acid or protein) for modulating an immune response, and/or for treating hyperproliferative disease, disorder, or condition such as cancer.
  • the disease, disorder, or condition is selected from a hyperproliferative disorder, viral or microbial infection, autoimmune disease, allergic condition, inflammatory disease, cardiovascular disease, metabolic disease, or neurodegenerative disease.
  • Any therapeutic nucleic acids and/or therapeutic proteins known in the art can be delivered to a subject by the approach disclosed herein. Examples are provided in Tables 1-4 above.
  • the modified GVs are administered to a subject in need of the treatment.
  • the viral particle carries one or more GOIs that can produce one or more agents of interest, e.g., therapeutic nucleic acids or therapeutic proteins as also disclosed herein.
  • the GVs may be loaded with AAV viral particles of a particular serotype that can infect enterocytes (e.g., AAVT, AAV6 or a variant thereof as disclosed herein
  • the agent of interest encoded by the GOIs carried in one or more viral particles such as AAV particles may be an antibody as disclosed herein.
  • the agent of interest encoded by the GOIs carried in one or more AAV viral particles such as one or more AAV6 variant particles may be a multi-chain protein such as a multichain antibody.
  • the coding sequences for the multiple chains of the multi-chain protein may be located in one nucleic acid molecule carried by an AAV viral particle (e.g., in a polycistronic setting).
  • each of the coding sequences may be located in one nucleic acid molecule carried by an AAV viral particle (e.g., in a monocistronic settling).
  • Such vector designs would depend on multiple factors, such as packaging capacity of a particular viral particle, which are known to those skilled in the art.
  • the modified GVs such as modified EVs carrying an AAV particle (e.g., AAV1, AAV6 or a variant thereof) may be used for delivering coding sequences of a multi-chain antibody.
  • AAV AAV1, AAV6 or a variant thereof
  • the coding sequences for the heavy chain and light chain of the antibodies are inserted into two AAV vectors.
  • the AAV viral particles carrying the coding sequences of the heavy chain and light, chain may be loaded into the same GVs or different.
  • a composition comprising GVs loaded with a first AAV particle for producing the heavy chain, and GV loaded with a second AAV particle for producing the light chain can be administered (e.g., orally) to a subject.
  • suitable host cells e.g., intestinal epithelium cells such as enterocytes
  • both the heavy chain and the light chain can be expressed and assembled to form the multichain antibody, which can be released to the circulation system of the subject, to exert the intended biological activity.
  • the first and second AAV particles can be loaded onto the same GV particles.
  • the first and second AAV particles can be loaded onto different GV particles.
  • GV particles loaded with the first AAV particle and GV particles loaded with the second A AV particle may be mixed in one formulation for administration to a subject.
  • Exemplary antibody agents that can be delivered to a subject using the AAV viral particle-loaded GV approach as disclosed herein are provided above.
  • any of the modified GVs described herein or pharmaceutically acceptable composition thereof may be administered to a patient in need thereof in combination with one or more additional therapeutic agents and/or therapeutic processes.
  • the modified GVs can be administered alone or in combination with one or more other therapeutic compounds, possible combination therapy taking the form of fixed combinations or the administration of the the modified GVs and one or more other therapeutic compounds being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic compounds.
  • the modified GVs can besides or in addition be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above.
  • Such additional agents may be administered separately from a provided composition comprising the modified GVs (e.g., EVs), as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together with the modified GVs in a single composition. If administered as part of a multiple dosage regime, the two active agents may be submitted simultaneously, sequentially or within a period of time from one another.
  • the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this disclosure.
  • the modified GVs such as modified EVs can be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form.
  • the present disclosure provides a single unit dosage form comprising the modified EVs, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • the additional agent is encapsulated in the same vesicle carrier as the modified GVs.
  • the additional agent is encapsulated in a different vesicle than the modified GVs.
  • the additional agent is not encapsulated in a vesicle.
  • the additional agent is formulated in a separate composition from the the modified GVs.
  • compositions of this disclosure should be formulated so that a dosage of between 0.01—100 mg/kg body weight/day of the viral particle-loaded GVs can be administered.
  • compositions which comprise an additional therapeutic agent that additional therapeutic agent and the modified GVs of the present disclosure may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent. In such compositions a dosage of between 0.01—1,000
  • the amount of additional therapeutic agent present in the compositions of this disclosure will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent.
  • the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.
  • agents with which the modified GVs of the present disclosure may be combined include, without limitation: treatments for Alzheimer’s Disease such as Aricept® and Excelon®; treatments for HIV such as ritonavir; treatments for Parkinson’s Disease such as L- DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine, pergolide, trihexephendyl, and amantadine; agents for treating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex® and Rebif®), Copaxone®, and mitoxantrone; treatments for asthma such as albuterol and Singulair' 8 '; agents for treating schizophrenia such as zyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agents such as corticosteroids, TNF blockers, IL-
  • immunomodulatory and immunosuppressive agents such as cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids, cyclophophamide, azathioprine, and sulfasalazine
  • neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anti- convulsants, ion channel blockers, riluzole, and anti-Parkinsonian agents: agents for treating cardiovascular disease such as beta- blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents for treating liver disease such as corticosteroids, cholestyramine, interferons, and anti-viral agents; agents for treating blood disorders such as corticosteroids, anti-leukemic agents, and growth factors;
  • combination therapies of the present disclosure include a monoclonal antibody or a siRNA therapeutic, which may or may not be encapsulated in a vesicle carrier such as WEVs.
  • the present disclosure provides a method of treating an inflammatory disease, disorder or condition by administering to a patient in need thereof a cargo-loaded GV such as EVs and one or more additional therapeutic agents.
  • additional therapeutic agents may be small molecules or a biologic and include, for example, acetaminophen, non-steroidal anti-inflammatory drugs (NS AIDS) such as aspirin, ibuprofen, naproxen, etodolac, and celecoxib, colchicine, corticosteroids such as prednisone, prednisolone, methylprednisolone, hydrocortisone, and the like, probenecid, allopurinol, febuxostat, and sulfasalazine.
  • NS AIDS non-steroidal anti-inflammatory drugs
  • corticosteroids such as prednisone, prednisolone, methylprednisolone, hydrocortisone, and the like, probenecid, allopurin
  • monoclonal antibodies such as tanezumab, anticoagulants such as heparin and warfarin, antidiarrheals such as diphenoxylate, and loperamide, bile acid binding agents such as cholestyramine, alosetron, and lubiprostone, anticholinergics or antispasmodics such as dicyclomine, beta-2 agonists such as albuterol and levalbuterol, anticholinergic agents such as ipratropium bromide and tiotropium, inhaled corticosteroids such as beclomethasone dipropionate and triamcinolone acetonide.
  • anticoagulants such as heparin and warfarin
  • antidiarrheals such as diphenoxylate
  • loperamide bile acid binding agents
  • anticholinergics or antispasmodics such as dicyclomine
  • beta-2 agonists such as albuterol and levalbuterol
  • anticholinergic agents such
  • the modified GVs disclosed herein may also be used to advantage in combination with an antiproliferative compound.
  • antiproliferative compounds include, but are not limited to, aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule active compounds; alkylating compounds; histone deacetylase inhibitors; compounds which induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors: mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity and further anti- angiogenic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonists; anti -androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitor
  • Conforma Therapeutics temozolomide (TemodaT®); kinesin spindle protein inhibitors, such as SB715992 or SB743921 from GlaxoSmithKline, or pentamidine/chlorpromazine from CombinatoRx; MEK inhibitors such as ARRY142886 from Array BioPharma, AZD6244 from AstraZeneca, PD181461 from Pfizer and leucovorin.
  • Additional therapeutic agents for co-use with the viral particle-loaded GVs such as EVs as described herein are known in the art and/or disclosed in WP2018102397 and the references cited therein, the relevant, dislcosures of each of which are incorporated by reference for the purposes or subject matter referenced herein.
  • Tills example illustrates an exemplary process for purifying extracellular vesicles from bovine whey (WEVs) that involves tangential flow filtration (TFF) and size exclusion chromatography (SEC).
  • WEVs bovine whey
  • TDF tangential flow filtration
  • SEC size exclusion chromatography
  • step 4 After the first, elution, switch over the Al line to the second 50L bottle of chilled IX PBS. Repeat step 3 with the remaining 500 mL of clarified TFF material. Pause the method and chase TFF material with IX PBS if necessary.
  • This example investigates the stability ot whey-denved extracellular vesicles (WEV) and AAV particles under a high shear force, such as microfluidization and probe sonication.
  • WEV extracellular vesicles
  • AAV stocks were prepared by triple-transfection of suspension-cultured FreeStyleTM 293-F cells (Thermo Fisher Scientific, Waltham, MA) with an adenoviral helper plasmid
  • pHelper Cell Biolabs, Inc., San Diego, CA
  • ITR-containing plasmid which optionally encodes a gene of interest
  • packaging plasmid encoding AAV2 Rep and the Cap gene for AAV 1 serotype.
  • Cells were harvested 72h post-transfection and lysed by freeze-thawing, DNAse- digested, then clarified by low-speed centrifugation and filtration through a 0.45um filter.
  • AAVs were purified from clarified lysate by affinity chromatography using pre-packed AAVX columns (Thermo Fisher Scientific, Waltham, MA) operated on an AktaPure FPLC system (Cytiva, Marlborough, MA). Purified AAVs were concentrated and buffer-exchanged into a storage buffer containing IX phosphate-buffered saline (PBS), supplemented with NaCl to a final concentration of 350 mM and 5% D-sorbitol.
  • PBS IX phosphate-buffered saline
  • the WEV or AAV particles were suspended in a solution and passed through a microfluidizer for 1-3 times. A constant pressure at. 10,000 psi or 30,000 psi was applied to the sample in each pass. The concentrations and mean particle sizes of WEV and AAV particles under the various microfluidization conditions were then analysed.
  • Nanoparticle tracking analysis was performed to measure particle size and particle concentration.
  • NTA is a method for visualizing and analyzing particles in liquids that relates the rate of Brownian motion to particle size.
  • FIG. 1A As shown in FIG. 1A, no significant WEV concentration changes was observed at the various microfluidization conditions, except for a slight decrease on multiple passes. The mean particle size was also maintained after microfluidization.
  • FIG. IB Intact particle morphology was observed under electromicroscopy after multiple passes through the microfluidizer.
  • FIGs. 1C and ID In sum, WEV particles showed good stability upon microfluidizer pressure.
  • the AAV particles showed similar stability upon microfluidizer pressure under a low pressures (e.g., 10,000 psi) upon one or more passes, but. not. by multiple passes at a high pressure (e.g., 3 passes at 30,000 psi), as determined by both AAV concentration (via both Zetasizer and ELISA assays) and particle size changes.
  • FIGs. 2A and 2B. (Hi) Stability Test by Probe Sonicator
  • This example illustrates an exemplary process of making AAV-loaded WEV particles by microfluidizer in the presence of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • AAV- WEV was mixed at 1:1 particle concentration ratio in PEG / lx PBS. A 2 mL of the total volume of this mixture was used in this study. Various PEG concentrations (10% and 30% w/v) and sizes (MW 8,000, 3,000, and 400 Da) were used in this study. Microfludization was performed at 20,000 psi, single pass. The samples were inserted into the chamber using 10 mL luer lock syringe by pressing and holding the retract button in LV 1 system. The extended button was then pressed and held to initiate a high-pressure stroke. Hie samples were collected and stored on an ice bath.
  • PEG was removed and exchanged to lx PBS by hollow' fiber small TFF system (MWCO 500 kDalton) using 20x volume of lx PBS vs sample volume.
  • Particle size, particle concentration, and surface charge were measured by NTA (Nanoparticle Tracking analysis), Zetasizer, and Spectradyne orthogonally.
  • the incorporation percentage was measured by AAVX affinity resin / ELISA assay, WEV Immunoprecipitation assay, and MSD assay orthogonally.
  • the final morphology was checked by cryo-TEM (transmission electron microscopy). Table 5 below shows the AAV incorporation efficiency under various PEG conditions.
  • This example illustrates an exemplary process of making AAV-loaded WEV particles by probe sonication in the presence of polyethylene glycol (PEG) (10% or 30% w/'v; MW 8,000 or 3,000 Da)
  • PEG polyethylene glycol
  • Table 6 below shows the AAV incorporation efficiency via probe sonication under various PEG conditions.
  • Example 5 Effects of Fusogenic Agents cm AAV Loading Effiency to WEV Particles This example investigates the impact of various fusogenic agents on loading efficiency of AAV into the WEV particles using different techniques, including microfluidization, probe sonication, and electroporation.
  • Various fusogenic agents including polyethylene glycol
  • Table 7 below shows the AAV-incorproation efficiency using the different techniques and fusogenic agents. See also FIG. 5.
  • Example 6 Preparation of A derm- Associated Vires Vectors Carrying Genes of Interest (GOI) Self-complementary (scAAV) or single-stranded (ssAAV) AAVs encoding green fluorescent protein (GFP) in serotypes AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV8, AAVS? and AAVrhlO were procured from the UNC Vector Core (Chapel Hill, NC). A schematic of the scAAV vector is provided in FIG. 6A.
  • scAAV Self-complementary
  • ssAAV single-stranded
  • GFP green fluorescent protein
  • AAV stocks were produced in adherent, HEK 293T cells by triple- transfection of AAV packaging, adenoviral helper, and ITR/transgene plasmids, then purified by two rounds of iodixanol gradient ultracentrifugation.
  • the genome -containing fraction was dialyzed into a storage buffer containing IX phosphate- buffered saline (PBS), supplemented with NaCl to a final concentration of 350 mM and 5% D- sorbitol.
  • PBS IX phosphate- buffered saline
  • scAAVs encoding human erythropoietin (EPO) and NanoLuc® Luciferase (NLuc, Promega, Madison, WI) in serotypes AAV1 , AAV6, and variants of AAV 6 (AAV6.2, AAV6_TM, AAV6.2FF, AAV6.2FVFF) were produced following conventional methods.
  • a schematic of the scAAV vector carrying the EPO and NLuc genes is provided in FIG. 6B.
  • AAV stocks were prepared by triple-transfection of suspension-cultured FreeStyleTM 293 -F cells (Thermo Fisher Scientific, Waltham, MA) with an adenoviral helper plasmid (pHelper, Cell Biolabs, Inc., San Diego, CA), a proprietary ITR-containing plasmid encoding EPO and NLuc, and a packaging plasmid encoding A AV2 Rep and the Cap gene for each serotype or variant tested.
  • pHelper Cell Biolabs, Inc., San Diego, CA
  • ITR-containing plasmid encoding EPO and NLuc
  • packaging plasmid encoding A AV2 Rep and the Cap gene for each serotype or variant tested.
  • AAVs were purified from clarified lysate by affinity chromatography using pre-packed AAVX columns (Thermo Fisher Scientific, Waltham, MA) operated on an AktaPure FPLC system (Cytiva, Marlborough, MA). Purified AAVs were concentrated and buffer-exchanged into a storage buffer containing IX phosphate-buffered saline (PBS), supplemented with NaCl to a final concentration of 350 mM and 5% D-sorbitol.
  • PBS IX phosphate-buffered saline
  • FIG. 7A A schematic of intestinal organoid architecture is provided in FIG. 7A.
  • organoids that had been passaged for at least two weeks were collected by disruption with Gentle Cell Dissociation Reagent (StemCell Technologies, Vancouver, Canada) seeded in Matrigel® Matrix (“Matrigel”, Coming, Elmira, NY) and cultured in IntestiCult Organoid Growth Medium (StemCell Technologies, Vancouver, Canada).
  • tissue-culture treated plates were first coated with a thin layer of Matrigel: Matrigel was diluted in Dulbecco’s phosphate buffered saline (DPBS) and added to the wells for one hour before gently aspirating the mixture.
  • DPBS phosphate buffered saline
  • Organoids that had been passaged for at least two weeks were collected by disruption with Gentle Cell Dissociation Reagent (StemCell Technologies, Vancouver, Canada), then trypsinized to generate single-cell suspensions, washed, and seeded in Organoid Growth Medium-Human (StemCell Technologies, Vancouver, Canada) with ROCK inhibitor Y-2763210 (StemCell Technologies, Vancouver, Canada). Media was changed daily, and ROCK inhibitor was removed after monolayer formation was visually apparent by light microscopy.
  • AAV vectors of various serotypes (AAV1 , AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, and AAVrhlO) carrying GFP prepared according to Example 1 above were incubated with intestinal organoids in the “3D” organoid culture or the “2D” culture disclosed in Example 7 above.
  • AAV-transduced intestinal organoid cells were imaged for expression of GFP by epifluorescence microscopy using an Axio Vert inverted fluorescence/phase-contrast microscope with a FITC filter (Zeiss, Jena, Germany) or an EVOS M7000 Imaging System (Thermo Fisher Scientific, Waltham, MA) with a GFP light cube. Samples within an experiment were always imaged with equivalent exposure times.
  • FIG. 7E Using the “3D” intestinal organoid culture, AAV1, AAV2, AAV2.5, AAV8, and AAVrhlO showed some degrees of transduction efficiency to intestinal cells, particularly, cells at the basolateral pole, with A AVI showed the highest transduction efficiency.
  • FIG. 7E Using the “2D” intestinal organoid culture, AAV 1, AAV2.5, and AAV6 showed some degrees of transduction efficiency to intestinal cells, particularly, cells at the apical pole. Both AAV1 and AAV6 showed high transduction efficiency.
  • FIG. 7F Using the “3D” intestinal organoid culture, AAV1, AAV2, AAV2.5, AAV8, and AAVrhlO showed some degrees of transduction efficiency to intestinal cells, particularly, cells at the basolateral pole, with A AVI showed the highest transduction efficiency.
  • FIG. 7E Using the “2D” intestinal organoid culture, AAV 1, AAV2.5, and AAV6 showed some degrees of transduction efficiency to intestinal cells, particularly, cells
  • AAV1 and AAV6 vectors carrying EPO and NLuc as illustrated in FIG. 6B were incubated with intestinal organoids in the “2D” trans- well organoid culture disclosed in Example 7 above.
  • ECL electrochemiluminescence
  • ECL electrochemiluminescence
  • a SULFO-tag conjugated Goat anti-human Fc antibody (Meso Scale Discovery® MSD, Rockville, MD) was then used to detect the presence of EPOR bound to EPO.
  • the plate was then read by an ECL plate reader (Meso Scale Discovery® MSD, Rockville, MD) in Gold Read Buffer (Meso Scale Discovery® MSD, Rockville, MD) to detect the ECL signal and quantify the amount of functional EPO in the supernatant samples.
  • Several washes with PBS- Tween were performed between each incubation to remove unbound components.
  • AAV6 showed similar transduction efficiency as AAV1 in the “2D” trans- well organoid culture as reflected by the expression levels of EPO and NLuc. Further, the data show that EPO and NLuc proteins were secreted bi-directionally from both AA1 and AAV6 transduced cells.
  • results from this example demonstrates efficient AAV transduction of intestinal epithelial cells with multiple serotypes using a physiologically relevant intestinal organoid model.
  • AAVl transduces the intestinal epithelium from both the basolateral and apical poles
  • AAV6 transduces such cells predominantly from the apical pole.
  • payload proteins e.g., EPO and NLuc
  • EPO and NLuc payload proteins
  • Example 9 Identification of AAV Variants Having Improved Transduction Efficiency to Intestinal Celts
  • AAV 6 capsid protein variants including AAV6.2, AAV6.2FF, AAV6TM, and AAV6.2FVFF variants, were produced by conventional recombinant technology.
  • a schematic of the mutations in the AAV6.2FVFF variant and the corresponding positions in AAVl is provided in FIG. 9. Sequences of the various AAV6 variants are provided above.
  • nLuc nanoluciferase
  • EPO erythropoietin
  • AAVl adeno-associated virus serotype 1
  • AAV6 serotype 6
  • FIG. 11A Levels of luminescence in the treated mice (indicative of nLuc expression) were examined and the results are provided in FIG. 11A. Both AAVl and AAV 6 can mediate expression of nLuc in mouse intestinal cells. The expression of EPO was examined by measuring the spleen size of the treated mice. As shown in FIG. 1 IB, mice treated by either the AAVl or the AAV6 particles showed enlarged spleen sizes as compared to the control mice.
  • Rodent (rats and mice) and human and non-human primate (NHP) fresh gastrointestinal (GI) tissues were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin.
  • Human GI tissues were fixed using the same approach. 5 mm sections were rehydrated and incubated with various biotinylated lectins diluted in PBS and followed by incubation with AF647- streptavidin. Slides were imaged using EVOS and various regions of GI tract were scored from no lectin binding (-) to strong lectin binding (+++). Lectin binding in rat and mouse intestine tissues are provided in Table 8 and lectin binding in human intestine tissues are provided in Table 9 below.
  • Table 10 list common lectins for preclinical validation.
  • Table 11 list the lectin binding to the brush border of villus enterocytes in the intestine segments of various species. A strong binding of lectins ECL and SB A to the brush border in the intestinal segments of the mouse was observed. A strong binding of lectins ECL and LEA- 1 to the brush border in the intestinal segments of rat, cynomolgus monkey and human was also observed.
  • mice were orally administered fluorescently labeled WGA (1 mg per dose or 0.125 mg per dose) in PBS solution. The animals were euthanized 2 hours post administration.
  • Intestine tissues were isolated from the animals and imaged using an in vivo imaging system (IVIS) to examine lectin binding. The results show that the orally administered WGA was retained in duodenum and jejunum for at least 2 hours.
  • IVIS in vivo imaging system
  • fluorescently labeled polystyrene beads were modified with WGA, using 2 kDa 3.4 kDa, or 5 kDa PEG as a linker.
  • WGA modified beads or unmodified beads were administered into mouse duodenum. Fluorescence intensity of was monitored by IVIS for 3 hours. As shown in Figure 12, retention of WGA modified beads (having the various PEG linkers) was observed in duodenum for at least 3 hours.
  • the WGA modified beads using the 2 kDa PEG linker showed better retention as relative to the WGA modified beads using the 3.4 kDa or the 5 kDa PEG linker at various time points.
  • Extracellular vesicles purified from whey (WEVs; 4.34 E+13, 462 uL), AAV1 particles carrying transgenes encoding a transgene (e.g., a luciferase reporter protein) (2.60 E+13, 7'70 uL) were initially mixed at the equal particles concentration to achieve the final concentration of 1.00E+13 particles/mL.
  • PEG MW400, 600 uL
  • 1XPBS buffer 168 uL was added to make up the total volume to 2000 uL. It was then put on Microfluidizer LV1 at 20,000 psi with single pass.
  • the collected formulation was then purified by using hollow fiber membrane (500kD mPES 0.5mm) to remove PEG. 200 uL of each lectins solution at 1 mg/mL was added to 1 mL of AAV-loaded WEV prepared as described above. It was then put on tumbler at 4 °C for 1 h. The resultant formulation was then used for further analysis without further purification.
  • hollow fiber membrane 500kD mPES 0.5mm
  • the resultant latinized, A AV-Loaded WEV particles were characterized to determine their sizes, polydispersity index (PDI), particle concentrations, and zeta potential (ZP) using conventional methods for measuring physical characteristics of particles. Briefly, samples for analysis were prepared by diluting particle -containing solutions ten times in O.lx PBS. Size, particle concentrations and zeta potentials were measured by Malvern Zetasizer Ultra. The results are provided in Table 12. Table 12. Characterization of Letinized AAV Loaded WEV
  • Lectinized and AAV-loaded WEV particles were given to mice via direct duodenum injection.
  • the particles investigated in this example are loaded with AAV1 particles carrying a transgene encoding NanoLuc luciferase as a reporter protein and lectin SB A or ECL, WEV/AAV1/SBA particles or WEV/AAV1/ECL particles.
  • Plasma samples were collected from the treated mice on Days 2, 4, and 10 post administration and subject to analysis by the NanoGlo 1 ® Plasma Nanoluc® assay.
  • the procedure to perform the NanoGlo® Plasma Nanoluc® assay is as follows. The procedure to perform the NanoGlo® Plasma Nanoluc® assay is as follows. The
  • NanoGlo® assay buffer (-20 °C) was thawed to room temperature. The mouse plasma samples (-80 °C) was thawed on ice. 40 uL of IX PBS (Room Temp) was added to each well of a 96-well, flat bottomed white plate. 10 uL of individual mouse plasma was added to each of three wells (triplicates) of the assay plate. NanoGlo® substrate was added to NanoGlo® Assay Buffer (1:50), e.g., 200 uL substrate to 9.8 mL assay buffer. After vortex, the NanoGlo® substrate solution was transferred to a reagent reservoir and add 50 uL to each well of the assay plate.
  • IX PBS Room Temp
  • the plate was incubated for 3 minutes at RT on an orbital shaker then transfer the plate to the SpectraMAX plate reader.
  • the plate configuration was set using the software.
  • the Luminescence Endpoint program was run on SpectraMax.
  • the samples were transferred from the white assay plate to a 96-well, flat-bottomed black assay plate.
  • the black plate was placed in the IVIS and image using Living Image Software on an open-filter bioluminescent well-plate program. The luminescent signal was quantitated using the Living Image Software.
  • NanoLuc®' was detected in plasma with mean total flux values of 43,358 ⁇ 59,759 photons/sec (p/s, mean + standard deviation) in 5/5 animals assayed.
  • OSO-0038 WEV/AAV1/ECL
  • NanoLuc® was detected in plasma with mean total flux values of 17,860 ⁇ 9463 p/s in 3/3 animals assayed.
  • DS-0634 EV/AAV1 particles with no lectin
  • NanoLuc® was detected in plasma with mean total flux values of 51,842 ⁇ 30,126 p/s in 4/4 animals assayed. Total flux values statistics for Day 2 post administration is as shown in FIG. 13A.
  • NanoLuc® was detected in plasma with mean total flux values of 17,080 ⁇ 12,381 p/s in 4/4 animals assayed.
  • NanoLuc® was detected in plasma with mean total flux values of 155,182 ⁇ 167,813 p/s in 5/5 animals assayed.
  • Samples collected from animals treated with OSO-0037 (SBA) were not of sufficient quality to be assayed. Total flux values statistics for Day 4 post administration is as shown in FIG. 13B.
  • NanoLuc® was detected in plasma with mean total flux values of 28,940 + 17,542 p/s in 5/5 animals assayed.
  • NanoLuc® was detected in plasma with mean total flux values of 56,092 + 78,856 p/s in 4/4 animals assayed.
  • NanoLuc® was detected in plasma with mean total flux values of 240,267 ⁇ 242,254 p/s in 5/5 animals assayed.
  • Total flux values statistics for Day 10 post administration is as shown in FIG. 13C.
  • the AAV1 particles loaded to EVs also carry a second trans gene encoding EPO.
  • the weights of spleens obtained on Day 10 post administration were analyzed and the results are shown in FIG. 14B.
  • Example 15 Investigation of AAV Serotypes for Transducing Intestinal Epithelial Monolayer via Apical Infection.
  • This example investigates transduction efficiency of various AAV serotype vectors to intestinal epithelial organoids as a monolayer for apical infection.
  • Mouse, rat, non-human primates (NHP) and human intestinal organoid monolayer cultures were prepared as per the following procedure towards ROP 2D intestinal epithelial monolayer growth and infection. See disclosures above.
  • MM Matrigel® Matrix
  • MM Matrigel® Matrix
  • the organoids were checked under microscope for density and viability.
  • Trypsin-EDTA (0.05%) from STEMCELL Technologies was warmed to 37°C. The viral constructs were thawed and kept on ice. Seeding and growing intestinal organoids as a 2D monolayer:
  • D-PBS from STEMCELL Technologies was maintained at 4°C. Appropriate amounts of this solution were dispensed in the desired number of wells into a conical tube as shown in Table 16 below.
  • Table 16 Volume of dilute matrigeL The thawed atrigel was added to cold D-PBS at a 1:50 ratio and mixed well. The diluted atrigel solution was added to coat the tissue culture-treated plate according to the recommended volumes shown in Table 16. The plate was tapped to disperse the liquid in the well. It is then incubated at 37°C for at least 1 hour. The plate was then tilted to allow' excess atrigel to collect at the edge and carefully aspirate the excess and ensured not to scratch the coated surface. The number of atrigel domes of intestinal organoids were evaluated for harvest according to the coated cultureware as shown in Table 17.
  • the domes and organoids in each well was disrupted by aspirating the gentle cell dissociation reagent row by row and mixing up and down at least 20 times. Make sure to move the tips around the well while mixing in order to effectively disrupt the domes and avoid bubbles.
  • the suspension was transferred from each well into a 50 mL conical tube.
  • the suspension was incubated at room temperature while rocking at 20 rpm for 10 minutes.
  • the suspension was centrifuged at 300 x g at 4 C C for 5 minutes.
  • the supernatant was carefully aspirated from the tubes ensuring not to disturb the pellet.
  • the pellet was washed by resuspending in 5 mL of cold DMEM/F-12 using a serological pipette.
  • the suspension was centrifuged at 300 x g at 4°C for 5 minutes.
  • the supernatant was carefully aspirated from the tubes ensuring not to disturb the pellet.
  • the organoids were resuspended in 2 mL of warm (37 C C) Trypsin-EDTA (0.05%) and mixed up and down thoroughly with a P1000 pipetor. Incubate the organoids at 37°C for 5-10 minutes. The organoids were removed from incubator and mixed thoroughly by vigorous pipetting or vortexing in order to disrupt the organoids as much as possible.
  • the volume of the organoid suspension was brought up to 5 mL with DMEM/F-12.
  • the suspension was then passed through a 100 urn Sterile Cell Strainer into a clean 50 ml. conical tube. Rinsed the original 50 ml. conical that contained the pellet with an additional 5 mL of DMEM/F-12 and passed the full volume through the Sterile Cell Strainer to collect any remaining cells. Capped the 50 mL conical containing the washed cells and gently mixed the tube by hand.
  • the filter step is optional and is only be used with high density/ amount of cells to remove excess debris.
  • the suspension was centrifuged at 300 x g at 4°C for 5 minutes. The supernatant was carefully aspirated from the tubes ensuring not to disturb the pellet.
  • the organoid fragments were resuspended in a monolayer growth medium as shown in Table 18. The cell suspension was slowly added to each well and care was taken not to disturb the monolayer underneath. This was then incubated at 37°C and 5% CO2. The growth was monitored daily, and the media was changed daily.
  • Viral constructs for transduction were prepared according to the viral titer and desired transduction dose by adding pre ⁇ determined volumes of virus and MM to a microcentrifuge tube. Appropriate volume of each prepared virus and MM solution was added in duplicate to desired wells directly on the IEC monolayer as shown in Table 18. The first pipette stop was only used. If using Transwell plate, add 600 uL of media to basolateral chamber of plate.
  • FIGs. 18A-18D Expression of transgenes nLuc, EPO, or GFP in mouse epithelial cells mediated by the tested AAV particles was shown in FIGs. 18A-18D.
  • AAV1, AAV6, and AAV6.2FVFF successfully transduced the transgenes into mouse epithelial cells, leading to expression of such transgenes in the target mouse ceils.
  • FIGs. 18E-18F Expression of transgenes nLuc or EPO in non-hunian primate (NHP) epithelial cells mediated by the tested AAV particles was shown in FIGs. 18E-18F.
  • AAV1, AAV6, and AAV6.2FVFF successfully transduced the transgenes into the NHPepithelial cells, leading to expression of such transgenes in the target NHP cells.
  • AAV6.2FVFF showed better results relative to AAV1 and AAV6 in infecting human cells (multiple donors; duodenal and jejunal), as shown in FIG. 18H.
  • AAV1, AAV6, and AAV6.2FVF variant were found to successfully mediate tragene gene (EPO) expression in rat intestinal epithelial cells (lECs) as shown in FIG. 19.
  • EPO ERTepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptepteptept
  • the example is to determine the transduction efficiency and kinetics of ectopic expression of the nanoluciferase reporter gene (nLuc) and human erythropoietin (huEPO) mediated by adeno-associated virus serotype 1 (AAV1) infection of luminal cells of the duodenum in fasted female C57B1/6 mice after a single direct administration of polystyrene microsphere and biotin adapter-glycocalyx vesicle formulation to the duodenum via intraduodenal injection or via permanent duodenal catheter.
  • AAV1 adeno-associated virus serotype 1
  • WEVs (as exemplay glycocalyx vesicles (G Vs)) were prepared following the procedures disclosed in Example 1 above and were randomly biotinylated and purified to remove free biotins.
  • AAV1 particles carrying the nLuc and EPO transgenes (AAV1-EPO- nLuc) see above disclosures) were mixed with the biotinylated WEVs in the absence or presence of a streptavidin-containing bridging molecule to form AAV-GV complexes.
  • Negative-stain electron microscopy assay showed that, in the presence of the bridging molecule, the biotinylated WEVs and AAVs form complexes efficiently.
  • AAVl-EPO-NLuc was mixed with SEC-purified biotinylated GVs at a ratio of 20:1
  • AAV:GV in the presence of the streptavidin-containing bridging molecule. 100 ul totaling 2E11 genome copies of AAV were injected into the duodenum of each mouse. Whole body luminescence from NLuc was measured at 2, 4, 6, and 7 days post-administration by intraperitoneal injection of NanoGio substrate (Promega) prior to imaging on an IVIS (Perkin- Elmer). As shown in FIG. 20, expression of NLuc was detected in mice treated with the AAV-GV complex.
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as “and/or” as defined above.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

Abstract

L'invention concerne des vésicules de glycocalyx (GV) telles que des vésicules extracellulaires (EV) chargées avec des particules virales portant un gène d'intérêt, et éventuellement modifiées par des lectines de surface qui se fixent à des cellules du tractus gastro-intestinal (GI). L'invention concerne également des procédés de production de telles vésicules extracellulaires et des procédés d'utilisation de celles-ci pour effectuer l'apport du gène d'intérêt à un compartiment du GI par administration orale, par exemple.
PCT/US2023/060261 2022-01-07 2023-01-06 Vésicules extracellulaires chargées avec des particules virales pour un apport de charge WO2023133527A2 (fr)

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