Compositions containing genetically modified plant-derived extracellular vesicles and their use as vaccines

Abstract

The present invention provides a composition comprising non-immunomodulatory, engineered, plant-derived extracellular vesicles (EVs) for use as a vaccine, said vesicles being loaded with an exogenous nucleic acid molecule encoding a protein antigen, and a method for preparing said composition, said method using one or more polycationic substances and one or more sugar molecules.

Description

[Technical Field]

[0001] Technical field The present invention relates to compositions comprising plant-derived extracellular vesicles for use as vaccines and / or for preventive purposes. More specifically, the present invention relates to compositions comprising non-immunomodulatory, genetically modified plant-derived extracellular vesicles (EVs) loaded with exogenous nucleic acid molecules. [Background technology]

[0002] background Vaccination is one of the most effective public health interventions for preventing and controlling infectious and non-infectious diseases. Vaccines come in various types, including live attenuated vaccines, inactivated vaccines, subunit vaccines or recombinant vaccines or conjugate vaccines, toxoid vaccines, and nucleic acid-based vaccines.

[0003] Nucleic acid-based vaccines include viral vectors, plasmid DNA, and mRNA. These vaccines have emerged as promising alternatives to conventional vaccine approaches because they have the ability to induce a broad protective immune response and can potentially be manufactured using rapid and flexible manufacturing processes. Among nucleic acid-based vaccines, RNA vaccines offer several advantages over others. In fact, RNA vaccines are characterized by their absence of eukaryotic contaminants. In contrast to DNA vaccines, RNA vaccines are safer because they do not need to reach the nucleus to act, and plasmid DNA vaccines can be integrated into the immune host's genome. In this respect, mRNA molecules induce an immune response to encoded antigens.

[0004] This mechanism has been demonstrated with various target genes, including reporter genes, viral antigens, tumor antigens, and allergens. However, mRNA instability, high innate immunogenicity, and inefficient in vivo delivery limit the clinical application of RNA vaccines. The main challenge these vaccines face is intracellular delivery. mRNA is highly unstable under physiological conditions in the body because it is highly sensitive to degrading enzymes.

[0005] To date, in clinical applications, mRNA molecules are enhanced by being complexed with cationic polymers or encapsulated in synthetic lipid nanoparticles (LNPs), also known as liposomes, cationic nanoparticles, EV-mimicking nanovesicles, or polypeptide-based vesicles. LNPs protect mRNA from enzymes, increase its stability, and improve RNA circulating lifetime and in vivo delivery. LNP particles are produced by mixing mRNA molecules with various synthetic lipids or polymers. However, LNPs are an inefficient delivery system.

[0006] In fact, LNPs can accumulate in unintended tissues, thereby limiting their effectiveness on the intended target tissue, and they are rapidly cleared by the reticuloendothelial system or mononuclear phagocyte system (Koppers-Lalic D., et al. Adv Drug Deliv Rev. 2013 Mar;65(3):348-56). Furthermore, LNPs can induce inflammatory responses and apoptosis in vivo.

[0007] Furthermore, intracellular uptake of LNPs can activate the cellular autophagy-lysosome pathway via endocytosis. There is ample evidence that endocytosis of nanoparticles generates autophagosomes, which then fuse with lysosomes for digestion of their contents.

[0008] In recent years, attempts have been made to overcome the limitations of LNPs using extracellular vesicles (EVs). In fact, EVs are naturally secreted by cells and are safer than synthetic nanomaterials like LNPs. By leveraging their natural mechanism of action, EVs can overcome some of the limitations of assembled particles, including immunogenicity, cytotoxicity, administration of foreign particles, limited cellular uptake, and chemical aggregation of particles. Unlike that of LNPs, the uptake pathway of EVs differs from that of LNPs, as they are released into the cytoplasm without being captured by lysosomes, making them less likely to induce the autophagy-lysosomal pathway. Furthermore, because of their small size, EVs can escape rapid phagocytosis and steadily carry and deliver nucleic acids while circulating through the vascular endothelium to target cells.

[0009] Furthermore, EVs exhibit several advantages compared to LNPs in terms of biocompatibility, low clearance in circulation, low toxicity, low safety concerns, and high specificity (Sancho-Albero M, et al (2020) “Use of exosome as vectors to carry advanced therapies”. RSC Adv 10, 23975-23987). Because EVs are naturally derived, their membranes are highly biocompatible and efficiently taken up by receptor cells. In addition, plant-derived EVs are resistant to the gastric environment and can reach the intestines after oral administration. EVs encapsulating nucleic acid molecules have been studied for multiple clinical applications, including RNA interference, RNA-based gene therapy for neurodegenerative diseases, cancer, and cancer vaccines using miRNA and siRNA molecules.

[0010] US20200069594 describes the use of plant-derived extracellular vesicles containing cationic polymers for delivering therapeutic agents containing encoded and non-encoding nucleic acid molecules.

[0011] It is well known in this art that extracellular molecules (EVs) can protect and deliver nucleic acid molecules. In the case of vaccine formulations, the beneficial activity of EVs is primarily based on their stimulating effects on innate and adaptive immune system cells, as demonstrated, for example, in the study by Jesus S. et al. on HBV vaccine formulations (Jesus S., et al “Exosome as adjuvants for the recombinant hepatitis B antigen: First report”. Eur J Pharm Biopharm. 2018 Dec; 133:1-11).

[0012] WO2020191361 describes the use of EVs as vaccines that induce cellular immune responses, and for the treatment and / or prevention of various diseases.

[0013] WO2020050808 describes the use of plant-derived exosomes as adjuvants in vaccine administration, along with the immunomodulatory properties of these vesicles that activate or suppress immune system cells.

[0014] Extracellular vesicles isolated from various plants have been shown to regulate immune system cells by suppressing inflammation in the intestines. Plant sources include curcumin (Ohno M., et al, “Nanoparticle curcumin ameliorates experimental colitis via modulation of gut microbiota and induction of regulatory T cells” PLoS One. (2017) Oct 6;12(10):e0185999), ginger (Zhang M., et al, (2016) “Edible ginger-derived nanoparticles: A novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis-associated cancer”, Biomaterials 101:321-40), orange (Berger E., et al, “Use of Nanovesicles from Orange Juice to Reverse Diet-Induced Gut Modifications in Diet-Induced Obese Mice” (2020) Mol Ther Methods Clin Dev. 18:880-892), grapes, grapefruit and carrots (Ju S, et al. “Grape One example is "Exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis" (2013) Mol Ther. 21(7):1345-57).Furthermore, blueberry-derived exosomes (EVs) have been shown to reduce the gene expression of inflammatory genes in TNF-α-stimulated endothelial cells, protecting them from TNF-induced cytotoxicity and oxidative stress (De Robertis M, et al. “Blueberry-Derived Exosome-Like Nanoparticles Counter the Response to TNF-α-Induced Change on Gene Expression in EA.hy926 Cells” (2020) Biomolecules 10(5):742).

[0015] Despite the beneficial effects of extracellular viable cells (EVs), the immunomodulatory properties of these vesicles can pose significant limitations in vaccine application. Indeed, nonspecific activation or inhibition of the immune system can be detrimental to vaccines. The use of EVs with immunosuppressive activity can significantly reduce vaccine efficiency by inhibiting the response of immune cells. Furthermore, the development of an immune response to EVs can accelerate vaccine clearance. Conversely, the enhancement of the immune system by immunostimulatory EVs is detrimental and can cause adverse activation and / or hyperreaction of the target immune system. Immune responses to vesicles can limit repeated vaccination. Additionally, innate immune sensing of vesicles can lead to inhibition of antigen expression and adversely affect the immune response (Pardi N, et al “mRNA vaccines - a new era in vaccinology” (2018) Nat Rev Drug Discov. 17(4):261-279). In summary, the effects of EVs on the immune system highlight significant drawbacks to using EVs in vaccine formulations.

[0016] To overcome the limitations and drawbacks of the prior art, the present invention provides a composition comprising non-immunomodulatory, genetically engineered, plant-derived extracellular vesicles (EVs) for use as a vaccine, as well as a method for preparing said composition as defined in the independent claims of the appended patent claims. The dependent claims specify further advantageous features of the claimed composition and method. The subject matter of the appended patent claims forms an essential part of this specification.

Summary of the Invention

[0017] Detailed description of the invention The present invention relates to a composition comprising non-immunomodulatory, genetically engineered, plant-derived extracellular vesicles (EVs) for use as a vaccine, wherein said extracellular vesicles (EVs) are delimited by a lipid bilayer membrane comprising an outer lipid layer and an inner lipid layer, encapsulate a foreign nucleic acid molecule encoding at least one protein antigen, and have a diameter in the range of 20 - 500 nm, preferably 200 - 300 nm, the membrane potential across the lipid bilayer of the EVs is in the range of +5 to -5 mV, and no more than 44% of the EVs in the composition contain phosphatidylserine in the outer layer of the lipid bilayer.

[0018] As used herein, the term "extracellular vesicles (EVs)" means a heterogeneous population of particles released by substantially all living cells, which are delimited or encapsulated by a phospholipid bilayer and carry lipids, proteins, nucleic acids, and other molecules derived from the cells from which they originate. These vesicles mainly include microvesicles released by budding of the cell membrane and exosomes derived from endosomal components. Extracellular vesicles are referred to as "particles", "microparticles", "nanovesicles", "microvesicles", "exosomes". Due to the EV-specific cell targeting properties determined by the lipid composition and protein content and the EV-specific stability in circulation, these vesicles (EVs) are qualified as vehicles for therapeutic drug delivery.

[0019] As used herein, the term "immunomodulation" refers to the process of altering the function of the immune system by enhancing (immunostimulating) or reducing (immunosuppressing) the immune response. Accordingly, as used herein, the expression "non-immunomodulatory EV" refers to an extracellular vesicle that does not exert either a stimulating or immunosuppressive effect on the immune system.

[0020] As used herein, the term “genetically modified EV” refers to an extracellular vesicle that has been in vitro modified to express heterologous components by adding exogenous nucleic acid molecules to a vesicle donor cell. Therefore, genetically modified EVs are intended to be vesicles that do not exist in nature.

[0021] In the context of this specification, the term “encapsulation” means introducing nucleic acid molecules into extracellular vesicles, such as plant-derived EVs, by means such as transfection, transformation, or transduction.

[0022] As used herein, the term “external nucleic acid molecule” refers to a heterogeneous nucleic acid molecule that is not part of the natural cargo of the EV of the present invention. The expression “heterogeneous” means a nucleic acid molecule derived from a different animal or plant species than the extracellular vesicles of the present invention, or a nucleic acid molecule derived from a different donor cell, under different conditions, or from genetically modified donor cells.

[0023] As used herein, the term "antigen protein" refers to a protein molecule capable of triggering an immune response.

[0024] According to the present invention, the foreign nucleic acid molecule added to the plant-derived EV is preferably selected from the group consisting of: DNA, cDNA, messenger RNA (mRNA), premRNA, long RNA, coding RNA, single-stranded RNA, double-stranded RNA, linear RNA, RNA oligonucleotide, self-replicating RNA (replicon RNA), retroviral RNA, and viral RNA (vRNA).

[0025] In a preferred embodiment of the present invention, the foreign nucleic acid molecule is a messenger RNA (mRNA) molecule. In the context of the present invention, the foreign mRNA molecule may include one or more modifications, such as a 5' cap structure, a 5' UTR, an open reading frame, a 3' UTR, or a poly-A tail.

[0026] According to the present invention, the EV in the composition may include a single nucleic acid molecule or a combination of two or more nucleic acid molecules.

[0027] In a preferred embodiment of the present invention, the content of added foreign nucleic acid molecules in EV is 20-200 ng / 10 9 EV, preferably 30-100 ng / 10 9 EV, more comfortably 40-60 ng / 10 9 It falls within the range of EVs.

[0028] The addition of foreign nucleic acid molecules to EVs according to the present invention can be achieved by many different techniques known in the art, including, for example, electroporation, sonication, lipofectamine intervention, microinjection, co-incubation, dialysis, and freeze-thaw cycles.

[0029] In this invention, extracellular vesicles having a diameter in the range of 20 to 500 nm, preferably 100 to 400 nm, and more preferably 200 to 300 nm are used.

[0030] According to the present invention, the membrane potential value across the lipid bilayer membrane of EV in the composition is in the range of +5 to -5 mV, preferably +2 to -4 mV, and more preferably 0 to -3 mV.

[0031] In a more preferred embodiment of the present invention, the membrane potential value of EV is -2mV.

[0032] In the composition according to the present invention, the amount of EV in the composition that is (≤) 44% or less of the total EV is phosphatidylserine in the outer layer of the lipid bilayer membrane.

[0033] Preferably, the amount of EV in the composition containing phosphatidylserine in the outer layer of the lipid bilayer membrane is within the range of 25% to 44% of the total EV, more preferably 35% to 44% of the total EV, and even more preferably 40% to 44% of the total EV.

[0034] The plant-derived extracellular viable (EV) used in the present invention is preferably derived from one or more plants selected from the following group: the citrus genus, including lemon and orange; the Actinidia genus, including kiwifruit; the pumpkin genus, including zucchini; the Brassica genus, including cabbage and kale; the Pomegranate genus, including pomegranate; the Vaccinium genus, including blueberry; and the Apium genus, including celery.

[0035] The scope of the present invention includes both compositions containing extracellular viable (EV) derived from a single plant species and compositions containing EV derived from multiple plant species. It is understood that plant-derived EV can be used in its original form or after chemical modification.

[0036] Preferably, the plant-derived extracellular viable (EV) in the composition according to the present invention is purified from fruit juice, a part of a plant, or a culture medium of plant cells. The plant cells or parts are derived from leaves, pulp, buds, or shoots.

[0037] Appropriate purification techniques for extracellular viable (EV) include, but are not limited to, ultracentrifugation, filtration, and tangential flow filtration. The selection of the most appropriate method for purifying plant-derived EV is within the scope of the knowledge and skill of those skilled in the art.

[0038] In one aspect of the present invention, the total protein content of EV in the composition of the present invention is 100-200 ng / 10 10 EV, comfort level 120-160 ng / 10 10 It falls within the range of EVs.

[0039] In another embodiment, the total RNA content in the composition of the present invention is 20-200 ng / 10 9 EV, comfort level 30-100 ng / 10 9 EV, even more preferably 40-60 ng / 10 9 It falls within the range of EVs.

[0040] The expression "total protein content" includes both endogenous protein cargo (content inside and in the membrane of the EV) and the loaded proteins in the EV used in this invention.

[0041] In this specification, the expression “total RNA content” includes both endogenous RNA cargo and loaded exogenous RNA in the EV of the present invention.

[0042] As will be further explained in the Examples section below, the inventors have surprisingly found that genetically modified plant-derived extracellular life (EVs) having the structural and functional characteristics defined above do not exhibit any immunomodulatory activity; that is, they have no ability to affect cells of the immune system, nor do they promote or diminish the activation and efficacy of these cells.

[0043] Unlike naturally occurring plant-derived EVs, the non-naturally derived EVs of the present invention are advantageous in that they can deliver antigen molecules to target cells without affecting immune system cells in any way. Therefore, the use of EVs according to the present invention overcomes safety concerns associated with EV-based vaccine formulations, avoids adverse activation or inhibition of the immune system, and thereby enhances vaccine efficacy.

[0044] Furthermore, the EV of the present invention has been proven to efficiently deliver nucleic acid drugs to receptor cells and protect them from environmental degradation. In particular, its high resistance to the gastric environment allows for oral administration of the composition according to the present invention.

[0045] After administration, interaction between the loaded extracellular matrix (EV) and antigen-presenting cells (APCs), including macrophages and dendritic cells, enables the transfer of nucleic acid molecules to the antigen-presenting cells. In the target cells, nucleic acid molecules, including DNA and mRNA molecules, are expressed, leading to the translation of protein antigens. Subsequently, the antigen is presented on the surface of the APC, directly inducing the specific activation of immune cells against tumor cells or pathogens, enabling efficient immune defense.

[0046] Thanks to the advantageous characteristics of non-naturally derived EVs as exemplified above, the compositions of the present invention are particularly suitable for use as vaccines.

[0047] In the present invention, the compositions of the present invention can be used as vaccines for the treatment of existing diseases or prophylactically to prevent the onset of these diseases.

[0048] Examples of protein antigens encoded by exogenous nucleic acid molecules incorporated into the EV of the present invention include, but are not limited to, bacterial, viral, fungal, protozoan, and tumor antigens, their mammalian homologs, and homologs derived from animals of veterinary or industrial interest.

[0049] Therefore, the compositions of the present invention are particularly useful for the treatment or prevention of infectious diseases or cancer.

[0050] Similar cancers include, but are not limited to, bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer, ovarian cancer, lymphoma, liver cancer, glioblastoma, melanoma, myeloma, leukemia, and pancreatic cancer.

[0051] Examples of infectious diseases include, but are not limited to, viral, bacterial, fungal, or protozoal diseases, such as COVID-19, influenza, HPV infection, HIV infection, rhinovirus infection, hepatitis, flavivirus infection, encephalitis, meningitis, gastroenteritis, cholera, diphtheria, chlamydia, tuberculosis, typhoid fever, sexually transmitted infections (STIs), malaria, mycoses, and toxoplasmosis.

[0052] In one embodiment of the present invention, at least one antigen encoded by an exogenous nucleic acid molecule added to EV is: human kallikrein-related peptidase 3 (also known as prostate-specific antigen (PSA)), human prostate stem cell antigen (PSCA), human prostate-specific membrane antigen (PSMA), human metalloreductase (prostate 6-transmembrane epithelial antigen 1 (STEAP1)), human receptor tyrosine protein kinase erbB-2 (also known as tyrosine kinase-type cell surface receptor HER2), human cell surface-related mucin 1 protein (MUC1) (also known as breast cancer-related antigen DF3), human tyrosinase-related protein 2 (TRP-2), human serine / threonine protein kinase B-raf (also known as proto-oncogene B-Raf), human mast cell / stem cell growth factor receptor kit (also known as proto-oncogene c-Kit), human GTPase NRas (also known as transformed protein N-Ras), human melanoma-associated antigen 1, human melanoma-associated antigen 1 protein, human NY-ESO-1 protein, and any combination thereof are selected from the group of tumor antigens.

[0053] In another aspect of the present invention, at least one protein antigen is a bacterial antigen derived from a bacterium selected from the group consisting of Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, Streptococcus pyogenes, Streptococcus pneumoniae, Borrelia burgdorferi, Borrelia mayonii (e.g., Lyme disease), Klebsiella species, Pseudomonas aeruginosa, Enterococcus species, Proteus species (e.g., Proteus vargaris, Proteus mirabilis, Proteus penneri), Neisseria gonorrhoeae, Enterobacter species, Actinobacter species, coagulase-negative staphylococci (CoNS), Mycoplasma species, Clostridium difficile, Bacillus anthrosis, Vibriocholera, Clostridium botulinum, Clostridium tetani, Salmonella species, Treponema pallidum, Plasmodium species, and any combination thereof.

[0054] In yet another aspect of the present invention, at least one protein antigen is a fungal antigen derived from a fungus selected from the group consisting of Blastomyces, Cryptococcus gattii, Cryptococcus neoformans, Fusarium, Aspergillus, Candida, Candida albicans, Candida auris, Cryptococcus, Histoplasma, Blastomyces, Coccidioides, Mucormycetes, Pneumocystis irovecii, dermatophytes, Sporothrix, and any combination thereof.

[0055] In yet another embodiment, at least one protein antigen is found in Plasmozia species (e.g., *Platypleura fauriei* and *Platypleura erythrorhizon*), Giardia intestinalis, Hexamita salmonis, Histomonas meleagridis, Trichomonas fetus, Dientamoeba fragilis, Trichomonas vaginalis, Leishmania, Trypanosoma cruzi, Trypanosoma brucei rhodensiense, Trypanosoma brucei gambiense, Plasmodium parasite, Entamoeba histolytica These are protozoan antigens derived from protozoa selected from the group consisting of *Trichoplasma histolytica*, *Naegleria*, *Acanthamoeba*, *Oomycetes*, *Phytophthora infestans*, *Giardia lamblia*, *Giardia duodenalis*, *Toxoplasma gondii*, *Balantidium coli*, *Theileria parva*, *Theileria annulate*, *Phipicephalus appendiculatus*, *Prototheca moriformis*, and any combination thereof.

[0056] Preferably, the protozoan antigen is selected from the group consisting of Toxoplasma gondii dense granule protein 6 (GRA6), Loptori protein 2A (ROP2A), Loptori protein 18 (ROP18), surface antigen 1 (SAG1), surface antigen 2A (SAG2A), apical membrane antigen 1 (AMA1), and any combination thereof.

[0057] In a further embodiment, at least one protein antigen may be human papillomavirus (HPV), human immunodeficiency virus HIV (e.g., HIV-1, HIV-2), hepatitis A virus (HBV), hepatitis C virus, hepatitis D virus, hepatitis E virus, herpesvirus (human gamma herpesvirus 4 (Epstein-Barr virus), herpes simplex virus 2 (HSV2), human herpesvirus 8), influenza virus (e.g., influenza A virus, influenza B virus) Viruses, cytomegalovirus, Crimean-Congo hemorrhagic fever orthonair virus, coronavirus, human polyomavirus 2, BK virus, severe acute respiratory syndrome coronavirus (SARS-CoV, SARS-CoV-2, COVID-19), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), norovirus, filovirus (Cueba, Marburg, Ebola virus), chikungunya virus, human alpha-herpesvirus 3 (HHV-3) or varicella-zoster virus (VZV) Rubella virus, Merkel cell polyomavirus (MCV), bunyavirus (e.g., hantavirus), arenavirus (e.g., lymphocytic choriolarinitis mamma-arenavirus (LCMV) and lassa virus), flavivirus (dengue virus, Zika virus, Japanese encephalitis, West Nile, tick-borne encephalitis virus (TBEV) and yellow fever), rhinovirus, human parainfluenza virus (HPIV), enterovirus (e.g., polio), respiratory syncytial virus (RSV), mumps The viral antigen is derived from a virus selected from the group consisting of viruses, coxsackievirus, measles virus, astrovirus (e.g., gastroenteritis), rhabdoviridae (e.g., rabies), adenovirus, adeno-associated virus (AAV), human papillomavirus, hepatitis B virus, hepatitis C virus, Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, human T-lymphophilic virus, oncogenic viruses including Merkel cell polyomavirus, and any combination thereof.

[0058] According to a preferred embodiment of the present invention, the viral antigen is the spike protein, also known as the surface glycoprotein of severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 or COVID-19; the N protein, also known as the nucleocapside phosphorylated protein of severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 or COVID-19; the M protein, also known as the membrane glycoprotein of severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 or COVID-19; and the hema of influenza A virus H5N1. Hemagglutinin (HA) protein, hemagglutinin (HA) protein of influenza A virus H3N2, hemagglutinin (HA) protein of influenza A virus H1N1, hemagglutinin (HA) protein of influenza A virus H7N9, hemagglutinin (HA) protein of influenza A virus H1N1, hemagglutinin (HA) protein of influenza A virus H2N2, hemagglutinin (HA) protein of influenza B virus, neuraminidase of influenza A virus H5N1 ( NA) protein, neuraminidase (NA) protein of influenza A virus H1N1, neuraminidase (NA) protein of influenza A virus H3N2, neuraminidase (NA) protein of influenza A virus H7N9, neuraminidase (NA) protein of influenza A virus H9N2, neuraminidase (NA) protein of influenza A virus H2N2, neuraminidase (NA) protein of influenza A virus H1N1, neuraminidase (NA) protein of influenza B virus (NA) protein, envelope protein of human immunodeficiency virus (HIV1), envelope protein of human immunodeficiency virus (HIV2), major capsid protein L1 of human papillomavirus (HPV), minor capsid protein L2 of human papillomavirus (HPV), glycoprotein of rabies lyssavirus, glycoprotein of human cytomegalovirus, envelope glycoproteins E1 and E2 of hepatitis C virus, fusion protein (F) of respiratory syncytial virus (RSV), spike glycoprotein of Zaire Ebola virus,The protein is selected from the group consisting of Zika virus protein prM, Zika virus serine protease NS3, Zika virus serine protease subunit NS2B, Zika virus envelope protein E, Zika virus capsid protein C, SARS-CoV-2 spike (S)RBD protein, and any combination thereof.

[0059] In an exemplary embodiment, at least one protein antigen encoded above comprises, essentially consists of, or consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, 15, 16, 18 and 20-49.

[0060] In another aspect of the present invention, the foreign nucleic acid molecule added to the EV is an mRNA molecule containing or consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 17, 19, and 50. More specifically, SEQ ID NOs: 14, 17, 19, and 50 correspond to mRNA sequences encoding the SARS-CoV-2 S protein, N protein, M protein, and spike (S)RBD protein, respectively.

[0061] According to the present invention, the composition may include EVs derived from genetically modified plants loaded with a single foreign nucleic acid molecule, or a combination of EVs derived from genetically modified plants loaded with different foreign nucleic acid molecules.

[0062] It is understood that protein antigens within the scope of the present invention may include one or more modifications to improve antigenic immunogenicity and / or stability. Exemplary modifications include post-translational modifications.

[0063] The composition of the present invention can be used alone or in combination with other vaccines.

[0064] In one embodiment, the composition of the present invention further comprises one or more polycationic substances, the one or more polycationic substances being bonded to the outer lipid layer of the lipid bilayer film of EV via electrostatic interactions.

[0065] Preferably, the one or more polycationic substances include cationic proteins such as protamine, calcitonin peptide, plecstrin, lactoferrin, protamine-like proteins such as spermine or spermidine, nucleolin, histone, and cell-penetrating peptides (CPPs); cationic peptides such as histidine-rich peptides, arginine-rich peptides, lysine-rich peptides, and cationic arginine-rich peptides (CARPs); polypeptides including poly-arginine, poly-lysine, poly-histidine, histidine-rich peptides, arginine-rich peptides, and lysine-rich peptides; polysaccharides such as chitosan, glycosaminoglycans such as polysulfated glycosaminoglycans (PSGAG), and cationic dextran; and those selected from the group consisting of glycerol, polyethylene glycol (PEG), and the like.

[0066] A preferred polycationic substance is protamine.

[0067] Preferably, the content of the one or more polycationic substances in the composition is from 0.001 to 2 μg / 10 10 EV, more preferably from 0.05 to 1 μg / 10 10 EV, even more preferably from 0.1 to 0.4 μg / 10 10 EV.

[0068] According to the present invention, the one or more polycationic substances can be used alone or in combination. It is understood that the polycationic substances can be used in their original form or with chemical modifications. Such components can be used individually or in combination.

[0069] In another aspect of the present invention, the EVs in the composition of the present invention are further added with one or more sugar molecules, and the one or more sugar molecules are bound to the foreign nucleic acid molecules added to the EVs through electrostatic interaction and hydrogen bonding.

[0070] Preferably, one or more sugar molecules are selected from the group consisting of disaccharides including trehalose, maltose, lactose, sucrose, cellobiose, chitobiose, cozybiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, genthiobiose, trehalulose, turanose, maltulose, louscrose, isomaltulose, genthiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, and xylobiose; sugar alcohols such as arabitol, erythritol, glycerol, HSH, isomalt, lactitol, maltitol, mannitol, sorbitol, and xylitol; and polysaccharides such as starch, glycogen, galactogen, inulin, arabinoxylan, cellulose, chitin, and pectin.

[0071] A preferred sugar molecule is trehalose. Trehalose is a non-reducing disaccharide and is commonly used as a cytoprotective agent to stabilize proteins and nucleic acids. Furthermore, trehalose can degrade the secondary structure of RNA.

[0072] Preferably, the content of one or more sugar molecules in the EV of the present invention is 0.1 to 10 mg / 10 10 EV, comfortably 0.5-5 mg / 10 10 EV, even more comfortable at 1-2 mg / 10 10 It falls within the range of EVs.

[0073] In another embodiment, the content of one or more sugar molecules in the EV of the present invention is in the range of 0.1 to 20 mg / μg of loaded foreign nucleic acid, preferably 1 to 10 mg / μg of loaded foreign nucleic acid, and more preferably 2 to 6 mg / μg of loaded foreign nucleic acid.

[0074] It is understood that sugar molecules can be used in their original form or after chemical modification. Such components can be used individually or in combination.

[0075] Before use, non-naturally derived extracellular vitriol (EV) in the composition of the present invention can be freeze-dried and resuspended in water. Alternatively, the naturally derived EV used in the composition of the present invention may be freshly prepared or stored at 4°C, -20°C, or -80°C.

[0076] The compositions of the present invention can be formulated in several administerable forms, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions.

[0077] The compositions of the present invention may, if necessary, contain appropriate excipients, preservatives, solvents, or diluents in accordance with conventional methods.

[0078] Examples of excipients include sugars such as sucrose, D-mannose, D-fructose, dextrose, anhydrous lactose, D-trehalose, and D-sorbitol; proteins such as human serum albumin, hydrolyzed casein, MRC-5 cell protein, hydrolyzed gelatin, CRM197 carrier protein, and proteins derived from plants, yeast, bacteria, and eggs; essential and non-essential amino acids such as asparagine, phenylalanine, arginine, and histidine; sodium including sodium chloride, sodium bicarbonate, sodium carbonate, sodium borate, sodium benzoate, sodium taurodeoxycholate, sodium deoxycholate, monobasic sodium phosphate, dibasic sodium phosphate, and sodium metabisulfite; potassium including potassium phosphate, potassium polaritrin, monobasic and dibasic potassium phosphate, and potassium chloride;Magnesium stearate, calcium chloride, calcium phosphate, calcium silicate, glutamic acid, cellulose, microcrystalline cellulose, cellulose acetate phthalate, aluminum, aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate, potassium aluminum sulfate, citric acid, ferric ammonium citrate, castor oil, neomycin, streptomycin, aminoglycoside, kanamycin, gentamicin, chlortetracycline, amphotericin B, prasdon C, alcohol, acetone, benzethonium chloride, formaldehyde, glycerin Phosphorus, ascorbic acid, trometamol, urea, glutaraldehyde, 2-phenoxyethanol, polysorbate 80 (Tween 80), polymyxin B, ammonium thiocyanate, trometamine, host cell DNA benzoase, formalin, phosphate-buffered saline, polysorbate 20, deoxycholate, dibasic dodecahydrate, monobasic dehydrate, formalin, polymyxin B, β-propiolactone, hydrocortisone, squalene, sorbitan trioleate, barium, cetyltrimethylammonium bromide (CTAB), octoxynol-10 (TRITON Examples of substances containing PEG include, but are not limited to, X-100), α-tocopherol succinate, cetyltrimethylammonium bromide, β-propiolactone, thimerosal, ethylenediaminetetraacetic acid (EDTA), phenol, β-propiolactone, DMEM, HEPES, polydimethylsiloxane, vitamins, dioleoylphosphatidylcholine (DOPC), 3-O-deacylated-4'-monophosphoryl lipid A (MPL), lipids, cholesterol, panthenol, gums including guar gum, boric acid and borates including sodium tetraborate, glycerol, allantoin, triethanolamine, alginic acid, pluronic acid, poloxamers including P188 and P331; PEGs including PEG8000; glycols including ethylene glycol, propylene glycol, and glycerol; citicoline (cytidine-5-diphosphocholine; CDP-choline), and cholesterol.

[0079] Examples of preservatives suitable for use in the compositions of the present invention include parabens such as ethylparaben, methylparaben, and propylparaben; formaldehyde donors such as DMDM ​​hydantoin, imidazolidinyl urea, and glutaraldehyde; phenol derivatives; benzoic acid; and benzyl alcohol.

[0080] Suitable solvents or diluents used in this invention can be selected from purified water, ethanol, and benzyl alcohol.

[0081] According to the present invention, it is intended that an adjuvant can be added to the composition for use as a vaccine.

[0082] Examples of adjuvants suitable for use in the immunogenic compositions of the present invention include, but are not limited to, mineral compositions such as aluminum hydroxide, potassium aluminum phosphate, aluminum salts such as AS04, calcium salts, hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), and sulfates; emulsions including oil-in-water and water-in-oil emulsions such as Freund's adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, AF03, AS03, AS02, glucopyranoside lipid adjuvant (GLA-SE), and glucopyranosyl lipid adjuvant (GLA); non-toxic derivatives of enterobacteriaceae lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), lipid A, bacterial or microbial derivatives including E. coli-derived lipid A such as OM-174; immunostimulatory oligonucleotides containing nucleotide sequences containing a CpG motif, bacterial double-stranded RNA, oligonucleotides containing palindromic sequences or poly(dG) sequences, ADP-ribosylated toxins and detoxification derivatives, RC529; cyclic GMP-AMP adjuvants, STING agonists, CAF01, immunostimulatory complexes (ISCOM), ISCOMATRIX, AS01; polyoxyethylene ethers and polyoxyethylene ester preparations, polymer particles such as poly(lactide-co-glycolide) (PLG) microparticles, polyphosphazenes (PCPP), Smilax Saponin preparations such as saponins derived from ornata (sarsaparilla), Gypsophila paniculata (brysveil), and Saponaria officinalis (soaproot); purified preparations such as QS7, QS17, QS18, QS21, QH-A, QH-B, and QH-C; human immunomodulators containing cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.); interferons (e.g., interferon-γ); macrophage colony-stimulating factor; and tumor necrosis factor.Bioadhesives and adhesives including esterified hyaluronic acid microspheres, or adhesives such as poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, cross-linked derivatives of polysaccharides and carboxymethylcellulose, chitosan and their derivatives; muramyl peptides including N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE); imidazoquinolone compounds including imiquamod and its homologues; virosomes and virus-like particles (VLPs).

[0083] The compositions of the present invention may be administered by various routes, including oral, nasal, non-enteral (including subcutaneous), intraperitoneal, intravenous, intradermal, intramuscular, intrasplenic, and intranodular.

[0084] Preferably, the pharmaceutical composition of the present invention is in a form suitable for oral administration, nasal administration, or non-enteral administration.

[0085] The dosage, number of doses, and frequency of administration are determined according to various factors such as the disease to be treated or prevented, and the characteristics of the patient, and can be determined by a person skilled in the art using ordinary knowledge.

[0086] Furthermore, the compositions of the present invention can be freeze-dried and are stable without the need for cold chain storage.

[0087] A method for preparing a composition having the characteristics defined above is also within the scope of the present invention.

[0088] According to the present invention, the method of the present invention includes the following steps: (i) A step of contacting a suspension of extracellular vesicles (EVs) derived from plants with one or more polycationic substances and mixing them to obtain a first mixture; (ii) A step of contacting a preparation of nucleic acid molecules with one or more sugar molecules and mixing them to obtain a second mixture, wherein the nucleic acid molecules encode at least one protein antigen; (iii) the step of mixing the first mixture and the second mixture to obtain a third mixture; and (iv) A step of adding a predetermined amount of water to the third mixture, wherein the ratio of the predetermined amount of water to the amount of the third mixture is in the range of 5:1 to 15:1, preferably in the range of 8:1 to 12:1.

[0089] The preferred ratio of a predetermined amount of water to the amount of the third mixture is 10:1.

[0090] In short, the method of the present invention may further include concentrating the composition obtained in step (iv). Concentration techniques are well known and include, for example, filtration, ultracentrifugation, tangential flow filtration, chromatography, and precipitation. Those skilled in the art will know techniques for concentrating compositions and can use such suitable methods.

[0091] In one embodiment of the method of the present invention, step (i) further comprises a step of incubating the first mixture at a temperature in the range of 30 to 40°C, preferably 37°C, for a period of 30 minutes to 2 hours, preferably 1 hour.

[0092] In another aspect of the method of the present invention, step (ii) further comprises the step of incubating the second mixture at a temperature in the range of 0 to 25°C, preferably at 20°C, for 5 to 30 minutes, preferably 10 minutes.

[0093] In yet another aspect of the method of the present invention, step (iii) further comprises the step of incubating the third mixture at a temperature in the range of 30 to 40°C, preferably at 37°C, for a period of 1 to 5 hours, preferably 3 hours.

[0094] In a further embodiment of the method of the present invention, step (iv) further comprises an incubation step at a temperature in the range of 0 to 10°C, preferably 4°C, for a period of 5 to 24 hours, preferably 12 hours.

[0095] The polycationic substances and sugar molecules suitable for use in the method of the present invention are as described above with reference to the composition.

[0096] Without being bound by any theory, the inventors believe that polycationic substances alter the charge of the lipid bilayer membrane of plant-derived EVs, enabling the adsorption of nucleic acid molecules to the outer surface of the membrane. Furthermore, the inventors believe that the sugar plays a role in protecting the nucleic acid molecules, enabling the efficient introduction of these molecules into plant-derived EVs.

[0097] Preferably, the concentration of plant-derived EV in the first mixture is 5 x 10⁻¹⁰ of the total volume of the first mixture. 10 from 10 12 Within the range of EVs / ml, more preferably 1 x 10⁻¹⁶ of the total volume of the first mixture. 11 From 5x10 11 It is composed within the range of EVs / ml.

[0098] The first mixture according to the method of the present invention may further contain a salt, preferably NaCl, more preferably NaCl at a concentration of 0.9% (w / v) relative to the total volume of the first mixture.

[0099] In one embodiment of the method of the present invention, one or more polycationic substances are present in the first mixture at a concentration of 0.1 to 2 μg / ml, preferably 0.1 to 1 μg / ml, and more preferably 0.4 to 0.6 μg / ml, relative to the total volume of the first mixture.

[0100] In another aspect of the present invention, nucleic acid molecules are present in the second mixture at a concentration of 0.1 to 10 μg / ml, preferably 0.1 to 1 μg / ml, and more preferably 0.1 to 0.5 μg / ml, relative to the total volume of the second mixture.

[0101] In yet another embodiment of the present invention, the one or more sugar molecules are present in the second mixture at a concentration of 1 to 20% (w / v) of the total volume of the second mixture, preferably 1 to 10% (w / v) of the total volume of the second mixture, and more preferably 1 to 5% (w / v) of the total volume of the second mixture.

[0102] According to the method of the present invention, the mixing of the suspension containing plant-derived EVs with a polycationic substance in step (i), and / or the mixing of the nucleic acid molecule preparation with one or more sugar molecules in step (ii), can preferably be carried out by vortexing for at least 30 seconds.

[0103] According to the present invention, the method may include, but is not limited to, electroporation, sonication, transfection, incubation, cell extrusion, saponin-mediated permeabilization, and freeze-thaw cycles, as further operations to improve the addition of nucleic acid molecules to plant-derived extracellular matrix (EVs).

[0104] Another aspect of the present invention is a composition comprising non-immunomodulatory, engineered, plant-derived extracellular vesicles (EVs) obtained by the method defined above, for use as a vaccine.

[0105] Test example The following test sections are provided solely for the purpose of describing the tests performed and are not intended to limit the scope of the invention as defined in the attached claims. The following test sections refer to the attached drawings: [Brief explanation of the drawing]

[0106] [Figure 1] Figure 1 shows the characteristics of the genetically modified plant-derived extracellular life (EV) of the present invention in Example 1, compared with those of naturally derived plant-derived EV. Representative images obtained from nanosite analysis of natural kiwifruit EV (A) and the genetically modified EV of the present invention (B) are shown, demonstrating a statistically significant difference in size. Statistical analysis of the average diameter of n=3 preparations of naturally derived plant-derived EV and genetically modified plant-derived EV of the present invention, analyzed by nanosites (C). E1 = genetically modified EV derived from cabbage EV, E2 = genetically modified EV derived from blueberry EV. p:**** <0.001. [Figure 2] Figure 2 shows the membrane potential (Z potential) values ​​across the lipid bilayer measured in extracellular viable cells (EVs) in Example 1. Membrane potential was measured as mVolt (mV) in both natural EVs (native EVs) and genetically modified EVs derived from zucchini (E1) and blueberries (E2). Statistical significance was calculated by comparing the membrane potential measured in genetically modified plant-derived EVs with that measured in natural plant-derived EVs. p:*** <0.005. A trial of N=3 was conducted for each dataset. Data are shown as mean ± standard deviation (SD). [Figure 3]Figure 3A shows the protein content of naturally derived EVs (EVs) in Example 1 and genetically modified plant-derived EVs of the present invention, expressed in nanograms (ng) of protein per 10¹¹ EVs. Measurements were performed for naturally derived EVs (native EVs) and genetically modified plant-derived EVs from pomegranate (E1) and kiwifruit (E2). Statistical significance was calculated by comparing the protein content of genetically modified plant-derived EVs with the values ​​measured for naturally derived EVs. p:** <0.01. N=3 tests were performed for each dataset. Figure 3B shows the proportion of EVs in the composition of the present invention in Example 1, which contains phosphatidylserine in the outer layer of the lipid bilayer membrane. The presence of phosphatidylserine in the outer layer of the vesicle membrane was analyzed in compositions containing naturally derived EVs (native EVs) and compositions containing genetically modified plant-derived EVs from cabbage (E1) and blueberry (E2). In each sample, phosphatidylserine content was measured using staining for annexin V and cell fluorescence chromatography (FACS), and expressed as the percentage of fluorescence signals. Statistical significance was calculated by comparing the percentage of genetically modified plant-derived extracellular viable cells (EVs) containing phosphatidylserine with that of naturally occurring plant-derived EVs. p:** <0.01. A trial of N=3 was conducted for each dataset. Data are presented as mean ± standard deviation (SD).

[0107] [Figure 4]Figure 4 shows the results of the immunomodulatory assay of the genetically modified plant-derived EVs of the present invention in Example 1. PBMC cells were incubated with genetically modified plant-derived EVs from pomegranate (50,000 particles / cell) for 48 hours, and cell proliferation was measured by BrdU uptake. (A) The histogram shows the absorbance (mean ± SD) of untreated PBMCs (CTR) and PBMCs stimulated with the EVs of the present invention. Absorbance is directly proportional to cell proliferation. The proliferation rate of PBMCs stimulated with the EVs of the present invention did not change compared to the control (CTR) and was not statistically significant. Next, lymphocytes were activated with LPS (dose 100 ng / ml) and stimulated with the EVs of the present invention (dose 50,000 particles / cell) for 48 hours, and proliferation was measured by BrdU uptake. (B) The histograms show the absorbance (mean ± SD) of unstimulated PBMCs (CTR-), PBMCs treated with LPS (CTR+), PBMCs treated with LPS and the EV of the present invention derived from pomegranate (E1), and PBMCs treated with LPS and the EV of the present invention derived from kiwifruit (E2). Absorbance is directly proportional to cell proliferation. LPS significantly activated PBMC proliferation compared to untreated cells, but the proliferation rate of PBMCs stimulated with LPS and the EV of the present invention did not change compared to PBMCs treated with LPS and was not statistically significant. PBMC proliferation was also measured using the fluorescent dye CFSE. After PBMCs were stimulated with the EV of the present invention (dose of 50,000 vesicles / cell) for 24 hours, proliferation was analyzed by flow cytometry (C, D). The histogram (C) shows the fluorescence FITC intensity (mean ± SD) of untreated PBMCs (CTR) and PBMCs stimulated with the EVs of the present invention derived from cabbage (E1), celery (E2), and zucchini (E3). The proliferation rates of PBMCs stimulated with different samples of the EVs of the present invention did not change compared to CTR and were not statistically significant. In fact, the histograms of flow cytometry analyses for CTR, E1, E2, and E3 completely overlapped (D). p:* ns > 0.05. [Figure 5]Figure 5 shows the total RNA content of EVs in Example 1. The total RNA content of EVs derived from natural plants as well as genetically modified plants (EVs) from celery (E1), pomegranate (E2), and kiwifruit (E3) was measured. The total RNA amount was measured as the absolute amount of RNA contained in each sample after RNA extraction and expressed as RNA amount (ng) normalized to the number of vesicles (ng / 10⁹ vesicles). Statistical significance was calculated by comparing the total RNA content of each sample of the EVs of the present invention with that of EVs derived from natural plants. p: * <0.05, *** <0.005, **** <0.001. N=3 experiments were performed for each dataset. Data are shown as mean ± standard deviation (SD). [Figure 6] Figure 6 shows the amount of foreign nucleic acid molecules added to the modified plant-derived extracellular viable cells (EVs) of the present invention in Example 2. For the assay, mRNA molecules encoding the SARS-CoV-2 nucleocapsid (N) protein were used, and the added nucleic acid molecules were measured by qRT-PCR in plant-derived EVs (natural EVs) and genetically modified EVs of kiwifruit (E1, E3) and celery (E2, E4). Two mRNA doses were used: 0.1 μg / ml for samples E1 and E2, and 1 μg / ml for samples E3 and E4. The amount of added mRNA was expressed as an RQ value according to the method described. Statistical significance was calculated by comparing the amount of added nucleic acid in each sample of the EVs of the present invention with that of naturally derived plant EVs. p:**** <0.001. N=3 experiments were performed for each dataset. Data are shown as mean ± standard deviation (SD). [Figure 7]Figure 7 shows the resistance of nucleic acid molecules added to the genetically modified plant-derived extracellular viable cells (EVs) of the present invention to degradation environments in Example 3. mRNA molecules were used for the test and measured by qRT-PCR. The graph shows the percentage of mRNA molecules still present after the degradation assay compared to the starting material, indicating that 100% of the mRNA is preserved in the EVs of the present invention. (A) Resistance to enzymatic degradation was measured after treatment with RNAse, and (B) resistance to the gastrointestinal environment was evaluated after treatment with a stomach-like solution. Naked mRNA was used as a control in all experiments. The tests were performed using genetically modified plant-derived EVs from pomegranate (7A) and kiwifruit (7B). Statistical significance was calculated by comparing the percentage of nucleic acids preserved in the EVs of the present invention with that of naked mRNA. p:**** <0.001. N=3 experiments were performed for each dataset. Data are shown as mean ± standard deviation (SD). [Figure 8] Figure 8 shows the storage resistance of nucleic acid molecules added to genetically modified plant-derived extracellular viable cells (EVs) of the present invention in Example 4. In these experiments, mRNA molecules encoding the SARS-CoV-2 nucleocapsid (N) protein were added to EVs derived from celery (E1) and kiwifruit (E2). After lyophilization, the amount of mRNA stored in the EVs after storage at +4°C for 7 days was measured by qRT-PCR assay and expressed as a percentage of the starting amount. N=3 tests were performed for each dataset. Data are shown as mean ± standard deviation (SD).

[0108] [Figure 9]Figure 9 shows the transfer of nucleic acid molecules added to the genetically modified plant-derived EVs of the present invention to receptor cells in Example 5. EVs of the present invention, supplemented with mRNA molecules encoding the SARS-CoV-2 nucleocapsid (N) protein, were incubated with macrophages. After 24 hours, the amount of mRNA in receptor cells was measured using molecular analysis (qRT-PCR), normalized with GAPDH as housekeeping, and expressed as an RQ value as described in the Methods section. The RQ value was normalized relative to the control (untreated cells, NT), and an RQ value of 1 means that no mRNA was detected in the sample. Macrophages were treated with natural plant-derived EVs (native EVs), genetically modified plant-derived EVs (E1, E2, E3), plant-derived EVs incubated with mRNA without nucleic acid supplementation (EV+mRNA), and naked mRNA. Receptor cells were treated at a dose of 50,000 particles / cell. Tests were conducted using EVs derived from cabbage (E1), pomegranate (E2), and kiwifruit (E3). Statistical significance was calculated by comparing the mRNA RQ value of each sample to untreated cells as a control (NT). p:*** <0.005, **** <0.001. A trial of N=3 was performed for each dataset. Data are presented as mean ± standard deviation (SD). [Figure 10]Figure 10 shows the functionality of nucleic acid molecules carried by the genetically modified plant-derived EVs of the present invention in the receptor cells of Example 5. In these tests, EVs of the present invention, to which mRNA molecules encoding green fluorescent protein (GFP) were added, were incubated with (A) endothelial cells and (B) macrophages as receptor cells. After 24 hours of co-incubation, the expression of proteins encoded by exogenous mRNA in the receptor cells was detected as a fluorescent signal using cytofluorometry (FACS). Receptor cells were treated with native plant-derived EVs, the genetically modified plant-derived EVs of the present invention, or naked mRNA at a dose of 50,000 particles / cell. The tests were performed using plant-derived EVs from zucchini (Figure 10A) and kale (Figure 10B). Statistical significance was calculated by comparing the percentage of signal intensity for each sample with untreated cells (NT) as a control. p:*** <0.005, **** <0.001. N=3 tests were performed for each dataset. The data is shown as mean ± standard deviation (SD). [Figure 11]Figure 11 shows protein expression in target receptor cells treated with nucleic acid molecules carried by the genetically modified plant-derived EVs of the present invention in Example 5. For these tests, EVs of the present invention, supplemented with mRNA molecules encoding SARS-CoV-2 spike glycoprotein (S1) RBD protein (SEQ ID NO: 50), SARS-CoV-2 spike glycoprotein complete protein (SEQ ID NO: 14), or SARS-CoV-2 nucleocapsid protein (SEQ ID NO: 17), were incubated with endothelial cells as receptor cells. After 24 hours of co-incubation, the expression of the exogenous mRNA-encoded protein in the receptor cells was detected using a fluorescently labeled secondary antibody, and the fluorescence signal was measured by cytofluorometry (FACS). Receptor cells were treated with a dose of 1.2 x 10¹⁰ particles of either native plant-derived EVs or the genetically modified plant-derived EVs of the present invention. The tests were performed using orange-derived EVs. Statistical significance was calculated by comparing the percentage of signal intensity for each sample with untreated cells (NT) as a control. p:**** <0.001. An experiment with N=3 was conducted for each dataset. Data are shown as mean ± standard deviation (SD). [Figure 12]Figure 12 shows that the plant-derived extracellular matrix (EVs) of the present invention, genetically engineered with nucleic acids rather than natural EVs, can activate lymphocytes after being taken up by macrophages in Example 6. For these tests, EVs of the present invention, supplemented with mRNA molecules encoding SARS-CoV-2 spike glycoprotein (S1) RBD protein (SEQ ID NO: 50), SARS-CoV-2 spike glycoprotein complete protein (SEQ ID NO: 14), or SARS-CoV-2 nucleocapsid protein (SEQ ID NO: 17), were incubated with APC cells (macrophages) as receptor cells. After incorporating the EVs into the APCs, PBMC cells were added, and treatment with the genetically engineered EVs was repeated twice at 5-day intervals. Finally, lymphocytes were analyzed by cell fluorescence analysis (FACS). Lymphocytes identified by CD4+ expression were evaluated for their activation. Lymphocyte activation was measured as increased lymphocyte proliferation (A) and increased expression of the lymphocyte activation markers CD25+ (B) and HLADR+ (C) in CD4+ lymphocytes. Cells were treated with either naturally derived plant extracellular viable cells (EVs) (native EVs) or genetically modified plant EVs according to the present invention at a dose of 1.2 x 10¹⁰ particles. The tests were performed using orange-derived EVs. Statistical significance was calculated by comparing the percentage of signal intensity for each sample with untreated cells (NT) or cells treated with native EVs as controls. Positive controls were those treated with bead human T-activator CD3 / CD28 (CTR+) and purified protein (SARS-CoV-2 spike glycoprotein RBD protein (S protein) or SARS-CoV-2 nucleocapsid protein (N protein)). p: * <0.05, ** <0.01, *** <0.005, **** <0.001. N=3 tests were performed for each dataset. Data are presented as mean ± standard deviation (SD). [Figure 13]Figure 13 shows that in Example 7, the plant-derived extracellular matrix (EV) of the present invention, genetically modified with nucleic acids rather than natural EVs, can induce a specific immune response in mice. This graph shows the absorbance measurement of IgA immunoglobulin specific to the SARS-CoV-2 spike glycoprotein (S1) RBD protein induced by vaccination. In this study, mice were immunized on day 0 and day 21, and post-sacrifice serum was analyzed on day 35. Mice were treated with EVs genetically modified with mRNA molecules encoding SARS-CoV-2 spike glycoprotein RBD(S1) (native EVs) or EVs. Treatment was performed by intramuscular or oral administration. Humoral immune responses as IgA induction were evaluated by ELISA using ELISA plates coated with SARS-CoV-2 spike glycoprotein RBD(S1). For each administration route, the signal intensity of natural EVs and genetically modified EVs was compared, and statistical significance was calculated. p: * <0.05, ** <0.01. Each dataset consisted of N=3 animals. The data is shown as mean ± standard deviation (SD). [Modes for carrying out the invention]

[0109] material and method Isolation of extracellular vesicles Extracellular vesicles were isolated from fresh fruit juices (kiwifruit, pomegranate, blueberry, orange, lemon) or fresh plant extracts (zucchini, cabbage, kale, celery). The juices or extracts were filtered by progressively decreasing pore size to remove fibers. The extracellular vesicles were then purified by differential ultracentrifugation or tangential flow filtration. In differential ultracentrifugation, the juices were centrifuged at 1,500 g for 30 minutes to remove debris and other contaminants. The extracellular vesicles were then purified by ultracentrifugation at 10,000 g, followed by ultracentrifugation at 100,000 g for 1 hour at 4°C (Beck-man Coulter Optima L-90K). The final pellet was resuspended in phosphate-buffered saline with 1% DMSO and filtered through a 0.22 micrometer filter to sterilize.

[0110] Extracellular vesicles were either used or stored long-term at -80°C. For tangential flow filtration, the juice was first filtered using a Supracap 50 depth filter sheet disc (Pall) to remove fibers and debris and clarify the juice. Then, the filtered juice was concentrated and purified by diafiltration using a TFF Omega tangential flow filtration cassette (Pall Cadence). Finally, the residue (retentate) obtained from tangential flow filtration was filtered through a 0.2 nm filter and sterilized.

[0111] Nanoparticle tracking analysis method (NTA) Nanoparticle tracking analysis (NTA) was used to define the dimensions and profiles of EVs using a NanoSight LM10 system (Malvern) equipped with a 405 nm laser and NTA 3.1 analysis software. The Brownian motion of EVs in the laser-illuminated sample was recorded with a camera and converted into size and concentration parameters via the Stokes-Einstein equations using NTA. All camera levels were set to 16, and three 30-second videos were recorded for each sample. Briefly, purified EVs were diluted 1:2000 with 1 ml of vesicle-free physiological saline (Fresenius Kabi). After NTA acquisition, the settings were optimized and kept constant for all samples, and then each video was analyzed to measure the mean, mode, and concentration of EVs.

[0112] Production of EVs according to the present invention The genetically modified plant-derived extracellular viable cells (EVs) of the present invention were produced by the following sequential process. Briefly, plant-derived EVs were mixed with a cationic peptide and reacted at 37°C for 1 hour. The prepared nucleic acid molecules were mixed with sugar and reacted at 20°C for 10 minutes. Subsequently, the two solutions were mixed and reacted at 37°C for 3 hours. Water was then added to the reaction mixture and allowed to stand at 4°C for 12 hours. To purify the genetically modified plant-derived EVs from the remaining free nucleic acid molecules, the sample was washed by ultracentrifugation at 100,000 g at 4°C for 2 hours (Beckman Coulter Optima L-90 K, Fullerton, CA, USA), and the sample was resuspended in physiological saline.

[0113] Measurement of EV membrane potential The analysis was performed using a Zeta-sizer nanoparticle size analyzer (Malvern Instruments, Malvern, UK). All samples were analyzed in filtered (cutoff value = 200 nm) physiological saline solution at 25°C. The zeta potential (slip surface) is generated at a distance x from the vesicle and indicates the degree of electrostatic repulsion between adjacent, similarly charged vesicles in the dispersion.

[0114] Protein extraction and quantification Proteins were extracted from EV samples using RIPA buffer (150 nM NaCl, 20 nM Tris-HCl, 0.1% sodium dodecyl sulfate, 1% deoxycholic acid, 1% Triton X-100, pH 7.8) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, Missouri, USA). Protein content was quantified using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's protocol. Briefly, 10 μl of sample was dispensed into the wells of a 96-well plate, and the total protein concentration was measured using a linear standard curve established with bovine serum albumin (BSA).

[0115] Phosphatidylserine analysis For phosphatidylserine analysis, EV samples were stained with Annexin V FITC and FITC isotype (Miltenyi Biotec, Germany) for 30 minutes and diluted with physiological saline before acquisition. Samples were characterized by cytofluorometry analysis using a CytoFLEX flow cytometer (Beckman Coulter) with CytExpert software, and the signal positivity rate was measured for each sample with FITC isotype as background.

[0116] RNA extraction and quantification Using the miRNeasy mini-kit (Qiagen, Hilden, Germany), total RNA was isolated from extracellular proteins (EVs) and cells according to the manufacturer's protocol and resuspended in water. RNA concentration in the samples was quantified using a spectrophotometer (mySPEC, VWR, Radnor, PA, USA).

[0117] mRNA detection by qRT-PCR cDNA was obtained from RNA samples using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). 5 nanograms of cDNA were added to SYBR GREEN PCR Master Mix (Applied Biosystems) and performed on a 96-well QuantStudio12 K Flex Real-Time PCR (qRT-PCR) system (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as a housekeeping gene for the cell samples. The doubling of mRNA expression (Rq) across all samples was calculated as 2-ΔΔCt relative to the control sample.

[0118] cell culture Human microvascular endothelial cells (HMECs) were obtained by immortalizing primary human cutaneous microvascular endothelial cells with Simian virus 40. HMECs were cultured in endothelial basal medium supplemented with a bullet kit (EBM, Lonza, Basel, Switzerland) and 1 ml of Mycozap CL (Lonza). Macrophage MV-4-11 cell line (ATCC® CRL9591®) was obtained from ATCC and cultured in Iskov-modified Dulbecco's medium (ATCC, USA) supplemented with 10% fetal bovine serum. Peripheral blood mononuclear cells (PBMCs) were isolated as follows: whole blood from a healthy volunteer donor was diluted 1:1 with PBS, 30 ml was placed in a 50 ml centrifuge tube, and gently placed on top of a 15 ml Histopack (Sigma-Aldrich). The turbid layer containing PBMCs was collected in a 50 ml centrifuge tube, diluted with 40 ml of PBS, and centrifuged twice at 300 g for 5 minutes for washing. The pelleted cells were counted, and viability was estimated using trypan blue staining. The cells were cultured in RPMI in 24-well plates supplemented with 10% fetal bovine serum.

[0119] Nucleic acid delivery to receptor cells To evaluate the intracellular uptake of eGFP mRNA added to the genetically modified plant-derived EVs of the present invention, these vesicles were incubated with HMEC cells and macrophages. A total of 50,000 recipient cells / well were seeded in 24-well plates and stimulated with 50,000 vesicles / recipient cells. After 24 hours, the cells were thoroughly washed, detached with trypsin, and the fluorescence of the GFP protein translated by FACS was measured using a CytoFLEX flow cytometer (Beckman Coulter Optima L-90 K, Fullerton, CA, USA) equipped with CytExpert software.

[0120] In vitro nucleic acid degradation assay To investigate the resistance of nucleic acid molecules added to the EV of the present invention to enzymatic degradation, the inventors performed an RNAse assay. Briefly, the sample was treated with RNase A (Thermo Fisher Scientific) at a concentration of 0.4 mg / mL at 37°C for 30 minutes. The reaction was stopped using an RNase inhibitor (Thermo Fisher Scientific) according to the manufacturer's protocol, and the sample was washed by ultracentrifugation at 100,000 g for 2 hours at 4°C using a 10 mL polycarbonate tube (SW 90 Ti rotor, Beckman Coulter Optima L-90 K ultracentrifuge). Finally, the EV pellet sample was resuspended in physiological saline buffer and molecular analysis was performed.

[0121] To investigate the resistance of nucleic acid molecules added to the EV of the present invention to gastric digestion, the inventors performed a gastric digestion assay. Briefly, a gastric-like solution was prepared containing 18.5% w / v HCl (pH 2.0), 24 mg / mL bile extract, pepsin solution (80 mg / mL in 0.1N HCl, pH 2.0, Sigma), and 4 mg / mL pancreatin (Sigma) in 0.1N NaHCO3. One μl of each EV sample in aqueous solution was incubated with 1.34 μL of the gastric-like solution at 37°C for 60 minutes with slow rotation. The pH of the gastric-like solution was adjusted to 6.5 with 1N NaHCO3 and called intestinal fluid. The EV samples were then incubated in the intestinal fluid for a further 60 minutes. The stability of nucleic acid molecules added to the EV of the present invention was evaluated by molecular analysis as described above. Naked RNA was used as a control in all resistance tests.

[0122] Freeze-drying of the sample The samples were freeze-dried for 3 hours using a Heto lyolab 3000 (Thermo Fisher Scientific) and stored at 4°C for 7 days. After storage, the nucleic acid content of the extravalent samples was analyzed using molecular analysis. The measured content was compared to the starting amount before freeze-drying.

[0123] Immune cell activation assay To evaluate PBMC proliferation by flow cytometry, PBMCs were stained with CFSE dye from the CellTrace Cell Proliferation Kit (Invitrogen, ThermoFisher Scientific) according to the manufacturer's instructions. The PBMCs were then seeded in 48-well plates at a density of 50,000 cells / well. To assess whether the genetically modified plant-derived EVs of the present invention affect PBMC proliferation, the PBMCs were stimulated with these vesicles at a dose of 50,000 particles / cell. Unstimulated PBMCs were used as a control. After 24 hours of incubation, the PBMCs were harvested and fluorescence was measured using a CytoFLEX flow cytometer equipped with CytoExpert software (Beckman Coulter). CFSE dye is detected as FITC fluorescence.

[0124] To analyze PBMC growth using a bromodeoxyuridine (BrdU) uptake assay, PBMCs were seeded in 96-well plates at a density of 20,000 cells / well, and 10 μl of BrdU-labeled solution (BrdU colorimetric assay, Roche) was added to each well. To evaluate whether the genetically modified plant-derived EVs of the present invention affect PBMC growth, PBMCs were stimulated with these vesicles at a dose of 50,000 particles / cell. Unstimulated PBMCs were used as a control. Furthermore, to evaluate whether the EVs of the present invention reduce the growth of activated PBMCs, PBMCs were stimulated with these vesicles and 100 ng / ml LPS (derived from Escherichia coli, Sigma-Aldrich) at a dose of 50,000 particles / cell. Unstimulated PBMCs were used as a negative control. After 48 hours of incubation, the effects of stimulation were analyzed. The assay was performed according to the manufacturer's instructions. Absorbance was measured at 420 nm with a reference wavelength of 490 nm using an ELISA reader. The average absorbance for each condition was calculated. Absorbance is directly proportional to the growth rate.

[0125] Detection of protein expression To evaluate protein expression in target cells, endothelial cells were measured in 1.2 x 10⁶ cells. 10Cells were stimulated with EVs. The assayed cell samples included untreated cells (NT), cells treated with plant-derived natural EVs, and cells treated with the present invention's plant-derived EVs, genetically engineered with mRNA molecules encoding SARS-CoV-2 spike glycoprotein (S1) RBD protein (SEQ ID NO: 50), SARS-CoV-2 spike glycoprotein complete protein (SEQ ID NO: 14), or SARS-CoV-2 nucleocapsid protein (SEQ ID NO: 17). After 24 hours, the cells were thoroughly washed, detached with trypsin, and fixed and permeabilized according to the manufacturer's instructions (Inside Stain Kit, Miltenyi Biotec). Subsequently, the cells were stained at room temperature for 30 minutes with specific antibodies to detect protein expression (antibodies against SARS-CoV-2 spike glycoprotein and nucleocapsid protein, Invitrogen) to detect protein expression. After washing, a fluorescent secondary antibody (Alexa Fluor Plus 594 or 488, Invitrogen, ThermoFisher Scientific) was added at room temperature for 1 hour. After washing, the cells were resuspended in an appropriate buffer and acquired by FACS using a CytoFLEX flow cytometer equipped with CytExpert software (Beckman Coulter Optima L-90 K, Fullerton, CA, USA).

[0126] Lymphocyte activation assay To evaluate the ability of the genetically engineered EVs of the present invention to induce lymphocyte activation after integration into APC, macrophages were seeded in 24-well plates at a rate of 20,000 cells / well, and 1.2 x 10⁶ cells were used. 10Cells were stimulated with EVs. The assayed cell samples included untreated cells (NT), cells treated with plant-derived natural EVs, and cells treated with the plant-derived EVs of the present invention, genetically engineered with mRNA molecules encoding SARS-CoV-2 spike glycoprotein (S1) RBD protein (SEQ ID NO: 50), SARS-CoV-2 spike glycoprotein complete protein (SEQ ID NO: 14), or SARS-CoV-2 nucleocapsid protein (SEQ ID NO: 17). After incorporation, PBMCs were added at a concentration of 200,000 cells / well, and EV treatment was repeated after 5 days. Ten days after the initial treatment, cells were harvested and stained with fluorescent antibodies for CD4, CD25, and HLA DR using appropriate isotypes (Miltenyi Biotec) at room temperature for 30 minutes. After washing, cells were obtained.

[0127] For proliferation analysis, the PBMCs used were pre-stained with the CSFE dye from the CellTrace Cell Proliferation Kit (Invitrogen, ThermoFisher Scientific) according to the manufacturer's instructions.

[0128] Finally, the samples were subjected to FACS analysis using a CytoFLEX flow cytometer equipped with CytExpert software (Beckman Coulter Optima L-90 K, Fullerton, CA, USA).

[0129] Mouse vaccine Female BALB / cAnNCrl mice aged 6-10 weeks were immunized twice with the genetically modified plant-derived extracellular viable (EV) of the present invention at doses equivalent to 30 μg of mRNA, on days 0 and 21, and sacrificed on day 35. Mice were treated orally (enteral administration) and intramuscularly (right leg) with either naturally occurring EV of plant origin or plant-derived EV of the present invention modified with mRNA molecules encoding the SARS-CoV-2 spike glycoprotein RBD(S1). After sacrifice, blood was collected and serum was separated for antibody detection.

[0130] Antibody measurement SARS-CoV-2 specific IgA antibody titers in serum were measured using the ELISA method. Briefly, MaxiSorp ELISA plates (Nunc) were coated overnight at 4°C with 1 μg / ml of SARS-CoV-2 spike protein (Thermofisher Scientific) in 100 μl of 50 mM sodium carbonate / bicarbonate pH 9.6 buffer per well. The coated plates were washed three times with 200 μl of 1X PBS and saturated with 3% BSA in 200 μl of 1X PBS per well. The plates were washed three times with 1X PBS and incubated with 3% BSA and 100-fold diluted mouse serum for 2 hours. Subsequently, the plates were washed three times with 200 μl of 1X PBS per well and incubated with 100 μl of 1:10,000 diluted secondary donkey anti-mouse IgA HRP-binding antibody per well. After incubation with the secondary antibody, the plate was washed five times with 200 μl of 1X PBS per well, and then incubated with 100 μl of TMB (Thermofisher Scientific) per well for 30 minutes. The reaction was stopped by adding 100 μl of stop solution (Thermofisher Scientific) per well. The absorbance at 450 nm was read using a plate reader.

[0131] statistical analysis Data analysis was performed using the demo version of GraphPad 8. Results are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to demonstrate statistical differences between groups, and Student's t-test was used for comparisons between two samples. The minimum significance level was set at p < 0.05. [Examples]

[0132] Results / Examples Example 1 To investigate the feasibility of the present invention, the inventors genetically engineered extracellular vesicles (EVs) from different plants and characterized them according to many features commonly used to characterize EVs in terms of physical properties such as particle size and surface charge (Thery C. et al, (2018) “Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines”; Journal of Extracellular Vesicles, 7:1, 1535750, DOI: 10.1080 / 20013078.2018.1535750). Figure 1 shows the sizes of EVs from various plants, including kiwifruit, cabbage, and blueberries. Similar results were obtained for EVs from other plants, such as pomegranate, kale, celery, cholette, orange, and lemon. Particle size is a fundamental parameter of EVs. The data obtained showed that the EVs of the present invention have a larger average diameter compared to EVs derived from natural plants (Figure 1A, B, C). EV size was measured by the inventors using nanoparticle tracking analysis (NTA). NTA is the most commonly used method for EV size measurement, utilizing the properties of both light scattering and Brownian motion to obtain the nanoparticle size distribution of a sample in a liquid suspension. In particular, NTA works by tracking particle motion via light scattering to evaluate the mean square displacement of particles moving under Brownian motion in a sample chamber irradiated with a laser beam. The diffusion constant can be calculated by tracking the particles, which is used in the Stokes-Einstein equations to calculate the hydrodynamic diameter. The Stokes-Einstein equations also take into account the temperature and viscosity of the suspension.Analysis conducted by the present inventors showed that the EVs of the present invention are larger in size than those of naturally occurring plant-derived EVs. While the diameter of naturally occurring plant-derived EV vesicles was 100-150 nm with an average diameter of 134 ± 6 nm, the diameter of the EVs of the present invention ranged from 200-250 nm with an average diameter of 220 nm. The size distribution indicated that the diameter of the EVs of the present invention ranged from 20-500 nm, preferably in the range of 200-300 nm.

[0133] To characterize the membrane properties of the genetically modified plant-derived extracellular vesicles (EVs) of the present invention, vesicle membrane potentials were measured. In fact, zeta potential is a common method for measuring the surface potential of EVs and is used as an indicator of surface charge and colloidal stability. The surface charge of EVs changes depending on the properties of the molecules expressed on their surface, influencing EV interactions in dispersed systems such as the human body and regulating EV activity in biological processes. For example, surface charge is known to affect various particle-related biological processes, such as cellular uptake and cytotoxicity. Zeta potential is a measure of the magnitude of electrostatic or charge repulsion / attraction between particles and can be measured from electrophoretic mobility in a suspension (electrophoretic light scattering), which is determined by applying an electric field and measuring the resulting particle velocity (Midekessa G, et al. Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability. ACS Omega. 2020 Jun 30;5(27):16701-16710. doi: 10.1021 / acsomega.0c01582. PMID: 32685837; PMCID: PMC7364712.). As shown in Figure 2, plant-derived EVs are known to have a negative surface charge, and their membrane potential (Z potential) ranged from -10 to -15 mVolt, with an average value of -13 mVolt. In contrast, the genetically modified plant-derived extracellular viable cells (EVs) of the present invention utilized different membranes with membrane potentials ranging from 0 to -3 mVolt and an average value of -2 mVolt. The above tests were conducted using genetically modified plant-derived EVs from zucchini (E1) and blueberry (E2), but similar results were obtained with EVs from other plants such as kiwifruit, cabbage, kale, pomegranate, lemon, orange, and celery.

[0134] To further characterize the EVs of the present invention, the protein content of these vesicles and EVs derived from natural plants was measured (Figure 3A). From the data thus obtained, the EVs of the present invention had a protein content of 120-160 ng / 10 10While EV has a high protein content in the EV range, natural plant-derived EV has 50-100 ng / 10 10 It was confirmed that it was within the range of extracellular viable (EV). The inventors tested with genetically modified plant-derived EVs of pomegranate (E1) and kiwifruit (E2), but similar results were obtained with EVs from other plants such as zucchini, cabbage, kale, blueberry, lemon, orange, and celery.

[0135] Furthermore, the inventors conducted specialized tests to further characterize the EV membrane of the present invention. As is known in the art, in natural EVs, phosphatidylserine (PS) is mainly located on the outer surface of the cell membrane. Regardless of theory, the inventors believe that during membrane rearrangement due to osmotic stress, PS loses its asymmetric distribution in the phospholipid bilayer and moves to the inner side of the membrane in the EV of the present invention. Detection of phosphatidylserine on the extracellular membrane of vesicles was performed using fluorescently labeled annexin V. In fact, annexin V is known to specifically bind to PS on the vesicle membrane. The amount of fluorescence signal from annexin V reflects the PS content on the outer surface of the EV membrane (Montoro-Garcia S, et al. “An innovative flow cytometric approach for small-size platelet microparticles: influence of calcium”. Thromb Haemost. 2012 Aug;108(2):373-83). The results obtained by the inventors showed that in the composition of the present invention, ≤44% of EVs had phosphatidylserine in the outer layer of the membrane (Figure 3B), with the percentage ranging from 40% to 44% and an average of 43%. In contrast, in compositions containing EVs derived from natural plants, the percentage of vesicles having phosphatidylserine in the outer layer of the membrane ranged from 55% to 48%, with an average of 49%.

[0136] The above tests were conducted using genetically modified plant-derived extracellular life (EVs) from cabbage (E1) and blueberries (E2), but similar results were obtained with EVs from other plants such as kiwifruit, lemon, orange, zucchini, kale, pomegranate, and celery.

[0137] As a further evaluation, the inventors conducted a dedicated test to assess the immunomodulatory activity of the genetically modified plant-derived extracellular proteins (EVs) of the present invention. Briefly, PBMCs, i.e., a mixed population of human blood-derived lymphocytes, monocytes, and other immune cells, were stimulated with the EVs of the present invention, and the cell proliferation rate was measured.

[0138] As shown in Figure 4A, the proliferation rate of PBMCs stimulated for 48 hours with the pomegranate-derived EV of the present invention was the same as that of untreated PBMCs, suggesting that these vesicles do not promote PBMC proliferation and do not exhibit immunostimulatory effects. Furthermore, to verify whether the EV of the present invention has an immunosuppressive effect, the inventors treated PBMCs with LPS, which is known to induce inflammatory responses and promote lymphocyte proliferation, and then stimulated the cells with the genetically modified plant-derived EV of the present invention. As shown in Figure 4B, the proliferation rate of PBMCs activated by LPS was not affected by the EV of the present invention (derived from pomegranate (E1) and kiwifruit (E2)).

[0139] These results confirm that the EV of the present invention has neither immunostimulatory nor immunosuppressive effects.

[0140] Furthermore, the PBMCs were stained with a fluorescent dye (CFSE) that allows for detection of PBMC growth by flow cytometry. As shown in Figures 4C and 4D, the growth rate of PBMCs stimulated with genetically modified EVs from cabbage (E1), celery (E2), and zucchini (E3) was the same as that of unstimulated PBMCs. Similar results were obtained with genetically modified EVs from other plants, including blueberries, lemons, oranges, and kale.

[0141] In summary, the results above indicate that the genetically modified plant-derived EVs of the present invention neither stimulate nor suppress the activation and proliferation of immune cells, confirming the non-immunomodulatory properties of these vesicles regardless of their plant origin.

[0142] Finally, the total RNA content of the EVs of the present invention and EVs derived from natural plants was measured (Figure 5). From the obtained data, it was found that the EVs of the present invention had a higher RNA content than natural vesicles, ranging from 30 to 100 ng / 10 9 This is an EV, and the average value is 50ng / 10 9 It was an extracellular matrix (EV). The RNA content of naturally derived EVs is 5-15 ng / 10 9 In EV, the average value is 10ng / 10 9 These are extracellular genes (EVs). The tests were conducted on genetically modified EVs of celery (E1), pomegranate (E2), and kiwifruit (E3), but similar results were obtained with EVs from other plants, including zucchini, cabbage, kale, lemon, orange, and blueberry.

[0143] In summary, this data reveals that the genetically modified plant-derived extracellular vesicles (EVs) of the present invention differ significantly from those derived from natural plants. In particular, the addition of foreign nucleic acids causes remarkably unique changes in the structure and function of plant-derived EVs compared to natural vesicles. As a result, the average diameter increases, the surface charge increases, the phosphatidylserine content decreases, and the immunomodulatory effect on immune system cells is lost.

[0144] Example 2 To confirm that the EV of the present invention is suitable as a vehicle for nucleic acid delivery, genetically modified EVs were created by supporting mRNA molecules internally, and the amount of added mRNA was measured by qRT-PCR analysis (Figure 6). The results showed that the EV of the present invention can deliver an increased dose of nucleic acid molecules. In fact, genetically modified EVs were created from kiwifruit (E1 and E3) and celery (E2 and E4) using two different doses of mRNA (0.1 μg / ml for E1 and E2, and 1 μg / ml for E3 and E4). The increase in nucleic acid dose was detectable as an increase in the amount of mRNA within the vesicles (E3 and E4 compared to E1 and E2, respectively). Similar results were obtained with EVs derived from other plants, including zucchini, cabbage, kale, lemon, orange, pomegranate, and blueberry. In summary, these data confirm that the EV of the present invention can encapsulate added nucleic acid molecules and increase their quantity.

[0145] Example 3 The inventors conducted specialized tests to evaluate the ability of the EVs of the present invention to protect added nucleic acid molecules from degradation (Figure 7). In particular, these studies showed that the genetically modified plant-derived EVs of the present invention can protect added nucleic acid molecules from treatment with degrading enzymes (RNAse). In short, after EV treatment with RNAse, qRT-PCR analysis revealed that approximately 80% of the added mRNA still existed within the vesicles of the present invention, while the naked mRNA used as a control was almost completely degraded (Figure 7A).

[0146] Further experiments showed that when vesicles were treated with a stomach-like solution mimicking the gastrointestinal environment, approximately 90% of the added mRNA remained in the EVs, while the naked mRNA was almost completely degraded, demonstrating that the EVs of the present invention can protect added nucleic acid molecules (Figure 7B). The above tests were performed with genetically modified plant-derived EVs from pomegranate (Figure 6A) and kiwifruit (Figure 6B), but similar results were obtained with EVs from other plants such as zucchini, cabbage, kale, blueberry, lemon, orange, and celery.

[0147] In summary, this data shows that the EV of the present invention protects encapsulated exogenous nucleic acids from degradation conditions. Furthermore, protection from the gastrointestinal environment supports the oral administration of the composition for use in the present invention.

[0148] Example 4 The plant-derived extracellular viable cells (EVs) of the present invention can be efficiently freeze-dried and stored. In particular, even after freeze-drying the EVs and storing them at +4°C for 7 days, the content of added mRNA within the vesicles did not decrease compared to the initial state (Figure 8). The tests were performed on genetically modified EVs derived from celery (E1) and kiwifruit (E2), but similar results were obtained with EVs derived from other plants such as zucchini, cabbage, kale, blueberry, lemon, orange, and pomegranate.

[0149] These data confirm that the EV of the present invention does not require the use of ultra-low temperatures such as -80°C, which are common for the storage of nucleic acid drugs, and can be easily freeze-dried and efficiently stored at +4°C or room temperature.

[0150] Example 5 The inventors further confirmed that the genetically modified plant-derived extracellular genes (EVs) of the present invention are suitable for use in delivering added nucleic acids to receptor cells (Figure 9). In these tests, macrophages were used as exemplary receptor cells, and the transfer of mRNA molecules in these cells was measured by qRT-PCR analysis. In particular, the above tests confirmed that EVs of the present invention derived from different types of plants (E1, E2, E3) were able to transfer mRNA molecules to macrophages in untreated cells (untreated, NT), whereas no mRNA transfer was detected in naturally derived EVs (native EVs), plant-derived EVs co-incubated with mRNA without nucleic acid addition (EV+mRNA), and naked mRNA. The tests were performed on genetically modified plant-derived EVs from cabbage (E1), pomegranate (E2), and kiwifruit (E3), but similar results were obtained with EVs from other plants such as zucchini, celery, kale, lemon, orange, and blueberry.

[0151] To demonstrate that nucleic acids delivered to recipient cells maintain their functional activity, the EV of the present invention, containing an mRNA molecule encoding a GFP protein, was assayed. After integration into the receptor cell, if the mRNA is functional, it is translated into a GFP protein, and the fluorescence of the functional protein is detected within the cell.

[0152] According to tests conducted by the inventors, mRNA transported by the EVs of the present invention was functional and detectable as a fluorescent signal in endothelial cells (Figure 10A) and macrophages (Figure 10B). However, no functional mRNA transfer was observed when using naturally derived (native EVs, Figure 10A) or naked mRNA (naked mRNA, Figure 10B). The tests were conducted using genetically modified plant-derived EVs from zucchini (Figure 10A) and kale (Figure 10B), but similar results were obtained with EVs from other plants such as celery, cabbage, kiwifruit, blueberry, lemon, orange, and pomegranate.

[0153] Furthermore, the inventors confirmed that the genetically modified plant-derived EVs of the present invention can translocate functional mRNA, which is translated into a protein antigen, to recipient cells and express it as a correctly folded antigen (Figure 11). During the testing process, EVs of the present invention containing mRNA molecules encoding different sequences of viral protein antigens were assayed: SEQ ID NO: 50, SARS-CoV-2 spike glycoprotein (S1) RBD protein; SEQ ID NO: 14, SARS-CoV-2 spike glycoprotein complete protein; SEQ ID NO: 17, SARS-CoV-2 nucleocapsid protein. After integration into endothelial cells, the mRNA, if functional, is translated into a protein and detected by a specific antibody. Figure 11 shows that the genetically modified plant-derived EVs of the present invention can translocate mRNA to target cells, and the target cells translate the mRNA into a specific protein antigen, whereas the protein antigen was not detected in untreated cells (NT) or cells treated with naturally derived plant EVs instead of genetically modified ones.

[0154] In summary, these data confirm that the EV of the present invention can efficiently deliver foreign nucleic acid molecules to various types of receptor cells, including antigen-presenting cells (APCs) such as macrophages, while simultaneously retaining the function of the nucleic acid and its ability to be translated into a protein. Thus, a correctly folded expressed protein can function as an antigen that promotes immunity by APCs. Furthermore, the tests conducted as described above showed that the EV of the present invention is suitable for use with nucleic acid molecules encoding different proteins, such as viral antigens (SARS-CoV-2 nucleocapsid (N) protein and spike glycoprotein) or other proteins such as GFP.

[0155] Example 6 The inventors further confirmed that the genetically engineered plant-derived EVs of the present invention can deliver nucleic acids to antigen-expressing and presenting APC cells, stimulating specific immune activation (Figure 12). In these studies, macrophages were used as exemplary APC receptor cells and stimulated with the genetically engineered EVs of the present invention before incubation with PBMCs, i.e., a mixed population of lymphocytes, monocytes, and other immune cells in human blood. For the studies, the EVs of the present invention were assayed with mRNA molecules encoding different sequences of viral protein antigens used as examples: Sequence 1, SARS-CoV-2 spike glycoprotein (S1) RBD protein; Sequence 2, SARS-CoV-2 spike glycoprotein complete protein; Sequence 3, SARS-CoV-2 nucleocapsid protein. Lymphocyte activation was measured after 10 days using FACS analysis as increased proliferation of lymphocytes (CD4+ cells), and expression of the activation markers CD25 and HLA DR. To detect lymphocyte proliferation, PBMCs were also stained with a fluorescent dye (CFSE) that can detect PBMC proliferation by flow cytometry. As shown in Figure 12A, the proliferation rate of CD4+ lymphocytes stimulated with genetically modified plant-derived EVs was increased compared to negative controls, untreated cells (NTs), and cells treated with natural EVs. As expected, the positive controls stimulated lymphocyte proliferation: human T-activator CD3 / CD28 (CTR+) and purified proteins (SARS-CoV-2 spike glycoprotein RBD protein (S protein), or SARS-CoV-2 nucleocapsid protein (N protein)). The increased lymphocyte proliferation rate indicates its activation.

[0156] Furthermore, stimulation of APCs with the genetically modified plant-derived EVs of the present invention increased the expression of the activation markers CD25 (Figure 12B) and HLA DR (Figure 12C) in lymphocytes.

[0157] Stimulation with plant-derived EVs of the present invention induced increased expression of both activation markers in CD4+ lymphocytes, demonstrating activation of immune cells. This stimulation was compared to negative controls, untreated cells (NT), and cells treated with natural plant-derived EVs. As expected, positive controls stimulated lymphocyte proliferation: human T-activator CD3 / CD28 (CTR+) and purified proteins (SARS-CoV-2 spike glycoprotein RBD protein (S protein), or SARS-CoV-2 nucleocapsid protein (N protein)).

[0158] Overall, the results above demonstrate that the genetically modified plant-derived extracellular proteins (EVs) of the present invention activate an immune response after being taken up by APCs (such as macrophages) and can be assayed with different nucleic acid molecules encoding different protein antigens.

[0159] In summary, the plant-derived extracellular matrix (EVs) of the present invention can transmit functional mRNA to the APC, which is then translated into a correctly folded protein antigen, specifically activating the immune response. Notably, only the genetically modified plant-derived EVs of the present invention, and not those derived from natural plants, induced lymphocyte activation.

[0160] Example 7 To demonstrate the suitability of the EV according to the present invention as a vehicle for nucleic acid delivery for use as a vaccine, experiments were conducted using an in vivo mouse model.

[0161] In particular, mice were immunized twice (with a 3-week interval between the two treatments), and the presence of specific antibodies in the serum was measured 2 weeks after the last administration. Mice were administered either naturally derived EVs (native EVs) or the plant-derived EVs of the present invention (genetically modified EVs) genetically engineered with mRNA molecules encoding viral protein antigens such as the SARS-CoV-2 spike glycoprotein RBD(S1), via intramuscular or oral administration routes.

[0162] Figure 13 shows the measurement of IgA antibodies specific to the SARS-CoV-2 spike glycoprotein RBD(S1) in mouse serum. Vaccination with the genetically modified plant-derived EVs of the present invention induced the production of specific antibodies, both by oral and intramuscular administration, compared to vaccination with naturally derived plant-derived EVs. The antibody-positive reaction detected after oral administration indicates that the composition for use of the present invention is protected from the gastrointestinal environment.

[0163] In summary, this data shows that the plant-derived extracellular matrix (EV) of the present invention is suitable for use as a vaccine because it can support nucleic acids, transfer to APCs, translate into correctly folded antigens, activate an immune response, and produce specific antibodies in vivo. Since the EV of the present invention does not exhibit immunostimulatory or immunosuppressive effects, the activation of the immune response is antigen-specific. Furthermore, the genetically modified plant-derived EV of the present invention can efficiently protect nucleic acids from degradation, enabling vaccine administration via different routes. Further aspects of the present invention are described below: [Section 1] A composition comprising non-immunomodulatory, genetically modified, plant-derived extracellular vesicles (EVs) for use as a vaccine, The extracellular vesicles (EVs) are compartmentalized by a lipid bilayer comprising an outer lipid layer and an inner lipid layer; It contains at least one foreign nucleic acid molecule encoding a protein antigen; and Having a diameter in the range of 20 to 500 nm, preferably 200 to 300 nm; The membrane potential across the lipid bilayer of the aforementioned EV is in the range of +5 to -5 mV; and, A composition wherein 44% or less of the EV in the composition contains phosphatidylserine in the outer layer of the lipid bilayer membrane. [Section 2] A composition for use as described in item 1, wherein the loaded foreign nucleic acid molecule is selected from the group consisting of DNA, cDNA, messenger RNA (mRNA), premRNA, long RNA, coding RNA, single-stranded RNA, double-stranded RNA, linear RNA, RNA oligonucleotide, self-replicating RNA (replicon RNA), retroviral RNA, and viral RNA (vRNA). [Section 3] The composition for use according to item 2, wherein the loaded foreign nucleic acid molecule is an mRNA molecule containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 17, 19, and 50. [Section 4] A composition for use according to any one of claims 1 to 3, wherein at least one encoded protein antigen is selected from the group consisting of tumor antigens, viral antigens, bacterial antigens, fungal antigens, and protist antigens. [Section 5] At least one encoded protein antigen is prostate-specific antigen (PSA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate 6-transmembrane epithelial antigen 1 (STEAP1), receptor tyrosine-protein kinase erbB-2, cell surface-associated mucin 1 protein (MUC1), tyrosinase-associated protein 2 (TRP-2), proto-oncogene B-Raf, proto-oncogene c-Kit, GTPase NRas, melanoma-associated antigen 1, melanoma-associated antigen 1 protein, NY-ESO-1 protein, SARS-CoV-2 spike protein, SARS-CoV-2 N protein, SARS-CoV-2 M protein, influenza A virus hemagglutinin protein, influenza B virus hemagglutinin protein, influenza A virus neuraminidase protein, influenza B virus neuraminidase protein, HIV-1 envelope protein, HIV-2 envelope protein, HPV major capsid protein L1, HPV minor capsid protein L2, rabies lyssavirus glycoprotein, human cytomegalovirus glycoprotein A composition for use as described in item 4, selected from the group consisting of hepatitis C virus envelope glycoprotein E1E2, RSV fusion protein, Zaire Ebola virus spike glycoprotein, Zika virus protein prM, Zika virus serine protease NS3, Zika virus serine protease subunit NS2B, Zika virus envelope protein E, Zika virus capsid protein C, Toxoplasma gondii protein containing concentrated granule protein 6, Loptory protein 2A, Loptory protein 18, surface antigen 1, surface antigen 2A, Toxoplasma gondii apical membrane antigen 1, SARS-CoV-2 spike (S)RBD protein, and any combination thereof. [Section 6] A composition for use according to item 5, wherein at least one encoded protein antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, 15, 16, 18 and 20-49. [Section 7] The amount of loaded foreign nucleic acid molecules in EV is 20-200 ng / 10 9 EV, preferably 30-100 ng / 10 9 A composition for use according to any one of items 1 to 6, which is within the range of EV. [Section 8] A composition for use according to any one of claims 1 to 7, wherein EV is derived from one or more plants selected from the group consisting of the genus Citrus, including lemon and orange; the genus Actinidia, including kiwifruit; the genus Pumpkin, including zucchini; the genus Brassica, including cabbage and kale; the genus Pomegranate, including pomegranate; the genus Vaccinium, including blueberry; and the genus Apium, including celery. [Section 9] A composition for use according to any one of claims 1 to 8, further comprising one or more polycationic substances, wherein the one or more polycationic substances bond to the outer lipid layer of the lipid bilayer of EV via electrostatic interactions. [Section 10] One or more polycationic substances are selected from the group consisting of protamine, cationic peptides, polypeptides, polysaccharides, glycerol, polyethylene glycol (PEG), and cationic proteins comprising any combination thereof, and the amount of the one or more polycationic substances is preferably 0.001 to 2 μg / 10 10 A composition for use as described in item 9, wherein the composition contains in amounts within the range of EV. [Section 11] A composition for use according to any one of claims 1 to 10, wherein the EV is further loaded with one or more sugar molecules, and the one or more sugar molecules are bound to the foreign nucleic acid molecules loaded on the EV via electrostatic interactions and hydrogen bonds. [Section 12] One or more sugar molecules are selected from the group consisting of disaccharides, sugar alcohols, polysaccharides, and any combination thereof, and the content of one or more sugar molecules in EV is preferably 0.1 to 10 mg / 10 10 A composition for use as described in item 11, which is within the range of EV. [Section 13] A composition for use according to any one of claims 1 to 12, which is in a form suitable for oral, nasal, or parenteral administration. [Section 14] A method for producing the composition described in any one of items 1 to 12, (i) A step of contacting a suspension of extracellular vesicles (EVs) of plant origin with one or more polycationic substances and mixing them to obtain a first mixture; (ii) A step of contacting a preparation of nucleic acid molecules with one or more sugar molecules and mixing them to obtain a second mixture, wherein the nucleic acid molecules encode at least one protein antigen; (iii) the step of mixing the first mixture and the second mixture to obtain a third mixture; and (iv) A step of adding a predetermined amount of water to the third mixture, wherein the ratio of the predetermined amount of water to the amount of the third mixture is within the range of 5:1 to 15:1. Methods that include... [Section 15] The method according to claim 14, further comprising concentrating the composition obtained in step (iv) preferably by filtration. [Section 16] One or more polycationic substances are selected from the group consisting of: protamine-like proteins such as protamine, calcitonin peptide, plectacin, lactoferrin, spermine or spermidine; cationic proteins including nucleolin, histones, and cell-permeable peptides (CPPs); cationic peptides including histidine-enriched peptides, arginine-enriched peptides, lysine-enriched peptides, and cationic arginine-enriched peptides (CARPs); polypeptides including polyarginine, polylysine, polyhistidine, histidine-enriched peptides, arginine-enriched peptides, and lysine-enriched peptides; polysaccharides including chitosan, polysulfated glycosaminoglycans such as PSGAG, and cationic dextran; glycerol, polyethylene glycol (PEG), and any combination thereof, and / or one or more sugar molecules Trehalose, maltose, lactose, sucrose, cellobiose, chitobiose, cozybiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, trehalose, turanose, maltulose, louscrose, isomaltulose, gentiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, The method according to claim 14 or 15, selected from the group consisting of disaccharides including xylobiose; sugar alcohols including arabitol, erythritol, glycerol, HSH, isomalt, lactitol, maltitol, mannitol, sorbitol, and xylitol; polysaccharides including starch, glycogen, galactogen, inulin, arabinoxylan, cellulose, chitin, and pectin, and any combination thereof.

Claims

1. A composition for use as a vaccine, comprising non-immunomodulatory, genetically modified, plant-derived extracellular vesicles (EVs) and one or more polycationic substances, The extracellular vesicles (EVs) are compartmentalized by a lipid bilayer comprising an outer lipid layer and an inner lipid layer; It contains at least one foreign nucleic acid molecule encoding a protein antigen; and Having a diameter in the range of 20 to 500 nm; The membrane potential across the lipid bilayer of the aforementioned EV is in the range of +5 to -5 mV; and, A composition wherein 44% or less of the EV in the composition contains phosphatidylserine in the outer layer of the lipid bilayer membrane.

2. The composition according to claim 1, wherein the loaded foreign nucleic acid molecule is selected from the group consisting of DNA, cDNA, messenger RNA (mRNA), premRNA, long RNA, coding RNA, single-stranded RNA, double-stranded RNA, linear RNA, RNA oligonucleotide, self-replicating RNA (replicon RNA), retroviral RNA, and viral RNA (vRNA).

3. The composition according to claim 2, wherein the loaded foreign nucleic acid molecule is an mRNA molecule containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 17, 19, and 50.

4. The composition according to any one of claims 1 to 3, wherein at least one encoded protein antigen is selected from the group consisting of tumor antigens, viral antigens, bacterial antigens, fungal antigens, and protist antigens.

5. At least one encoded protein antigen is prostate-specific antigen (PSA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate 6-transmembrane epithelial antigen 1 (STEAP1), receptor tyrosine-protein kinase erbB-2, cell surface-associated mucin 1 protein (MUC1), tyrosinase-associated protein 2 (TRP-2), proto-oncogene B-Raf, proto-oncogene c-Kit, GTPase NRas, melanoma-associated antigen 1, melanoma-associated antigen 1 protein, NY-ESO-1 protein, SARS-CoV-2 spike protein, SARS-CoV-2 N protein, SARS-CoV-2 M protein, influenza A virus hemagglutinin protein, influenza B virus hemagglutinin protein, influenza A virus neuraminidase protein, influenza B virus neuraminidase protein, HIV-1 envelope protein, HIV-2 envelope protein, HPV major capsid protein L1, HPV minor capsid protein L2, rabies lyssavirus glycoprotein, human cytomegalovirus glycoprotein The composition according to claim 4, selected from the group consisting of protein, hepatitis C virus envelope glycoprotein E1E2, RSV fusion protein, Zaire Ebola virus spike glycoprotein, Zika virus protein prM, Zika virus serine protease NS3, Zika virus serine protease subunit NS2B, Zika virus envelope protein E, Zika virus capsid protein C, Toxoplasma gondii protein containing concentrated granule protein 6, Loptory protein 2A, Loptory protein 18, surface antigen 1, surface antigen 2A, Toxoplasma gondii apical membrane antigen 1, SARS-CoV-2 spike (S) RBD protein, and any combination thereof.

6. The composition according to claim 5, wherein at least one encoded protein antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, 15, 16, 18 and 20-49.

7. The amount of loaded foreign nucleic acid molecules in EV is 20-200 ng / 10 ９ A composition according to any one of claims 1 to 6, which is within the range of EV.

8. The composition according to any one of claims 1 to 7, wherein EV is derived from one or more plants selected from the group consisting of the genus Citrus, which includes lemon and orange; the genus Actinidia, which includes kiwi fruit; the genus Pumpkin, which includes zucchini; the genus Brassica, which includes cabbage and kale; the genus Pomegranate, which includes pomegranate; the genus Vaccinium, which includes blueberry; and the genus Apium, which includes celery.

9. The composition according to any one of claims 1 to 8, wherein the one or more polycationic substances are bonded to the outer lipid layer of the lipid bilayer of EV via electrostatic interactions.

10. The composition according to claim 9, wherein one or more polycationic substances are selected from the group consisting of protamine, cationic peptide, polypeptide, polysaccharide, glycerol, polyethylene glycol (PEG), and cationic proteins comprising any combination thereof.

11. The composition according to any one of claims 1 to 10, wherein one or more polycationic substances are present in the composition in an amount in the range of 0.001 to 2 μg / 10¹⁰ EV.

12. The composition according to any one of claims 1 to 11, wherein the EV is further loaded with one or more sugar molecules, and the one or more sugar molecules are bound to the foreign nucleic acid molecules loaded on the EV via electrostatic interactions and hydrogen bonds.

13. The composition according to claim 12, wherein one or more sugar molecules are selected from the group consisting of disaccharides, sugar alcohols, polysaccharides, and any combination thereof.

14. The composition according to any one of claims 1 to 13, wherein the content of one or more sugar molecules in EV is in the range of 0.1 to 10 mg / 10 EV.

15. A composition according to any one of claims 1 to 14, which is in a form suitable for oral, nasal, or parenteral administration.

16. A method for producing the composition according to any one of claims 1 to 14, (i) A step of contacting a suspension of extracellular vesicles (EVs) derived from plants with one or more polycationic substances and mixing them to obtain a first mixture; (ii) A step of contacting a preparation of nucleic acid molecules with one or more sugar molecules and mixing to obtain a second mixture, wherein the nucleic acid molecules encode at least one protein antigen; (iii) the step of mixing the first mixture and the second mixture to obtain a third mixture; and (iv) A step of adding a predetermined amount of water to the third mixture, wherein the ratio of the predetermined amount of water to the amount of the third mixture is within the range of 5:1 to 15:

1. Methods that include...

17. The method according to claim 16, further comprising concentrating the composition obtained in step (iv).

18. One or more polycationic substances are selected from the group consisting of: cationic proteins containing protamine, calcitonin peptide, plectacin, lactoferrin, spermine or spermidine, nucleolin, histone, cell permeable peptide (CPP); cationic peptides containing histidine-enriched peptide, arginine-enriched peptide, lysine-enriched peptide, cationic arginine-enriched peptide (CARP); polypeptides containing polyarginine, polylysine, polyhistidine, histidine-enriched peptide, arginine-enriched peptide, lysine-enriched peptide; polysaccharides containing chitosan, glycosaminoglycans such as polysulfated glycosaminoglycans (PSGAG), cationic dextran; glycerol, polyethylene glycol (PEG), and any combination thereof, and / or one or more sugar molecules are trehalose, ma The method according to claim 16 or 17, selected from the group consisting of disaccharides including rutose, lactose, sucrose, cellobiose, chitobiose, cozybiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, genthiobiose, trehalose, turanose, maltulose, louscrose, isomaltulose, genthiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, and xylobiose; sugar alcohols including arabitol, erythritol, glycerol, HSH, isomalt, lactitol, maltitol, mannitol, sorbitol, and xylitol; polysaccharides including starch, glycogen, galactogen, inulin, arabinoxylan, cellulose, chitin, and pectin, and any combination thereof.

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