Isolated extracellular vesicles and their use

Abstract

The current invention relates to isolated Extracellular Vesicles (EVs) wherein said EVs comprise the miRNA markers hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p.

Description

[0001] ISOLATED EXTRACELLULAR VESICLES AND THEIR USE

[0002] FIELD OF THE INVENTION

[0003] The invention relates to isolated extracellular vesicles (EVs) characterized by the presence of specific microRNA (miRNA) markers. The invention encompasses methods for producing these isolated EVs and their use in the prevention or treatment of various disorders such as skin disorders.

[0004] BACKGROUND

[0005] Extracellular vesicles (EVs) are nano-sized, lipid bilayer-enclosed particles naturally released from cells into the extracellular environment. These vesicles play a critical role in intercellular communication and have emerged as potent therapeutic tools due to their ability to transport bioactive molecules, such as proteins, lipids, and nucleic acids, including microRNAs (miRNAs). EVs are effective in regenerative medicine, immune modulation, and disease treatment.

[0006] MicroRNAs are small, non-coding RNA molecules that regulate gene expression post-transcriptionally, that influence a wide array of cellular processes. Specific miRNA profiles in EVs have been associated with targeted therapeutic outcomes, including tissue repair, modulation of immune responses, and attenuation of pathological processes. However, the variability in miRNA content among EVs has presented challenges in standardizing their therapeutic efficacy. There is thus a need to isolated and identify EVs with enriched specific miRNAs that confer targeted biological activities.

[0007] Skin repair and wound healing present significant challenges in both therapeutic and cosmetic contexts. Chronic wounds, such as diabetic ulcers and pressure sores, often fail to progress through the normal phases of healing due to impaired cellular responses, inflammation, and poor tissue regeneration. Similarly, post-surgical scars and traumatic skin injuries can lead to excessive fibrosis, scar tissue formation, and cosmetic concerns, which significantly impact patient quality of life. From a cosmetic perspective, hyperpigmentation, aging-related skin damage, and scarring remain difficult to manage with current non invasive and no surgical therapies. Addressing these challenges requires innovative approaches that enhance cellular repair, modulate inflammation, and reduce excessive scar formation while maintaining the skin's structural integrity and appearance.EVs are potent in skin repair due to their ability to promote cell proliferation, migration, and extracellular matrix remodeling, which are essential processes in wound healing. Zhang et al., 2021 showed that hypoxic EVs enriched in hsa-miR-125b-5p induce proliferation and migration of endothelial cells. Additionally, Xu et al 2024 showed that adipose-derived stem cell (ADSC) exosomes with high levels of miR-125b-5p speed up wound healing and improve healing quality but also prevent scar formation. EVs are thus promising therapeutics for complex skin conditions, including chronic wounds, diabetic ulcers, and post-surgical scars.

[0008] The know studies focus on EVs enriched in one or at least a few miRNAs. US2020072843 describes cancer cells derived EVs comprising various microRNAs, including miR-125b-5p, miR-199a-3p, miR-199b-3p and miR-16-5p. US2023107241 and W02023082012 disclose stem or somatic cells derived EVs comprising broad and heterogeneous microRNA profiles for medical use. Furuta Taisuke et al., 2016 describes mesenchymal stem cell (MSC)-derived exosomes promoting tissue repair, wherein certain microRNAs such as miR-125b-5p are expressed at higher levels than others. Fang Shuo et al., 2016 discloses umbilical cord-derived MSC EVs for wound healing. However, these documents do not disclose or suggest isolated extracellular vesicles characterized by a defined and concurrent enrichment of the specific microRNA combination described herein for targeted skin repair, wound healing applications or ARDS.

[0009] There remains a need for EVs with high levels of multiple miRNA involved in various pathways involved in several disorders, including wound healing, skin repair and ARDS. The present invention addresses this need by providing isolated EVs enriched with a defined set of miRNAs, including hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, and hsa-miR-16-5p, and optionally additional miRNAs such as hsa-miR-125a-5p, hsa-let-7a-5p, and hsa-miR-29a-3p. These EVs demonstrate enhanced therapeutic potential in treating a variety of conditions, including skin disorders.

[0010] SUMMARY OF THE INVENTION

[0011] The invention relates to isolated extracellular vesicles (EVs) characterized by the presence of specific miRNA markers, namely hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, and hsa-miR-16-5p according to claim 1. Preferred embodimentsof the EVs are shown in any of the claims 2 to 5. The disclosure further pertains to pharmaceutical compositions comprising isolated EVs, according to claim 6.

[0012] The disclosure also relates to the use of the isolated EVs therapeutic, prophylactic, and cosmetic applications according to claim 7. Preferred embodiments of the use related to the treatment of various skin disorders, lung disorders, inflammatory bowel disease, or ocular surface diseases according to claims 8 to 14.

[0013] In a final aspect, the invention relates to methods of producing isolated EVs possessing the specific miRNA markers profile according to claim 15. The method's ability to enrich EVs with specific miRNAs provides a promising approach for improving the effectiveness of treatments in the various medical fields detailed above.

[0014] DESCRIPTION OF FIGURES

[0015] Figure 1: representation of the in vitro scratch assay model of wound healing using epidermal Keratinocytes and the EVs according to the invention.

[0016] Figure 2: Representative pictures of the scratch wound assay from Figure 1 of five different conditions. In each micrograph, dark grey area shows the scratch wound empty space in a keratinocyte monolayer. Each picture at 20h is a picture of the exact same well at Oh. Conditions include, Negative control, Positive control (EGF), and MSC-derived EVs batches were used as test compounds CD007-06-01 (Compound 1), CD009-03-01_02 (Compound 2), CD004-02-15_19 (Compound 3).

[0017] Figure 3: represents relative wound density in human keratinocytes at 0, 4, 12, 16 and 20h, depicted as % in three different experiments. 100% means that the wound is completely closed, while 0 means the wound is completely open. Values depicted as means of 3 technical replicates (wells) ± standard deviation. Conditions include, Negative control (Neg control), positive control (pos control (EGF)), Compound 1 (Cl) corresponds to donor 1 - CD007-06-01; Compound 2 (C2) is donor 2-CD009-03-01_02, and Compound 3 (C3) is donor 3- CD004-02-15_19.Figure 4: representative graphs of relative wound density with time in human keratinocytes from Experiment 1. RWD at 0, 4, 12, 16 and 20h, depicted as %. 100% means that the wound is completely closed, while 0 means the wound is completely open. Ligne graph depicted as means of 3 technical replicates (wells) ± standard deviation. Each compound test includes five conditions, Negative control (Neg control), positive control (pos control (EGF)), and three MSC EVs at concentration i.e 1.0E+7, 1.0E+8 and 1.0E+9 EVs per well. A. Compound 1, B. Compound 2, C. Compound 3.

[0018] Figure 5: represents a graphical overview of the three different experiments (n=3). The bar graph show the relative wound density (RWD) at 4, 8 and 12 hours of 3 different experiments and the standard error of the means. *p <0.05 compared to negative control of the same time point.

[0019] Figure 6: represents the timelines of the clinical study assessing the safety, tolerability and preliminary efficacy of the EVs according to the invention in skin regeneration and repair.

[0020] Figure 7: represents design of the curative model of ARDS experiment using the EVs of the invention. "T" represents hours. Vehicle: PBS.

[0021] Figure 8: represents body temperature readouts in the curative model of ARDS experiment using the EVs of the invention. Groups 5, 7, 9, 11 - controls (LPS only); groups 6, 8, 10, 12 - experimental groups (1st dose of EVs 4E+10 particles / kg coadministered with LPS of indicated concentrations; 2nd dose of EVs 8E+10 particles / kg without LPS). LPS is expected to decrease body temperature. EVs statistically significantly protect over temperature loss after 1 mg / kg LPS challenge. Readout 5h after first dosing.

[0022] Figure 9: represents BAL total protein readouts in the curative model of ARDS experiment using EVs of the invention. Groups 5, 7, 9, 11 - controls (LPS only); groups 6, 8, 10, 12 - experimental groups (1st dose of EVs 4E+10 particles / kg coadministered with LPS of indicated concentrations; 2nd dose of EVs 8E+10 particles / kg without LPS). LPS is expected to increase protein levels in bronchoalveolar lavage (BAL). EVs statistically significantly reduce protein level in BAL of animals after 1 mg / kg LPS challenge. BAL - bronchoalveolar lavage.Figure 10: represents immune system cells in BAL in the curative model of ARDS experiment using the EVs of the invention. Groups 5, 7, 9, 11 - controls (LPS only); groups 6, 8, 10, 12 - experimental groups (1st dose of EVs 4E+10 particles / kg coadministered with LPS of indicated concentration; 2nd dose of EVs 8E+10 particles / kg without LPS). EVs statistically significantly reduce BAL lymphocyte numbers after 0.1 mg / kg LPS challenge. BAL - bronchoalveolar lavage.

[0023] Figure 11: representation of the Dry Eye Disease (DED) study timeline. Four days of artificial tears occurred before the model induction. At day 0, Fluo + Light (F+L) pictures were taken, as well as the Phenol Red Thread Test (FRT) was performed. BAK was applied twice a day from day 0 to 6 to induce the Dry Eye Disease (DED) model; when the treatment 35 started, from day 7 to 14, BAK was applied once a day. F+L pictures were taken on day 0, 3, 5, 7, 10, 12 and 14. FRT was performed on days 0, 7 and 14.

[0024] Figure 12: representation of BAK effect on mouse model of DED. A) Fluorescein staining score (NEI index) on day 7 compared to day 0, indicating clear corneal epitheliopathy progression. B) Tear secretion on day 7 compared to day 0, indicating lower tear secretion.

[0025] Figure 13: representation of tear secretion in BAK-induced DED mouse treated by MSC-derived EVs of the invention (Cl), cytokine-primed MSC-derived EVs (C2) and saline (placebo, C4). Tear secretion was quantified using phenol red test (FRT).

[0026] Figure 14: A) Representative pictures of CD45 staining on cornea samples of mice treated with compounds 1 (MSC-derived EVs of the invention), 2 (cytokine-primed MSCs derived EVs) and 4 (saline). B) Image quantification of CD45+ cells / field showed that Compound 1 significantly reduced CD45+ leukocyte infiltration in the whole cornea.

[0027] Figure 15: A) Corneal sections of mice treated with Compound 1 (MSC-derived EVs of the invention), 2 (cytokines-primed EVs) and 4 (saline) stained with CD31 and LYVE1 are represented. B) Quantification of the positive signal. Compound 1 and 2 did not show significant difference from placebo both with CD31 and LYVE1 staining.DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention concerns isolated extracellular vesicles (EVs) characterized by the presence of specific microRNA (miRNA) and the use of said EVs or compositions comprising said EVs in the treatment of disorders such as skin disorders.

[0029] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0030] As used herein, the following terms have the following meanings:

[0031] "A", "an", and "the" as used herein refer to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0032] "About" as used herein refers to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0033] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0034] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understoodthat the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0035] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0036] The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0037] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0038] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0039] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.For the purpose of the current invention, the terms "extracellular vesicles" or "EVs" are interchangeable and is to be understood as micro or nano-meter-sized particles secreted by different types of cells in vivo and in vitro, often with proteins associated with said EVs. EVs comprise proteins, growth factors, miRNA and other molecules encapsulated in a lipid bi-layer sphere. EVs can be classified according to their size and intracellular origin.

[0040] Exosomes are a subgroup of EVs that are typically 0.1 microns or smaller in diameter. Exosomes are derived from multivesicular bodies, a late endosomal compartment, which are secreted via the fusion of multivesicular bodies with the plasma membrane. Another type of EV is the shedding vesicles (also known as microvesicles) which are a heterogenous population of membrane vesicles up to 1 micron in size directly released from the cell membrane through the disruption of the cortical cytoskeleton. All types of vesicles secreted by cells are defined in general as EVs.

[0041] The term "microneedling" refers in the present invention to a dermal treatment technique utilizing a device equipped with multiple tiny needles to puncture the skin surface, thereby enhancing the absorption of topical treatments and promoting skin rejuvenation. Microneedling also creates micro-injuries in the skin that stimulate collagen and elastin production.

[0042] The term "wrinkles" refers in the present invention to any visible lines or creases on the skin surface, which may result from ageing, sun damage, or repetitive facial expressions, and which can affect the texture and appearance of the skin. Wrinkles are a hallmark of skin ageing, influenced by a combination of extrinsic factors (such as ultraviolet (UV) exposure, air pollution, smoking, and nutritional deficiencies) and intrinsic factors, often referred to as chronological ageing. These factors contribute to a decline in skin functionality and regenerative potential. Intrinsic ageing typically manifests as smooth, pale skin with fine wrinkles, diminished elasticity, and increased dryness.

[0043] The term "scars" refers in the present invention to the fibrous tissue that replaces normal skin after an injury or wound has healed. Scars result from the natural healing and reparative processes following dermal inflammation and fibrotic scar formation. They can be classified based on their clinical and histological appearance into various types, including keloid, hypertrophic, stretched, and atrophic scars. Atrophic scars, which are depressed areas of skin caused by trauma or surgicalprocedures, are characterized by a loss of tissue. Histologically, these scars display reduced collagen and elastic fibres, decreased vascularity, lower fibroblast activity, and a thinner epidermis, resulting in a smooth, sunken appearance with colour variations compared to surrounding skin characterized by a different texture and appearance compared to the surrounding skin.

[0044] The term "cell medium", "cell culture medium" or "medium" refers to an aqueous solution of nutrients and other components of defined composition, which can be used for maintenance or growth of cells.

[0045] The term "pharmaceutically acceptable carrier" as used herein, refers to a carrier or a diluent that does not cause significant irritation to a human subject and does not abrogate the biological activity and properties of the administered composition. As an example, the pharmaceutically acceptable carrier may act as a stabilizer and / or as an adjuvant. Examples, without limitations, of carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with / or water.

[0046] The term "sufficient amount" means an amount sufficient to produce a desired and measurable effect, e.g., an amount sufficient to alter a protein expression profile.

[0047] The term "therapeutically effective amount" is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy.

[0048] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. The term "treating" refers to reversing, preventing, alleviating or inhibiting the progress of a disease, disorder or condition, or one or more symptoms of a disease, disorder or condition. As used herein, "treating" may also refer to decreasing the probability or incidence of the occurrence of a disease, disorder or condition in a mammal as compared to an untreated control population, or as compared to the same mammal prior to treatment. For example, as used herein, "treating" may refer to preventing a disease, disorder or condition, and may include delaying or preventing the onset of a disease, disorder or condition, or delaying or preventing the symptoms of a disease, disorder or condition. As used herein, "treating" may also refer to reducing the severity of a disease, disorder orcondition or symptoms of such disease, disorder or condition prior to affliction with the disease, disorder or condition. Such prevention or reduction of the severity of a disease, disorder or condition prior to affliction relates to the administration of the composition of the present technology, as described herein, to a human subject that is not at the time of administration afflicted with the disease, disorder or condition. As used herein "treating" may also further refer to preventing the recurrence of a disease, disorder or condition or of one or more symptoms of such disease, disorder or condition. The terms "therapy," "treatment," and "therapeutically," as used herein, refer to the act of treating as defined above.

[0049] The terms "composition" and "pharmaceutical composition" are herein used interchangeably, and refer to compositions at any stage of the manufacturing process, including the final pharmaceutically acceptable product and any in-process intermediates thereof.

[0050] For the purpose of the current invention, the terms "mesenchymal stem cell" and "mesenchymal stromal cell" or "MSCs" are herein used interchangeably, and it is to be understood as meaning stromal adherent cells able to differentiate into various cell types. Sources of MSCs are typically from bone marrow, cord cells, adipose tissue, amniotic fluid, mammary glands, or blood.

[0051] Extracellular vesicles

[0052] The current disclosure relates to isolated Extracellular Vesicles (EVs) wherein said EVs comprise a specific miRNA marker profile. In an embodiment, said EVs comprise at least one miRNA marker selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p.

[0053] In another or further embodiment, EVs comprise hsa-miR-125b-5p and at least one other miRNA chosen from hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p andhsa-miR-221-3p, wherein the level of hsa-miR-125b-5p in said EVs is at least 3.5 higher than the individual levels of said other miRNA.In an embodiment, said hsa-miR-125b-5p levels are between 3.5 and 15 higher, between 3.5 and 14 higher, between 3.5 and 13 higher, between 3.5 and 12 higher, between 3.5 and 11 higher, between 3.5 and 10 higher, between 3.5 and 9 higher, between 3.5 and 9 higher, between 3.5 and 8 higher, between 3.5 and 7 higher, between 3.5 and 6 higher, between 3.5 and 5 higher, or between 3.5 and 4 higher than the individual levels of said other miRNA. Alternatively, said hsa-miR-125b-5p levels are between 4 and 15 higher, between 5 and 15 higher, between 6 and 15 higher, between 7 and 15 higher, between 8 and 15 higher, between 9 and 15 higher, between 10 and 15 higher, between 11 and 15 higher, between 12 and 15 higher, between 13 and 15 higher, or between 14 and 15 higher than the individual levels of said other miRNA. In yet another alternative embodiment said hsa-miR-125b-5p levels are between 4 and 14 higher, between 5 and 13 higher, between 6 and 12 higher, between 7 and 11 higher, between 8 and 10 higher, or between 7 and 9 higher than the individual levels of said other miRNA.

[0054] In an embodiment, said EVs comprise at least 2 or 3 miRNA markers selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p. In an embodiment, the EVs comprise hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p.

[0055] In an embodiment, the EVs comprise at least one, preferably all, miRNA markers selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p and further comprise one or more miRNAs selected from the group consisting of hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p.

[0056] In an embodiment, said EVs comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or all miRNAs selected from the group consisting of hsa-miR-125a-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p.

[0057] In an embodiment of the isolated EVs as disclosed herein, said EVs comprise at least one miRNA marker selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p. In an embodiment, said EVscomprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or all markers selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p.

[0058] The EVs of the invention comprise high levels of hsa-miR-125b-5p, especially compared to other miRNAs present.

[0059] In some embodiments, the level of hsa-miR-125b-5p in said EVs is 3.5 to 15 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p hsa-let-7a-5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

[0060] In an embodiment, the level of hsa-miR-125b-5p in said EVs is 3.5 to 6 fold higher, 3.8 to 4-fold higher, 4 to 5.5 fold higher, 4.5 to 5 fold higher, or 5 to 5.7 than the individual levels of hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, and / or hsa-let-7a-5p.

[0061] In an embodiment of the isolated EVs as disclosed herein the level of hsa-miR-125b-5p in said EVs is 6.5 fold to 15 fold higher than the individual level of miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and / or hsa-miR-221-3p. In yet an embodiment, the level of hsa-miR-125b-5p is 7.4 to 14.4 fold higher, 7 to 14 fold higher, 8 to 13 fold higher, 9 to 12 fold higher, 9 to 10 fold, 9 to 11 fold, 10 to 11 fold, 10 to 12 fold higher or 10 to 14.4 fold higher than the individual levels of miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

[0062] The individual level of miRNA of the EVs as disclosed herein is determined using any suitable method known in the art. A non-limiting method for establishing the level of miRNAs in EVs starts with EVs RNA isolation using appropriate RNA extraction kits, followed by quantification using fluorescence-based methods, such as Ribogreen assays, and quality assessment using bioanalyzers. For profiling and quantification, small RNA sequencing may be employed which comprises the preparation of RNA libraries, sequencing, and subsequent bioinformatics analysis to map and normalize the miRNA reads. The level of miRNA is typically expressed in terms of sequencing reads, which represent the abundance of each miRNA in the sample. Alternatively,the miRNA level may be determined using methods known in the art such as quantitative real-time PCR (qRT-PCR), microarray-based analysis, northern blotting, droplet digital PCR (ddPCR) or NanoString technology.

[0063] The inventors have found that the EVs as disclosed herein, having a particular miRNAs profile which are present at specific levels, promote wound healing and skin repair. Skin repair as used throughout the disclosure is a complex biological process that restores the integrity of damaged skin tissue after a break in the skin caused by injury, surgery, or disease but includes restoration after other forms of damage, such as ageing, hyperpigmentation, scarring, or cosmetic imperfections. Skin repair encompasses aesthetic concerns and functional recovery. It consists of four overlapping phases: hemostasis, inflammation, proliferation, and remodelling. During hemostasis, platelet activation leads to blood clot formation, which provides a temporary barrier and releases growth factors. The inflammatory phase involves immune cell recruitment, which clears pathogens and debris while signalling the start of tissue repair. In the proliferation phase, keratinocytes, fibroblasts, and endothelial cells promote tissue regeneration through re-epithelialization, extracellular matrix deposition, and angiogenesis. Finally, during remodelling, collagen synthesis and tissue contraction restore the skin's tensile strength and structural integrity.

[0064] Wound healing as used through the disclosure is a subtype of a skin repair mechanism that restored the integrity of damaged skin tissue after a break in the skin caused by injury, surgery, or disease.

[0065] Cosmetic skin repair as used throughout the disclosure is a subtype of a skin repair mechanism that restores the integrity of skin that shows signs of cosmetic damage such as ageing, hyperpigmentation, scarring or other cosmetic imperfections.

[0066] The EVs disclosed herein promote wound healing and cosmetic skin repair by significantly decreasing the time of at least one of the skin repair phases, preferably by decreasing the time proliferation phase. In other embodiments, the EVs decrease the time of both the proliferation phase and the repair phase. In yet another embodiment, the EVs decrease the time of inflammation, proliferation, and remodelling phase. In other embodiments, the EVs decrease the time for: hemostasis, inflammation, proliferation, and remodeling.

[0067] The inventors also found that the EVs of the invention with the specific miRNAs profile have strong anti-inflammatory activity in the lungs promoting epithelial repairand reducing fibrosis, thereby improving pulmonary function. The EVs of the invention target intestinal inflammation, promote mucosal healing and restore the integrity of the intestinal barrier. The EVs disclosed herein are promising therapeutics for inflammatory diseases, including bronchopulmonary dysplasia (BPD) and Crohn's disease.

[0068] The isolated EVs as disclosed herein are isolated from MSCs (Mesenchymal Stem Cells). Said MSCs are human-derived and may originate from bone marrow, umbilical cord, Wharton's jelly, cord blood, amniotic membrane, bone marrow, adipose tissue, dental pulp, peripheral blood, fallopian tube, mammary gland, liver and lung tissue. In a preferred embodiment, said MSCs are derived from umbilical cord (UC-MSCs).

[0069] In an embodiment, the isolated EVs have a size below 1 pm. In a preferred embodiment, the EVs have a size of about 750 nm, preferably below 500 nm, preferably below 400 nm, preferably below 300 nm. In another or further preferred embodiment, the EVs have a size of at least 5 nm, more preferably at least 10 nm, more preferably at least 25 nm, or more preferably at least 50 nm. In another or further preferred embodiment, the EVs have a size of between 25 and 500 nm, preferably between 25 and 400 nm, or more preferably between about 50 and about 300 nm. In a preferred embodiment, said EVs have a mean particle size of below 120 nm, such as between 95 and 110 nm.

[0070] In an embodiment, the isolated EVs as described are associated with albumin and / or Annexin V.

[0071] In an embodiment, the isolated EVs as disclosed herein are positive for CD44 and CD29. In a further embodiment, the isolated EVs as disclosed herein are further positive for one or more markers chosen from CD105, CD9, CD63, CD81, SSEA4, HLA1, CD49e or MSCP.

[0072] In an embodiment, the isolated EVs as disclosed herein are negative for one or more markers selected from CDllb, CD14, CD34, CD45, or HLA-DR.

[0073] In an embodiment of the isolated EVs as disclosed herein, said EVs are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.Pharmaceutical compositions, use and administration

[0074] The current disclosure also relates to a pharmaceutical composition comprising a therapeutically effective amount of isolated EVs. The pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, and compatible carriers, and may optionally comprise other (i.e., secondary) therapeutic agents.

[0075] A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a prophylactically or therapeutically active agent. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethene glycol; esters, such as ethyl oleate and ethyl laurate; buffering agents, such as magnesium hydroxide and aluminium hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0076] The pharmaceutical composition comprising isolated EVs as disclosed herein is formulated for intravenous, intranasal, intratracheal, oral, subcutaneous, intramuscular, cutaneous or transdermal, preferably cutaneous administration to a patient.

[0077] In some embodiments of the pharmaceutical composition, said EVs are loaded with and / or said pharmaceutical composition is formulated with further molecules, proteins, and / or substances that have therapeutic or cosmetic properties. Non limiting examples of such further molecules include nucleic acids such as DNA or RNA, peptides, vitamins, such as vitamin A, B3, C or E and / or derivatives thereof such as retinoic acid, retinol, retinaldehyde, or niacinamide preferably vitamin C. In some embodiments said EVs may be loaded with or said pharmaceutical composition may be formulated with organic acids such as glycolic acid, salicylic acid, lactic acid or ferulic acid.In an embodiment of the pharmaceutical composition as disclosed herein, said pharmaceutical composition is formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

[0078] Indications

[0079] The disclosure also relates to the use (e.g., administered) of the isolated EVs or pharmaceutical compositions comprising EVs, for therapeutic or prophylacticuse.

[0080] The disclosure also relates to methods for cosmetic treatment or prevention of wrinkles, scars, and / or hyperpigmentation, said method comprises a step of providing the EVs or the pharmaceutical composition as disclosed herein to a patient, in need thereof, said providing preferably comprises

[0081] The EVs as disclosed herein being involved in wound healing and cosmetic skin repair are suitable for both therapeutic and cosmetic administration.

[0082] In some embodiments, the isolated EVs or pharmaceutical composition are used in the prevention or treatment of skin disorders. In some embodiments said skin disorders are selected from skin wounds, skin hyperreactivity with hyperkeratosis, skin pigmentation disorders, and atrophic scars.

[0083] In a further embodiment, the isolated EVs or the pharmaceutical composition as disclosed herein are used for treating diabetic foot, acute, scars post debridement, scleroderma, psoriasis, Vitiligo, melasma, traumatic scars and surgical scars.

[0084] The EVs and the compositions comprising said EVs of the inventions are highly effective in skin repair such as wound healing and cosmetic skin repair. Due to the acceleration of the skin repair process, the EVs as disclosed herein are effective in treating skin disorders and cosmetic conditions that benefit from the acceleration of any one or more phases selected from hemostasis, inflammation, proliferation, and remodelling of the skin.

[0085] In addition, the EVs enhance collagen synthesis and deposition by activating fibroblasts, which improves the tensile strength and elasticity of the skin. This makes the EVs disclosed herein suitable in methods of treating wrinkles, atrophic scars, and other structural defects caused by ageing or trauma.The EVs disclosed herein also contribute to the regulation of melanin production by modulating melanocyte activity, making them suitable for treating skin pigmentation disorders such as melasma and Vitiligo.

[0086] The anti-inflammatory properties of the EVs as disclosed herein reduce chronic inflammation, rendering them suitable for treating conditions such as psoriasis and skin hyperreactivity with hyperkeratosis.

[0087] In another embodiment, the EVs or pharmaceutical compositions as disclosed herein are used in the prevention or treatment of lung disorders, inflammatory bowel disease, or ocular surface disease. Due to their specific miRNA profile and levels, they have strong anti-inflammatory properties suited for the treatment of these diseases.

[0088] In an embodiment, the EVs or the compositions comprising said EVs are used in the prevention or treatment of lung disorders. In an embodiment, said lung disorder may be an inflammatory lung disease, lung vascular disease, or acute lung injury. More preferably, said inflammatory lung disease is pulmonary hypertension which is also referred to as pulmonary artery hypertension (PAH), asthma, bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), allergy, idiopathic pulmonary fibrosis or pneumonia. Said inflammatory lung disease may be caused by an infection, such as a viral infection. In another embodiment, said acute lung injury is linked with sepsis or acute respiratory distress syndrome (ARDS).

[0089] These diseases also include lung vascular diseases which may not have an inflammatory component. Still, other pulmonary conditions that may be treated according to the invention include acute lung injury which may be linked with sepsis or with ventilation. An example of this latter condition is acute respiratory distress syndrome.

[0090] Pulmonary hypertension is a lung disease characterized by blood pressure in the pulmonary artery that is far above normal levels. Symptoms include shortness of breath, chest pain particularly during physical activity, weakness, fatigue, fainting, lightheadedness particularly during exercise, dizziness, abnormal heart sounds and murmurs, engorgement of the jugular vein, retention of fluid in the abdomen, legs and ankles, and bluish colouring in the nail bed.Bronchopulmonary dysplasia is a condition that afflicts neonates who have been given oxygen or have been on ventilators, or neonates born prematurely particularly those born very prematurely (e.g., those born before 32 weeks of gestation). It is also referred to as neonatal chronic lung disease. Causes of BPD include mechanical injury for example as a result of ventilation, oxygen toxicity for example as a result of oxygen therapy, and infection. The disease may progress from non-inflammatory to inflammatory with time. Symptoms include bluish skin, chronic cough, rapid breathing, and shortness of breath. Subjects having BPD are more susceptible to infections such as respiratory syncytial virus infection. Subjects having BPD may develop pulmonary hypertension.

[0091] Acute respiratory distress syndrome (ARDS), also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome is a condition that arises as a result of injury to the lungs or acute illness. The injury to the lung may be a result of ventilation, trauma, burns, and / or aspiration. The acute illness may be infectious pneumonia or sepsis. It is considered a severe form of acute lung injury, and it is often fatal. It is characterized by lung inflammation, impaired gas exchange, and release of inflammatory mediators, hypoxemia, and multiple organ failure. ARDS can also be defined as the ratio of arterial partial oxygen tension (PaO?) as a fraction of inspired oxygen (FiOz) below 200 mmHg in the presence of bilateral infiltrates on the chest x-ray. A PaO2 / FiO2 ratio less than 300 mmHg with bilateral infiltrates indicates acute lung injury, which is often a precursor to ARDS. Symptoms of ARDS include shortness of breath, tachypnea, and mental confusion due to low oxygen levels.

[0092] Idiopathic pulmonary fibrosis is characterized by scarring or thickening of the lungs without a known cause. It occurs most often in persons 50-70 years of age. Its symptoms include shortness of breath, regular cough (typically a dry cough), chest pain, and decreased activity level.

[0093] The subjects may be those that have a lung disease (or condition) amenable to treatment using the EVs of the invention, or they may be those that are at risk of developing such a disease (or condition). Such subjects include neonates and particularly neonates born at low gestational age. As used herein, a human neonate refers to a human from the time of birth to about 4 weeks of age. As used herein, a human infant refers to a human from about the age of 4 weeks of age to about 3 years of age. As used herein, low gestational age refers to birth (or delivery) that occurs before a normal gestational term for a given species. In humans, a fullgestational term is about 40 weeks and may range from 37 weeks to more than 40 weeks. Low gestational age, in humans, akin to a premature birth is defined as birth that occurs before 37 weeks of gestation. The invention therefore contemplates prevention and / or treatment of subjects born before 37 weeks of gestation, including those born at even shorter gestational terms (e.g., before 36, before 35, before 34, before 33, before 32, before 31, before 30, before 29, before 28, before 27, before 26, or before 25 weeks of gestation). Typically such premature infants will be treated as neonates, however, the invention contemplates their treatment even beyond the neonate stage and into childhood and / or adulthood. Certain subjects may have a genetic predisposition to certain forms of lung disease such as for example pulmonary hypertension, and those subjects may also be treated according to the invention.

[0094] The disclosure further contemplates the administration of EVs even in the absence of symptoms indicative of a lung disease such as but not limited to BPD.

[0095] The EVs target multiple mechanisms of lung injury including hyper-inflammation and cytokine storm, fibrosis, oxidative stress caused by (mechanical) ventilation and epithelial cell apoptosis due to viral activity and inflammatory reaction.

[0096] In another embodiment, said composition according to any of the embodiments above is used in the prevention or treatment of inflammatory bowel diseases (IBD) such as Crohn's Disease or ulcerative colitis. IBDs are a collection of diseases that result in an inflammation of the gastrointestinal (GI) tract, and the two most common IBDs are ulcerative colitis (UC) and Crohn's disease (CD). UC is a disease that causes long-lasting inflammation and sores in the innermost lining of the colon and rectum. CD can develop anywhere in the digestive tract and can penetrate into the deep layers of the affected tissue. A symptom of CD is the development of perianal fistulas. The diseases are similar, however, in that, both can cause abdominal pain, severe diarrhea, fatigue and weight loss.

[0097] In other embodiments, the EVs or compositions as disclosed herein are used for the treatment of patients who suffer from an Ocular Surface Disease (OSD). Ocular surface diseases are conditions that affect and damage the surface layers of the eyes. The parts of the eye most affected by OSDs are the cornea, the conjunctiva, and the glandular network.In an embodiment, said OSD can be Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction. In an embodiment, the patient has undergone at least one prior treatment for ocular surface disease. Said prior treatment could be chosen from eye drops or artificial tears, corticosteroids, NSAIDS, anti-VEGF therapy, or surgical corneal transplantation. In an embodiment, said prior treatment failed or did not provide long-lasting results.

[0098] In some embodiments, the EVs used for the treatment and prevention of OSD are isolated from MSCs that are primed with one or more cytokines. The term "primed" or "primed MSCs," as used herein, refers to the intentional activation of mesenchymal stem cells (MSCs) through exposure to specific cytokines. This process results in changes to the functional properties of the MSCs and alters the characteristics of the extracellular vesicles (EVs) they produce.

[0099] In an embodiment, the concentration of each cytokine may be between 10 and 30 ng / ml, such as 20 ng / ml or 25 ng / ml. In some embodiments, said MSCs are primed with one or more cytokines chosen from TNFa, ILip, IL6, or combinations thereof. In an embodiment, said EVs are primed with a cocktail of TNFa, ILip and IL6. In an embodiment, said cocktail comprises between 10 and 30 ng / ml TNFa, between 10 and 30 ng / ml ILip and / or between 10 and 30 ng / ml IL6.

[0100] In an embodiment, the EVs or the compositions comprising said EVs are used in the prevention or treatment of infertility or symptoms thereof in a subject. Preferably, the composition is used for the treatment of female infertility. Female infertility can result from genetic conditions, gonadotoxic chemotherapeutics, environmental and lifestyle exposures (e.g., smoking, pollutants), natural aging processes and iatrogenic causes.

[0101] In a preferred embodiment, said infertility is endometriosis-associated infertility or symptoms thereof. In some embodiments, the subject has a history of infertility. In some embodiments, the subject is from 21 to 50 years of age. In some embodiments, the subject has a history of one or more failed in vitro fertilization (IVF) cycles. In some embodiments, the subject with a history of infertility may have a lower level of anti-Mullerian hormone as compared to a subject without a history of infertility. In some embodiments, the subject is not pregnant. In some embodiments, the subject is not menopausal. In some embodiments, the subject is not post-menopausal. In some embodiments, the subject has one or more of: ovulatory disorders (such as polycystic ovary syndrome (PCOS), premature ovarianfailure (POF), ovarian insufficiency or hormonal imbalances), endometriosis, a thyroid disorder, a blocked or damaged fallopian tube (such as caused by pelvic inflammatory disease (PID), past infections, or surgeries) , pelvic inflammatory disease (PID), a high follicle-stimulating hormone (FSH) level, a low estrogen level, a fibroid, a polyp, an autoimmune disorder, a uterine abnormality (such as structural problems, fibroids, polyps, or scarring inside the uterus such as Asherman's syndrome or thin endometrium), a congenital uterine abnormality, a sexually transmitted infection (STI), a hormonal imbalance, a vulvo vaginal atrophy and / or one or more failed repetitive IVF cycles (such as recurrent implantation failure). In some embodiments, a hormonal imbalance may comprise a prolactin imbalance, a cortisol imbalance, and / or a gonadotropin-releasing hormone (GnRH) deficiency. In some embodiments, the subject may not have cancer. In some embodiments, symptoms of infertility may comprise but are not limited to inability to conceive, irregular menstrual periods, absent menstrual periods, painful menstrual periods, cramping, heavy or prolonged menstrual bleeding, endometriosis, hormonal fluctuations, mood disturbances, sudden fluctuations in weight, sleep disturbances, and fatigue.

[0102] Formulation

[0103] The EVs or pharmaceutical composition for use in skin disorders or in methods of cosmetic treatments are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum. These formulations are specifically designed to ensure that EVs are accessible to the wound site or penetrate the dermis effectively. For example, creams, gels, and serums provide a medium for gradual and targeted absorption at the epidermal level, while sprays and patches ensure even distribution and prolonged contact with the skin. These delivery methods allow the therapeutic EVs to reach deeper layers of the skin, enhancing collagen production, promoting epidermal restructuring, and facilitating the healing of scars, wrinkles, and pigmentation disorders. These formulations may include carriers and additives that enhance the absorption of EVs into the skin and ensure their stability. Stabilizing agents such as antioxidants, cryoprotectants, or surfactants help preserve the structural integrity and bioactivity of EVs during storage and application. Said composition may also be formulated to be used externally, alone or in combination with hydrogels, polymers or polymer medical devices for slow release of EVs.In some embodiments the of the use of said EVs in skin disorders treatment or in methods of cosmetic treatment, said EVs are loaded with and / or associated with further molecules, proteins, and / or substances that have therapeutic or cosmetic properties. Non limiting examples of such further molecules include nucleic acids such as DNA or RNA, peptides, vitamins, such as vitamin A, B3, C or E and / or derivatives thereof such as retinoic acid, retinol, retinaldehyde, or niacinamide preferably vitamin C. In some embodiments said EVs may be loaded with organic acids such as glycolic acid, salicylic acid, lactic acid or ferulic acid.

[0104] The EVs or compositions for use in the treatment of lung disorders or inflammatory bowel disease may take such forms as water-soluble suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase solubility. Alternatively, the exosomes may be in lyophilized or other powder or solid form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0105] The EVs or compositions comprising said EVs, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, including for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative.

[0106] In some embodiments of the invention disclosed herein, said EVs or the pharmaceutical composition are formulated for topical delivery to the eye, preferably to the conjunctival sac. The compositions may take such forms as water-soluble suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase solubility. Alternatively, the MSC-derived EVs or exosomes may be frozen or in lyophilized orother powder or solid form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0107] Routes of administration

[0108] The isolated EVs or pharmaceutical composition for use in skin disorders or in methods of cosmetic treatments is applied topically to the surface of the skin. In a further embodiment, the isolated EVs or pharmaceutical composition is applied by intradermal injection. In some embodiments, the isolated EVs or pharmaceutical composition comprising said EVs are administered subsequent to microneedling.

[0109] Microneedling has gained traction as an effective treatment for atrophic scars and wrinkles, leveraging micro-injuries to stimulate collagen production and improve skin texture. Several studies have demonstrated that microneedling enhances skin rejuvenation, resulting in improved wrinkles and restoration of skin thickness and firmness. Given the commonality of atrophic scars and wrinkles as aesthetic concerns, which can significantly impact an individual's quality of life, traditional treatments often yield limited results. However, microneedling's ability to promote skin remodelling through collagen induction has garnered attention. Exosome therapy, a cutting-edge technique in regenerative medicine, shows promise in enhancing healing and tissue regeneration. The combination of EVs of the present invention with microneedling and intradermal injections improves collagen deposition, thereby enhancing scar appearance and achieving greater structural integrity, resilience, and elasticity in the skin.

[0110] In the case of administration of EVs in the context of a lung disease, the preferred way of administration will be intratracheal infusion or inhalation. In the context of neurology diseases, this will preferably be a systemic infusion, intranasal infusion or inhalation. In the context of Crohn's disease fistulas, ulcers or cartilage repair, this will preferably be via local injection(s). In the case of treatment of wound healing, burns and / or ulcers, the EVs will preferably be administered at the external side of the body, preferably in combination with hydrogels or polymer medical devices for slow release of EVs.

[0111] The EVs or compositions comprising said EVs may be administered by any route that ensures the delivery to the lungs or the gastrointestinal tract. Systemic administration routes such as intravenous bolus injection or continuous infusion are suitable. More direct routes such as intranasal administration, intratrachealadministration (e.g., via intubation), and inhalation (e.g., via an aerosol through the mouth or nose) are also contemplated by the invention and in some instances may be more appropriate particularly where rapid action is necessary. As used herein, an aerosol is a suspension of liquid dispersed as small particles in a gas, and it includes a fine mist or a spray containing such particles. As used herein, aerosolization is the process of producing of an aerosol by transforming a liquid suspension into small particles or droplets. This may be done using an aerosol delivery system such as a pressurized pack or a nebulizer. Nebulizers include air-jet (i.e., pneumatic), ultrasonic, and vibrating-mesh nebulizers, for example with the use of a suitable propellant such as but not limited to dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In addition to nebulizers, other devices for pulmonary delivery include but are not limited to metered dose inhalers (MDIs) and dry powder inhalers (DPIs). Capsules and cartridges of for example gelatin for use in an inhaler or insufflator may be formulated containing lyophilized exosomes and a suitable powder base such as lactose or starch.

[0112] Frequency of administration

[0113] In an embodiment, the isolated EVs or pharmaceutical composition as disclosed herein, are administered one time or repeatedly.

[0114] The disclosure also contemplates repeated administration of EVs, including two, three, four, five or more administrations. In some instances, said EVs may be administered continuously. Repeated or continuous administration may occur over a period of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18, or 1-24 hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6 days, or 1-7 days) or several weeks (e.g., 1-2 weeks, 1-3 weeks, or 1-4 weeks) depending on the severity of the condition being treated. If administration is repeated but not continuous, the time in between administrations may be hours (e.g., 4 hours, 6 hours, or 12 hours), days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days), or weeks (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks). The time between administrations may be the same or they may differ. As an example, if the symptoms of the disease appear to be worsening the EVs may be administered more frequently, and then once the symptoms are stabilized or diminishing the EVs may be administered less frequently. In an embodiment, administration is once or twice a day.In some embodiments, the isolated EVs or pharmaceutical composition for use in skin disorders or in methods of cosmetic treatment as disclosed herein are administered at least 3 times. In another embodiment, said EVs or pharmaceutical composition are administered multiple times, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0115] In an embodiment said EVs or pharmaceutical composition for use in the treatment of skin disorders or in methods of cosmetic treatment are administered at least once every 3 weeks. In other embodiments said EVs or pharmaceutical composition are administered three times a day, twice a day, daily, every other day, every 5 days, weekly, every 10 days, every 2 weeks, or monthly. In some embodiments, said EVs or pharmaceutical composition are administered once every 3, 4, 5, 6, 7, 8, 9 or 10 weeks.

[0116] With respect to treating BPD in neonates and particularly low gestation age neonates, the invention contemplates administration of EVs within 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours, or 1 hour of birth. In some important instances, the MSC exosomes are administered within 1 hour of birth.

[0117] In an embodiment of the use for therapeutic, prophylactic or cosmetic use, at least two doses of isolated EVs or composition comprising said EVs are administered with a window of at least 1 to 24 hours between each administration. In other embodiments, 2 or 3 doses of isolated MSC-derived EVs or composition comprising said EVs are delivered. In some embodiments, more than 3 doses are administered such as 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses, 13 doses, 14 doses, 15 doses, 16 doses, 17 doses, 18 doses, 19 doses, 20 doses, 25 doses, 30 doses, 35 doses, 40 doses, 45 doses, or 50 doses. The number of administrated doses depends on the condition of the patient and / or on the mode of administration.

[0118] In some instances of the use for therapeutic, prophylactic or cosmetic use, repeated intravenous administration of low doses of EVs may occur. Accordingly, the disclosure contemplates repeated administration of low-dosage forms of EVs as well as single administrations of high-dosage forms of EVs. Low dosage forms may range from, without limitation, 1010- 1011EVs per kilogram or per local infusion, while high dosage forms may range from, without limitation, 1011- 1012per kilogram or per local infusion. It will be understood that, depending on the severity of thedisease, the health of the subject, and the route of administration, inter alia, the single or repeated administration of low or high-dose EVs is contemplated.

[0119] Dose

[0120] The isolated EVs or pharmaceutical compositions as disclosed herein, for use in skin disorders or in methods of cosmetic treatment, are administered at a dose of between 1.2x 109EVs / mL and 1.2x 1012EVs / mL. In some embodiments, the isolated EVs or pharmaceutical composition are applied at a dose of between 1.2x 109EVs / mL and 1.2X1011EVs / mL, between 1.2xl09EVs / mL and 1.2xlO10EVs / mL, between 1.2x IO10EVs / mL and 1.2x 1012EVs / mL, or between 1.2x 1011EVs / mL and 1.2x 1012EVs / mL.

[0121] The isolated EVs or pharmaceutical composition as disclosed herein are administered at a dose of between 20 pl and 50 pl per cm2of skin or between 30 pl and 40 pl per cm2of skin. In some embodiments, the isolated EVs or pharmaceutical composition are applied at a dose of between 30 pl and 50 pl per cm2of skin, between 30 pl and 45 pl per cm2of skin, between 30 pl and 40 pl per cm2of skin, or between 30 pl and 35 pl per cm2of skin. Alternatively, the isolated EVs or pharmaceutical composition are applied at a dose of between 20 pl and 40 pl per cm2of skin, between 25 pl and 40 pl per cm2of skin, or between 35 pl and 40 pl per cm2of skin.

[0122] The EVs or compositions for use in treating lung disorders, inflammatory bowel disease or infertility, as disclosed herein, are administered at a dose of 109EVs / kg to 1012EVs / kg of said patient, or at a dose of 1010EVs / kg to 1012EVs / kg of said patient. In a further embodiment, dosage ranges from 109EVs / kg to 1011EVs / kg in children (from the age of 0 months to 12 years). In teenagers and adults, said dose may range from 109 EVs / kg to 1012 EVs / kg in adults.

[0123] In an embodiment of the current invention, said EVs are administered at a dose of about 1010to about 1012EVs per patient for the treatment of CD perianal fistulas.

[0124] In the embodiments where the EVs or pharmaceutical composition comprising said EVs are used in the treatment of OSD, said EVs or pharmaceutical composition are administered in a volume of between about 0.010 ml to about 2 ml, eg by one or more eye drops, such as two eye drops, of between 0.010 to 0.070 ml.The EVs or pharmaceutical compositions comprising said EVs for use in the treatment of OSD, may be administered with one or more secondary therapeutic agents. As used herein, a therapeutic agent refers to any agent which can be used in the treatment of ocular surface disease or of one of its underlying causes.

[0125] The EVs or composition comprising said EVs for use in the treatment of OSD are administered at a single dose of between 1.2 x 107and 1.2 x 1012EVs / eye, between 1.2 x 108and 1.2 x 1011EVs / eye, or between 1.2 x 109and 1.2 x 1010EVs / eye. Alternatively, said MSC-derived EVs or composition comprising said EVs are administered at a single dose of between 1.2 x 107and 1.2 x 1011EVs / eye, between 1.2 x 107and 1.2 x IO10EVs / eye, between 1.2 x 107and 1.2 x 109EVs / eye, or between 1.2 x 107and 1.2 x 108EVs / eye. In yet another alternative embodiment, said MSC-derived EVs or composition comprising said EVs are administered at a single dose of between 1.2 x 108and 1.2 x 1012EVs / eye, between 1.2 x 109and 1.2 x 1012EVs / eye, between 1.2 x IO10and 1.2 x 1012EVs / eye, or between 1.2 x 1011and 1.2 x 1012EVs / eye.

[0126] In yet another or further embodiment, said EVs or composition comprising said EVs are administered to both eyes at a daily total dose of between 7.2 x 107and 7.2 x 1012EVs for both eyes, between 7.2 x 108and 7.2 x 1011EVs for both eyes or 7.2 x 109and 7.2 x 1010EVs for both eyes. The total dose is administered over three administrations per day. Alternatively, said MSC-derived EVs or composition comprising said EVs are administered to both eyes at a daily total dose of between 7.2 x 107and 7.2 x 1011EVs for both eyes, between 7.2 x 107and 7.2 x 1010EVs for both eyes, between 7.2 x 107and 7.2 x 109EVs for both eyes or between 7.2 x 107and 7.2 x 108for both eyes. In yet another alternative embodiment, said MSC-derived EVs or composition comprising said EVs are administered to both eyes at a daily total dose of between 7.2 x 108and 7.2 x 1012EVs for both eyes, between 7.2 x 109and 7.2 x 1012EVs for both eyes, between 7.2 x 1010and 7.2 x 1012EVs for both eyes or between 7.2 x 1011and 7.2 x 1012EVs for both eyes.

[0127] Methods of EV production

[0128] In a last aspect, the disclosure relates to methods of production of isolated EVs comprising the specific marker profile described in any of the previous embodiments.

[0129] The EVs are produced using human mesenchymal stromal cells (MSCs) cultures, derived from umbilical cord tissue (UCT) by filtration.In an embodiment of the method of producing isolated EVs, as disclosed herein, the method comprises isolating EVs from a conditioned cell culture medium, preferably from a culture of mesenchymal stem cells; wherein the isolated EVs comprise hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p.

[0130] In another embodiment of the method of producing isolated EVs as disclosed herein, said method comprises:

[0131] • isolating EVs from a conditioned cell culture medium, preferably from a culture of mesenchymal stem cells, and

[0132] • selecting said EVs that comprise hsa-miR-125b-5p, hsa-miR-199a-3p, hsa- miR-199b-3p and hsa-miR-16-5p.

[0133] In a further embodiment of any of the methods, said EVs comprise at least 2 or 3 miRNA markers selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p. In yet another embodiment, the EVs comprise hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p.

[0134] In yet a further embodiment of any of the methods, the EVs that comprise at least one miRNA marker selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p, further comprise one or more of the miRNAs selected from the group consisting of hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p. In another embodiment, said EVs comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or all miRNAs selected from the group consisting of hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p.

[0135] In an embodiment the method as disclosed herein, said EVs comprise at least one miRNA marker selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p. In a further embodiment, said EVs comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 marker selected from the group consisting of hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p.

[0136] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0137] EXAMPLES

[0138] Example 1 : Production of Mesenchymal Stromal Cells-derived Extracellular Vesicles (EVs) and characterization of their miRNA content.

[0139] Production of naive Extracellular Vesicles (MSC-EVs).

[0140] Extracellular vesicles (EVs) were produced using human mesenchymal stromal cells (MSCs) derived from umbilical cord tissue (UCT).

[0141] MSCs were enzymatically extracted from slices of umbilical cord using different enzymes. Digested slices were immediately placed into 2D culture plates to initiate the expansion of MSCs.

[0142] After MSCs expansion, MSC-EVs production was performed in 3D stirring bioreactor culture systems using microcarrier beads. MSCs, mixed with the microcarriers and defined culture medium, were inoculated into the bioreactor system. The supernatants were harvested and stored at 4°C-8°C.

[0143] EV concentration and characterization.

[0144] Cross flow filtration was performed using cassettes with a lOOkDa molecular weight cut-off membrane. EVs were larger than lOOkDa and could be concentrated in the retentate. An additional single wash step was performed after pre-concentration using saline.

[0145] After washing the EVs were re-concentrated and nanoparticle tracking (NTA) (ZetaView®, PMX220 TWIN (Particle Metrics)) was performed to confirm a target concentration of 1.2E+11 particles / mL used here.

[0146] Subsequently, EVs were aliquoted. The vials with EVs were transferred into cryoboxes and frozen. EVs were ready to use immediately after thawing.Characterization of MSC-derived EVs miRNA content.

[0147] The miRNA expression profile of MSC-EVs produced as described above was determined by small RNA sequencing. miRNAs of MSC-EVs were extracted from 5 EV batches. miRNAs were isolated from 2.84E+10 particles of each EV batch by miRNeasy Mini Kit (Qiagen), quantified by Ribogreen (Life Technologies), and qualified by Agilent 2100 Bioanalyzer system using Eukaryote Total RNA Pico chip. A total of 20 ng RNA from each sample was used for small RNA library construction using QIAseq miRNA Library Kit (Qiagen). Library pre-sequencing QC was performed using QIAxcel DNA Screening Kit (Qiagen), and library quantification was evaluated with qPCR. During library preparation, libraries were indexed, pooled, and sequenced together. Small RNA sequencing was conducted by Novaseq 6000 (Illumina) using the Novaseq 6000 SI Reagent Kit (100) single read standard workflow ). After sequencing and demultiplexing, 2 FASTQ files were generated for each sample.

[0148] The small RNA sequencing data processing and analyses, including filtering, trimming, mapping, quantification of the sequenced reads, and normalization of the resulting read counts were carried out by BioLizard NV using a Nextflow pipeline (https: / / nf-co.re / smrnaseq / !.1.0). Before the processing, all FASTQ files of the same samples were merged. Quality control of the raw sequencing data was checked using the FastQC tool, followed by MultiQC, and low-quality reads were filtered out. Unique molecular identifier (UMI) sequences were trimmed, and duplicate reads were removed using UMI-tools. The processed reads were mapped to the mature miRNA reference sequences from miRbase and against the host reference genome. Normalization factors were calculated for the raw read counts based on a normalization of the library sizes using the Trimmed Mean of M-values (TMM) method as implemented in the R package edgeR.

[0149] A list of human mature microRNAs was identified in MSC-EVs. The most abundant ones are shown in Table 1.Table 1. The most abundant mature microRNAs identified in MSC-EVs of the invention. Quantity expressed as sum of reads in 5 EV batches tested. Cut-off: >100 reads in each EV batch. Accession number in the miRbase (https: / / www.mirbase.orQ / ).

[0150] >

[0151]

[0152] Importance of the miRNA expressed in the MSC-derived EVs of the invention for their biological activity.

[0153] miRNA activity can be inhibited Hairpin inhibitors, like antisense oligonucleotides. These single-strand RNA oligonucleotides bind and specifically sequester their complementary mature miRNA strands and avoid the mRNA targeting. To validate the specific activity of the miRNA contained in the EVs of the present invention, EVs and a cocktail of complementary Hairpin inhibitors for the 14 selected most abundant miRNA displayed in table 1 are delivered to macrophages at the same time.

[0154] Briefly, Macrophage raw 264 (CLS, 400319) are seeded at a density of 180.000 cells per well in a 24 well plate in 500ul of DMEM (Gibco, 21063029) + 1% PS + 5% FBS (Gibco, A3160801). 24h after seeding, Ml proinflammatory phenotype is stimulated by adding lOng / ml Lipopolysaccharide in each well (LPS, L4391 Merck). Inhibitory control is mimicked by lug / ml Dexamethasone (Dexa, D4902 Merck) treatment. EVs treatments are normalized to particle number. Hairpin inhibitors are delivered in parallel to EVs treatment by classical lipofectamine (Lipofectamine RNAi MAX 13778100 Thermo Fisher) transfection, following manufacturer instructions. Alltreatments are added simultaneously to the cells. After 16h, the media is collected to investigate Interleukin-6 (IL-6) or Tumor Necrosis Factor-a proteins levels (ELISA). ELISA are performed accordingly to manufacturer protocol.

[0155] The EVs having the specific miRNA profile as disclosed herein prevent Ml proinflammatory phenotype induction in macrophages. Delivery of hairpin inhibitors against the miRNA expressed in the EVs abolish or prevent the anti-inflammatory activity of the EVs.

[0156] Example 2 : Efficacy of the Mesenchymal Stromal Cells-derived Extracellular Vesicles (EVs) of the invention in the treatment of wound healing and in wound regeneration using in vitro scratch assay model of wound healing.

[0157] A significant area of interest is the use of UC-MSC-derived extracellular vesicles (EVs) in skin wound healing. The use of UC-MSC-derived EVs represents a cutting-edge strategy in the field of skin wounds, including acute injuries and chronic, non-healing wounds like diabetic ulcers and burns. The EVs according to the invention were tested for their capacity to heal and repair wounds.

[0158] Materials and Methods

[0159] In vitro scratch assay model of wound healing

[0160] Reagents:

[0161] Cells used: NHEK-Ad - Human Epidermal Keratinocytes, Adult, Single Donor, Catalogue #: 00192627, Lonza; Cells between passages 2-6 were used for the experiments.

[0162] Cell media: KGM™ Gold Keratinocyte Growth Medium BulletKit™, Catalog # 00192060, Lonza.

[0163] Lonza Trypsin / EDTA, trypsin neutralizing solution (TNS), and HEPES, 'ReagentPack™ Subculture Reagents, 100 mL', Catalog #: CC-5034.

[0164] EGF was purchased from Gibco™ Human EGF Recombinant Protein Supplier: Gibco (available and purchased from Fisher Scientific Belgium)

[0165] Catalog / ref nr: PGH0311L (10125414 is the ref number for Fisher Scientific) For each biological replicate, 8 pl of the stock was needed (8 for the E9 condition and 3 to make the masterstock 1 dilution, see further). Stock was kept at -80°C until day of experiment and never re-frozen.

[0166] Test substances:EV samples are produced as described above and provided in a stock solution of 1.2 Ell particles / ml. Three different batches were used (see table 2).

[0167] Table 2: Test Substance - Same batches of EVs were used for three independent experiments. n = 3

[0168]

[0169] Design of experiment and study samples

[0170] 11 conditions were tested, in three technical repeats:

[0171] 1. Negative control KGM medium 0% FBS + Saline

[0172] 2. Positive control KGM medium 0% FBS+ lOng / ml EGF

[0173] 3. KGM medium + Compound 1, 1 E+9 per well

[0174] 4. KGM medium + Compound 1 1E+8 per well

[0175] 5. KGM medium + Compound 1 1E+7 per well

[0176] 6. KGM medium + Compound 2 1E+9 per well

[0177] 7. KGM medium + Compound 2 1E+8 per well

[0178] 8. KGM medium + Compound 2 1E+7 per well

[0179] 9. KGM medium + Compound 3 1E+9 per well

[0180] 10. KGM medium + Compound 3 1E+8 per well

[0181] 11. KGM medium + Compound 3 1E+7 per well

[0182] Compounds were thawed and diluted in saline, each well will contain KGM medium 0% FBS.

[0183] 11 conditions in triplicate means at least 33 wells were seeded.

[0184] Protocol

[0185] Day 1:

[0186] • NHEK cells, cultured in KGM gold medium 10% FBS, are detached using a first washing 2 times with HEPES-BSS and then incubated using 2 ml of trypsin / EDTA solution at room temperature.

[0187] • 4 minutes later, 3 ml of room temperature TNS is added and cells are centrifuged 220 x g for 5 minutes to pellet the cells.

[0188] • NHEK cells are resuspended in KGM medium 10% and counted.

[0189] • Cell suspension is made and pipetted (50 OOOcells / well), 200 pl per well. • Cells are placed in incubator 37°C and 5% CO2.Day 2:

[0190] • All media compounds are preheated at 37°C.

[0191] • Cells are inspected microscopically for confluency, if cells are confluent, the experiment can continue.

[0192] • Preparation of the dilutions:

[0193] Each compound of table 2 is delivered in a 1.2 Ell particles / ml solution (i.e. 1.2 E8 particles per pl or 1.2 E9 particles per 10 pl)

[0194] Preparation of 2 master stock solutions:

[0195] Masterstock 1: 1.2 E+10 / ml and

[0196] Masterstock 2: 1.2 E+9 / ml

[0197] Masterstock 1 is made by 1 / 10 dilution of the 1.2 Ell particles / ml solution (using 7 pl of stock + 63p I of saline in a total of 70 pl).

[0198] Masterstock 2 is made by a 1 / 10 dilution of the masterstock 1 1.2 E+10 particles / ml solution (again using 7 pl of this stock + 63p I of saline, in a total volume of 70 pl).

[0199] Next, following dilutions were made. Briefly, Eppendorf tubes containing 1000 pl of media were prepared. From these tubes, 200pl were pipetted in the wells after making the scratch.

[0200] Table 3: Sample Preparation

[0201]

[0202] • The Sartorius wound maker (Sartorius, catalogue number BA-04858) was sterilized using 70% ethanol, washed 5 minutes in Milli-Q water and 5 minutes using ethanol.• A scratch in the confluent cell layer is made.

[0203] • Cells are washed 2x with HBSS.

[0204] • Conditions are added (200 pl / well).

[0205] • Plate is placed in incucyte (Incucyte® S3 System). Incucyte is an automated live-cell imaging and analysis platform, used primarily in biological cell culture. It is mainly used for real time long term, live cell imaging, and it provides quantitative metrics for cell behaviours such as growth (proliferation), movement (migration), death (cytotoxicity), responses to different treatments. Pictures are made with following settings:

[0206] • Channel selection: phase.

[0207] • Scan type: scratch wound.

[0208] • Vessel type: 96-well Essen ImageLock.

[0209] • Scan interval: one picture / well, every lh, for 20h

[0210] Day 3-4:

[0211] Every hour, lOx pictures were made with the incucyte. Afterwards, the incucyte software was used to assess the wound thickness and wound confluency. For 3 biological replicates performed, statistical analysis was performed using Graphpad Prism software 5.03 (Graphpad Software, La Jolla, CA). Data normality was tested with D'Agostino & Pearson normality test. When Gaussian distribution was reached, experimental groups were compared using a one-way analysis of variance (ANOVA) with a Bonferroni post-test. Non-parametric data were evaluated with a Kruskall-Wallis test combined with Dunn's post-test or Friedman test (for ’paired’ data). All the data are expressed as mean ± standard error of the mean (SEM).

[0212] Results

[0213] Ability of MSC-derived EVs of the invention to repair wound.

[0214] To assess the wound healing ability of MSC-derived EVs of the invention, we used in vitro scratch assay using human keratinocytes as described in Figure 1. This assay has been widely used to access, screen and evaluate the wound healing regenerative capacities of various compounds and growth factors.

[0215] Three different donor batches were included in the study (see table 2). EVs were produced as described in example 1.

[0216] • Donor 1- Compound 1 - CD007-06-01,

[0217] • Donor 2 -Compound 2 -CD009-03-01_02, and

[0218] • Donor 3-Compound 3 -CD004-02-15_19.On day two of the cell culturing assay setup, a scratch was made in the keratinocyte monolayer and the test compounds were added. The plate was inserted in the incucyte and at each hour, a picture of the wound was made. At each timepoint the relative wound density (RWD) was calculated based on the following formula:

[0219] <

[0220] >

[0221]

[0222] wfl) = Density of wound region at time; ftj

[0223] c(ij ~ Density of cell region at time, fl)

[0224] Specifically, the RWD metric refers to the density of the wound region at a certain timepoint compared by the timepoint 0, i.e. at the start of the experiment. 0% means that the wound is completely open while 100% means the wound is closed and entirely repopulated by cells.

[0225] The experiment was successful, as 20h after the start of the experiment full closure of the wound (or scratch) was observed in the positive conditions (treated with EGF) and some conditions containing the highest number of EVs of the invention, while in the negative conditions the wound was still open (see Figure 2).

[0226] As depicted in Figure 3 A, B and C, the RWD gradually increases over time, for example in experiment 1 for the positive control it changes from 14,6% after 4h towards 91% after 20h. All compounds at the highest concentrations (1E+9) dramatically increased the RWD, 100% for compound 1, 89% for compound 2 and 99% for compound 3 after 20h.

[0227] In each experiment, we observe that the wound closes over time (see Figure 3). For example, in the first experiment, at 20h, the wound is closed for 20% in negative control, while for the positive control, the wound is closed for 80% (Figure 3A). In the third experiment, the wound closure is very fast as 20h, the wound is closed in all conditions, even in the negative control (Figure 3C). This is probably caused by patient and passage variability of cell lines.

[0228] In each of the three experiments, Compound 1 and 3 at the highest dose 1E+9 particles / well induce fast closure of the wounds. For example, at timepoint 16h at the first experiment, the wound closure of both compounds is around 95% and this is even higher as the positive control EGF (reaching 80%) (Figure 3A). In the secondexperiment this is also observed in the same timepoint (Figure 3B), while in the third experiment they have a higher RWD compared to negative control at 8 and 12 h (Figure 3C). Compound 2 at the highest dose 1E+9 has a higher RWD compared to negative control in experiment 1 and 2 (Figure 3A and B), but not in experiment 3 (Figure 3C).

[0229] The concentration of 1E+7 per well was for any compound too low to induce wound closure better than the negative control. For the medium concentration of 1E+8 particles per well, we observe mostly a slightly better wound healing capacity, but this effect is rather modest.

[0230] Figures 4 A, B and C focus more on each compound. Representative graphs from Experiment 1 allow a better visualisation of the wound closure over time of each compound. We observe for each compound that the amount of 1E+7 particles per well was not enough to increase wound healing as the relative wound density was the same as in negative conditions. We also observed that the amount of 1E+8 particles per well of all 3 different compounds increased the RWD but not as dramatically as 1E+9 particles per well. The effect of Compound 1 at concentration

[0231] 1E+8 was the best as after 20h the RWD is 54% for Compound 1, 43% for Compound

[0232] 2 and 49% for Compound 3.

[0233] Statistical analysis

[0234] Given the heterogeneity of the wound closure, statistical analysis was performed on timepoints 4, 8 and 12h after start of the experiment. To have enough power, this was done on the datasets of the negative and positive controls and of each compound at 1E+9 particles per well (see table 4 and 5)

[0235] Table 4: Summary table. Each value represents the mean Relative wound density (RWD) in % of the technical replicates at each timepoint at each condition of the three independent experiments (n = 3).

[0236]

[0237] Table 5: Means and standard deviation of the 3 independent experiments together (n=3).

[0238]

[0239] A graphical overview of the three different experiments (n = 3) is provided in Figure

[0240] 5.

[0241] In addition, statical analysis were performed on the means of the 3 independent experiment (Table 5). Using graphpad prism 7 software, one-way ANOVA analysis was performed (using paired, nongausian distribution, also known as Friedman test) in which all conditions were compared to the negative control. Compound 1 and 3, at a concentration of e9 were found to be statistically different as compared to the negative control.

[0242] Table 6. Statistical summary of experiments (n = 3)

[0243] >

[0244]

[0245] Conclusions

[0246] The study was performed in three independent biological repeats experiments (n = 3), and three technical repeats in each experiment. Batches from three different donors were used in each experiment. We conclude that:

[0247] • MSC-derived EVs according to the invention significantly improved wound closure in scratch assay using human skin derived cell model.

[0248] • Compounds 1 and 3 show significant improvement at concentration of 1E+9 particles per well demonstrating batch to batch reproducibility.

[0249] • Dose-dependent effect was confirmed, with the medium dose of 1E+8 particles per well of the Compounds 1 and 3 showed a trend but were not significant.

[0250] • Highest dose of 1E+9 particles per well effect was even greater when comparing to a positive control.• Compound 2 have improving effect in the experiment 1 and experiment 2 compared to the negative control.

[0251] Example 3 : Phase I, monocentric, double-blind, placebo-controlled clinical study assessing the safety, tolerability and preliminary efficacy of the Mesenchymal Stromal Cells-derived Extracellular Vesicles (EVs) of the invention in skin regeneration and repair.

[0252] The clinical study investigates the efficacy of the Mesenchymal Stromal Cells-derived EVs of the invention, in promoting skin repair. This Phase I trial evaluates the safety, tolerability, and preliminary efficacy of said EVs specifically for the treatment of atrophic scars (traumatic and surgical) and wrinkles.

[0253] Given the commonality of atrophic scars and wrinkles as aesthetic concerns, which can significantly impact an individual's quality of life, traditional treatments often yield limited results. However, microneedling's ability to promote skin remodelling through collagen induction has garnered attention. Exosome therapy, a cutting-edge technique in regenerative medicine, shows promise in enhancing healing and tissue regeneration.

[0254] The combination of EVs of the present invention with microneedling and intradermal injections improves collagen deposition, thereby enhancing scar appearance and achieving greater structural integrity, resilience, and elasticity in the skin.

[0255] On the one hand, microneedling creates controlled micro-injuries in the dermis, triggering a natural wound-healing response that stimulates fibroblast activity to produce collagen and elastin. This process improves skin texture and reduces scars and fine lines. On the other hand, EVs according to the present invention is the investigational medicinal product (IMP) consisting of a population of allogeneic umbilical cord mesenchymal stromal cells-derived (MSC) EVs expressing a defined list of miRNAs and formulated as a suspension of 1.2E+11 particles / mL in saline for cutaneous and transdermal administration.

[0256] The proposed trial assesses the safety and treatment effect of the EVs of the invention on the skin. Therefore, volunteers receiving EVs / placebo undergo frequent clinical assessments, so that adverse reactions related to EVs / placebo or related to the administration technique is promptly identified, and recorded, and the condition alleviated by taking appropriate measures.This trial involves the evaluation of the safety of EVs / placebo in skin regeneration divided into 2 distinct sub-studies:

[0257] Cohort 1: Improvement of mature scars.

[0258] Cohort 2: Wrinkle attenuation in the cleavage area (decollete).

[0259] A preliminary efficacy assessment is performed by comparing the treated zone with EVs of the invention and with saline and the evolution of the treatment from baseline to the end of the study (follow-up). The double-dummy study design, with blinding both participants and investigators, is chosen to minimize bias and maximize accuracy of the readouts.

[0260] Six female and / or male adult subjects are enrolled per group and evaluated by the same Investigator / Physician throughout the study. The physician performs the treatment is different from the observer physician. Subjects receive a total of 3 administrations of 1.2E + 11 particles of EVs / mL in a volume determined by the treated surface, following the guidelines of 0.1ml of suspension for every 2.5cm2. Subjects are blinded to the treatment (EVs or placebo).

[0261] The overall procedure includes microneedling passes in the scar or the decollete before the intradermal administration of EVs or placebo using a mesotherapy gun. The treatment areas will be numbed with local anaesthesia if the subjects require it.

[0262] Rationale

[0263] Wrinkles and scars are common forms of skin damage observed in a significant portion of the population, affecting both men and women. These signs of ageing can appear on the face, decollete, or body as a result of natural ageing or following surgical procedures and injuries. Regardless of their cause or appearance, both wrinkles and scars disrupt the tissue and alter the local environment. They are characterised by a reduction in collagen, decreased vascularity, and alterations in elastic fibres.

[0264] In this study, six female adult volunteers are enrolled in group 2 and six female and / or male adult volunteers in group 1. Both are treated by the same investigator throughout the trial. Each volunteer receives three administrations of 1.2E+11 EVs particles, with the volume determined by the treated surface area, following a guideline of 0.1 ml of suspension for every 2.5 cm2. The clinical assessment is done by another physician to guarantee the blinding.The repetitive procedures of microneedling and intradermal injection of EVs occurs three times, with one treatment every three weeks. This regimen produces more pronounced and longer-lasting effects, effectively addressing both superficial and deeper skin concerns.

[0265] The treatment period lasts six weeks, followed by an eight-week follow-up period. During this 14-week timeframe, each subject is examined by the investigator / physician seven times, with one visit every three weeks.

[0266] The present trial integrates 2 cohorts (see figure 6):

[0267] • Cohort 1: Safety and preliminary efficacy of EVs of the invention vs placebo in improving scars on the skin body.

[0268] • Cohort 2: Safety and preliminary efficacy of EVs of the invention vs placebo in attenuating wrinkles in the decollete.

[0269] Objectives and endpoints / outcome measures

[0270] Primary objective

[0271] To assess the safety and tolerability of 3 intradermal administrations of Evs of the invention and NaCI 0.9% solution for infusion (placebo) with microneedling, given once every 3 weeks.

[0272] Secondary objectives

[0273] Cohort 1: Skin scar improvement

[0274] 1. To assess the preliminary efficacy of EVs vs placebo in scar improvement from screening to Visit 6 at Week 12 or, optionally, at Visit 7 or Week 14 (end of the study).

[0275] 2. To score scars using the Patient and Observer Scar Assessment Scale Scores (POSAS) from screening up to Visit 6 at Week 12.

[0276] 3. To assess the full skin thickness (measurement of infiltrate depth) and the extent of inflammatory infiltrates by ultrasound from screening to Visit 6 at Week 12.

[0277] 4. To measure thetexture, volume, redness and colour of the scar using the Antera 3D technique from screening to Visit 6 at Week 12.

[0278] 5. To assess the skin elasticity, hydration, and firmness in the scar from screening to Visit 6 at Week 12.

[0279] 6. To evaluate the Global Aesthetic Improvement Scale (GAIS) by the volunteer and the Investigator from screening to Visit 6 at Week 12.7. To perform a histological examination of the treated areas (skin collagen reorganisation, fibrosis, and re-epithelisation) before EVs treatment (Visit 2, Baseline) and 6 weeks after the last EVs treatment (Visit 6).

[0280] 8. To evaluate at Visit 7 the skin healing of the puncture biopsy performed at Visit 6.

[0281] Cohort 2: Wrinkle improvement in the decollete

[0282] 1. To assess the preliminary efficacy of EVs of the invention in wrinkles in the cleavage area (decollete) from screening to Visit 6 at Week 12.

[0283] 2. To score wrinkles using the Fabi-Bolton scale from screening to Visit 6 at Week 12.

[0284] 3. To measure the inflammatory infiltrate depth and skin thickness by ultrasound from screening to Visit 6 at Week 12.

[0285] 4. To measure wrinkles, folds, fine lines, redness and colour in the decollete using the Antera 3D technique from screening to Visit 6 at Week 12. 5. To assess the skin elasticity, hydration, and firmness in the decollete from screening to Visit 6 at Week 12.

[0286] 6. To evaluate the Global Aesthetic Improvement Scale (GAIS) by the volunteer and the Investigator from screening to Visit 6 at Week 12. 7. To perform a histological examination of the treated areas (skin collagen reorganisation, fibrosis, and re-epithelisation) before EVS treatment (Visit 2, Baseline) and 6 weeks after the last EVs treatment (Visit 6).

[0287] 8. To evaluate at Visit 7 the skin healing of the puncture biopsy performed in Visit 6.

[0288] Primary endpoint

[0289] The number of treatment-emergent adverse events (TEAEs) assessed from after injection of EVs and NaCI 0.9% solution for infusion (placebo) to the end of the study by the physician and the subject.

[0290] Secondary endpoints

[0291] Cohort 1: Skin scar improvement

[0292] 1. The proportion of subjects presenting an improvement in scar scoring using the POSAS from screening to Visit 6 at Week 12.

[0293] 2. The number of subjects with an improvement in texture, volume, redness and colour of the scar from screening to Visit 6 at Week 12.

[0294] 3. The number of subjects exhibiting an improvement in skin elasticity, hydration, and firmness in the scar from screening to Visit 6 at Week 12.4. The number of subjects showing a score improvement of the GAIS assessed by the subject and the Investigator from screening to Visit 6 at Week 12.

[0295] 5. The proportion of subjects showing a decrease in the inflammatory infiltrate depth and skin thickness in the scar from screening to Visit 6 at Week 12 or, optionally, at Visit 7 or Week 14 (end of the study).

[0296] 6. The proportion of subjects exhibiting an improvement in collagen organisation, a decrease in inflammatory infiltrates and skin improvement at 6 weeks after the last EXOB-OOl treatment (Visit 6).

[0297] Cohort 2: Wrinkle improvement in the decollete

[0298] 1. The proportion of subjects exhibiting improvement of wrinkle score using the Fabi-Bolton scale from screening to Visit 6 at Week 12 or, optionally, at Visit 7 or Week 14 (end of the study).

[0299] 2. The proportion of subjects with improvement of wrinkles, folds, fine lines, redness and colour in the decollete from screening to Visit 6 at Week 12 or, optionally, at Visit 7 or Week 14 (end of the study).

[0300] 3. The number of subjects exhibiting an improvement in skin elasticity, hydration, and firmness in the decollete from screening to Visit 6 at Week 12 or, optionally, at Visit 7 or Week 14 (end of the study).

[0301] 4. The number of subjects showing a score improvement of the GAIS assessed by the subject and the Investigator from screening to Visit 6 at Week 12 or, optionally, at Visit 7 or Week 14 (end of the study).

[0302] 5. The proportion of subjects showing a decrease in the inflammatory infiltrate depth and skin thickness in the decollete from screening to Visit 6 at Week 12 or, optionally, at Visit 7 or Week 14 (end of the study).

[0303] 6. The proportion of subjects exhibiting an improvement in collagen organisation, a decrease in inflammatory infiltrates and skin improvement at 6 weeks after the last EVs and placebo treatment (Visit 6).

[0304] Trial design and duration

[0305] The trial is an exploratory, double-dummy study on skin improvement after intradermal injections of EVs according to the invention compared to placebo.

[0306] The trial is a phase 1, interventional, monocentric, double-blind, placebo-controlled (double-dummy) clinical trial to study the effect of EVs of the invention in skin repair (scar improvement and wrinkle attenuation).Cohort 1 consists of evaluating the safety and preliminary efficacy of EVs of the invention in scar improvement and cohort 2 will study the safety and preliminary efficacy of EVs of the invention in wrinkle attenuation in the decollete.

[0307] A total of 12 subjects are enrolled in 2 cohorts, 6 volunteers are enrolled in cohort 1 to study scar improvement and 6 volunteers are enrolled in cohort 2 to study wrinkle attenuation in the decollete.

[0308] The treatment consists of microneedling (array of 12 needles) of the treatment area followed by the injection of 1 dose of 1.2E+11 particle / mL of EVs of the invention and placebo (NaCI 0.9%) via the intradermal route using a mesotherapy gun. The treatment visits include baseline (Visit, Week 0), Visit 3 (Week 3) and Visit 4 (Week 6), making a total of 3 administrations with EVs and 3 with saline. Two biopsies will be performed at baseline before EVs treatment and at visit 6, 6 weeks after the last EVs treatment.

[0309] The total duration of the study for each volunteer is a minimum of 14 weeks from screening to the last follow-up visit. Follow-up visits include Visit 5 (Week 9 and 3 weeks after the last EVs treatment) and 6 (Week 12 and 6 weeks after the last EVs treatment). An additional in-clinic visit is performed at week 14 to evaluate the healing of the biopsy performed at visit 6.

[0310] Study Duration

[0311] The total duration of the study for each volunteer is a minimum of 14 weeks divided into the following visits:

[0312] Screening: Screening of subject can be done separately or simultaneously with the baseline visit after evaluating and confirming the subject's eligibility.

[0313] Treatment in-clinic visits: From baseline to visit 4. The maximal duration of the treatment with EVs is 6 weeks.

[0314] Follow-up in-clinic visits: Includes Visit 5 (Week 9 and 3 weeks after the last EVs treatment) and Visit 6 (Week 12 and 6 weeks after the last EVs treatment). An additional in-clinic visit is performed at week 14 to evaluate the healing of the biopsy performed at Visit 6. The maximum duration of the Follow-up will be 8 weeks.Inclusion criteria

[0315] Subjects meet specific eligibility criteria and are enrolled into one of the two groups, cohort 1 or 2.

[0316] Cohort 1: Skin scar improvement

[0317] • Female and male volunteers willing to sign the informed consent.

[0318] • Volunteers between 18 and 70 years old

[0319] • Volunteers with normotrophic or atrophic scars, that include C-section, surgical, stretch marks and concave scars.

[0320] • Scars with more than 1 year old (mature scars).

[0321] • Volunteers with 2 comparable scars (same type of scars and similar thickness) or one scar with a minimum of 15 cm long.

[0322] • The subject agrees to discontinue any topical use of cosmetic creams in the scar region.

[0323] Cohort 2: Wrinkle improvement in the decollete

[0324] • Female volunteers willing to sign the informed consent.

[0325] • Volunteers with grade 3 wrinkles according to the Fabi-Bolton grading score in the decollete.

[0326] • Volunteers between 50 and 70 years old.

[0327] • The subject agrees to discontinue any topical use of cosmetic creams in the decollete area.

[0328] Exclusion criteria

[0329] Volunteers are excluded from the study if any of the following criteria apply: • Pregnant or nursing women.

[0330] • Volunteers who have received any cosmetic or dermatological treatment (lasers, dermabrasion, chemical peels, etc) in the previous 6 months in the scar (cohort 1) or the decollete area (cohort 2).

[0331] • Subjects allergic to the local anaesthetic and not willing to perform the intervention without anaesthetic.

[0332] • Subjects with inflammatory skin disease in the intervention site.

[0333] • Subjects taking anticoagulants and at risk of bleeding.

[0334] • Subjects with psychiatric problemsand drug addiction.

[0335] • History of malignant neoplasm within the past 5 years, or stage 3 or 4 of any cancer at any time.

[0336] • History of melanoma, leukaemia, or lymphoma (any stage).

[0337] • Participation in any other interventional clinical study.• Prior treatment with stem cells or exosome therapy.

[0338] • Positive for hepatitis B, C or HIV.

[0339] • Volunteers who are immunocompromised.

[0340] • Tattoos that cover the proposed treatment sites.

[0341] • Volunteers with hypertrophic, keloid, burn and acne scars (specific for volunteers enrolled in cohort 1).

[0342] • Wounds or local disease in the treatment area (specific for volunteers enrolled in cohort 2).

[0343] Administration of the Investigational Medical Product (IMP)

[0344] The IMP consists of a sterile saline suspension of allogeneic EVs derived from cultured human umbilical cord MSCs having a defined miRNA profile as an active principle donated by mothers at full-term pregnancy with a scheduled caesarean section. Caesarean section significantly increases the sterility of the cord tissue samples and the reproducibility of the manufacturing process. Donors are checked against an extensive list ofexclusion criteria related to past and active metabolic, infectious, and malignant diseases.

[0345] The IMP is the "off-the-shelf" drug product consisting of a population of EVs smaller than 0.22 pm in diameter, enclosed in a double layer of phospholipids with integral and surface-bound proteins as the main components, and expressing a list of miRNAs as disclosed herein.

[0346] Placebo consists of a saline solution for infusion of NaCI 0.9%. The saline may be aliquoted manually and cryopreserved at -20°C.

[0347] Volunteers receive 3 intradermal administrations of 1.2E+11 EVs / mL in saline 0.9%, given once every 3 weeks (Visit 2, 3 and 4).

[0348] The intervention field (scar and decollete) is anaesthetised with atopical application of 5% lidocaine (EMLA cream or EMLA patch) for approximately 30 minutes before the procedure. Afterwards, the skin is disinfected with Diaseptyl® (0,5% chlorhexidine digluconate, aqueous solution of 0,2% Chlorhexidine, purified water, glycerol and triethanolamine).

[0349] Volunteers undergo microneedling (Dr Pen, Hydrapen H3: 12 needles) of the treatment field with a depth varying according to the epidermis thickness measured by ultrasound to be sure that needles reach the dermis. The procedure is performed according to the manufacturer's instructions on the use, handle and procedure.Intradermal injections of EVs or saline is done with a mesotherapy gun. Each injection delivers 0.1 mL of EVs, totalling 9 injections in the scar or 25 cm2 in the decollete. The mesogun procedure on the use, handle, and administration will be provided according to the commercial device.

[0350] 1. Administration of EVs in cohort 1

[0351] The intervention area for EVs treatment is the scar whose types are mentioned in the eligibility criteria. Volunteers are included if they have two comparable scars or one scar with a length superior to 15 cm.

[0352] Regardless of the number of scars, the intervention consists of microneedling (Dr Pen) and EVs or saline injection by a mesotherapy gun.

[0353] If the volunteers have two comparable scars, each scar must be 5 cm in length. One scar receives EVs and the other is treated with saline. Two biopsies are performed per scar with a minimum distance of 5 mm. The first biopsy is performed before treatment with EVs or saline and the second 6 weeks after the last treatment.

[0354] If a volunteer has only one scar, it must be at least 15 cm long. The scar is divided into 2 distinct areas, one receives EVs and the other half is treated with saline. The 2 treatment fields must be 5 cm apart to avoid diffusion and contamination of the opposite treatment field. Two biopsies on opposite sides of the scar are performed, one before the first treatment and the other 6 weeks after the last treatment.

[0355] 2. Administration of EVs in cohort 2

[0356] The intervention area in the decollete is located on the left and right side of the decollete and below the manubrium (horizontal dashed line) and 2.5 cm away from the sternum (vertical dashed line). Each intervention area consists of 25 cm2 and undergos microneedling and injection of EVs or saline by mesotherapy gun. The total content of the vial inserted in mesotherapy gun container is distributed throughout the limited area, making 9 injections of 0.1 mL / 2.5 cm2.

[0357] Two biopsies per side are performed, one before the first treatment and the other 6 weeks after the last treatment.Comparators IMP

[0358] NaCI 0.9% (saline solution for infusion) is used to produce the placebo.

[0359] The administration of a placebo is similar to EVs. Volunteers undergo microneedling (Dr Pen, Hydrapen H3: 12 needles) in the treatment field with a depth varying according to the epidermis thickness measured by ultrasound to be sure that needles reach the dermis. Then, volunteers receive a placebo contained in a mesotherapy gun via the intradermal route. Each injection delivers 0.1 mL of placebo making a total of 9 injections in the scar or an area of 25 cm2 in the decollete.

[0360] Placebo is administered using a mesotherapy gun to be delivered to the dermis and after the microneedling procedure.

[0361] The placebo is tested, handled and stored under the instructions of the manufacturer and Good Manufacturing Practice (GMP) requirements for clinical trials.

[0362] The saline solution is NaCI 0,9%, Sodium chloride, solution for injection (Mini-Plasco® ® of 10ml or 20ml), manufactured by B. Braun.

[0363] Example 4: EVs activity in in vitro assays for intended use in treatment of BPD EVs were produced and characterized according to example 1 and are used in the treatment of major symptoms of BPD:

[0364] a) Inflammation

[0365] EV internalization assay. Fluorescently-labelled EVs of the invention are co-cultured with human peripheral blood mononuclear cells (PBMCs). Flow cytometry is performed to quantify populations of immune cells with internalized labelled EVs. The following markers are investigated: anti-CD4, anti-CD8, anti-CDIIc, anti-CD14, anti-CD19, anti-CD56 and anti-CD15. Analysis is performed after 0-1-6-12-24 hrs and after 6 hrs for neutrophils. EVs produced by fibroblasts are used as negative control.

[0366] Lymphocyte migration assay. EVs of the invention are co-cultured with PBMC in a trans-well system. Cell migration is investigated towards SDF-1 chemokine. Cell phenotypes and quantification are performed with flow cytometry.

[0367] B cell assay. EVs of the invention are co-cultured with CpG-stimulated human peripheral blood mononuclear cell. Cell proliferation and differentiation to plasmacells are investigated, phenotypes and quantification are performed with flow cytometry. Quantification of cytokines and antibodies are performed with ELISA. T cell assay. EVs of the invention are co-cultured with human peripheral blood mononuclear cell and T cell activator CD3 / CD28 beads. CD4+ T cell proliferation and CD4+ T cell apoptosis are quantified using flow cytometry. Treg / Teff ratio is calculated. Treg proliferation is quantified using flow cytometry. Quantification of following cytokines is performed with ELISA: IL10, TGF-b, galectin-1, HGF, PGE2, GM-CSF, IL2, TNF-a, IFN-y.

[0368] CD cells assay: CD cell activation is investigated in co-culture with EVs of the invention using flow cytometry. Upregulation of co-stimulatory molecules CD80 and CD86 and maturation marker CD83 are quantified using flow cytometry. Quantification of following cytokines is performed with ELISA: IL6, IL8, IL12, CCL3, CCL4, IL10, TGF-b. Phagocytosis assay is performed (incubation with FITC-dextran) using flow cytometry. Migration assay is performed using trans-well assay and flow cytometry.

[0369] Macrophage assay. EVs of the invention are co-cultured with Ml-stimulated (LPS) and M2-stimulated (IL4 / IL13) macrophages. Ml to M2 ration is calculated using flow cytometry. Quantification of following cytokines are performed with ELISA: IL6, TNFa, IFNy, ILip, IL12, IL10, VEGF, MCP1, TGF-b, FGF and confirmed with qPCR gene expression analysis. Trans-well migration assay (towards fMLP) is performed and quantified using flow cytometry.

[0370] NK-cell assay. EVs of the invention are co-cultured with PBMC derived NK cells. Quantification of following cytokines are performed with ELISA: TNFa, IFNy. Maturation markers CD27, CDIIb, CD107a, IFN-g and cell proliferation are quantified using flow cytometry.

[0371] b) Fibrosis

[0372] Experiments are performed using commercial fibrosis assay with normal human lung fibroblasts and human epithelial cells. Human lung fibroblasts are co-cultured with EVs of the invention delivered at different timepoints, with multiple doses and different concentrations. Subsequently, analyses of alpha-SMA and collagen I I fibronectin are performed.

[0373] In another experiment the EVs of the invention influence on epithelial-to-mesenchymal transition (EMT) in primary human bronchial epithelial cells ismeasured. EMT is investigated using commercial assays following the manufacturer protocol. In one experiment the experimental primary cells derived from healthy tissue or from idiopathic pulmonary fibrosis patients are stimulated with TGF-b to induce EMT, and non-stimulated cells are used as control. Subsequently, human epithelial cells are co-cultured with EVs of the invention delivered at different timepoints, with multiple doses and different concentrations. EMT is detected and quantified using FACS and E-cadherin and fibronectin expression.

[0374] In another experiment the EVs of the invention influence on fibroblasts-to-myofibroblasts transition (FMT) in primary human bronchial fibroblasts cells is measured. FMT is investigated using commercial assays following the manufacturer protocol. In one experiment the experimental primary cells derived from healthy tissue or from idiopathic pulmonary fibrosis patients are stimulated with TGF-b to induce FMT, and non-stimulated cells are used as control. Subsequently, human fibroblast cells are co-cultured with EVs of the invention delivered at different timepoints, with multiple doses and different concentrations. FMT is detected and quantified using alpha-smooth muscle actin marker.

[0375] c) Oxidative stress and cell apoptosis

[0376] Assays and cell lines described for investigation of fibrosis are used for quantification of cell apoptosis and oxidative stress (ELISA, flow cytometry).

[0377] Oxidative stress is induced using H2O2 or other reagents. Cells are exposed to the oxidative stress during different time periods, also different doses of the oxidative stress inducers are used. Subsequently, cells are co-cultured with EVs of the invention delivered at different timepoints, with multiple doses and different concentrations. Direct effects of oxidative stress are measured by detection and quantification of reactive oxygen species (ROS) and indirect effects by measuring nucleic acids damage, lipids peroxidation and protein oxidation. Detection and quantification of direct and indirect markers of oxidative stress will be performed using ELISA and FACS.

[0378] Cell apoptosis in response to the oxidative stress is detected and quantified using commercial assays following the manufacturer protocol. In one experiment LIVE / DEAD™ (ThermoFisher) is used for detection of cell viability using FACS. In other experiments, Annexin V immunofluorescent staining detects an early apoptosis and is quantified using FACS.In other experiments, an early apoptosis is detected by quantification of the activated caspase-3 and caspase-7 using FACS.

[0379] Example 5: Efficacy of the Mesenchymal Stromal Cells-derived Extracellular Vesicles (EVs) of the invention in the treatment of Acute Respiratory Distress Syndrome (ARDS).

[0380] Materials and Methods

[0381] EVs production

[0382] EVs were produced and characterized according to example 1.

[0383] LPS (lipopolysaccharide) -induced ARDS rat model.

[0384] Sprague Dawley male rats were used in all experimental settings at the Charles River Laboratories Edinburgh Ltd (UK). ARDS was induced with LPS (from E. coli 026: B6, Sigma Aldrich). Inducer, vehicle, and test items were administered directly to the lung by oropharyngeal (OP) aspiration. During this procedure animals were lightly anaesthetized with an inhaled anaesthetic (e.g. isoflurane) and recovered consciousness within a couple of minutes. Heating pads assisted the animals in maintaining body temperature homeostasis throughout the OP aspiration dosing regimen. Temperature was maintained between 25 to 35°C. In-life readouts taken: body weight, body temperature; clinical signs: respiratory assessments (using eDacq data acquisition system), pulse oximetry, gait, fur morphology, eyes openness, activity. Animals were euthanized after 4h or 6h by exposure to pentobarbital (IP injection) to collect bronchoalveolar lavage (BAL) (for WBC counts and protein content).

[0385] The following experiment was performed (curative model). 2 doses of EVs were administered by OP aspiration: 1st dose of 4 x 1010particles / kg co-infused with LPS (cone. 0.05, 0.1 mg, 0.5, or 1 mg / kg), 2nd dose of 8 x 1010particles / kg was administered after 3h without LPS after the 1st dose. Control: LPS in saline. Necropsy was performed at 6h (Figure 7).

[0386] Results

[0387] One of the most common ARDS animal models are rodents administered with LPS. We have studied the effect of the EVs according to the invention in LPS-induced ARDS adult rat model, with oropharyngeal aspiration administration (see Materials and Methods). The model revealed full clinical symptoms of ARDS including respiratory problems (data not shown). One experiment using 1 EVs batch wasperformed. This experiment used four different LPS doses to test a response in a mild and severe lung injury. The experiment indicated no toxic effect with the tested doses (4-8E+10 particles I kg body weight). EVs were effective and improved different physiological parameters: it improved such clinical symptoms of disease as changed fur morphology, partial eye closeness, gait, and decreased activity. EVs did not improve the increased respiratory rate (table 7).

[0388]

[0389] Table 7: Safety and efficacy of EVs of the invention. Representative readouts of clinical signs in curative model of ARDS. Group 11 - control (LPS; n=4), group 12 -experimental group (1st dose of EVs 4E+10 particles / kg co-administered with LPS of 1 mg / kg; 2nd dose of EVs 8E+10 particles / kg without LPS; n=4). Pre - pretrial period, IPD - investigational product delivery moment, "X" - timepoint when specific readout was positive, - timepoint when specific readout was negative. Numbers indicate the number of animals in each group expressing the specific readout (clinical symptom).

[0390] Moreover, EVs statistically significantly prevented body temperature loss (Figure 8) and statistically significantly reduced bronchoalveolar lavage (BAL) total protein content in severe ARDS model (Figure 9). Improvement in the immune system response was observed in mild ARDS model: EVs statistically significantly reduced numbers of BAL lymphocytes (Figure 10). These results support the rationale to envision ARDS treatment with EVs of the invention.

[0391] Example 6 : Efficacy and safety of Mesenchymal Stromal Cells-derived Extracellular Vesicles (EVs) of the invention in the treatment of an ocular surface disease.

[0392] Materials and MethodsEVs production

[0393] EVs were produced and characterized according to example 1.

[0394] BAK-induced dry eye disease animal model and experimental set up.

[0395] The animal model was generated as described in Suanno et al. (Exp Eye Res 2023, Jul:232: 109516). Briefly, one week twice a day topical benzalkonium chloride (BAK) administration (induction phase) was followed by one week once a day topical BAK administration. BAK induces high levels of proinflammatory cytokines in the cornea and conjunctiva, along with epithelial cell apoptosis and reduction of mucins, which leads to tear film instability, thereby successfully simulating Ocular Surface Disease such as Dry Eye Disease (DED).

[0396] The following experimental groups were defined:

[0397] 1. 1st generation extracellular vesicles - blinded group

[0398] 2. Extracellular vesicles derived from cytokines-primed MSCs - blinded group 3. saline (placebo) - blinded group

[0399] Concentration of the drug(s): one maximum concentration was tested for each formulation: 1.2E+11 particles / mL. Concentration of the labelled product could be slightly lower due to the losses during washing steps.

[0400] Dose: one drop (lOpL) was instilled in the conjunctival sac 3 times per day.

[0401] • Single administration volume app. lOpL and EVs dose 1.2E+9 particles / administration (eye).

[0402] • Total dose (48 administrations per animal / 24 per eye): 480uL and EVs per mouse.

[0403] • Cumulative dose per eye: 2.9E+10

[0404] • Cumulative dose per animal: 5.8E+10

[0405] EVs administration: topical (eye drops) in the conjunctival sac, 3 times per day for a period of 8 days (total 24 per eye).

[0406] Animals' well-being was monitored during the entire experiment. Disease activity was monitored by biomicroscopy.

[0407] To avoid stress, animals were allowed to acclimatize to their environment for 1 week prior to experimentation and were kept in a standard environment throughout the study: temperature ~25°C, humidity ~60%, alternating 12 h light-dark cycles (8 AM to 8 PM). To reduce pain after surgery, all animals received a single dose of carprofen 25 at 5 mg / kg subcutaneously.

[0408] All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Each animal was deeply anesthetized before all surgical procedures. At the end of experiment, animals were euthanized. EV toxicity had been already tested using a non-clinical animal model of bronchopulmonary dysplasia. No toxic effect at maximum dose and followingmultiple injections was observed and the EVs product is considered as save with no risk of addition harm to the animals.

[0409] Corneal damage quantification

[0410] Corneal damage was quantified using fluorescein staining. At days 0, 3, 5, 7, 12, and 14 in vivo fluorescein staining was used to evaluate defects on corneal epithelium during BAK treatment. Eyes were photographed with the slit-lamp microscope SL 990 (C.S.O., Florence, Italy). Corneal fluorescein staining was graded with a scale system based on the area of corneal staining).

[0411] Tear secretion quantification

[0412] Tear secretion was quantified using phenol red test. Tear fluid production was measured with the phenol red thread test 1 h after the last BAK administration. Briefly, a cotton thread was held with forceps and gently placed in the lateral canthus of the conjunctival fornixes for 15 s. The length of the moistened thread was evaluated in both eyes, and the average was recorded. The measurements were performed on both eyes consecutively without topical anaesthesia.

[0413] Leukocyte infiltration quantification.

[0414] Leukocyte infiltration was quantified using CD45 staining.

[0415] To measure leukocyte infiltration, the corneas were dissected and washed in PBS and fixed in acetone at 4 °C for 15 min. Nonspecific staining was blocked with 2% BSA and 5% NDS and then immunostained with anti-CD45 primary antibody followed by incubation with fluorescent secondary antibody. Six peripheral and six central fields per cornea were taken with a DeltaVision™ Ultra microscope. Leukocyte infiltration was quantified by counting CD45+ cells per field by using Image! software. The data are expressed as cells / field.

[0416] Lymph-angiogenesis quantification.

[0417] Lymph-angiogenesis was quantified by CD31 and LYVE1 staining.

[0418] On day 14, corneas were carefully dissected and rinsed in PBS. The corneal epithelium was subsequently scraped off after EDTA treatment for 30 minutes at 37°C. Fixation of the tissue was conducted with iced acetone for 15 minutes following 2 hours of blocking in PBS / 2%BSA. For visualization of blood and lymphatic vessels, corneas were immunostained with anti-mouse CD31 and anti-mouse LYVE-1 primary antibodies at 4°C overnight, respectively, and subsequently with fluorophore conjugated secondary antibodies at room temperature. This was followed by threerinses in PBS. Corneas were flat mounted on glass slides using Vectashield mounting medium. Positive signal was quantified using fluorescent microscope.

[0419] Results

[0420] Study timeline and Dry Eye Disease induction

[0421] The study timeline is represented in Figure 11.

[0422] Our data showed that the BAK model effectively recapitulated DED pathological changes. Fluorescein staining score (NEI index) was significantly increased on day 7 compared to day 0, indicating clear corneal epitheliopathy progression. Tear secretion was significantly decreased on day 7 compared to day 0, indicating lower tear secretion (Figure 12).

[0423] Tear secretion quantification

[0424] The phenol red test (PRT) used to evaluate tear secretion was performed 1 h after the last BAK administration. Results show that mice treated with Compound 1 (naive MSC-derived EVs) did improve tear secretion compared to the placebo group. Mice treated with Compound 2 (cytokine-primed MSC-EVs) significantly improved tear secretion compared to the placebo-treated group (Compound 4, Figure 13).

[0425] CD45 staining (leukocyte infiltration)

[0426] Markers of leukocyte infiltration indicating the inflammation have been considered on whole cornea samples to evaluate the efficacy of the treatment Compounds (Figure 14A). Mice treated with Compound 1 (MSC-derived EVs of the invention) had a significantly reduced infiltration of CD45+ leukocyte compared to the placebo group, as opposed to mice treated with Compound 2 (cytokine-primed derived EVs) which did not report a significant difference (Figure 14B). This confirms immunomodulatory and anti-inflammatory activity of MSC-derived EVs.

[0427] CD31 and LYVE1 Staining (lymph-angiogenesis)

[0428] Corneal blood and lymphatic vessels were marked with CD31 and LYVE1, respectively (Figure 15A). No significant differences were found between Compound 1 and 2 with Compound 4, demonstrating the MSC-derived EVs did not induce vessel growth in the cornea and lack of adverse effect of the EVs in the cornea (Figure 15A and B).Example 7 : Efficacy and safety of Mesenchymal Stromal Cells-derived Extracellular Vesicles (EVs) in the treatment of infertility.

[0429] EVs production

[0430] EVs were produced and characterized according to example 1.

[0431] Treatment of Infertility and Associated Symptoms in Subjects Treated with EVs according to the invention.

[0432] EVs is administered via intravenous infusion to subjects with a history of infertility. Subjects between the ages of 21 and 50 are selected for treatment. Several subjects have a history of multiple failed IVF cycles and inability to conceive. Prior to administration, subjects are found to have relatively low levels of anti-Mullerian hormone and estrogen and a relatively high level of follicle-stimulating hormone relative to subjects who do not have a history of infertility. After administration, subjects are found to have increased levels of anti-Mullerian hormone and estrogen and a reduced level of follicle-stimulating hormone compared to before administration. Subjects report a reduction in symptoms including cramping, mood disturbance, and irregular menstrual cycle. Several subjects become pregnant within six months of administration of the therapeutic product.

[0433] Other embodiments

[0434] Various aspects of the invention are described in the following numbered paragraphs.

[0435] 1. Isolated Extracellular Vesicles (EVs) wherein said EVs comprise the miRNA markers hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa- miR-16-5p.

[0436] 2. Isolated EVs wherein said EVs comprise hsa-miR-125b-5p and at least one miRNA chosen from hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a- 5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p, and wherein the level of hsa-miR-125b-5p in said EVs is at least 3.5 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR- 199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i- 5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

[0437] 3. Isolated EVs according to any of the embodiments 1 or 2 wherein the level of hsa-miR-125b-5p in said EVs is at least 3.5 to 6 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR-199b-3p or hsa-miR-16-5p.4. The isolated EVs according to any of the embodiments 1 to 3 wherein said EVs further comprise one or more of the miRNAs selected from the group consisting of hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f- 5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa- miR-127-3p and hsa-miR-221-3p.

[0438] 5. The isolated EVs according to embodiments 1 to 4, wherein said EVs comprise the miRNA markers hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR- 199b-3p and hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR- 29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p.

[0439] 6. The isolated EVs according to embodiment 5, wherein the level of hsa-miR- 125b-5p in said EVs is at least 3.5 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191- 5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

[0440] 7. The isolated EVs according to embodiment 2, wherein said EVs comprise hsa- miR-125b-5p and at least one miRNA chosen from hsa-miR-199a-3p, hsa- miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, and hsa-let-7a-5p, and wherein the level of hsa-miR-125b-5p in said EVs is 3.5 to 6 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, and / or hsa-let-7a-5p.

[0441] 8. The isolated EVs according to embodiment 2, wherein said EVs comprise hsa- miR-125b-5p and at least one miRNA chosen from miR-29a-3p, hsa-let-7f- 5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa- miR-127-3p and / or hsa-miR-221-3p, wherein the level of hsa-miR-125b-5p in said EVs is 6.5 fold to 15 fold higher than the individual level of miR-29a- 3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa- let-7i-5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

[0442] 9. Isolated EVs, wherein said EVs comprise at least one, preferably all, miRNA markers selected from the group consisting of hsa-miR-125b-5p, hsa-miR- 199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p and further comprise one or more miRNAs selected from the group consisting of hsa-miR-125a-5p, hsa- let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191- 5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p. 10. The EVs according to any of the embodiments 1 to 9 wherein said EVs are isolated from MSCs (Mesenchymal Stem Cells), preferably umbilical cord MSCs.11. A pharmaceutical composition comprising a therapeutically effective amount of isolated EVs according to any of the embodiments 1 to 10.

[0443] 12. The pharmaceutical composition according to embodiment 11, further comprising at least one pharmaceutically acceptable carrier.

[0444] 13. The pharmaceutical composition according to any of the embodiments 11 or 12, wherein said composition is formulated for intravenous, intranasal, intratracheal, oral, subcutaneous, intramuscular, cutaneous or transdermal, preferably cutaneous administration to a patient.

[0445] 14. The isolated EVs according to any of the embodiments 1 to 10 or pharmaceutical composition according to embodiments 11 to 13, wherein the isolated EVs or composition are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

[0446] 15. The isolated EVs according to any of the embodiments 1 to 10 or pharmaceutical composition according to embodiments 11 to 13, wherein the isolated EVs are loaded with vitamin C.

[0447] 16. Isolated EVs according to embodiments 1 to 10 and / or 14 to 15 or pharmaceutical composition according to any of the previous embodiments 11 to 15 for therapeutic or prophylactic use.

[0448] 17. The isolated EVs or pharmaceutical composition for use according to embodiment 16 in the prevention or treatment of skin disorders.

[0449] 18. The isolated EVs or pharmaceutical composition for use according to embodiment 17, wherein said skin disorder is selected from skin wounds, skin hyperreactivity with hyperkeratosis, skin pigmentation disorders, atrophic scars.

[0450] 19. The isolated EVs or pharmaceutical composition for use according to embodiment 18 wherein the skin disorders are selected from diabetic foot, acute scars post debridement, scleroderma, psoriasis, Vitiligo, melasma, traumatic scars and surgical scars.

[0451] 20. The isolated EVs or pharmaceutical composition for use according to any of the embodiments 17 to 19, wherein the isolated EVs or composition is applied topical to the surface of the skin.

[0452] 21. The isolated EVs or pharmaceutical composition for use according to any of the embodiments 17 to 21, wherein the isolated EVs or composition are applied by intradermal injection.

[0453] 22. The isolated EVs or pharmaceutical composition for use according to embodiment 21, wherein the isolated EVs or composition are applied subsequently to microneedling.23. The isolated EVs or pharmaceutical composition for use according to any of the embodiments 17 to 22, wherein the isolated EVs or composition are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

[0454] 24. The isolated EVs or pharmaceutical composition for use according to any of the embodiments 17 to 23, wherein the isolated EVs or composition are applied at a dose of between 1.2xl09EVs / mL and 1.2xl012EVs / mL.

[0455] 25. The isolated EVs or pharmaceutical composition for use according to embodiment 17 to 24, wherein the isolated EVs or composition are applied at a dose of between 20 and 50 pl per cm2of skin.

[0456] 26. The isolated EVs or pharmaceutical composition for use according to any of the embodiments 17 to 25, wherein the isolated EVs or composition is to be administered one time or repeatedly.

[0457] 27. The isolated EVs or pharmaceutical composition for use according to any of the embodiments 17 to 26, wherein the isolated EVs or composition is to be administered at least 3 times.

[0458] 28. The isolated EVs or pharmaceutical composition for use according to embodiments 17 to 27, wherein the isolated EVs or composition is to be administered at least once every 3 weeks.

[0459] 29. Method for cosmetic treatment or prevention of wrinkles, scars, and / or hyperpigmentation, said method comprises a step of providing the EVs according to any of the embodiments 1 to 10 and / or 14 to 15 or the pharmaceutical composition according to claim 11 to 15 to a patient, in need thereof.

[0460] 30. The method according to embodiment 29, wherein the isolated EVs or composition is applied topical to the surface of the skin.

[0461] 31. The method according to any of the embodiment 29 or 30, wherein the isolated EVs or composition are applied by intradermal injection.

[0462] 32. The method according to any of the embodiments 29 to 31, wherein the isolated EVs or composition are applied subsequently to microneedling. 33. The method according to any of the embodiment 29 to 32, wherein the isolated EVs or composition are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

[0463] 34. The method according to any of the embodiments 29 to 33, wherein the isolated EVs or composition are applied at a dose of between 1.2x 109EVs / mL and 1.2xl012EVs / mL.35. The method according to any of the embodiments 29 to 34, wherein the isolated EVs or composition are applied at a dose of between 20 and 50 pl per cm2of skin.

[0464] 36. The method according to any of the embodiment 29 to 35, wherein the isolated EVs or composition is to be administered one time or repeatedly. 37. The method according to any of the embodiment 29 to 36, wherein the isolated EVs or composition is to be administered at least 3 times.

[0465] 38. The method according to any of the embodiment 29 to 37, wherein the isolated EVs or composition is to be administered at least once every 3 weeks.

[0466] 39. The EVs or pharmaceutical composition for use according to embodiment 16 in the prevention or treatment of lung disorders, inflammatory bowel disease, an ocular surface disease, or infertility.

[0467] 40. The EVs or pharmaceutical composition for use according to embodiment 39 for use in the prevention or treatment of pulmonary hypertension, asthma, bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), allergy, pneumonia, acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis, Crohn's Disease, Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction. 41. The EVs or pharmaceutical composition for use according to embodiment 40, wherein the infertility is endometriosis-associated infertility.

[0468] 42. A method for producing isolated EVs, said method comprising:

[0469] - isolating EVs from a conditioned cell culture medium, preferably from a culture of mesenchymal stem cells; wherein the isolated EVs comprise hsa-miR- 125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p.

[0470] 43. A method of producing isolated EVs according to any of the embodiments 1 to 10 and / or 14 to 15, comprising:

[0471] - isolating EVs from a conditioned cell culture medium, preferably from a culture of mesenchymal stem cells

[0472] - selecting said EVs that comprise hsa-miR-125b-5p, hsa-miR-199a-3p, hsa- miR-199b-3p and hsa-miR-16-5p.

[0473] 44. Method of treatment of lung disorders, inflammatory bowel disease, an ocular surface disease, or infertility said method comprises a step of providing the EVs according to any of the embodiments 1 to 10 and / or 14 to 15 or the pharmaceutical composition according to claim 11 to 15 to a patient, in need thereof.

[0474] 45. The method according to embodiment 44 wherein the lung disorders, inflammatory bowel disease, or ocular surface disease are selected from pulmonary hypertension, asthma, bronchopulmonary dysplasia (BPD),chronic obstructive pulmonary disease (COPD), allergy, pneumonia, acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis, Crohn's Disease, Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction.

[0475] 46. Method of prevention or treatment of skin disorders said method comprises a step of providing the EVs according to any of the embodiments 1 to 10 and / or 14 to 15 or the pharmaceutical composition according to claim 11 to 15 to a patient, in need thereof.

[0476] 47. The method according to embodiment 46 wherein said skin disorder is selected from skin wounds, skin hyperreactivity with hyperkeratosis, skin pigmentation disorders, atrophic scars.

[0477] 48. The method according to embodiment 47 wherein said skin disorder is selected from diabetic foot, acute scars post debridement, scleroderma, psoriasis, Vitiligo, melasma, traumatic scars and surgical scars.

[0478] 49. The method according to embodiments 46 to 48 wherein the isolated EVs or composition is applied topical to the surface of the skin.

[0479] 50. The method according to embodiments 46 to 49, wherein the isolated EVs or composition are applied by intradermal injection.

[0480] 51. The method according to embodiments 46 to 50 wherein the isolated EVs or composition are applied subsequently to microneedling.

[0481] 52. The method according to embodiments 46 to 51, wherein the isolated EVs or composition are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

[0482] 53. The method according to embodiments 46 to 52, wherein the isolated EVs or composition are applied at a dose of between 1.2x 109EVs / mL and 1.2x 1012EVs / mL.

[0483] 54. The method according to embodiments 46 to 53, wherein the isolated EVs or composition are applied at a dose of between 20 and 50 pl per cm2of skin.

[0484] 55. The method according to embodiments 46 to 54, wherein the isolated EVs or composition is to be administered one time or repeatedly.

[0485] 56. The method according to embodiments 46 to 55, wherein the isolated EVs or composition is to be administered at least 3 times.

[0486] 57. The method according to embodiment 44, wherein the infertility is endometriosis-associated infertility.

[0487] 58. The method according to embodiment 57, wherein treated subject is from 21 to 50 years of age

[0488] 59. The method according to any of the embodiments 57 or 58, wherein the treated subject is female.The method according to any of the embodiments 57 to 59, wherein the subject has a history of one or more failed in vitro fertilization (IVF) cycles. The method according to any of the embodiments 57 to 60, wherein the treated subject has a lower level of anti-Mullerian hormone as compared to a subject without a history of infertility.

[0489] The method according to any of the embodiments 57 to 61, wherein the treated subject has premature ovarian failure (POF) and / or polycystic ovary syndrome (PCOS)

Claims

1. CLAIMS1. Isolated Extracellular Vesicles (EVs) wherein said EVs comprise the miRNA markers hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa- miR-16-5p.

2. Isolated EVs wherein said EVs comprise hsa-miR-125b-5p and at least one miRNA chosen from hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a- 5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR-221-3p, and wherein the level of hsa-miR-125b-5p in said EVs is at least 3.5 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR- 199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i- 5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

3. Isolated EVs according to any of the claims 1 or 2 wherein the level of hsa- miR-125b-5p in said EVs is at least 3.5 to 6 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR-199b-3p or hsa-miR-16-5p.

4. The isolated EVs according to claim 1 wherein said EVs further comprise one or more of the miRNAs selected from the group consisting of hsa-miR-125a- 5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa- miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and hsa-miR- 221-3p.

5. The isolated EVs according to any of the previous claims wherein said EVs comprise miRNA markers hsa-miR-125b-5p, hsa-miR-199a-3p, hsa-miR- 199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a-5p, hsa-miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i- 5p, hsa-miR-127-3p and hsa-miR-221-3p.

6. The isolated EVs according to claim 5, wherein the level of hsa-miR-125b-5p in said EVs is at least 3.5 fold higher than the individual levels of hsa-miR- 199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, hsa-let-7a- 5p, miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR- 21-5p, hsa-let-7i-5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

7. The isolated EVs according to claim 2, wherein said EVs comprise hsa-miR- 125b-5p and at least one miRNA chosen from hsa-miR-199a-3p, hsa-miR- 199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, and hsa-let-7a-5p, and wherein the level of hsa-miR-125b-5p in said EVs is 3.5 to 6 fold higher than the individual levels of hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-miR-16-5p, hsa-miR-125a-5p, and / or hsa-let-7a-5p.

8. The isolated EVs according to claim 2, wherein said EVs comprise hsa-miR- 125b-5p and at least one miRNA chosen from miR-29a-3p, hsa-let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR- 127-3p and / or hsa-miR-221-3p, wherein the level of hsa-miR-125b-5p in said EVs is 6.5 fold to 15 fold higher than the individual level of miR-29a-3p, hsa- let-7f-5p, hsa-miR-34a-5p, hsa-miR-191-5p, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-127-3p and / or hsa-miR-221-3p.

9. The EVs according to any of the previous claims wherein said EVs are isolated from MSCs (Mesenchymal Stem Cells), preferably umbilical cord MSCs.

10. A pharmaceutical composition comprising a therapeutically effective amount of isolated EVs according to any of the claims 1 to 9.

11. The pharmaceutical composition according to claim 10, further comprising at least one pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to any of the claims 10 or 11, wherein said composition is formulated for intravenous, intranasal, intratracheal, oral, subcutaneous, intramuscular, cutaneous or transdermal, preferably cutaneous administration to a patient.

13. The isolated EVs according to any of the claims 1 to 9 or pharmaceutical composition according to claims 10 to 12, wherein the isolated EVs or composition are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

14. The isolated EVs according to any of the claims 1 to 9 and / or 13 or pharmaceutical composition according to claims 10 to 13, wherein the isolated EVs are loaded with vitamin C.

15. Isolated EVs according to claims 1 to 9, 13 and / or 14 or pharmaceutical composition according to claims 10 to 14 for therapeutic or prophylactic use.

16. The isolated EVs or pharmaceutical composition for use according to claim 15 in the prevention or treatment of a skin disorder, said use comprises a step of providing the EVs according to any of the embodiments 1 to 9 and / or 13 to 14 or the pharmaceutical composition according to claim 10 to 14 to a patient, in need thereof.

17. The isolated EVs or pharmaceutical composition for use according to claim 16, wherein said skin disorder is selected from skin wounds, skin hyperreactivity with hyperkeratosis, skin pigmentation disorders, atrophic scars.

18. The isolated EVs or pharmaceutical composition for use according to claim 17 wherein the skin disorders are selected from diabetic foot, acute scars postdebridement, scleroderma, psoriasis, Vitiligo, melasma, traumatic scars and surgical scars.

19. The isolated EVs or pharmaceutical composition for use according to any of the claims 16 to 18, wherein the isolated EVs or composition is applied topical to the surface of the skin.

20. The isolated EVs or pharmaceutical composition for use according to any of the claims 16 to 19, wherein the isolated EVs or composition are applied by intradermal injection.

21. The isolated EVs or pharmaceutical composition for use according to claim 20, wherein the isolated EVs or composition are applied subsequently to microneedling.

22. The isolated EVs or pharmaceutical composition for use according to any of the claims 16 to 21, wherein the isolated EVs or composition are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

23. The isolated EVs or pharmaceutical composition for use according to any of the claims 16 to 22, wherein the isolated EVs or composition are applied at a dose of between 1.2xl09EVs / mL and 1.2x 1012EVs / mL.

24. The isolated EVs or pharmaceutical composition for use according to claims 16 to 23, wherein the isolated EVs or composition are applied at a dose of between 20 and 50 pl per cm2of skin.

25. The isolated EVs or pharmaceutical composition for use according to any of the claims 16 to 24, wherein the isolated EVs or composition is to be administered one time or repeatedly.

26. The isolated EVs or pharmaceutical composition for use according to any of the claims 16 to 25, wherein the isolated EVs or composition is to be administered at least 3 times.

27. The isolated EVs or pharmaceutical composition for use according to claims 16 to 26, wherein the isolated EVs or composition is to be administered at least once every 3 weeks.

28. The EVs or pharmaceutical composition for use according to claim 15 in the prevention or treatment of a lung disorder, said use comprises a step of providing the EVs according to any of the embodiments 1 to 9 and / or 13 to 14 or the pharmaceutical composition according to claim 10 to 14 to a patient, in need thereof.

29. The EVs or pharmaceutical composition for use according to claim 28, wherein the lung disorder is acute respiratory distress syndrome (ARDS).

30. The EVs or pharmaceutical composition for use according to claim 28, wherein the lung disorder is selected from pulmonary hypertension, asthma,bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), allergy, pneumonia, or idiopathic pulmonary fibrosis.

31. The EVs or pharmaceutical composition for use according to claim 15 in the prevention or treatment of an inflammatory bowel disease, said use comprises a step of providing the EVs according to any of the embodiments 1 to 9 and / or 13 to 14 or the pharmaceutical composition according to claim 10 to 14 to a patient, in need thereof.

32. The EVs or pharmaceutical composition for use according to claim 31, wherein the inflammatory bowel disease is Crohn's Disease.

33. The EVs or pharmaceutical composition for use according to claim 15 in the prevention or treatment of an ocular surface disease, said use comprises a step of providing the EVs according to any of the embodiments 1 to 9 and / or 13 to 14 or the pharmaceutical composition according to claim 10 to 14 to a patient, in need thereof.

34. The EVs or pharmaceutical composition for use according to claim 33, wherein the ocular surface disease is Dry Eye Disorder.

35. The EVs or pharmaceutical composition for use according to claim 33, wherein the ocular surface disease is selected from Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction in a patient.

36. The EVs or pharmaceutical composition for use according to any of the claims 33 to 35, wherein the EVs are isolated from MSCs that are primed with one or more cytokines.

37. The EVs or pharmaceutical composition for use according to claim 36 wherein said MSCs are primed with one or more cytokines chosen from TNFa, ILip, IL6, or a combination thereof.

38. The EVs or pharmaceutical composition for use according to claim 15 in the prevention or treatment of infertility, said use comprises a step of providing the EVs according to any of the embodiments 1 to 9 and / or 13 to 14 or the pharmaceutical composition according to claim 10 to 14 to a patient, in need thereof.

39. The EVs or pharmaceutical composition for use according to claim 38, wherein the infertility is endometriosis-associated infertility.

40. The EVs or pharmaceutical composition for use according to any of the claims 38 or 39, wherein treated subject is from 21 to 50 years of age41. The EVs or pharmaceutical composition for use according any of the claims 38 to 40, wherein the treated subject is female.

42. The EVs or pharmaceutical composition for use, according to any of the claims 38 to 41, wherein the subject has a history of one or more failed in vitro fertilization (IVF) cycles.

43. The EVs or pharmaceutical composition for use, according to any of the claims 38 to 42, wherein the treated subject has a lower level of anti-Mullerian hormone as compared to a subject without a history of infertility.

44. The EVs or pharmaceutical composition for use, according to any of the claims 38 to 43, wherein the treated subject has premature ovarian failure (POF) and / or polycystic ovary syndrome (PCOS).

45. Method for cosmetic treatment or prevention of wrinkles, scars, and / or hyperpigmentation, said method comprises a step of providing the EVs according to any of the claims 1 to 9 and / or 13 to 14 or the pharmaceutical composition according to claim 10 to 14 to a patient, in need thereof.

46. The method, according to claim 45, wherein the isolated EVs or composition is applied topical to the surface of the skin.

47. The method, according to any of the claims 45 or 46, wherein the isolated EVs or composition are applied by intradermal injection.

48. The method according to any of the claims 45 to 47, wherein the isolated EVs or composition are applied after preparation of the skin, such as by microneedling.

49. The method, according to any of the claims 45 to 48, wherein the isolated EVs or composition are formulated in a cream, gel, lotion, ointment, spray, patch, injectable or serum.

50. The method according to any of the claims 45 to 49, wherein said isolated EVs or composition are provided at a dose of between 1.2xl09EVs / mL and 1.2xl012EVs / mL.

51. The method according to any of the claims 45 to 50, wherein the isolated EVs or composition are applied at a dose of between 20 and 50 pl per cm2of skin.

52. The method, according to any of the claims 45 to 51, wherein the isolated EVs or composition is to be administered one time or repeatedly.

53. The method, according to any of the claims 45 to 52, wherein the isolated EVs or composition is to be administered at least 3 times.

54. The method, according to any of the claims 45 to 53, wherein the isolated EVs or composition is to be administered at least once every 3 weeks.

55. A method for producing isolated EVs, said method comprising:- isolating EVs from a conditioned cell culture medium, preferably from a culture of mesenchymal stem cells; wherein the isolated EVs comprise hsa-miR-125b- 5p, hsa-miR-199a-3p, hsa-miR-199b-3p and hsa-miR-16-5p.

56. A method of producing isolated EVs according to any of the claims 1 to 9 and / or 13 to 14, comprising:- isolating EVs from a conditioned cell culture medium, preferably from a culture of mesenchymal stem cells, and- selecting said EVs that comprise hsa-miR-125b-5p, hsa-miR-199a-3p, hsa- miR-199b-3p and hsa-miR-16-5p.

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