US-20260124156-A1 - PLANT-DERIVED EXTRACELLULAR VESICLE (EVS) COMPOSITIONS AND USES THEREOF

Abstract

The invention relates to a composition comprising a population of plant-derived extracellular vesicles (EVs) having a diameter ranging from 10 to 500 nm and showing pro-angiogenic, and anti-bacterial activity, for use in therapeutic applications. The invention also relates to a method of loading one or more negatively-charged biologically-active molecules into said population of EVs.

Inventors

• Giovanni Camussi
• Chiara GAI
• Margherita Alba Carlotta POMATTO

Assignees

• EV Biosolutions S.p.A.

Dates

Publication Date

20260507

Application Date

20251117

Priority Date

20190313

Claims (12)

1. 1 . A method for loading one or more negatively charged biologically active molecules into a population of plant-derived extracellular vesicles (EVs), comprising the steps of: (i) contacting and co-incubating a population of plant-derived EVs with a polycationic substance and one or more negatively charged biologically active molecules; and (ii) purifying the loaded EVs obtained in step (i) from the polycationic substance and the remaining one or more free negatively charged active molecules; (iii) obtaining plant-derived EVs loaded with negatively charged biologically active molecules.
2. 2 . The method of claim 1 , wherein said plant-derived EVs are delimited by a lipid bilayer membrane and have a diameter ranging from 10 to 500 nm, a protein content in the range of from 1 to 55 ng/10 9 EVs, and an RNA content in the range of from 10 to 60 ng/10 10 EVs.
3. 3 . The method of claim 1 , wherein the polycationic substance is selected from the group consisting of protamine, polylysine, cationic dextrans and combinations thereof.
4. 4 . The method of claim 1 , wherein the one or more negatively charged biologically active molecules is selected from the group consisting of drugs, nucleic acid molecules and lipophilic molecules, including lipophilic vitamins, wherein the nucleic acid molecules comprise miRNA, mRNA, tRNA, rRNA, siRNA, regulating RNA, non-coding and coding RNA, DNA fragments and DNA plasmids.
5. 5 . A method of promoting lesions closure in a subject in need thereof, said method comprising administering to said subject a composition comprising a population of plant-derived EVs, the plant-derived EVs in said population being delimited by a lipid bilayer membrane and having a diameter ranging from 10 to 500 nm, a protein content in the range of from 1 to 55 ng/10 9 EVs, and an RNA content in the range of from 10 to 60 ng/10 10 EVs, said EVs being engineered with miRNA miR-21.
6. 6 . A method of inhibiting angiogenesis in a subject in need thereof, said method comprising administering to said subject a composition comprising a population of plant-derived EVs, the plant-derived extracellular EVs in said population being delimited by a lipid bilayer membrane and having a diameter ranging from 10 to 500 nm, a protein content in the range of from 1 to 55 ng/10 9 EVs, and an RNA content in the range of from 10 to 60 ng/10 10 EVs, said EVs being engineered with miR-221, miR-223, miR-145 3, miR-29, miR-126 and/or miR-31.
7. 7 . The method of claim 5 , wherein the lesions are mucosal lesions selected from the group consisting of traumatic lesions due to prosthesis, diabetic, mouth, decubital or genital mucosal lesions.
8. 8 . The method of claim 5 , wherein the EVs population is derived from one or more plants selected from the group consisting of: the family Rutaceae, the family Rosaceae, the family Vitaceae, the family Brassicaceae, the family Selaginellaceae, the family Asteraceae, the family Oleaceae, the family Xanthorrhoeaceae, the family Nelumbonaceae, the family Araliaceae, the family Lamiaceae, the family Hypericaceae, the family Pedaliaceae, the family Ginkgoaceae, the family Piperaceae, and the family Rubiaceae.
9. 9 . The method of claim 8 , wherein the EVs population is derived from one or more plants selected from the group consisting of: the genus Citrus , including lemon, orange, tangerine, clementine, bergamot, and pompia; Malus pumila; Vitis vinifera; Anastatica hierochuntica; Selaginella lepidophylla; Calendula officinalis; Olea europaea; Aloe vera; Nelumbo ; the subgenus Panax; Lavandula; Hypericum perforatum; Harpagophytum procumbens; Ginkgo biloba; Piper kadsura, Piper futokadsura ; and Hedyotis diffusa.
10. 10 . The method of claim 5 , wherein the composition is formulated as a pharmaceutical composition for topic application, local injection or oral administration, or is formulated as a food supplement preparation.
11. 11 . The method of claim 6 , wherein the EVs population is derived from one or more plants selected from the group consisting of: the family Rutaceae, the family Rosaceae, the family Vitaceae, the family Brassicaceae, the family Selaginellaceae, the family Asteraceae, the family Oleaceae, the family Xanthorrhoeaceae, the family Nelumbonaceae, the family Araliaceae, the family Lamiaceae, the family Hypericaceae, the family Pedaliaceae, the family Ginkgoaceae, the family Piperaceae, and the family Rubiaceae.
12. 12 . The method of claim 6 , wherein the composition is formulated as a pharmaceutical composition for topic application, local injection or oral administration, or is formulated as a food supplement preparation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part application of U.S. application Ser. No. 17/438,020 filed Sep. 10, 2021, which is a U.S. National Phase filing of PCT International Patent Application No. PCT/EP2020/056632, having an international filing date of Mar. 12, 2020, which claims priority to Italian Patent Application No. 102019000003639, filed Mar. 13, 2019 each of which is hereby incorporated by reference in its entirety TECHNICAL FIELD The present invention relates to plant-derived extracellular vesicle (EVs) compositions and their therapeutic applications. BACKGROUND Extracellular vesicles (EVs) are a heterogeneous population of particles released by virtually all living cells. They have been purified from nearly all mammalian cell types and body fluids, as well as from lower eukaryotes, prokaryotes and plants. They mainly include microvesicles, released through the budding of the plasma membrane, and exosomes, derived from the endosomal compartment. Extracellular vesicles are referred to as “particles”, “microparticles”, “nanovesicles”, “microvesicles” and “exosomes” [Yáñlez-Mó M, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015 May 14; 4:27066 doi: 10.3402/jev.v4.27066; Lötvall J, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014 Dec. 22; 3:26913. doi: 10.3402/jev.v3.26913.; Harrison P, et al. Extracellular Vesicles in Health and Disease. CRC Press, pages 1-5, 2014]. EVs contain a complex and variable cargo of cytoplasmic proteins, surface receptors, certain lipid-interacting proteins, DNA and RNA molecules. By transferring their cargo, EVs play a key role as mediators of intercellular communication. Edible plant-derived EVs in their native form, not loaded with exogenous molecules, will be herein referred to as “native EVs”. Native EVs are known to be effective for the treatment of leukemia [WO2016166716A1] and colitis [Ju S, et al. Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol Ther. 2013 July; 21(7):1345-57. doi: 10.1038/mt.2013.64.] by oral administration. Native nanovesicles derived from grapes, grapefruit, ginger and carrots have shown anti-inflammatory effects in chronic inflammatory bowel disease [Zhang M, et al. Edible ginger-derived nanoparticles: A novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis-associated cancer. Biomaterials. 2016 September; 101:321-40. doi:10.1016/j.biomaterials.16.06.018; Ju S, et al. Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol Ther. 2013 July; 21(7):1345-57. doi: 10.1038/mt.2013.64]. WO2017/052267 discloses the use of topically administered edible native plant-derived EV to promote skin improvement in terms of wrinkle formation, moisturization, whitening, epithelial cell proliferation and collagen deposition. To the inventors' knowledge, the prior art does not disclose plant-derived EVs effects on angiogenesis and bacteria viability when administered topically on wound and skin lesions characterized by ischemia and impaired angiogenesis, or increased exposition to bacterial infection. Since EVs naturally protect and transfer their cargo to target cells, they represent a useful alternative to synthetic and exogenous particles, such as liposomes, cationic nanoparticles, EV-mimetic nanovesicles and polypeptide-based vesicles to convey therapeutic agents. EVs can exploit their natural mechanism of action and overcome some of the limitations of assembled-particles, including immunogenicity, toxicity, administration of exogenous particles, limited cell uptake and chemical assemblage of particles. In recent years, numerous techniques have been investigated to transfer different molecules (RNAs, DNAs, drugs) into EVs. EV-associated nucleic acids are protected from degrading enzymes present in the microenvironment and could be delivered to target cells. Methods aimed to introduce molecules into EVs include electroporation, sonication, transfection, incubation, cell extrusion, saponin-mediated permeabilization, and freeze-thawing. WO2017/004526A1 discloses the use of microvesicles derived from grapes, grapefruit as carriers for miR18a and miR17 to be used as anticancer drugs, or for tracers to be used for diagnosis. To overcome the limitations and drawbacks of prior art, the present invention provides a composition comprising a population of plant-derived extracellular vesicles (EVs) as well as a method for loading one or more biologically active molecule into the population of plant-derived EVs, as defined in the appended independent claims. The dependent claims identify further advantageous features of the claimed composition and met