WO2021237297A1 - Vésicules extracellulaires anti-virales, leurs procédés de préparation et leurs utilisations - Google Patents

Vésicules extracellulaires anti-virales, leurs procédés de préparation et leurs utilisations Download PDF

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WO2021237297A1
WO2021237297A1 PCT/AU2021/050513 AU2021050513W WO2021237297A1 WO 2021237297 A1 WO2021237297 A1 WO 2021237297A1 AU 2021050513 W AU2021050513 W AU 2021050513W WO 2021237297 A1 WO2021237297 A1 WO 2021237297A1
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viral
virus
sequence
therapeutic agent
viral infection
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Andrew Mark Coley
Lieven Huang
Ian Edward Dixon
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Exopharm Limited
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Priority claimed from AU2020901723A external-priority patent/AU2020901723A0/en
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Publication of WO2021237297A1 publication Critical patent/WO2021237297A1/fr

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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K47/6917—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 the form being a lipoprotein vesicle, e.g. HDL or LDL proteins
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C07K2319/00—Fusion polypeptide
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
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    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
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    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Anti-viral extracellular vesicles their methods of preparation and uses
  • the present invention relates to anti-viral extracellular vesicles, pharmaceutical compositions including them, methods of preparation, and their use for the prevention or treatment of viral infections and associated diseases or conditions.
  • viruses attach to target cells via a specific ligand to receptor interaction (e.g. Spike protein of SARS-CoV-2 and ACE2) to tether to the cell.
  • a specific ligand to receptor interaction e.g. Spike protein of SARS-CoV-2 and ACE2
  • the conjugate of the virion and the endogenous receptor are internalised and the replication stage commences.
  • Extracellular vesicles which include exosomes, are a natural mechanism by which cells communicate and share material.
  • EVs are a heterogeneous collection of biological structures bound by membranes. These structures have a lipid bilayer and they may reside within a cell or in an extracellular environment. EVs range in size from about 20 nm to 1000 nm. EVs efficiently exchange information between cells and transfer biologically active proteins, lipids, and various nucleic acids including mRNA, miRNA, rRNA, IncRNA and DNA. Endogenous EVs naturally occur in vivo. EVs may also be produced ex vivo from a variety of sources - exogenous EVs. Endogenous and exogenous EVs from selected sources (e.g. blood platelets, stem cells) have been shown to be poorly immunogenic, so are a well-tolerated material if given to a patient and may be given more than one time as a therapy, unlike other nanoparticles such as liposomes.
  • sources e.
  • Exogenous EVs may be harvested from sources such as the secretome of platelets and the secretome of stem cells (e.g. mesenchymal type cells or MSCs).
  • sources such as the secretome of platelets and the secretome of stem cells (e.g. mesenchymal type cells or MSCs).
  • stem cells e.g. mesenchymal type cells or MSCs.
  • Engineered EVs have been utilised as a natural delivery vehicle packed with small molecules, nucleic acids, peptides and proteins. EVs have the opportunity to provide tailored personalised medicine, in which EVs can be isolated from the plasma of a patient, loaded with a therapeutic agent, and administered back to the same patient as an autologous product. Exogenous EVs purified from cell sources unmatched to the recipient may provide off-the-shelf EVs as an allogeneic product. Allogeneic EVs offer a scalable product for ready use.
  • Engineered EVs may be produced in a number of ways.
  • the cells secreting the engineered EVs may be caused to produce a particular material (e.g. protein, enzyme) and some of that particular material will be loaded into EVs secreted by the cells.
  • Another method to produce engineered EVs is by incubating a therapeutic agent with cells being cultured. In this way, the cells excrete EVs with the therapeutic agent encapsulated.
  • Another method to produce engineered EVs is to incubate a therapeutic agent with the EVs post secretion. In this way, the EVs end up with the therapeutic agent encapsulated.
  • engineered EVs used to deliver therapeutic agents include delivering miRNA to macrophages, and siRNA cells in the brain. EVs have also been used to prepare vaccines by binding immunogenic viral peptides to the exosome surface and presenting these antigens to induce neutralising antibody tiers.
  • the present invention provides an extracellular vesicle (EV) comprising:
  • transmembrane protein displaying an attachment site on the surface of the EV
  • an anti-viral therapeutic agent located in or on the inner-vesicle space.
  • the present invention provides an EV comprising:
  • an anti-viral therapeutic agent located in or on the inner-vesicle space.
  • EVs according to the present invention may be prepared by adding:
  • an EV incorporating a transmembrane protein on the surface of the EV wherein a targeting molecule attached to the transmembrane protein targets the EV to a target thereby forming a mixture; and reacting the mixture to incorporate the anti-viral therapeutic agent in or on an inner vesicle space of the EV.
  • composition of EVs according to the present invention may be prepared by adding:
  • transmembrane protein wherein a targeting molecule is attached to an attachment site of the transmembrane protein
  • the targeting molecule targets the EV to a target cell
  • composition of EVs according to the present invention may be prepared by:
  • transmembrane protein wherein a targeting molecule is attached to an attachment site of the transmembrane protein
  • the targeting molecule targets the EV to a target cell
  • the present invention provides pharmaceutical compositions comprising EVs of the invention and a pharmaceutically acceptable excipient.
  • the present invention provides a kit for use in a therapeutic application mentioned herein, the kit including:
  • the present invention provides a method of preventing a viral infection, and/or a disease or condition associated with a viral infection, in an individual comprising, consisting essentially of or consisting of the steps of: administering a therapeutically effective amount of a composition of EVs according to the invention to the individual; wherein the individual is at risk of having or contracting a viral infection, and/or a disease or condition associated with a viral infection; thereby preventing the infection, and/or the disease or condition associated with the viral infection, in the individual.
  • the present invention provides a method of treating a viral infection, and/or a disease or condition associated with a viral infection, in an individual comprising, consisting essentially of or consisting of the steps of: administering a therapeutically effective amount of a composition of EVs according to the invention to the individual; wherein the individual is diagnosed with, or suspected of having, a viral infection, and/or a disease or condition associated with a viral infection; thereby treating the infection, and/or the disease or condition associated with the viral infection, in the individual.
  • the present invention provides use of a composition of EVs of the invention in the manufacture of a medicament for: • preventing a viral infection, and/or a disease or condition associated with a viral infection in an individual, wherein the individual is at risk of having or contracting a viral infection, and/or a disease or condition associated with a viral infection;
  • the present invention also provides a composition of EVs of the present invention for use in:
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of, or consisting of EVs of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient for use in:
  • Figure 1 Representative VSVG-target molecule constructs; A1 corresponds to the target molecule signal sequence, A2 corresponds to the VSVG signal sequence, B1 corresponds to the target molecule not including the target molecule’s signal sequence or transmembrane domain, C1 corresponds to a VSVG transmembrane domain fragment, ad C2 corresponds to full length VSVG not including the VSVG signal sequence.
  • FIG. 2 Confocal microscopy analysis of HEK293 cells transfected with parental vector ‘AcGFP-N1’ showing expression of cytoplasmic soluble GFP.
  • Top left panel shows nuclei specific staining from Hoechst 33342; top right shows cellular staining from AcGFP-N1; bottom left shows transmitted light image; bottom right shows overlay of top left, top right and bottom left images.
  • FIG. 3 Confocal microscopy analysis of HEK293 cells transfected with parental vector ‘DsRed-NT showing expression of cytoplasmic soluble RFP.
  • Top left panel shows nuclei specific staining from Hoechst 33342; top right shows cellular staining from DsRed-N1; bottom left shows transmitted light image; bottom right shows overlay of top left, top right and bottom left images.
  • FIG. 4 Confocal microscopy analysis of HEK293 cells transfected with ‘fVSVG-AcGFP’ construct. Top left panel shows nuclei specific staining from Hoechst 33342; top right shows cellular staining from fVSVG-AcGFP; bottom left shows transmitted light image; bottom right shows overlay of top left, top right and bottom left images. Full-length VSVG fused to AcGFP results in more punctate intracellular distribution than soluble AcGFP (cf Figure 2) consistent with that of a secreted protein.
  • FIG. 5 Confocal microscopy analysis of HEK293 cells transfected with ‘CovS- mVSVG-AcGFP’ construct.
  • Top left panel shows nuclei specific staining from Hoechst 33342;
  • top right shows cellular staining from CovS-mVSVG-AcGFP;
  • bottom left shows transmitted light image;
  • bottom right shows overlay of top left, top right and bottom left images.
  • the CovS-mVSVG-AcGFP fusion protein results in a punctate intracellular distribution consistent with that of a secreted protein.
  • FIG. 6 Confocal microscopy analysis of HEK293 cells transfected with ‘CovS- mVSVG-DsRed’ construct.
  • Top left panel shows nuclei specific staining from Hoechst 33342;
  • top right shows cellular staining from CovS-mVSVG-DsRed;
  • bottom left shows transmitted light image;
  • bottom right shows overlay of top left, top right and bottom left images.
  • the CovS-mVSVG-DsRed fusion protein results in a punctate intracellular distribution consistent with that of a secreted protein.
  • Figure 7 Western blot analysis of CovS-mVSVG expression. 1° Antibody used was anti-RBD. Lane 1 - molecular weight markers, Lane 2 - untransfected HEK293 control, Lane 3 - DS-Red transfected HEK293, Lane 4 - CovS-mVSVG transfected HEK293, Lane 5 - recombinant RBD +ve control, Lane 6 - molecular weight markers.
  • Figure 8 Western blot analysis of CovS-mVSVG expression. 1° Antibody used was anti-RBD. Lane 1 - molecular weight markers, Lane 2 - untransfected HEK293 control, Lane 3 - AcGFP-N1 transfected HEK293, Lane 4 - CovS-mVSVG transfected HEK293, Lane 5 - recombinant RBD +ve control, Lane 6 - molecular weight markers.
  • Figure 9 Analysis of EVs by imaging flow cytometry (Amnis, Luminex).
  • Panels A-C represent EVs isolated from non-transfected HEK293 cells; D-F represent EVs purified from HEK293 cells transfected with the parental vector, AcGFP-N1; G-l represent EVs purified from HEK293 cells transfected with ‘CovS-mVSVG-AcGFP’ Construct.
  • Panels A, D and G represent the particles present in the conditioned media from each cell type.
  • the top left quadrant (R1) contains discrete EVs.
  • Panels B, E and H represent the EVs present in region R1 with an ‘R2’ gate representing EV population to be studied.
  • Panels C, F and I represent the EVs present in R2 and show their respective GFP fluorescence when excited at 488 nm wavelength light. 0% of EVs were fluorescent in Panel C, 63% of the EVs contained GFP in the vector control and 16% contained CovS-mVSVG-AcGF fusion protein in panel I.
  • Figure 10 18 siRNA sequences were designed and synthesised, and incorporated into SARS-CoV-2 viral cultures in Vero cells.
  • Figure 11 Tropism of ‘CovS-mVSVG-AcGFP’ extracellular vesicles for ACE2+ Calu3 ‘target’ cells indicated by uptake of AcGFP signal (arrowed). The blue signal indicates HEK293 nuclei.
  • Figure 12 Transfection of siRNA into HEK293 cells. Cy5-conjugated siRNA was electroporated into HEK393 cells. Here the colocalization of the siRNA (red) and CD63, a marker of nascent EVs (green) can be seen indicating loading of the siRNA into the EVs prior to their secretion. The blue signal indicates HEK293 nuclei.
  • Sequence information Table 1 Sequences of exemplary PCR oligonucleotide primers incorporated into construct generation.
  • Table 2 Exemplary target molecules, transmembrane proteins, and transmembrane-target molecule fusion proteins.
  • Table 3 Exemplary anti-viral therapeutic molecules.
  • Extracellular vesicles (EVs) or micro vesicles (MVs) generally refer to a heterogeneous in vivo collection of membrane bound biological structures having a diameter from about 20 to about 1000 nm.
  • the term “about” includes and describes the value or parameter per se.
  • “about x” includes and describes “x” perse.
  • “about 20” in some embodiments includes 18 - 22.
  • adding does not limit the order, method or how the materials being added are combined, unless indicated otherwise.
  • “adding A to B” may also describe “adding B to A”.
  • “adding A and B to C” may also describe the various other combinations such as “adding A to B and C”, “adding A and C to B”, “adding B to A and C”, “adding B and C to A”, and “adding C to A and B”.
  • the present invention permits the rapid design and manufacture of anti-viral EVs that incorporate (i) an externally presented targeting molecule that corresponds to a ligand used by the virus to tether to host cells (‘viral ligand’) (and is therefore capable of targeting the EV to cells that the virus preferentially tethers to and infects) and (ii) an anti-viral therapeutic agent including small molecule drugs, selective anti-viral therapeutic agents such as specific RNAi and siRNA that inhibit the genome of the specific virus, non-selective anti-viral therapeutic agents such as miRNA that support the infected cell to activate its anti-viral defences (e.g. STING), and combinations thereof.
  • an externally presented targeting molecule that corresponds to a ligand used by the virus to tether to host cells (‘viral ligand’) (and is therefore capable of targeting the EV to cells that the virus preferentially tethers to and infects)
  • an anti-viral therapeutic agent including small molecule drugs,
  • the present invention allows engineered EVs to be rapidly deployed to reduce the effect of novel or known viral infections.
  • the present invention also provides the opportunity to respond to an emerging threat of a particular virus, preferably to humans.
  • the present invention provides an extracellular vesicle (EV) comprising:
  • the present invention provides an EV comprising:
  • an anti-viral therapeutic agent located in or on the inner-vesicle space.
  • the present invention is applicable to any virus or virion.
  • the virus is selected from: coronaviruses, respiratory syncytial virus, hepatitis virus, influenza viruses, dengue virus, rhinoviruses, rotaviruses, Herpes viruses (HSV), Human Papillomaviruses (HPV), Human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Ebola virus, Echovirus, Flavivirus, Morbilivirus, and Hantavirus.
  • the virus may include viruses that cross species from time to time (Xeno viruses).
  • the viral infection is caused by a virus selected from: coronavirus, respiratory syncytial virus, hepatitis virus, influenza virus, and dengue virus.
  • the virus is coronavirus, respiratory syncytial virus or influenza virus. Even more preferably, the virus is severe acute respiratory syndrome coronavirus (SARS- CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), most preferably SARS-CoV-2.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the anti-viral therapeutic agent is effect against a virus described herein.
  • the anti-viral therapeutic agent may include a selective anti-viral therapeutic agent, a non-selective anti-viral therapeutic agent, or combinations thereof.
  • a selective anti-viral therapeutic agent as used herein refers to an anti-viral therapeutic agent that selectively inhibits viral replication of a specific virus in an infected cell.
  • the selective anti-viral therapeutic agent is effective against a virus selected from: coronaviruses, respiratory syncytial virus, hepatitis virus, influenza viruses, dengue virus, rhinoviruses, rotaviruses, Herpes viruses (HSV), Human Papillomaviruses (HPV), Human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Ebola virus, Echovirus, Flavivirus, Morbilivirus, and Hantavirus.
  • the selective anti-viral therapeutic agent is effective against a virus selected from: coronavirus, respiratory syncytial virus, hepatitis virus, influenza virus, and dengue virus. More preferably, the virus is coronavirus or influenza.
  • the virus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), most preferably SARS-CoV-2.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the selective anti-viral therapeutic agent is selected from: a small molecule, enzyme, transcription factor, antibody fragment, antibody, protein, mRNA, miRNA, pre-miRNA, rRNA, siRNA, shRNA, IncRNA, sncRNA, tRNA, cDNA, plasmid vector DNA, Cas9/gRNA, CRISPR and combinations thereof.
  • the selective anti-viral therapeutic agent inhibits viral replicase activity.
  • the anti-viral therapeutic comprises siRNA, wherein the siRNA comprises a sense strand and a complementary anti-sense strand wherein the sense and the antisense strand are substantially complementary to each other to form a double-stranded structure or duplex, and wherein the antisense strand comprises a nucleotide sequence sufficiently complementary to a target SARS-CoV-2 nucleotide sequence.
  • a nucleotide sequence sufficiently complementary to a target SARS-CoV-2 nucleotide sequence includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to the target SARS-CoV-2 nucleotide sequence, wherein the sufficiently complementary sequence is capable of inhibiting expression of the target sequence in vitro and/or in vivo, preferably in vivo.
  • expression of the target sequence is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
  • the siRNA comprises a sense strand and an antisense strand, wherein the sense and the antisense strands are substantially complementary to each other to form a double-stranded structure or duplex, and wherein the sense strand comprises a nucleotide sequence substantially identical to a target SARS-CoV-2 nucleotide sequence.
  • a nucleotide sequence substantially identical to a target SARS-CoV-2 nucleotide sequence includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the target SARS-CoV-2 nucleotide sequence.
  • the target SARS-CoV-2 nucleotide sequence comprises a portion of the genomic sequence of the SARS-CoV-2 virus.
  • the target SARS-CoV-2 nucleotide sequence substantially corresponds to a replicase region of the genomic sequence of the SARS-CoV-2 virus.
  • the target SARS- CoV-2 nucleotide sequence substantially corresponds to the spike glycoprotein (S) region, the membrane protein (M) region, the envelope protein (E) region, or the nucleocapsid protein (N) region of the SARS-CoV-2 viral genome.
  • the target SARS-CoV-2 nucleotide sequence may comprise, consist of, or consist essentially of a sequence as set forth in any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, wherein the sequence does not include any non-natural modifications.
  • the target SARS-CoV-2 nucleotide sequence may include natural mutations of the viral nucleotide sequence (for example, natural mutations present in different strains of SARS-CoV-2), such that the target SARS-CoV-2 nucleotide sequence comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence selected from the group of sequences set forth in any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, wherein the sequence does not include any non-natural modifications.
  • the siRNAs are capable of inhibiting expression of the target sequence in vitro and/or in vivo, preferably in vivo.
  • Each strand of the siRNA can range from 12-30 nucleotides in length.
  • each strand can be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 25-27 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.
  • the sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”).
  • the duplex region of the siRNA may be 12-30 nucleotide pairs in length.
  • the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17 - 23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length.
  • the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.
  • the siRNAs can be blunt at both ends, or have an overhang at one end and a blunt end at the other end, or have an overhang at both ends. More specifically, the siRNAs may contain one or more overhang regions and/or capping groups of siRNA at 3’-end, or 5’-end or both ends of a strand.
  • the overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length.
  • the overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.
  • the overhang can form a mismatch with the target RNA or it can be complementary to the gene sequences being targeted or can be other sequence.
  • the first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
  • the siRNAs can be modified on the sense and/or antisense strands by one or more modifications.
  • the modification, or modified nucleobase may be a sugar modification or backbone modification, such as those described herein.
  • alternating nucleotides on the sense and/or antisense strands can be modified.
  • the alternating nucleotides on both the sense and antisense strands are modified at the 2'-hydroxyl group of the ribose ring and the modification is amino, fluoro, O-methyl, alkoxy or alkyl.
  • one or more phosphodiester groups may be modified to a phosphorothioate group.
  • each of the odd numbered nucleotides are modified in the antisense strand numbering from 5 ’ to 3’ and each of the even numbered nucleotides are modified in the anti-parallel sense strand numbering from 3 ’ to 5 ⁇
  • a first stretch of the sense strand and a second stretch of the antisense strand each consist of contiguous alternating single 2'-0-methyl modified and single unmodified ribonucleotides, wherein each modified ribonucleotide in the first stretch is base paired with the unmodified ribonucleotide in the second stretch.
  • the sense strand comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74.
  • the antisense strand comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 , 73 and 75.
  • the siRNA comprises a sense strand and a complementary anti-sense strand.
  • the siRNA comprises sequences at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to sequences selected from the group comprising SEQ ID NOs: 42 and 43, 44 and 45, 46 and 47, 48 and 49, 50 and 51 , 52 and 53, 54 and 55, 56 and 57, 58 and 59, 60 and 61, 62 and 63, 64 and 65, 66 and 67, 68 and 69, 70 and 71 , 72 and 73, and 74 and 75.
  • the sense strand comprises a sequence having at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to at least 15, 18, 23 contiguous nucleotides of a sequence set forth in any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74.
  • the antisense strand comprises a sequence having at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to at least 15, 18, 23 contiguous nucleotides of a sequence set forth in any one of SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73 and 75.
  • the sense strand comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74.
  • the anti-sense strand comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75.
  • the siRNA comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 42 and 43, 44 and 45, 46 and 47, 48 and 49, 50 and 51, 52 and 53, 54 and 55, 56 and 57, 58 and 59, 60 and 61 , 62 and 63, 64 and 65, 66 and 67, 68 and 69, 70 and 71, 72 and 73, 74 and 75.
  • the sense strand comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 44, 52, 54, and 66.
  • the antisense strand comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 45, 53, 55, and 67.
  • the siRNA comprises sequences at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to sequences selected from the group comprising SEQ ID NOs: 44 and 45, 52 and 53, 54 and 55, and 66 and 67.
  • the sense strand comprises a sequence having at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to at least 15, 18, 23 contiguous nucleotides of a sequence set forth in any one of SEQ ID NOs: 44, 52, 54, and 66.
  • the antisense strand comprises a sequence having at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to at least 15, 18, 23 contiguous nucleotides of a sequence set forth in any one of SEQ ID NOs: 45, 53, 55, and 67.
  • the sense strand may comprise, consist of, or consist essentially of a sequence as set forth in any one of SEQ ID NOs: 44, 52, 54, and 66.
  • the anti- sense strand comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 45, 53, 55, and 67.
  • the siRNA comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 44 and 45, 52 and 53, 54 and 55, and 66 and 67.
  • the siRNAs may be synthetic, recombinant, isolated, substantially purified or purified.
  • a non-selective anti-viral therapeutic agent as used herein may refer to an anti viral therapeutic agent that may assist an infected cell to respond to viral infection.
  • a non-selective anti-viral therapeutic agent as used herein may refer to a broad-spectrum anti-viral therapeutic agent that is effective against more than one virus, preferably 2, 3, 4, 5, 6, 7 or more viruses.
  • the non-selective anti-viral therapeutic agent is selected from: small molecule, enzyme, transcription factor, antibody fragment, antibody, protein, mRNA, miRNA, pre-miRNA, rRNA, siRNA, shRNA, IncRNA, sncRNA, tRNA, cDNA, plasmid vector DNA, Cas9/gRNA, CRISPR and combinations thereof.
  • the non- selective anti-viral therapeutic agent is miRNA.
  • the non-selective anti-viral therapeutic agent is a STING agonist.
  • the target cell is a particular cell or tissue that is preferentially infected by the virus. In embodiments wherein the virus is coronavirus or influenza virus, including SARS-CoV, MERS-CoV, and SARS-CoV-2, the target cell is preferably a cell located in the upper respiratory system.
  • the targeting molecule is a molecule that has the ability to target a particular tissue or cell that is preferentially infected by virus.
  • the targeting molecule may be a polypeptide, such as a protein, a peptide, (e.g. a targeting peptide that is used to target cells infected by virus), or a viral antigen epitope.
  • the targeting molecule is a ligand for a receptor on a particular tissue or cell that is preferentially infected by virus.
  • the ligand may be a host ligand, or a biological fragment or derivative thereof, that substantially corresponds to a ligand for the receptor derived from the host, for example for the SARS-CoV-2 virus the host ligand may be an ACE2 ligand.
  • the ligand may be a viral ligand, or a biological fragment or derivative thereof, derived from the virus, for example for the SARS-CoV-2 virus the ligand may be the spike S protein, or S1 domain thereof, of SARS-CoV-2 that is capable of targeting the EV to the target cell.
  • the ligand may include a full length ligand, a full length ligand not including a signal sequence, or a biologically active fragment or derivative of a ligand.
  • the targeting molecule may therefore be selected from: a host ligand, a viral ligand, biological fragments or derivatives thereof, and combinations thereof.
  • the targeting molecule may be determined based on the nucleotide sequence of the target virus.
  • the target molecule may be determined by sequencing the nucleotide sequence of the target virus and selecting the targeting molecule.
  • the targeting molecule comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 21 , 22 or 23.
  • the targeting molecule comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 21 , 22 or 23.
  • the transmembrane protein includes proteins that are capable of being incorporated in an EV lipid bilayer membrane, for example, including but not limited to: vesicular stomatitis virus glycoprotein (VSVG), Lamp-1, Lamp-2, CD13, CD86, Flotillin, Syntaxin-3, CD40, CD40L, CD44, ICAM-1, Integrin 1B, CD9, CD37, CD53, CD63, CD81, CD82, CX MHC-II components, TCR beta, tetraspanins, PDGFR, GPI anchor proteins, lactadherin, LAMP2B, biologically active fragments or derivatives thereof, and combinations thereof.
  • VSVG vesicular stomatitis virus glycoprotein
  • Lamp-1 Lamp-2
  • CD13 CD86
  • Flotillin Syntaxin-3
  • CD40, CD40L, CD44 ICAM-1
  • Integrin 1B Integrin 1B
  • CD9 Integrin 1B
  • CD9 Integrin 1B
  • the transmembrane protein is VSVG, or a biologically active fragment or derivative thereof.
  • the VSVG may comprise full length VSVG, full length VSVG not including the VSVG signal sequence, a biologically active fragment of VSVG for example a VSVG transmembrane and membrane-proximal domain fragment that is capable of incorporating into the EV membrane.
  • the transmembrane protein comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 24, 25, or 26.
  • the transmembrane protein comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 24, 25, or 26.
  • the targeting molecule attached to the attachment site of the VSVG may comprise (i) the targeting molecule, which may include biologically active fragments thereof; and (ii) VSVG, wherein the VSVG may comprise full length VSVG not including the VSVG signal sequence, or a biologically active fragment of VSVG for example a VSVG transmembrane domain fragment that is capable of incorporating into the EV membrane.
  • the targeting molecule attached to the attachment site of the transmembrane protein may optionally further comprise a linker sequence, wherein the linker sequence is located between the target molecule and transmembrane protein.
  • the targeting molecule attached to the attachment site of the transmembrane protein may optionally further comprise a signal sequence, wherein the signal sequence is derived from the targeting molecule or the transmembrane protein.
  • the transmembrane protein and targeting molecule form a fusion protein.
  • the fusion protein may comprise the targeting molecule attached to the attachment site of the transmembrane protein, or the targeting molecule incorporated within the transmembrane protein.
  • the fusion protein comprises a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a sequence set forth in any one of SEQ ID NOs: 27 - 41.
  • the fusion protein comprises, consists of, or consists essentially of a sequence as set forth in any one of SEQ ID NOs: 27 - 41.
  • the EV is substantially non-immunogenic. More particularly, the concentration of targeting molecule incorporated in the EV does not substantially induce an immune response.
  • the targeting molecule is selected from: the spike S protein of SARS-CoV-2, an ACE-2 ligand and combinations thereof.
  • the EV is derived from a cell selected from: stem cells, platelets, immortalised cell lines, primary cells, plant cells, yeast cells, bacterial cells, and combinations thereof.
  • Reference to a “protein” includes reference to a peptide, polypeptide or protein or parts thereof.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference hereinafter to a “protein” includes a peptide comprising a sequence of amino acids as well as a peptide associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • “Derivatives” include fragments, parts, portions and variants from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of the subject protein or molecule. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • An example of substitutional amino acid variants are conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and/or hydropathicity and/or hydrophilicity.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), b-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic
  • the present invention contemplates mutant forms of transmembrane proteins, target molecules and anti-viral therapeutics (in the form of enzyme, transcription factor, antibody fragment, antibody, protein, and combinations thereof) of the invention comprising one or more conservative amino acid substitutions compared to a sequence set forth herein.
  • the transmembrane protein, target molecule or anti viral therapeutic comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions.
  • the present invention also contemplates non-conservative amino acid changes.
  • non-conservative amino acid changes For example, of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or positively charged amino acids.
  • the transmembrane protein, target molecule or anti-viral therapeutic comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.
  • Chemical and functional equivalents of the subject protein or molecule should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
  • Analogues contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues. Mutants include molecules which exhibit modified functional activity.
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • biologically active fragment describes a portion or sub sequence of the subject protein or molecule, including a domain thereof, that has no less than 10%, preferably no less than 25%, more preferably no less than 50%, and even more preferably no less than 75%, 80%, 85%, 90%, or 95% of a biological activity of the subject protein or molecule. Such activity may be evaluated using standard testing methods and bioassays recognizable by the skilled artisan in the field as generally being useful for identifying such activity.
  • a protein variant shares at least 70%, preferably at least 75%, 80% or 85% and more preferably at least 90%, 95%, 98%, or 99% sequence identity with a reference amino acid sequence such as SEQ ID NO: 21 - 75.
  • sequence identity is measured over at least 60%, more preferably over at least 75%, more preferably over at least 90% or more preferably over at least 95%, 98% or substantially the full length of the reference sequence.
  • Exemplary methods for determining biological activity of the mutant transmembrane proteins, target molecules or anti-viral therapeutics of the invention will be apparent to the skilled artisan and/or described herein. For example, methods for determining target molecule binding, competitive inhibition of binding, affinity, association, dissociation and therapeutic efficacy are described herein.
  • the invention also provides a nucleic acid encoding a transmembrane protein, target molecule or anti-viral therapeutic (in the form of enzyme, transcription factor, antibody fragment, antibody, protein, and combinations thereof) as described herein.
  • the invention also provides a nucleic acid anti-viral therapeutic (in the form of mRNA, miRNA, pre-miRNA, rRNA, siRNA, shRNA, IncRNA, sncRNA, tRNA, cDNA, plasmid vector DNA, Cas9/gRNA, CRISPR and combinations thereof).
  • a nucleic acid anti-viral therapeutic in the form of mRNA, miRNA, pre-miRNA, rRNA, siRNA, shRNA, IncRNA, sncRNA, tRNA, cDNA, plasmid vector DNA, Cas9/gRNA, CRISPR and combinations thereof.
  • such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter.
  • an expression construct can be in a vector, e.g., a plasmid.
  • the expression construct may comprise a promoter linked to a nucleic acid encoding that protein.
  • the present invention also provides a transmembrane protein, target molecule or anti-viral therapeutic or a nucleic acid encoding same having at least 80% identity to a sequence disclosed herein.
  • a transmembrane protein, target molecule or anti-viral therapeutic or nucleic acid of the invention comprises sequence at least about 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.
  • a nucleic acid of the invention comprises a sequence at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to a sequence encoding a transmembrane protein, target molecule or anti-viral therapeutic having a function as described herein according to any example.
  • the present invention also encompasses nucleic acids encoding a transmembrane protein, target molecule or anti viral therapeutic of the invention, which differs from a sequence exemplified herein as a result of degeneracy of the genetic code.
  • composition of EVs according to the present invention may be prepared by adding:
  • an anti-viral therapeutic agent and - an EV incorporating a transmembrane protein on the surface of the EV, wherein a targeting molecule attached to the transmembrane protein targets the EV to a target cell; thereby forming a mixture; and reacting the mixture to incorporate the anti-viral therapeutic agent in or on an inner vesicle space of the EV.
  • the reaction may include: direct incubation, sonication, electroporation, calcium assisted uptake, and combinations thereof.
  • composition of EVs according to the present invention may be prepared by adding:
  • transmembrane protein wherein a targeting molecule is attached to an attachment site of the transmembrane protein
  • the targeting molecule targets the EV to a target cell
  • the reaction may include: direct incubation, sonication, electroporation, calcium assisted uptake, and combinations thereof.
  • a composition of EVs according to the present invention may be prepared by:
  • transmembrane protein wherein a targeting molecule is attached to an attachment site of the transmembrane protein
  • the targeting molecule targets the EV to a target cell
  • the methods may further comprise determining the targeting molecule based on the nucleotide sequence of a target virus.
  • determining the targeting molecule may include sequencing the nucleotide sequence of the target virus and selecting the targeting molecule.
  • compositions of EVs according to the invention may be purified and isolated according to methods described in WO 2019241836, the contents of which are incorporated herein by reference.
  • compositions comprising, formulations and modes of administration
  • the EVs of the invention will normally, but not necessarily, be formulated into a pharmaceutical composition prior to administration to a patient. Accordingly, in another aspect the invention is directed to pharmaceutical compositions comprising EVs of the invention and a pharmaceutically acceptable excipient.
  • compositions of the invention may be prepared and packaged in bulk form wherein an effective amount of EVs of the invention can be extracted and then given to the patient such as with powders, syrups, solutions for injection, inhalation and other routes of administration.
  • the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains an effective amount of EVs of the invention.
  • the pharmaceutical compositions of the invention typically contain from 1 ng to 1000 mg of EVs of the invention.
  • compositions of the invention may optionally further comprise one or more additional pharmaceutically active compounds.
  • the pharmaceutical compositions of the invention typically include more than one pharmaceutically acceptable excipient. However, in certain embodiments, the pharmaceutical compositions of the invention contain one pharmaceutically acceptable excipient.
  • the phrase ‘therapeutically effective amount’ generally refers to an amount of EVs that (i) prevents viral infection in an individual (ii) prevents a particular disease, condition, or disorder associated with viral infection in an individual, (iii) treats the particular disease, condition, or disorder, (iv) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (v) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
  • the "therapeutically effective amount” may vary depending on the EV, how the EV is administered, the disease or condition and its seventy and the history, age, weight, family history, genetic makeup, stage of pathological processes associated with the viral infection, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
  • a composition of the invention, or composition for use in a method or use of the invention contains about 10e 10 to about 10e 20 EVs, including any number within that range for example about 10e 11 , about 10e 12 , about 10e 13 , about 10e 14 , about 10e 15 , about 10e 16 , about 10e 17 , about 10e 18 , and about 10e 19 .
  • the composition contains about 10e 14 EVs.
  • the amount of EVs present in a composition, e.g. for inhalation administration is up to about 10% w/v, preferably up to about 10% w/v, preferably about 5% w/v, preferably about 2% w/v.
  • the concentration (by weight) in any composition described herein is at least about 0.1% up to about 10% or more, and all combinations and sub-combinations of ranges therein.
  • the compositions can be formulated to contain EVs in a concentration of from about 0.1 to less than about 20%, for example, about 19, 18, 17, 16, 15, 14, 13, 12, 11 and 10%, with concentrations of from greater than about 0.1%, for example, about 0.2, 0.3, 0.4 or 0.5%, to less than about 10%, for example, about 9, 8, 7, 6, 5, 4, 3, 2, or 1%.
  • compositions may contain from about 0.5% to less than about 10%, for example, about 9, 8, 7, 6, 5, 4, 3, 2, or 1 %, with concentrations of from greater than about 0.5%, for example, about 0.6, 0.7, 0.8, 0.9 or 1%, to less than about 20%, for example, about 19, 18, 17, 1 6, 1 5, 14, 13, 12, 11 or 10%.
  • the compositions can contain from greater than about 1% for example, about 2%, to less than about 10%, for example about 9 or 8%, including concentrations of greater than about 2%, for example, about 3 or 4%, to less than about 8%, for example, about 7 or 6%.
  • the EVs can, for example, be present in a concentration of about 2% or 5%. In all cases, amounts may be adjusted to compensate for differences in amounts of EVs actually delivered to the treated tissue.
  • Frequency of application of any one of the EVs or compositions described herein includes up to about 12 times a day.
  • the EVs or compositions described herein may be applied twice a day at an 8 hr interval.
  • the composition is applied every 1, 2 or 3 hours.
  • the frequency of application may be progressively reduced as symptoms improve.
  • the frequency of application is at least maintained at a level such that at least one biochemically or clinically observable symptoms is improved compared to the start of treatment.
  • pharmaceutically acceptable excipient means a pharmaceutically acceptable material which is included in the composition for a purpose other than pharmaceutical efficacy (this is not intended to exclude materials which may have some biological effect).
  • an excipient may be involved in giving form or consistency to the pharmaceutical composition, such as forming a vehicle or carrier for an EV of the invention.
  • Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the EVs of the invention when administered to a patient and interactions which would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided.
  • each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.
  • Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen.
  • suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition.
  • certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms.
  • Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms.
  • Certain pharmaceutically-acceptable excipients may be chosen for their ability to facilitate the carrying or transporting of the EVs of the invention (or other compounds) once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.
  • Suitable pharmaceutically-acceptable excipients include the following types of excipients: diluents, fillers, binders, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, and buffering agents.
  • suitable pharmaceutically-acceptable excipients maintain the EV structure, and do not cause the EV lipid bilayer to disperse.
  • Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention.
  • resources that are available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company, e.g., 18 th Ed.), Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wlkins.
  • compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company, e.g., 18 th Ed).
  • dosage forms include those adapted for (1) inhalation such as aerosols and solutions; (2) intranasal administration such as aerosol spray, solution, powder, suspension, emulsion, drops, single or multi-dose, with or without a preservative; (3) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; and (4) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets.
  • inhalation such as aerosols and solutions
  • intranasal administration such as aerosol spray, solution, powder, suspension, emulsion, drops, single or multi-dose, with or without a preservative
  • parenteral administration such as sterile solutions, suspensions, and powders for reconstitution
  • oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions
  • a pharmaceutical composition may be formulated as inhaled formulations, including sprays, mists, or aerosols.
  • inhalation formulations the composition or combination provided herein may be delivered via any inhalation methods known to a person skilled in the art.
  • inhalation methods and devices include, but are not limited to, metered dose inhalers with propellants such as CFC or HFA or propellants that are physiologically and environmentally acceptable.
  • propellants such as CFC or HFA or propellants that are physiologically and environmentally acceptable.
  • Other suitable devices are breath operated inhalers, multidose dry powder inhalers and aerosol nebulizers.
  • Aerosol formulations for use in the subject method typically include propellants, and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.
  • Inhalant compositions may comprise liquid or powdered compositions containing the EVs that are suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses.
  • Suitable liquid compositions comprise the EVs in an aqueous, pharmaceutically acceptable inhalant solvent such as isotonic saline or bacteriostatic water.
  • the solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's lungs.
  • Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous solutions of the EVs.
  • parenteral as used herein includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique.
  • Suitable oral forms include, for example, tablets, troches, lozenges, aqueous suspensions, dispersible powders or granules, hard or soft capsules, or syrups or elixirs.
  • compositions provided herein may be formulated as a lyophilizate.
  • the various dosage units are each preferably provided as a discrete dosage tablet, capsules, lozenge, dragee, gum, or other type of solid formulation.
  • Capsules may encapsulate a powder, liquid, or gel.
  • the solid formulation may be swallowed, or may be of a suckable or chewable type (either frangible or gum-like).
  • the present invention contemplates dosage unit retaining devices other than blister packs; for example, packages such as bottles, tubes, canisters, packets.
  • the dosage units may further include conventional excipients well-known in pharmaceutical formulation practice, such as binding agents, gellants, fillers, tableting lubricants, and colorants; and for suckable or chewable formulations.
  • Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavouring agents, colouring agents and/or preserving agents in order to provide appealing and palatable preparations.
  • Tablets contain the EVs in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating agents such as corn starch or alginic acid, binding agents such as starch, gelatine or acacia, and lubricating agents such as magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatine capsules wherein the EVs are mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the EVs are mixed with water.
  • an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin
  • soft gelatine capsules wherein the EVs are mixed with water.
  • Aqueous suspensions contain the EVs in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the EVs in admixture with one or more preservatives. Additional excipients, such as sweetening, flavouring and colouring agents, may also be present.
  • Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavouring agents and/or colouring agents.
  • sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also comprise one or more demulcents, preservatives, flavouring agents and/or colouring agents.
  • kit for use in a therapeutic application mentioned herein including:
  • kit may contain one or more further active principles or ingredients for treatment of viral infection, and/or associated diseases or conditions.
  • the kit or “article of manufacture” may comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a therapeutic composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the therapeutic composition is used for treating the condition of choice.
  • the label or package insert includes instructions for use and indicates that the therapeutic composition can be used to treat a viral infection, and/or associated diseases or conditions described herein.
  • the kit may comprise (a) a therapeutic composition; and (b) a second container with a second active principle or ingredient contained therein.
  • the kit in this embodiment of the invention may further comprise a package insert indicating that the therapeutic composition and other active principle can be used to treat a disorder or prevent a complication stemming from a viral infection, and/or associated diseases or conditions described herein.
  • the kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • the therapeutic composition may be provided in the form of a device, disposable or reusable, including a receptacle for holding the therapeutic or pharmaceutical composition.
  • the device is a tube.
  • the device may hold 10-100 ml_ of the therapeutic composition.
  • the therapeutic composition may be provided in the device in a state that is ready for use or in a state requiring mixing or addition of further components.
  • the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific EVs employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the patient), and the severity of the particular disorder undergoing therapy.
  • the EVs of the invention may be administered by any suitable route of administration, including systemic administration or direct administration.
  • Systemic administration includes administration by inhalation, intranasal administration, parenteral administration, and oral administration. Inhalation refers to administration into the patient's lungs whether inhaled through the mouth or through the nasal passages.
  • Parenteral administration refers to routes of administration other than enteral, transdermal, or by inhalation, and is typically by injection or infusion.
  • Parenteral administration includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique.
  • Suitable oral forms include, for example, tablets, troches, lozenges, aqueous suspensions, dispersible powders or granules, hard or soft capsules, or syrups or elixirs.
  • compositions provided herein may be formulated as a lyophilizate.
  • compositions in a form suitable for inhalation, intranasal, parenteral, or oral use are preferred.
  • the EVs of the invention may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for EVs of the invention depend on the pharmacokinetic properties of said EVs, such as absorption, distribution, and half-life, which can be determined by the skilled artisan.
  • suitable dosing regimens including the duration such regimens are administered, for EVs of the invention depend on the condition being treated, the severity of the condition being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated and any comorbidities, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual patient's response to the dosing regimen or over time as individual patient needs change. Typical daily dosages may vary depending upon the particular route of administration chosen. Typical dosages for oral administration range from 1 mg to 1000 mg per person per dose.
  • the EVs and compositions according to the invention are useful for preventing or treating a viral infection, and/or a disease or condition associated with a viral infection.
  • the viral infection is caused by a virus selected from: coronaviruses, respiratory syncytial virus, hepatitis virus, influenza viruses, dengue virus, rhinoviruses, rotaviruses, Herpes viruses (HSV), Human Papillomaviruses (HPV), Human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Ebola virus, Echovirus, Flavivirus, Morbilivirus, and Hantavirus.
  • the virus may include viruses that cross species from time to time (Xeno viruses).
  • the viral infection is caused by a virus selected from: coronavirus, respiratory syncytial virus, hepatitis virus, influenza virus, and dengue virus. More preferably, the virus is coronavirus, respiratory syncytial virus or influenza. Even more preferably, the virus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), most preferably SARS-CoV-2.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the present invention provides a method of preventing a viral infection, and/or a disease or condition associated with a viral infection, in an individual comprising, consisting essentially of or consisting of the steps of: administering a therapeutically effective amount of a composition of EVs according to the invention to the individual; wherein the individual is at risk of having or contracting a viral infection, and/or a disease or condition associated with a viral infection; thereby preventing the infection, and/or the disease or condition associated with the viral infection, in the individual.
  • the present invention provides a method of treating a viral infection, and/or a disease or condition associated with a viral infection, in an individual comprising, consisting essentially of or consisting of the steps of: administering a therapeutically effective amount of a composition of EVs according to the invention to the individual; thereby treating the infection, and/or the disease or condition associated with the viral infection, in the individual.
  • the present invention provides a method of treating a viral infection, and/or a disease or condition associated with a viral infection, in an individual comprising, consisting essentially of or consisting of the steps of: administering a therapeutically effective amount of a composition of EVs according to the invention to the individual; wherein the individual is diagnosed with, or suspected of having, a viral infection, and/or a disease or condition associated with a viral infection, thereby treating the infection, and/or the disease or condition associated with the viral infection, in the individual.
  • the present invention provides a method for the treatment of a viral infection, and/or a disease or condition associated with a viral infection, in an individual comprising, consisting essentially of or consisting of the steps of: identifying a subject having, or suspected of having, a viral infection, and/or a disease or condition associated with a viral infection; and administering to the individual in need thereof a therapeutically effective amount of a composition of EVs according to the invention, thereby treating the infection, and/or the disease or condition associated with the viral infection, in the individual.
  • the invention provides a method for the treatment or reduction of the severity of a symptom of a viral infection, and/or a disease or condition associated with a viral infection in an individual comprising, consisting essentially of or consisting of the steps of: identifying a subject having, or suspected of having, a viral infection, and/or a disease or condition associated with a viral infection; and administering to the individual in need thereof a therapeutically effective amount of a composition of EVs according to the invention, thereby treating, or reducing the severity of a symptom of the viral infection, and/or disease or condition associated with the viral infection in the individual.
  • the invention provides methods of inhibiting replication of a virus in a cell.
  • the methods include contacting a cell with an EV of the invention in an amount effective to inhibit replication of the virus in the cell, thereby inhibiting replication of the virus in the cell.
  • Contacting of a cell with an EV may be done in vitro or in vivo. Contacting a cell in vivo with the EV includes contacting a cell or group of cells within a subject, e.g., a human subject, with the EV. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as described herein.
  • inhibiting is used interchangeably with reducing, “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.
  • “Inhibiting replication of a virus” includes any level of inhibition of replication of the virus, e.g., at least partial suppression of the virus replication.
  • the replication of the virus may be assessed based on the level, or the change in the level, of any variable associated with the virus replication, e.g., viral level, viral RNA level, viral protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.
  • Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with virus replication compared with a control level.
  • the control level may be any type of control level that is utilized in the art, e.g., a. predose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).
  • a positive control may include a reference level in a subject, cell, or sample that has not been infected with the virus.
  • replication of the virus is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
  • Inhibition of virus replication may be manifested by a reduction of the amount of viral RNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a viral gene is present and which has or have been treated (e.g., by contacting the cell or cells with an EV of the invention, or by administering an EV of the invention to a subject in which the cells are or were present) such that the replication of the virus is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)).
  • the inhibition is assessed by expressing the level of viral RNA in treated cells as a percentage of the level of viral RNA in control cells.
  • inhibition of virus replication may be assessed in terms of a reduction of a parameter that is functionally linked to virus replication, e.g., viral protein expression, infectious viral particle production by plaque assay, replication of the viral genome, or replication of viral RNA genes.
  • Viral gene silencing may be determined in any cell replicating the virus, either constitutively or by genomic engineering, and by any assay known in the art.
  • Inhibition of the expression of a viral protein may be manifested by a reduction in the level of the viral protein, RNA, or infectious virus that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject).
  • the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.
  • a control cell or group of cells that may be used to assess the inhibition of the expression of a viral gene includes a cell or group of cells that has not yet been contacted with an EV of the invention.
  • the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an EV.
  • a positive control cell or group of cells that may be used as a reference level to assess the inhibition of the expression of a viral gene includes a cell or group of cells that has not been infected with the virus.
  • the level of viral RNA that is expressed by a cell or group of cells, or the level of circulating viral RNA, may be determined using any method known in the art for assessing RNA expression.
  • the invention provides methods of treating a disease or condition associated with a virus in a subject.
  • the disease or condition associated with the virus may be caused by, induced by or result from the viral infection.
  • the method includes administering to the subject a therapeutically effective amount of an EV of the invention as described herein or a pharmaceutical composition as described herein, thereby treating or preventing the viral-associated disease or condition in the subject.
  • the subject may be a subject at risk of viral infection and/or developing a viral-associated disease and the effective amount is a prophylactically effective amount.
  • the present invention provides a method of treating and/or preventing a disease or condition associated with, or caused by, a virus, the method comprising administering to a subject in need thereof an EV of the invention as described herein or a pharmaceutical composition as described herein, thereby treating and/or preventing a disease or condition associated with, or caused by, the virus.
  • the present invention provides a method of treating and/or preventing a respiratory disease or condition associated with SARS-CoV-2 infection, the method comprising administering to a subject in need thereof an EV of the invention as described herein or a pharmaceutical composition as described herein, thereby treating and/or preventing a respiratory disease or condition associated with SARS-CoV-2 infection.
  • the present invention provides a method for reducing airway inflammation associated with, or caused by, SARS-CoV-2, the method comprising administering to a subject in need thereof an EV of the invention as described herein or a pharmaceutical composition as described herein, thereby reducing airway inflammation associated with, or caused by, SARS- CoV-2.
  • the present invention also provides a method of improving the ability of a subject to control a respiratory disease or condition during SARS-CoV-2 infection, the method comprising administering to a subject in need thereof an EV of the invention as described herein or a pharmaceutical composition as described herein, thereby improving the ability of a subject to control a respiratory disease or condition during the SARS-CoV-2 infection.
  • the present invention provides for use of an EV of the invention as described herein or a pharmaceutical composition as described herein, in the preparation of a medicament for treating and/or preventing a disease or condition caused by a virus.
  • the present invention further provides for use of an EV of the invention as described herein or a pharmaceutical composition as described herein, in the preparation of a medicament for treating and/or preventing a respiratory disease or condition associated with SARS-CoV-2 infection in a subject.
  • the present invention further provides for use of an EV of the invention as described herein or a pharmaceutical composition as described herein, in the preparation of a medicament for treating and/or preventing SARS-CoV-2 infection in a subject.
  • the present invention further provides use of an EV of the invention as described herein or a pharmaceutical composition as described herein, in the preparation of a medicament for reducing airway inflammation in a subject diagnosed with, or suspected of having, SARS-CoV-2 infection.
  • the present invention further provides use of an EV of the invention as described herein or a pharmaceutical composition as described herein, in the preparation of a medicament for improving the ability of a subject to control a respiratory disease or condition during SARS-CoV-2 infection.
  • the present invention provides for an EV of the invention as described herein or a pharmaceutical composition as described herein, for use in treating and/or preventing a disease or condition caused by a virus in a subject.
  • the present invention provides for an EV of the invention as described herein or a pharmaceutical composition as described herein, for use in treating and/or preventing a respiratory disease or condition associated with SARS-CoV-2 infection in a subject.
  • the invention provides an EV of the invention as described herein or a pharmaceutical composition as described herein, for use in reducing airway inflammation in a subject diagnosed with, or suspected of having, SARS-CoV-2 infection.
  • the invention provides an EV of the invention as described herein or a pharmaceutical composition as described herein, for use in controlling a respiratory disease or condition during SARS-CoV-2 infection in a subject.
  • the SARS-CoV-2-associated disease or condition is selected from the group consisting of: elevated body temperature, elevated heart rate, elevated respiratory rate, headache, discomfort, body aches and chills, sore throat, cough, pneumonia, difficulty breathing, shortness of breath, loss of smell and/or taste, general malaise, hypoxia, acute respiratory distress syndrome (ARDS), and combinations thereof.
  • elevated body temperature elevated heart rate, elevated respiratory rate, headache, discomfort, body aches and chills, sore throat, cough, pneumonia, difficulty breathing, shortness of breath, loss of smell and/or taste, general malaise, hypoxia, acute respiratory distress syndrome (ARDS), and combinations thereof.
  • ARDS acute respiratory distress syndrome
  • the EV may be administered to the subject by an administration means selected from the group consisting of inhalation, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combinations thereof.
  • the virus is SARS-CoV-2
  • the EV is preferably administered to the subject via inhalation administration.
  • the EV is administered to the subject such that the EV is delivered to a specific site within the subject.
  • the virus is SARS-CoV- 2
  • the site is the lungs.
  • the EV is administered in two or more doses.
  • the EV is administered at intervals selected from the group consisting of once every about 2 hours, once every about 3 hours, once every about 4 hours, once every about 6 hours, once every about 8 hours, once every about 12 hours, once every about 24 hours, once every about 48 hours, once every about 72 hours, once every about 96 hours, once every about 120 hours, once every about 144 hours, once every about 168 hours, once every about 240 hours, once every about 336 hours, once every about 504 hours, once every about 672 hours and once every about 720 hours.
  • the EV is administered once daily or once weekly.
  • the EV of the invention as described herein or the pharmaceutical composition as described herein is administered to the subject before any clinically or biochemically detectable symptoms of viral infection.
  • administration of the EV of the invention as described herein or the pharmaceutical composition as described herein to a subject reduces viral load in a subject.
  • the viral load is reduced in the respiratory tract, for example the upper and/or lower respiratory tract.
  • the viral load is reduced in the nasal cavity and pharynx (i.e throat).
  • administration of the EV of the invention as described herein or the pharmaceutical composition as described herein to a subject asymptomatic for a viral infection may prevent or reduce the progression to symptomatic phase.
  • the present invention provides a method of reducing the severity of a viral infection, or reducing the period in which a subject displays one or more symptoms of a viral infection, the method comprising administering to a subject in need thereof an EV of the invention as described herein or a pharmaceutical composition as described herein, thereby reducing the severity of the viral infection, or reducing the period in which a subject displays one or more symptoms of the viral infection.
  • a reduction in viral infection may be determined using any method known in the art or described herein, including measuring viral load in a sample from the subject after treatment and comparing it to viral load in a sample from the same subject before treatment.
  • the viral load is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
  • the present invention also provides a method for inhibiting or minimising the progression of a symptom of a viral infection, and/or a disease or condition associated with a viral infection in an individual comprising, consisting essentially of or consisting of the steps of: administering a therapeutically effective amount of a composition of EVs according to the invention to an individual who is experiencing a symptom of a viral infection, and/or a disease or condition associated with a viral infection; thereby inhibiting or minimising the progression of the symptom the viral infection, and/or the disease or condition associated with the viral infection in the individual.
  • the symptom of the viral infection, and/or disease associated with the viral infection is selected from: pharyngitis, anosmia, elevated body temperature, elevated heart rate, elevated respiratory rate, abnormal white blood cell count, low blood pressure, hypoxemia, tissue hypoxia, hypoperfusion and combinations thereof.
  • the individual is identified as having, or suspected of having, at least 2 symptoms of viral infection, and/or a disease associated with a viral infection.
  • An individual may be identified as experiencing a symptom of a viral infection, and/or a disease or condition associated with a viral infection by any biochemical or clinical method or test as described herein.
  • Methods of prevention or treatment of the invention may be achieved using the EVs of the invention as a monotherapy, or in dual or multiple combination therapy with one or more therapeutic agents or therapies.
  • one or more EVs of the invention may be used in combination.
  • One or more EVs of the invention may also be used with one or more other therapeutic agents or therapies.
  • the one or more other therapeutic agents of therapies include but are not limited to: antiviral small molecule drugs, immunotherapy, immunosuppressive therapy, non-steroidal anti-inflammatory drugs, corticostreoids, and combinations thereof.
  • Symptoms of viral infection identified by biochemical or clinical tests known to those skilled in the art may include, but are not limited to:
  • Successful treatment may be determined by: decreased viral titre; • treatment of, or a decrease in the severity of one or more symptoms of, a viral infection, and/or a disease or condition associated with viral infection;
  • preventing or “prevention” is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or condition (i.e., causing at least one of the clinical symptoms of the disease not to develop in an individual that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).
  • Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians.
  • prevention is not an absolute term.
  • the methods of the present invention can be to prevent or reduce the severity, or inhibit or minimize progression, of a symptom of a disease or condition as described herein. As such, the methods of the present invention have utility as treatments as well as prophylaxes.
  • treatment includes delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition.
  • treating refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the individual; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating.
  • Treatment may not necessarily result in the complete clearance of a disease or disorder but may reduce or minimize complications and side effects of infection and the progression of a disease or disorder.
  • the success or otherwise of treatment may be monitored by, amongst other things, physical examination of the individual, blood biomarkers, resolution of one or more symptoms associated with the viral infection, and combinations thereof.
  • Treatment of a disease or condition described herein may include a visibly, clinically or biochemically detectable change in the subject in any one or more of the following: temperature, resolution of a cough, cyanosis, increased blood oxygen saturation, decreased viral titre, blood biomarkers including pro-inflammatory markers, and combinations thereof.
  • a “subject” herein is preferably a human subject. It will be understood that the terms “subject” and “individual” are interchangeable in relation to an individual requiring treatment according to the present invention.
  • the invention finds application in humans, the invention is also useful for therapeutic veterinary purposes.
  • the invention is useful for domestic or farm animals such as cattle, sheep, horses and poultry; for companion animals such as cats and dogs; and for zoo animals.
  • an effective amount in reference to EVs of the invention or other pharmaceutical ly-active agent means an amount of EVs sufficient to treat the patient's condition but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment.
  • An effective amount of EVs will vary with the particular EVs chosen (e.g. consider the potency, efficacy, and half-life of therapeutic agent); the route of administration chosen; the condition being treated; the severity of the condition being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be routinely determined by the skilled artisan.
  • nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an "isolated" nucleic acid molecule such as a cDNA molecule or an RNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding polypeptides/proteins of the invention are isolated or purified.
  • isolated nucleic acid molecule does not include a nucleic acid that is a member of a library that has not been purified away from other library clones containing other nucleic acid molecules.
  • portion refers to a fragment of a nucleic acid molecule containing at least about 5, 10, 15, 20, 25, or more contiguous nucleic acids in length of the relevant nucleic acid molecule and having at least one functional feature of the nucleic acid molecule ( or the encoded protein has one functional feature of the protein encoded by the nucleic acid molecule.
  • target sequence refers to a contiguous portion of the nucleotide sequence of a viral gene or genome.
  • strand comprising a sequence refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
  • G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively.
  • T and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine, 2’-deoxythymidine or thymidine.
  • ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
  • siRNA silencing RNA
  • dsRNA agent dsRNA agent
  • iRNA agent a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined below, nucleic acid strands.
  • nucleic acid strands the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide.
  • an “siRNA” may include ribonucleotides with chemical modifications; an siRNA may include substantial modifications at multiple nucleotides. Such modifications may include all types of modifications disclosed herein or known in the art.
  • antisense strand refers to the strand of a double stranded siRNA which includes a region that is substantially complementary to a target sequence (e.g., a SARS-CoV-2 RNA).
  • sense strand refers to the strand of a siRNA that includes a region that is substantially complementary to a region of the antisense strand.
  • duplex includes a region of complementarity between two regions of two or more polynucleotides that comprise separate strands, such as a sense strand and an antisense strand, or between two regions of a single contiguous polynucleotide.
  • the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.
  • Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing.
  • Sequences can be “fully complementary” with respect to each when there is base-pairing of the nucleotides of the first nucleotide sequence with the nucleotides of the second nucleotide sequence over the entire length of the first and second nucleotide sequences.
  • a first sequence is referred to as “substantially complementary” with respect to a second sequence herein
  • the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application.
  • oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity.
  • a siRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.
  • “Complementary” sequences may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
  • Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.
  • contacting a cell with an EV includes contacting a cell by any possible means.
  • Contacting a cell with an EV includes contacting a cell in vitro with the EV or contacting a cell in vivo with the EV.
  • the contacting may be done directly or indirectly.
  • the EV may be put into physical contact with the cell by the individual performing the method, or alternatively, the EV may be put into a situation that will permit or cause it to subsequently come into contact with the cell.
  • Contacting a cell in vitro may be done, for example, by incubating the cell with the EV.
  • Contacting a cell in vivo may be done, for example, by injecting the EV into or near the tissue where the cell is located, or by injecting the EV into another area, e.g., the bloodstream or the subcutaneous space, such that the EV will subsequently reach the tissue where the cell to be contacted is located.
  • Combinations of in vitro and in vivo methods of contacting are also possible.
  • a cell might also be contacted in vitro with an EV and subsequently transplanted into a subject.
  • sample includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.
  • biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like.
  • Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs.
  • samples may be derived from the lung.
  • a “sample derived from a subject” refers to blood or plasma drawn from the subject.
  • a “sample derived from a subject” refers to lung tissue (or subcomponents thereof) derived from the subject.
  • A. EVs comprising a targeting molecule attached to a transmembrane protein
  • Vectors encoding AcGFP and DsRed were purchased. Template DNA encoding SARS-CoV-2 Spike (S) protein, VSVg surface protein was also purchased. Constructs were assembled using a combination of polymerase chain reaction (PCR) and ligation of the desired molecular arrangements via unique restriction sites. Reverse and forward primers containing unique restriction sites flanking the gene of interest were designed, ordered and reconstituted to 100 mM. PCR was performed using the GoTaq® Green MasterMix (Promega, Wisconsin, USA) according to manufacturer’s protocol. PCR product was analysed via gel electrophoresis to confirm correct size and DNA clean-up performed using a column-based reaction (Qiagen, Hilden, Germany). Duplex DNA sequences comprising of restriction sites tagged to the VSV-G signal sequence were designed and ordered accordingly (IDT, Singapore).
  • PCR polymerase chain reaction
  • PCR products and duplex DNA were digested using restriction enzymes specific for unique sites designed in the flanking regions with Agel, Notl, Sad (New England Biolabs, MA, USA) and Apal (Takara Bio Inc., USA) utilised. Digests were performed according to manufacturer’s protocol. DNA were resolved via 1% agarose (Sigma- Aldrich, MO, USA) in 1x Tris-acetate-EDTA (TAE) buffer (Bio-Rad Laboratories, Inc., CA, USA) at 100V for 45 minutes. Digested products were carefully excised, and gel clean-up performed using a column-based kit (Qiagen, Hilden, Germany).
  • TAE Tris-acetate-EDTA
  • the resultant DNA was quantified for size and ligated in a combination of relevant inserts using the T4 DNA ligase enzyme (New England Biolabs, MA, USA).
  • Ligated product was transformed into One Shot® MAX Efficiency® DH5aTM-T1® competent cells (Invitrogen Life Technologies, CA, USA) and plated onto LB kanamycin agar, with colonies inspected the following day.
  • Colony PCR was performed using the CMV-F (5- CGCAAATGGGCGGTAGGCGTG-3’) and EBV-R reverse complementary (5- GATGAGTTTGGACAAACCAC-3’) primers.
  • Colonies of interest were expanded and purified via Midiprep (Giagen, Hilden, Germany), with the resultant DNA checked for size accuracy via enzyme digest, and, quantified using the NanoDropTM 2000c spectrophotometer (Thermo Fisher Scientific, MA, USA). Sanger sequencing was performed (Australian Genome Research Facility, Melbourne, USA) and sequences were checked for alignment using the National Center for Biotechnology Information’s Basic Local Alignment Search Tool (NIH, MD,USA).
  • HEK-293T (293 LTV) cells Cell Biolabs, Inc. CA, USA
  • 293fectinTM transfection reagent Thermo Fisher Scientific, MA, USA
  • Cells were incubated in a humidified chamber at 37°C (5% C02) and analysed using the EVOS M5000 imaging system (Thermo Fisher Scientific, MA, USA) at 24, 48 and 72 hours post-transfection.
  • ‘Target’ cells were seeded at a density of 1E+05 / mL (500 pL per chamber in 4- well chamber-slides). 10 pL of the enriched particles were added to chambers in the 4- well chamber slides. The chamber slides were washed with 1x PBS, fixed with 4% PFA for 5 minutes, washed with 1x PBS containing 0.05% Triton X-100, stained with DAPI for 5-mins and washed with 1.0 mL PBS. Cells were then mounted with glycerol on a coverslip for microscopic visualisation.
  • transmembrane construct expression transfected cells were seeded at a density of 1E+05 / ml_ (500 pi ⁇ per chamber in 4-well chamber-slides) and incubated overnight in DM EM, 10% FCS, 5% C02 37oC.
  • the chamber slides were washed with 1x PBS, fixed with 4% PFA for 5 minutes, washed with 1x PBS containing 0.05% Triton X-100, stained with DAPI for 5- mins and washed with 1.0 ml_ PBS. Cells were then mounted with glycerol on a coverslip for microscopic visualisation.
  • Cell lysates were prepared for SDS-PAGE by pelleting cells at 550 xg for 5 minutes at 4°C. The supernatant was aspirated and the pelleted cells resuspended in 1 mL ice-cold 1x DPBS. Cells were lysed in 300 uL 1x RIPA lysis buffer (Thermo #89900), including Roche completeTM, Mini, EDTA-free Protease Inhibitor Cocktail (#04693 132001). The cells were incubated on ice for 15 mins to achieve full lysis and Centrifuged at -14,000 c g (4°C) for 15 minutes to pellet the cell debris. The supernatant was then processed for SDS-PAGE and western blotting after protein concentration estimation by BCA assay.
  • the samples were solubilised in In 1x loading dye (BoltTM LDS Sample Buffer) and 1x reducing agent (BoltTM Reducing Agent) according to manufacturer’s instructions and heated to 70°C for 10 minutes. Samples were loaded onto SDS-PAGE precast gels and they were electrophoretically separated for 50 mins at 200V. Western transfer to PVDS membrane was carried out using an iBIot 2 device.
  • In 1x loading dye BoltTM LDS Sample Buffer
  • 1x reducing agent BoltTM Reducing Agent
  • the membrane was blocked using 5% BSA in TBST for 30 mins at ambient temperature, washed, and 1° antibody was applied at 1:2000 in blocking solution overnight.
  • the membrane was washed x 5 for 5 mins each the 2° antibody was applied anti-mouse HRP-conjugated at 1:10,000 dilution for 2 hrs rocking at room temperature.
  • the membrane was then washed x5 for 5 mins each and visualised using the PierceTM ECL Western Blotting Substrate (# 32209) using the Bio-Rad XRS Chemidoc System.
  • the Total Exosome Isolation Kit (ThermoFisher) may be used to isolate the EVs from culture supernatant and resuspend in 100 uL PBS.
  • the EVs may be solubilised in In 1x loading dye (BoltTM LDS Sample Buffer) and 1x reducing agent (BoltTM Reducing Agent) according to manufacturer’s instructions and heated to 70°C for 10 minutes. Samples may be loaded onto SDS-PAGE precast gels and electrophoretically separated for 50 mins at 200V. Western transfer to PVDS membrane may then be carried out using an iBIot 2 device.
  • the Total Exosome Isolation Kit (ThermoFisher) was used according to the manufacturer’s instructions. Briefly, EV isolation solution was added to HEK293 cell free supernatant. The culture media/reagent was mixed and incubated at 4°C overnight. The samples were centrifuged at 10,000 c g for 1 hour at 2°C to 8°C and the supernatant was aspirated. The pelleted EVs were resuspended in 1 ml_ PBS or similar buffer.
  • EVs were washed in PBS followed by concentration by ultracentrifugation (1 x 105 x overnight at 4°C).
  • the EVs were either processed directly for Amnis Imaging Flow Cytometry (Luminex) or stained with EV-specific or fusion-mVSVG-GFP/DsRed protein- specific fluorophore-conjugated antibodies then Amnis Imaging Flow Cytometry was carried out.
  • siRNA was designed to anneal to RNA of interest. This process involved accessing the SARS-CoV-2 genome sequences of interest by interrogation of publicly- available genome databases and using a computer program to design appropriate single stranded ‘coding’ siRNA sequences identical to the coding sequences of the target RNA sequence.
  • the computer program also provides ‘noncoding’ siRNA sequence complementary to the siRNA coding sequence with additional features specific to all ‘non-coding’ siRNA sequences.
  • the coding and non-coding siRNA was synthesised by a commercial supplier, incorporating a ‘Locked Nucleic Acid’ version of the 5’ nucleic acid of the ‘coding’ siRNA to aid siRNA stability. Annealing of coding and non-coding strands was carried out to generate mature siRNA.
  • Vero E6/TMPRSS2 cells were cultured in 96 well to 50% confluency.
  • siRNA was formulated with Lipofectamine (ThermoFisher) and added to the Vero E6 wells to a final concentration of 100 nM/well. Each siRNA was assayed in triplicate. After 3-hours the wells were washed with culture media and SARS-CoV-2 virus was added to each well at a multiplicity of infection of 0.05. The cultures were incubated for 24 hours, the supernatant aspirated and qRTPCR performed on the culture supernatant from each well using primers specific for the SARS-CoV-2 E-protein DNA sequence.
  • results of test siRNA inhibition were calculated as a percentage of the value of the assay incorporating an irrelevant siRNA. Additional controls including no siRNA added and the antiviral compound Remdesivir were carried out. In some cases the concentration of siRNA will be titrated to provide dose response data.
  • RNA constructs Single stranded RNA constructs were designed.
  • the sense strand was annealed to the anti-sense strand that was labelled by a fluorescent reporter on the 5’ end.
  • the sequences were prepared at 100uM stock in IDT duplex buffer.
  • the siRNA sense and antisense strand were annealed at equal volumes for 2 minutes at 94°C followed by slow cooling over 1 hour.
  • 10 mM dsRNA sequence was electroporated into the cell line of interest using Lonza’s Nucleofector X unit and following manufacturer’s instructions.
  • HEK293 cells were grown in DMEM (Gibco) supplemented 10% serum. All cells were maintained at 37°C 5% CO2.
  • EVs containing the siRNA were isolated from the HEK293 cell culture supernatant. Efficient EV loading of siRNA was confirmed by qRTPCR or BioAnalyser (Agilent) automated electrophoresis analysis.
  • the Total Exosome Isolation Kit (ThermoFisher) was used according to the manufacturer’s instructions. Briefly, EV isolation solution was added to HEK293 cell free supernatant. The culture media/reagent was mixed and incubated at 4°C overnight. The samples were centrifuged at 10,000 c g for 1 hour at 2°C to 8°C and the supernatant was aspirated. The pelleted EVs were resuspended in 1 ml_ PBS or similar buffer.
  • ‘Target’ cells were seeded at a density of 1E+05 / ml_ (500 pl_ per chamber in 4- well chamber-slides). 10 pL of the enriched particles were added to chambers in the 4- well chamber slides. The chamber slides were washed with 1x PBS, fixed with 4% PFA for 5 minutes, washed with 1x PBS containing 0.05% Triton X-100, stained with DAPI for 5-mins and washed with 1.0 ml_ PBS. Cells were then mounted with glycerol on a coverslip for microscopic visualisation.
  • Vero E6/TMPRSS2 cells will be cultured in 96 well to 50% confluency.
  • siRNA will be loaded into HEK293 cells previously transfected with the gene encoding the targeting transmembrane protein by electroporation and the cells cultured for 24 hrs.
  • EVs will be purified from the culture and the amount of loaded siRNA established by qRTPCR.
  • EVs containing siRNA will be added to the Vero E6 cell culture wells such that a final concentration of 100 nM siRNA/well. Each siRNA will be assayed in triplicate. After 3- hours the wells will be washed with culture media and SARS-CoV-2 virus will be added to each well at a multiplicity of infection of 0.05.
  • the cultures will be incubated for 24 hours, the supernatant aspirated and qRTPCR performed on the culture supernatant from each well using primers specific for the SARS-CoV-2 E-protein DNA sequence. Results of test siRNA inhibition are calculated as a percentage of the value of the assay incorporating an irrelevant siRNA. Additional controls including no siRNA added and the antiviral compound Remdesivir will be carried out. In some cases, the concentration of siRNA will be titrated to provide dose response data. In some cases, the tropic element fused to mVSVG will be derived from alternative viruses.
  • gp350 from the Epstein Barr virus (EBV) will be fused to mVSVG and transfected into EV-producing cells to produce a fusion protein of gp350-mVSVG which will be located on the EVs and facilitate EV tropism to CD21 (the receptor for gp350)-expressing target cells.
  • the EVs will be loaded with siRNA that inhibits translation of EBV-critical gene product(s) and thereby inhibit the proliferation of EBV in cells.
  • the gp350 will direct the EVs containing the EBV siRNA to cells that are susceptible to, or already infected with, EBV. This will result in a reduction of EBV proliferation in the CD21+ cells.
  • the exosomes described herein will be incorporated into anti-viral therapy for the treatment of an acute or chronic viral infection.
  • EVs will be engineered by the method and approaches described herein to express a single or multiple tropic molecules on their surface.
  • the engineered EVs will also be loaded with regulatory RNA, DNA, small molecule drugs or other compounds that inhibit intracellular viral replication.
  • the virus- specific EVs will be manufactured to sufficient quality and quantity to be used clinically for the treatment of disease in a mammal caused by that cognate virus.
  • the administration route and the dose of the engineered EV preparation will depend on the identity of the virus, the nature and severity of the infection, clinical presentation of the subject to be treated and judgement of the treating medical professional.
  • the engineered EVs can be administered via the inhalation route to directly access the infected pulmonary cells. Alternatively, the tending physician may recommend systemic delivery of the same engineered EVs, or both routes simultaneously.
  • the engineered EVs may be administered intraocularly. If the disease is neurological and / or cerebral, the engineered EVs may be delivered intranasally, intrathecally or intravenously to affect access to the central nervous system via the cranial nerves, directly or via the blood brain barrier respectively. Treatment using the engineered EVs will be continued as regularly as deemed appropriate by the treating physician and may be used in combination with other appropriate therapeutic interventions as deemed appropriate by the treating physician.

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Abstract

La présente invention concerne une vésicule extracellulaire (VE) comprenant une VE définissant un espace vésiculaire interne, une protéine transmembranaire affichant un site de fixation sur la surface de la VE, une molécule de ciblage fixée au site de fixation de la protéine transmembranaire, ce qui permet de cibler la VE sur une cellule cible, et un agent thérapeutique antiviral situé dans ou sur l'espace vésiculaire interne. La présente invention concerne des procédés de préparation de ces VE et leurs procédés d'utilisation.
PCT/AU2021/050513 2020-05-27 2021-05-27 Vésicules extracellulaires anti-virales, leurs procédés de préparation et leurs utilisations WO2021237297A1 (fr)

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CN114469996A (zh) * 2021-12-23 2022-05-13 中国医学科学院医学生物学研究所 一种包含miR-135b-5p的外泌体及在抗轮状病毒感染中的应用
CN114469996B (zh) * 2021-12-23 2023-10-20 中国医学科学院医学生物学研究所 一种包含miR-135b-5p的外泌体及在抗轮状病毒感染中的应用
WO2023145884A1 (fr) * 2022-01-28 2023-08-03 国立大学法人京都大学 POLYNUCLÉOTIDE DÉRIVÉ DU SARS-CoV-2 ET SON UTILISATION
CN116574685A (zh) * 2023-03-14 2023-08-11 苏州大学 一种装载抗菌肽ll-37的外泌体及其在抗寨卡病毒中的应用
CN116574685B (zh) * 2023-03-14 2024-05-31 苏州大学 一种装载抗菌肽ll-37的外泌体及其在抗寨卡病毒中的应用

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