Extracellular vesicles for the treatment of ocular surface disease

Abstract

The current invention relates to isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use in the treatment of an ocular surface disease, wherein said EVs are administered to the eye of a patient suffering from said ocular surface disease, wherein said EVs are MSC (Mesenchymal Stem Cells) derived EVs, preferably umbilical cord MSC-derived EVs, 10 wherein said EVs are associated with albumin and Annexin V, and wherein said EVs are positive for CD44 and CD29.

Description

[0001] EXTRACELLULAR VESICLES FOR THE TREATMENT OF OCULAR SURFACE DISEASE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the use of isolated Extracellular Vesicles (EVs) for the treatment of ocular surface diseases.

[0004] BACKGROUND

[0005] Ocular Surface Disease (OSD) can result from various types of damage to the eye surface, including mechanical trauma, chemical and thermal burns, or severe inflammatory conditions such as Graft-Versus-Host Disease (GVHD) following allogeneic hematopoietic stem cell (HSC) transplantation. Numerous other factors can also harm the cornea, potentially leading to reduced vision or blindness. The underlying pathology typically involves an inflammatory response that progresses to fibrotic scarring and neovascularization— termed Corneal Neovascularization Disease (CNV)— which results in the loss of visual acuity due to diminished optical clarity and corneal immune privilege. Animal models further suggest that inflammation from ocular surface damage can extend into brain structures.

[0006] OSD significantly affects global health, with approximately 180 million people suffering from severe visual impairment, including 45 million affected bilaterally. The prevalence is rising, particularly in regions like the USA and China, due to increased use of contact lenses. Current treatment options for CNV are limited and often inadequate, with no single intervention proving fully effective. Standard therapeutic approaches include: a) Corticosteroids: These have variable therapeutic efficacy and are associated with severe side effects such as cataract formation, increased intraocular pressure (IOP), delayed healing, higher infection risk, and corneal thinning. b) Non-steroidal Anti-inflammatory Drugs (NSAIDs): Generally less effective compared to corticosteroids and carry risks such as corneal melting, making them unsuitable for widespread use in CNV treatment. c) Anti-VEGF Therapy: Currently under clinical evaluation and not yet available on the market. d) Surgical Corneal Transplantation: Often performed as a last resort at a high cost (15,000-30,000 Euros). However, CNV and associated scarring can significantly reduce transplantation outcomes, with a 25% rejection rate and up to 40% of low- risk transplants developing CNV within 6-9 months.

[0007] Due to the limited efficacy and significant drawbacks of existing treatments, corneal injury and its associated conditions could be classified as orphan diseases and represent an unmet medical need.

[0008] Dry Eye Syndrome (DES), also known as Dry Eye Disease (DED), is an example of an ocular surface disease, typically resulting from insufficient tear production or altered lipid composition, which accelerates tear evaporation and causes eye surface damage. The prevalence of DES varies between 7% and 15% of the population, depending on geographic region, and is significantly higher in women and older individuals. DES is also frequently associated with contact lens use. In patients with chronic GVHD, DES is highly prevalent, affecting 60% to 90% of cases.

[0009] DES is an idiopathic inflammatory condition, often seen in the context of GVHD- associated DES. Severe cases can lead to fibrotic scarring and vascularization of the cornea, further compromising vision. DES is a chronic condition primarily managed with symptomatic treatments such as artificial tears and anti-inflammatory medications like corticosteroids and cyclosporine. Unfortunately, corticosteroids have limited long-term efficacy and prolonged use can result in serious side effects, underscoring the need for new therapeutic strategies.

[0010] There is a need for a better or alternative treatment of OSD such as Dry Eye Syndrome, especially if conventional treatments fail.

[0011] Extracellular vesicles (EVs), also known as exosomes, are lipid bilayer-enclosed particles that are naturally secreted by cells. Unlike cells, EVs cannot replicate, but they carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from their parent cells, which gives them significant therapeutic potential. W02022008657 describes a specific set of EVs that can be used for treating of inflammatory diseases such as lung or gastro-intestinal disorders. This document does not describe the potential of using EVs in a treatment of OSD. SUMMARY OF THE INVENTION

[0012] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a treatment of ocular surface disease such as dry eye according to claim 1. In some embodiments, the EVs disclosed herein are positive for CD44 and CD29. In further embodiments, the EVs are positive for CD44 and CD29 and are associated or comprise Annexin V and albumin. The MSC-derived EVs or compositions comprising the latter as described herein are shown to induce tear and / or mucin secretion in the treated eye or part thereof. It is believed that the marker expression profile of said EVs plays an important role in the functionality of said EVs and their usefulness for the therapy described herein.

[0013] Preferred embodiments of the invention are shown in any of the claims 2 to 16. Summarized, the invention pertains at least to the following embodiments:

[0014] 1. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use in the treatment of an ocular surface disease, wherein said EVs are administered to the eye of a patient suffering from said ocular surface disease.

[0015] 2. Use according to embodiment 1, wherein the EV is positive for any one of the markers selected from CD44, CD29, CD105, CD9, CD63, CD81, SSEA4, HLA1, CD49e or MSCP.

[0016] 3. Use according to any of the previous embodiments, wherein the EVs are associated with albumin or Annexin V.

[0017] 4. Use according to embodiment 1, for treatment of ocular inflammation or tissue repair.

[0018] 5. Use according to any of the previous embodiments, for the treatment of Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction.

[0019] 6. Use according to any of the previous embodiments, wherein the EVs are primed with cytokines to enhance their anti-inflammatory properties.

[0020] 7. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use in the treatment of an ocular surface disease, wherein said EVs are administered to the eye of a patient suffering from said ocular surface disease, wherein, wherein said EVs are MSC (Mesenchymal Stem Cells) derived EVs, preferably umbilical cord MSC-derived EVs, wherein said EVs are associated with albumin and Annexin V and wherein said EVs are positive for CD44 and CD29.

[0021] 8. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to embodiment

[0022] 7, wherein said EVs are further positive for one or more markers chosen from CD105, CD9, CD63, CD81, SSEA4, HLA1, CD49e or MSCP.

[0023] 9. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the embodiments 7 or 9, wherein said EVs express CD105, CD29, CD9, CD63, CD81, SSEA4, HLA1, MSCP, CD49e and CD44.

[0024] 10. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to embodiment

[0025] 8, wherein the respective expression level of CD44 and CD29 is between 2.5- and 5-fold higher than the level of expression of CD105, CD49e or MSCP when normalized with CD9, CD63 and CD81 expression.

[0026] 11. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the embodiments 7 to 10 wherein the ratio between EV-associated Annexin and EV-associated albumin is between 5 and 222 pg Annexin V per g albumin

[0027] 12. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the embodiments 7 to 11, wherein said EVs have a mean particle size of below 120 nm, such as between 95 and 110 nm.

[0028] 13. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the embodiments 7 to 12, wherein said EVs are negative for one or more markers chosen from HLA class II, CDllb, CD 14, CD19, CD34, HLA-DR or CD45.

[0029] 14. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the embodiments 7 to 13 wherein said MSCs are primed with one or more cytokines.

[0030] 15. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the embodiments 7 to 14, wherein said MSCs are primed with one or more cytokines chosen from TNFa, ILip or IL6.

[0031] 16. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to embodiments 14 or 15, wherein the levels of expression of CD29 and CD44 in said primed EVs are higher than in said naive EVs, preferably at least 2 times higher.

[0032] 17. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to embodiments 7 to 16, wherein said ocular surface disease is Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction, preferably Dry Eye Disorder.

[0033] 18. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiments 7 to 17, wherein said EVs or composition is formulated for topical delivery to the eye, preferably to the conjunctival sac.

[0034] 19. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiments 7 to 18, wherein said EVs are to be reconstituted in a buffer prior to said administration.

[0035] 20. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiments 7 to 19 wherein said EVs are administered at a dose of between 1.2 x 107and 1.2 x 1012EVs / eye.

[0036] 21. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiments 7 to 20, wherein said EVs or composition is to be administered one time, repeatedly, or continuously.

[0037] 22. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiment 20, wherein said administration is repeated, preferably with at least 1 to 24 hours between each administration, more preferably once or twice a day.

[0038] 23. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiments 7 to 22, wherein said isolated MSC-derived EVs are autologous to the patient, or wherein the isolated MSC-derived EVs are allogeneic to the patient.

[0039] 24. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiments 7 to 23, wherein said patient has received at least one prior treatment, said prior treatment being chosen from eye drops or artificial tears, corticosteroid therapy, NSAID therapy, anti-VEGF therapy, or surgical corneal transplantation.

[0040] 25. Isolated MSC-derived EVs or the pharmaceutical composition for use according to embodiments 7 to 24, wherein said EVs when administered induce tear and / or mucin secretion in the eye or part of said eye.

[0041] 26. A method for treating an ocular surface disease in a patient in need thereof, comprising administering an effective amount of isolated Mesenchymal Stem Cell (MSC)-derived extracellular vesicles (EVs) to the eye of said patient. 27. The method according to embodiment 26, wherein the EV is positive for any one of the markers selected from CD44, CD29, CD105, CD9, CD63, CD81, SSEA4, HLA1, CD49e or MSCP.

[0042] 28. The method according to any of the embodiments 26 or 27, wherein the EVs are associated with albumin or Annexin V

[0043] 29. The method according to any of the embodiments 26 to 28, for treating of Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction.

[0044] 30. Method according to embodiment 28 wherein the ratio between EV- associated Annexin and EV-associated albumin is between 5 and 222 pg Annexin V per g albumin and wherein said EVs are positive for CD44 and CD29.

[0045] 31. Method according to any of the embodiments 26 to 30 wherein said EVs express CD105, CD29, CD9, CD63, CD81, SSEA4, HLA1, MSCP, CD49e and CD44.

[0046] 32. The method according to embodiment 31 wherein the respective expression level of CD44 and CD29 is between 2.5- and 5-fold higher than the level of expression of CD105, CD49e or MSCP when normalized with CD9, CD63 and CD81 expression.

[0047] 33. The method according to any of the embodiments 26 to 32 wherein said EVs have a mean particle size of below 120 nm, such as between 95 and 110 nm.

[0048] 34. The method according to any of the embodiments 26 to 33 wherein said EVs are negative for one or more markers chosen from HLA class II, CDllb, CD 14, CD19, CD34, HLA-DR or CD45.

[0049] 35. The method according to any of the embodiments 26 to 34 wherein said MSCs are primed with one or more cytokines.

[0050] 36. The method according to any of the embodiments 26 to 35 wherein said MSCs are primed with one or more cytokines chosen from TNFa, ILip or IL6.

[0051] 37. The method according to any of the embodiments 35 or 36, wherein the levels of expression of CD29 and CD44 in said primed EVs are higher than in said naive EVs, preferably at least 2 times higher.

[0052] 38. Method according to any of the embodiments 26 to 37, wherein said ocular surface disease is Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction, preferably Dry Eye Disorder.

[0053] 39. Method according to any of the embodiments 26 to 38, wherein said EVs or composition is formulated for topical delivery to the eye, preferably to the conjunctival sac. 40. Method according to any of the embodiments 26 to 39, wherein said EVs are to be reconstituted in a buffer prior to said administration.

[0054] 41. Method according to any of the embodiments 26 to 40, wherein said EVs are administered at a dose of between 1.2 x 107and 1.2 x 1012EVs / eye.

[0055] 42. Method according to any of the embodiments 26 to 41 wherein said EVs or composition is to be administered one time, repeatedly, or continuously.

[0056] 43. Method according to embodiment 41, wherein said administration is repeated, preferably with at least 1 to 24 hours between each administration, more preferably once or twice a day.

[0057] 44. Method according to any of the embodiments 26 to 43 wherein said isolated MSC-derived EVs are autologous to the patient, or wherein the isolated MSC- derived EVs are allogeneic to the patient.

[0058] 45. Method according to any of the embodiments 26 to 44, wherein said patient has received at least one prior treatment, said prior treatment being chosen from eye drops or artificial tears, corticosteroid therapy, NSAID therapy, anti- VEGF therapy, or surgical corneal transplantation.

[0059] 46. Method according to any of the embodiments 26 to 45, wherein said EVs when administered induce tear and / or mucin secretion in the eye or part of said eye.

[0060] 47. A pharmaceutical composition comprising an effective amount of isolated extracellular vesicles (EVs) derived from Mesenchymal Stem Cells (MSCs) and a pharmaceutically acceptable carrier for topical administration to the eye.

[0061] 48. The pharmaceutical composition of embodiment 46, wherein said EVs are positive for any one of the markers selected from CD44, CD29, CD105, CD9, CD63, CD81, SSEA4, HLA1, CD49e or MSCP.

[0062] 49. The pharmaceutical composition of embodiments 47 or 48, wherein said EVs are associated with albumin or Annexin V.

[0063] 50. The pharmaceutical composition of embodiment 49, wherein the ratio between EV-associated Annexin and EV-associated albumin is between 5 and 222 pg Annexin V per g albumin and wherein said EVs are positive for CD44 and CD29.

[0064] 51. The pharmaceutical composition of embodiments 47 to 50, wherein said EVs express CD105, CD29, CD9, CD63, CD81, SSEA4, HLA1, MSCP, CD49e and CD44.

[0065] 52. The pharmaceutical composition of embodiments 48 to 51, wherein the respective expression level of CD44 and CD29 is between 2.5- and 5-fold higher than the level of expression of CD105, CD49e or MSCP when normalized with CD9, CD63 and CD81 expression. 53. The pharmaceutical composition of embodiments 47 to 52 wherein said EVs have a mean particle size of below 120 nm, such as between 95 and 110 nm.

[0066] 54. The pharmaceutical composition of embodiments 47 to 53 wherein said EVs are negative for one or more markers chosen from HLA class II, CDllb, CD 14, CD19, CD34, HLA-DR or CD45.

[0067] 55. The pharmaceutical composition of embodiments 47 to 54 wherein said MSCs are primed with one or more cytokines.

[0068] 56. The pharmaceutical composition of embodiments 47 to 55 wherein said MSCs are primed with one or more cytokines chosen from TNFa, ILip or IL6.

[0069] 57. The pharmaceutical composition of embodiments 55 or 56, wherein the levels of expression of CD29 and CD44 in said primed EVs are higher than in said naive EVs, preferably at least 2 times higher.

[0070] 58. The pharmaceutical composition of embodiments 47 to 57, wherein said pharmaceutical composition is formulated for topical delivery to the eye, preferably to the conjunctival sac.

[0071] 59. The pharmaceutical composition of embodiments 47 to 58, wherein said pharmaceutical composition comprised between 1.2 x 107and 1.2 x 1012EVs / eye.

[0072] 60. The pharmaceutical composition of embodiments 47 to 59, wherein said pharmaceutical composition is for single, repeatedly, or continuously administration.

[0073] 61. The pharmaceutical composition of embodiment 59, wherein said administration is repeated, preferably with at least 1 to 24 hours between each administration, more preferably once or twice a day.

[0074] 62. The pharmaceutical composition of embodiments 47 to 61 wherein said isolated MSC-derived EVs are autologous to the patient, or wherein the isolated MSC-derived EVs are allogeneic to the patient.

[0075] DESCRIPTION OF FIGURES

[0076] Figure 1. Representation of the 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 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. Figure 2. Representation of BAK effect on mouse model. 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.

[0077] Figure 3. Representation of tear secretion in BAK-induced DED mouse treated by MSC-derived EVs (Cl), cytokine-primed MSC-derived EVs (C2) and saline (placebo, C4). Tear secretion was quantified using phenol red test (FRT).

[0078] Figure 4. A) Representative pictures of CD45 staining on cornea samples of mice treated with compounds 1 (MSC-derived EVs), 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.

[0079] Figure 5. A) Corneal sections of mice treated with Compound 1 (MSC-derived EVs), 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.

[0080] DETAILED DESCRIPTION OF THE INVENTION

[0081] The present invention concerns a treatment of an ocular surface disease such as dry eye by means of Extracellular Vesicles (EVs) or a pharmaceutical composition comprising said EVs.

[0082] 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.

[0083] As used herein, the following terms have the following meanings: "A", "an", and "the" as used herein refers 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.

[0084] "About" as used herein referring 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.

[0085] "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.

[0086] 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 understood that 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.

[0087] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0088] 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.

[0089] 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. 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.

[0090] 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.

[0091] For the purpose of the current invention, the term "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.

[0092] 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. The term "allogeneic" as used herein refers to EVs that are produced by one individual or organism and then used or applied to another individual or organism of the same species.

[0093] The term "autologous" as used herein refers to EVs that are derived from the same individual or organism as the one receiving or utilizing them. In the context of autologous EVs, the vesicles are obtained from the subject's cells, tissues, or biological material.

[0094] The term "associated with EV(s)" in relation to substances means that said substance is either a) attached to or bound to the surface layer of the EVs (by any type of binding, such as covalent or non-covalent binding) preferably by means of a non- covalent bond; and / or b) attached to or bound within the surface layer of said EVs; and / or c) is internalized within said EVs. Said substances associated with EVs may be any type of substances, such as, but not limited to molecules including amino acids, proteins, peptides, nucleic acids such as RIMA and DNA (e.g. noncoding RNA, miRNA, mRNA), sugars, carbohydrates, fats, vitamins, growth factors, pro- angiogenic molecules, cardioprotective enzymes, antibodies, anti-inflammatory molecules, anti-fibrotic molecules, anti-oxidative molecules, pro-neurogenic molecules and anti-viral molecules; and ions such as metal ions or calcium ions.

[0095] 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.

[0096] The term "serum-free" cell culture medium refers to a cell culture medium that does not contain animal or human serum, plasma or hemolymph. Said serum-free medium may contain ingredients that are processed or derived from blood, serum or plasma such as albumin, transferrin, low-density lipids and hormones. It may also contain other biological ingredients, preferably of known and fully reproducible composition and concentration, that are not serum, plasma or hemolymph (e.g., growth factors, hormones and carrier proteins). The term "xeno-free" cell culture medium is to be understood as cell media that does not contain any components that are directly derived from non-human animals, or recombinant components manufactured from non-human animal DNA sequences.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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 or condition 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.

[0101] The terms "composition" and "pharmaceutical compositon" 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.

[0102] 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.

[0103] For the purpose of the current invention, the term "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.

[0104] Extracellular vesicles and their preparation

[0105] EVs according to the current disclosure are preferably prepared according to the method disclosed in W02022008652 which is considered to be incorporated in its entirety herein. In short, MSCs will be cultured and expanded in a vessel, preferably a bioreactor, more preferably a stirred-type tank bioreactor, for the use of the production of EVs. To that purpose, MSCs will be allowed to grow and expand in a culture medium that is serum- and xeno-free, supplemented with human albumin and optionally transferrin. By making use of serum- and xeno-free growth medium for culturing and expanding said MSCs, the presence of unwanted contaminants in the EVs is avoided. Such contaminants may interfere with the further clinical use of the resulting EV product. Serum and platelet lysate generally comprise albumin and cell growth is often influenced by the level of albumin in the medium. The levels of albumin as for any other ingredient in serum and platelet lysate are however variable. In order to obtain clinical-grade EVs, the use of serum and / or platelet lysate is to be avoided.

[0106] Albumin is however a crucial component of the end-product as it aids in the stability and functionality of said EVs. "Stability" of a product, such as EVs, in present context refers to the capability of a particular formulation or product in a specific environment, such as a container or closed system, to remain within a predefined range of values of parameters of physical, chemical, microbiological, toxicological and functional specifications or characteristics, for a given time period. Non-limiting examples of such parameters are particle number, particle size, leakage of internal components and activity.

[0107] MSCs may grow and expand on microcarriers or microbeads present in said bioreactor. In an embodiment, said medium therefore comprises human albumin, either recombinant or purified albumin, e.g. purified from human plasma. In a further embodiment, said albumin is present in said medium at a concentration of between 1 g / l and 5 g / l. The latter concentration was shown to be particularly useful in obtaining a good and stable end product.

[0108] The cell growth medium further comprises transferrin, preferably recombinant or purified transferrin, e.g. purified from plasma. Transferrins make up an extensive micro-heterogeneous group of single-chain glycoprotein isotypes with approximate molecular weights of 78 to 80 kDa. Transferrin is the physiologically appropriate method for providing iron to cells in culture as it facilitates extracellular iron storage and transport. Transferrins have been reported to aid in the cellular uptake of the EVs in vivo, see for instance W02013084001. In addition similar to albumin, transferrins can aid in the stabilization of the final EV product. In an embodiment, said transferrin is present in the growth medium at a concentration of between 50 mg / l and 100 mg / l, preferably between 55 mg / l and 100 mg / l, preferably between 50 mg / l and 70 mg / l, preferably between 55 mg / l and 70 mg / l. The latter concentration was found optimal for the quality of the EV product.

[0109] The current protocol enables the presence of both albumin and transferrin in the end product, where the majority of the albumin and transferrin will be associated with said EVs.

[0110] In a further embodiment, said medium is a basic mixture of inorganic metal salts, nutrients such as amino acids, and vitamins, with a pH in the range from pH 7.0 to 7.4, such as known from the composition of Dulbecco's Modified Eagle Medium (DMEM) or DMEM / F-12. The media may also contain glutamine or glutamate, or a precursor thereof such as L-alanyl- L-glutamine, and glucose at a level of not more than 4500 mg / l and one or more fatty acids.

[0111] MSCs may grow and expand on microcarriers or microbeads present in said bioreactor. Such microcarriers or beads are known in the art and commercially available. In an embodiment, said microcarriers or beads may be coated with an extracellular matrix protein, such as fibronectin, laminin, hyaluronic acid, their mimetics or a combination thereof. In a preferred embodiment, said microcarriers or beads are negatively charged.

[0112] Once the MSCs have reached a desired concentration and / or confluency, cell supernatant comprising said EVs is collected for further processing. In the context of the current invention, said cell supernatant is the cell medium in which the MSCs were allowed to grow and expand. In an embodiment, said supernatant is collected when said MSCs reach a minimal concentration of at least 40xl06cells / l. Cell concentration and viability may be defined by means of cell counting, such as for instance by means of a hemacytometer such as Burker Counting Chamber and trypan blue staining. Under the culturing conditions described above, said MSCs will produce at least 0.25 x 109particles / mL cell medium in a period of 18 to 24 hours of culturing said MSCs.

[0113] "Particle" as used above can be any particle having a particle size of preferably between 0.05 and 0.22 microns, among which are EVs. Other examples of particles can be proteins or peptide aggregates. The source of said particles is MSCs or the cell medium for culturing and expanding MSCs. Therefore said particle can be any particle that is normally present or part of said cell medium or MSC. Preferably, when using the method as described herein, at least 90% of said particles have a size of between 0.05 and 0.22 microns are EVs, referred to as "D90".

[0114] In a subsequent step, said cell supernatant is to be filtered in order to remove contaminants present in said cell medium. The latter will contribute to the purity and stability of the end-product. In a preferred embodiment, said filtration will comprise at least two steps of filtration. In an embodiment, at least one of said filtration steps will be dead-end filtration. In a further embodiment, both filtration steps are performed by means of dead-end filtration. Preferably, at least one of the filtration steps is also used as a product sterilization means, in order to be compliant with GMP regulations for sterile products for human administration. . Sequential deadend filtration was in some cases found necessary to sufficiently remove impurities from the supernatant. In some cases, it was found that single filtration often resulted in blockage of the filter used and reduced the purity and quantity of the end product.

[0115] In the first filtration step, cell supernatant is filtered through a dead-end filter. In a further preferred embodiment, said filtration occurs by means of bringing said supernatant over a filter with a mesh size of between 1 and 5 microns, more preferably between 1 and 3 microns. In an embodiment, said first filtration step is performed by means of dead-end filtration, preferably in a closed system with a peristaltic pump providing constant flow through the filter, preferably at 100 ml / min.

[0116] In an embodiment, the filtrate of the first filtration will be brought over a second filter, this time a filter with a pore size that is smaller than the pore size used in the first filtration step. Preferably, this second filtration step is a sterilization step, in order to render the final product compliant with GMP regulations as defined above.

[0117] In a more preferred embodiment, said filter has a mesh size below 1 micron, more preferably between 0.05 and 1 micron, even more preferably between 0.1 and 0.5 microns, and most preferably between 0.1 and 0.22 microns. In an embodiment, said second filtration step is performed by means of dead-end filtration, preferably in a closed system interconnected with the first step of filtration, and with a peristaltic pump providing constant flow through the filter, preferably at 100 ml / min. The cell supernatant or filtrate is thus cleared of vesicles and aggregates over 220 nanometer in size.

[0118] The filtrate of said filtration steps will comprise the EVs according to the current invention. In a final step, said EVs will be washed and concentrated. Washing and concentration may occur by conventional means known in the art such as membrane filtration, or micro- or ultrafiltration. Concentration is a process that involves removing fluid from a solution while retaining the solute particulate components and molecules.

[0119] Membrane filtration is a separation technique widely used in the life science laboratory. Depending on membrane porosity, it can be classified as a microfiltration or ultrafiltration process. Microfiltration membranes, with pore sizes typically between 0.1 pm and 10 pm, are generally used for clarification, sterilization, and removal of microparticulate or for cell harvesting. Ultrafiltration membranes, with much smaller pore sizes between 0.001 pm and 0.1 pm, are used for concentrating and desalting dissolved molecules (proteins, peptides, nucleic acids, carbohydrates, and other biomolecules), exchanging buffers, and gross fractionation. Ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. There are two main membrane filtration modes which can use either microfiltration or ultrafiltration membranes: 1) Direct Flow Filtration (DFF), also known as "dead-end" filtration, applies the feed stream perpendicular to the membrane face and attempts to pass 100% of the fluid through the membrane, and 2) Tangential Flow Filtration (TFF), also known as crossflow filtration, where the feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is recirculated back to the feed reservoir.

[0120] By preference said washing and concentration will be performed by means of TFF. TFF or Crossflow Filtration is a process where the feed stream flows parallel to the membrane face. The applied pressure causes one portion of the flow stream to pass through a membrane (filtrate or permeate) while the remainder (retentate) is recirculated back to the feed reservoir. In an embodiment, the filtrate of one or more dead-end filtration steps will be used in a TFF concentration step. In a further preferred embodiment, the TFF will have a cut-off of 100 kDa and will remove the majority if not all particles and components with a molecular weight lower than 100 kDa into the TFF permeate. Consequently, said final composition, which will remain in the retentate, will be depleted from free (that is: not EV-associated) constituents, components or substances with a molecular weight below 100 kDa. Said constituents, components or substances can be any particle such as a protein or a peptide that is normally present or part of said cell medium. The retentate will be recirculated in the TFF device until the required retentate concentration has been reached. During the recirculation, the retentate can be washed using a washing medium or washing buffer, preferably a saline washing buffer, in order to remove unwanted components. The concentrated retentate can subsequently be collected in a collection vessel, such as but not limited to a collection bag or cryotubes, either kept at a low temperature of below 10 °C, preferably 4°C, or frozen at -20°C to - 196°C, preferably at -40°C to - 196°C, even more preferably at -80°C to -196°C.

[0121] In an embodiment, the 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.

[0122] Optical techniques are routinely used to size and count EVs. In an embodiment of the current invention, the particle size of said EVs is measured by Nanoparticle Tracking Analysis (NTA), which is a preferred method for quantification and sizing of nanoparticles suspended in liquid buffers. A suitable apparatus is the ZetaView® Nanoparticle Tracking Analysers PMX220 TWIN (Particle Metrics). In another further embodiment, the particle size is measured using Tunable Resistive Pulse Sensing (TRPS) which is used as a reference method to NTA. In another or further embodiment, the particle size is measured using High-Resolution Flow Cytometry.

[0123] In a preferred embodiment, the EVs are measured as a sample population by NTA and exhibit a number-weighted mean diameter or average particle size of 220 nm or less, or 200 nm or less, or 180 nm or less, or 160 nm or less, or 140 nm or less, or 120 nm or less. Suitably, said EVs exhibit a mean particle size of between 95 and 110 nm, or around 100 nm or 106 nm, and preferably exhibit a normal distribution. Alternatively, said sample population of EVs exhibit a mode diameter of 220 nm or less, or 200 nm or less, or 180 nm or less, or 160 nm or less, or 140 nm or less, or 120 nm or less, or 100 nm or less.

[0124] In an embodiment, the EVs according to the current invention are defined by being isolated MSC-derived EVs, preferably umbilical cord MSC-derived EVs and are positive for CD44 and CD29. In a further embodiment, said EVs are further positive for one or more markers chosen from CD105, CD9, CD63, CD81, SSEA4, HLA1, CD49e or MSCP. The term "are positive for," as used herein, indicates that the EVs described in this disclosure exhibit the presence of specific markers on their surface or within them. These markers can be detected using various laboratory techniques, such as flow cytometry or immunostaining.

[0125] In a preferred embodiment, said EVs express CD105, CD29, CD9, CD63, CD81, SSEA4, HLA1, MSCP, CD49e and CD44. The term "EVs express the markers" indicates that the EVs are actively producing the specified marker, or that the marker is part of the standard set of molecules generated by the EVs or cells.

[0126] In an embodiment of the EVs as disclosed herein, the respective expression level of CD44 and CD29 is between 2.5 and 5-fold higher than the level of expression of CD105, CD49e or MSCP when normalized with CD9, CD63 and CD81 expression.

[0127] In some embodiments, the expression level of CD44 and CD29 is between 2.7 and 5-fold, between 2.9 and 5-fold, between 3 and 5-fold, between 3.2 and 5-fold, between 3.5 and 5-fold, between 3.8 and 5-fold, between 4 and 5-fold, between 4.2 and 5-fold, between 4.4 and 5-fold, between 4.5 and 5-fold, or between 4.8 and 5- fold higher than the level of expression of CD105, CD49e or MSCP when normalized with CD9, CD63 and CD81 expression. In alternative embodiments, the expression level of CD44 and CD29 is between 2.5- fold and 4.8-fold, between 2.5-fold and 4.5-fold, between 2.5-fold and 4.5-fold, between 2.5-fold and 4.4-fold, between 2.5-fold and 4.2-fold, between 2.5-fold and 4-fold, between 2.5-fold and 3.8-fold, between 2.5-fold and 3.5-fold, between 2.5- fold and 3.2-fold, between 2.5-fold and 3-fold or between 2.5-fold and 2.8-fold.

[0128] The expression levels of the EV markers are preferably normalized to the reference markers CD9, CD63, and CD81. These tetraspanins are major components of extracellular vesicles, making their expression levels ideal as a baseline or standard for comparing other markers. This normalization ensures that any differences in marker expression reflect true biological variation, rather than being influenced by sample size, detection efficiency, or experimental conditions.

[0129] In an embodiment said EVs are defined as being HLA class II negative, and preferably also class I negative. The human leukocyte antigen (HLA) system or complex is a group of related proteins that are encoded by the major histocompatibility complex (MHC) gene complex in humans. These cell-surface proteins are responsible for the regulation of the immune system. Different classes have different functions. HLAs corresponding to MHC class I present peptides from inside the cell, while HLAs corresponding to MHC class II present antigens from outside of the cell to T- lymphocytes. HLAs corresponding to MHC class III encode components of the complement system.

[0130] In an embodiment, the EVs according to the current invention are defined by being negative for one or more markers chosen from HLA class II, CDllb, CD 14, CD19, CD34, HLA-DR or CD45.

[0131] In some embodiments, when an EV is positive for a particular marker, it means that the EV contains detectable levels (e.g., as determined by Western blotting) of the marker and / or levels sufficient to elicit a certain response in a target cell or tissue or elicit a certain response in a human subject in the context of methods of treatment as described herein. In some embodiments, when an EV is negative for a particular marker, it is meant that the EV contains none of or only insignificant amounts of the particular marker. For example, an insignificant amount may be an amount that is undetectable or an amount that is detectable at only trace amounts.

[0132] The presence or absence of a particular marker in the EVs is determined by any methods known in the art that include although not limited to Western blotting, immunohistochemical staining, flow cytometry, NTA, ELISA, or RT-PCR. In a preferred embodiment, a MACSPex EV Kit® is being used, to measure surface epitopes present on EVs by utilizing labeled bead populations. The MACSPlex EV Kit® is designed to detect multiple EV surface markers by using fluorescently labeled beads that bind to specific epitopes on the EV surface.

[0133] In an embodiment, the EVs according to the current invention are defined by being positive for one or more of the following: intra-vesicular Annexin V or external phosphatidylserine.

[0134] In an embodiment, the EVs according to the current invention are defined by being positive for one or more of the following marker proteins associated with the cytosolic compartment: Heat shock protein HSP 90-alpha [HSP90AA1], Heat shock protein HSC70 [HSPA8], Heat shock protein beta-1 [HSPB1], Serpin B6 [SERPINB6], Peroxiredoxin-1 [PRDX1], Annexin A2 [ANXA2] or Annexin A5 [ANXA5]. In an embodiment, the EVs according to the current invention are defined by being positive for one or more of the following transmembrane markers: Integrin beta-1 [ITGB1] or Integrin alpha-2 [ITGA2].

[0135] In an embodiment, the EVs according to the current invention are associated with albumin. In an embodiment, the EVs according to the current invention are associated with Annexin V. In an embodiment, the EVs according to the current invention are associated with both albumin and Annexin V.

[0136] Human albumin has a molecular weight of about 66 kDa. A concentration step of the EVs in supernatant in the EV production method can be chosen so that, for example, particles with a molecular weight of below 100 kDa, which are not associated with the EVs will be removed from the EVs. As the molecular weight of human albumin is below 100 kDa, albumin not associated with EVs is therefore removed by this concentration step, the remaining albumin in the composition being associated with the EVs. In a further preferred embodiment, at least 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, more preferably 99% of the albumin in said composition is associated with EVs.

[0137] Annexins are calcium-dependent phospholipid binding proteins for which association with EVs increases the anti-inflammatory properties of EVs. Some annexins, such as Annexin V, are linked with pro-inflammatory activity. However, it was shown that the association of Annexin V with EVs increased the anti-inflammatory activity of EVs. Annexin V has a molecular weight of about 37 kDa.

[0138] In an embodiment of the current invention, the ratio between EV-associated albumin and EV-associated Annexin V will be between 4500: 1 to 200000: 1. In a further embodiment, said composition comprises intra-vesicular Annexin V and human albumin at a ratio of between 5 and 222 pg Annexin V per g human albumin.

[0139] In some embodiments, the EVs as disclosed herein are associated with albumin and Annexin V, and the ratio between EV-associated Annexin and EV-associated albumin is between 5 and 222 pg Annexin V per g albumin and said EVs are further positive for CD44 and CD29.

[0140] Preferably, said EVs are defined by the markers as described above in any of the embodiments. Preferably, said EVs have a size as described above in any of the embodiments. Preferably the EVs are derived from mesenchymal stem cells (MSCs). Nonetheless, it would be obvious to the skilled person that EVs originating from other sources, such as but not limited to bone marrow, Wharton's jelly, cord blood, amniotic membrane, bone marrow, adipose tissue, dental pulp, peripheral blood, fallopian tube, mammary gland, liver or lung tissue, can deviate from the abovedescribed characteristics, without departure from the scope of the invention.

[0141] In an embodiment, said EVs are naive EVs, derived from naive MSCs. In another embodiment, said MSCs are primed with one or more cytokines, resulting in primed MSCs and primed EVs. In an embodiment, said cytokines are added to the medium of the cultured MSCs. Said cytokines for priming are preferably chosen from TNFa, ILip, IL6 or a combination thereof.

[0142] 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.

[0143] In a preferred 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.

[0144] In an embodiment, said the levels of expression of CD 29 and CD44 in said primed EVs are higher than in said naive EVs, preferably at least 2 times more, more preferably at least 3 times more. Said levels are preferably measured via MACSplex

[0145] EV kit.

[0146] Subjects

[0147] The subjects may be those patients who suffer from an Ocular Surface Disease. 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.

[0148] In an embodiment, said OSD can be Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction. In a preferred embodiment said OSD is Dry Eye Disorder.

[0149] In a preferred embodiment, said patient is a non-human or human patient, preferably a human patient.

[0150] 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.

[0151] In an embodiment, said prior treatment failed or did not provide long-lasting results.

[0152] Administration, Pharmaceutical Compositions, Effective Amounts

[0153] The current disclosure also relates to the use of said EVs or a pharmaceutical composition comprising an effective amount of said isolated EVs, in the treatment of ocular surface disease.

[0154] The EVs may be used (e.g., administered) in pharmaceutically acceptable preparations (or pharmaceutically acceptable compositions), typically when combined with a 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. 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 aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0155] The EVs 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.

[0156] In an embodiment of the invention disclosed herein, the MSC-derived EVs or composition comprising said EVs are administered one time.

[0157] In an embodiment of the invention disclosed herein, at least two doses of isolated MSC-derived 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.

[0158] The disclosure also contemplates repeated administration of MSC-derived EVs or a composition comprising said EVs, including two, three, four, five or more administrations of MSC-derived EVs or a composition comprising said EVs. In some instances, the MSC-derived EVs or composition 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, 12 hours or 24 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 or composition 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.

[0159] The preparations of the invention are administered in effective amounts. An effective amount is the amount of an agent that alone stimulates the desired outcome. The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual patient parameters including age, physical condition, size, weight, and the stage of the disease. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

[0160] In an embodiment of the invention disclosed herein, said isolated MSC-derived EVs or composition comprising said EVs are administered at a dose of between 1 x 1010and 1 x 1012, between 1 x 10nand 1 x 1012, or between 1 x 1010and 1 x 1011EVs per kg bodyweight.

[0161] In an embodiment of the invention disclosed herein, said isolated MSC-derived EVs or composition comprising said EVs are administered at a dose of between 1 x 1010and 1 x 1012, between 1 x 10nand 1 x 1012, or between 1 x 1010and 1 x 1011EVs per ml.

[0162] In some embodiments of the pharmaceutical composition for use as disclosed herein, said composition is formulated as a suspension, comprising at least 1 x 1011, 1.1 x 1011, 1.2 x 1011, 1.5 x 1011, 2 x 1011, 5 x 1011, 1 x 1012, or 1 x 1013EVs per ml.

[0163] In other or further embodiments, said isolated MSC-derived EVs or composition comprising said EVs 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 IO10EVs / 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 1011 EVs / 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

[0164] 1.2 x 109and 1.2 x 1012EVs / eye, between 1.2 x 1010and 1.2 x 1012EVs / eye, or between 1.2 x 1011and 1.2 x 1012EVs / eye.

[0165] In yet another or further embodiment, said isolated MSC-derived EVs or composition comprising said EVs are administered to both eyes at a daily total dose of between

[0166] 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 IO10EVs 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 IO10EVs 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 IO10and 7.2 x 1012EVs for both eyes, or between 7.2 x 1011and 7.2 x 1012EVs for both eyes.

[0167] The isolated MSC-derived EVs or composition comprising said EVs may be administered by any route that allows delivery to the eye. Systemic administration routes such as injection, intravenous bolus injection or continuous infusion are suitable. More direct routes such as topical administration are also contemplated by the invention and may be more appropriate. In some embodiments of the invention disclosed herein, said MSC-derived EVs or the pharmaceutical composition are formulated for topical delivery to the eye, preferably to the conjunctival sac.

[0168] 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 or other powder or solid form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0169] It is to be understood that other agents to be administered to subjects being treated according to the disclosure may be administered by any suitable route including oral administration, injection, intravenous administration, topical administration etc. Those of ordinary skill in the art will know the customary routes of administration for such secondary agents.

[0170] In embodiments of the pharmaceutical composition for use as disclosed herein, said isolated MSC-derived EVs are autologous to the human subject, or said isolated MSC- derived EVs are allogeneic to the human subject.

[0171] In another embodiment, the isolated MSC-derived EVs or pharmaceutical composition comprising said EVs, are formulated and administered by injection in a volume of between about 1.0 mL and about 2.0 mL.

[0172] In a further embodiment, an appropriate dose of EVs is prepared in a suitable syringe, such as a 2.5mL VY-P syringe (made in PVC, LATEX free). Once the final volume of EVs is in said syringe, e.g. 2.5 mL VY-syringe, the empty part of the syringe is filled with room air, in order to increase the air volume, which is important for a better injection.

[0173] In another embodiment, the isolated MSC-derived EVs or a pharmaceutical composition comprising said EVs, 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.

[0174] Kits

[0175] The disclosure also encompasses a packaged and labeled pharmaceutical product. This article of manufacture or kit includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial, a plastic ampoule or other containers that are hermetically sealed. The unit dosage form should be suitable for delivery to the eye for example by topical administration or injection. Preferably, the article of manufacture or kit further comprises instructions on how to use including how to administer the pharmaceutical product. The instructions may further contain informational material that advises a medical practitioner, technician or subject on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instructions indicating or suggesting a dosing regimen for use including but not limited to actual doses, monitoring procedures, and other monitoring information.

[0176] As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipment.

[0177] The kits may include MSC-derived EVs in sterile aqueous suspensions that may be used directly or may be diluted with normal or half-normal saline for injection or topical application or use in a nebulizer, or dilution with normal or half-normal saline.

[0178] In another embodiment, said kits include MSC-derived EVs that are frozen. These EVs are to be thawed before use and / or are to then to be reconstituted in a suitable diluent or buffer.

[0179] In yet another embodiment, said kits include MSC-derived EVs that are lyophilized, such as freeze-dried and stored at 4°C or frozen at -20°C before reconstitution. These EVs are then to be reconstituted in a suitable diluent or buffer.

[0180] The kits may therefore also contain a diluent solution or agent, such as saline or surfactant. In some embodiments, the kit comprises an EV suspension of between 1 X 1010and 1.2 X 1011particles / mL in saline.

[0181] The kit may also include a catheter, injection needles, supplies for IV administration, or for administration to the eye, such as pipette.

[0182] The present invention will be now described in more details, referring to examples that are not limitative.

[0183] EXAMPLES

[0184] With as a goal illustrating better the properties of the invention the following presents, as an example and limiting in no way other potential applications, a description of a number of preferred applications of the method for examining the state of the grout used in a mechanical connection based on the invention, wherein : Example 1 : Efficacy and safety of Mesenchymal Stromal Cells-derived Extracellular Vesicles (EVs) in the treatment of an ocular surface disease.

[0185] Materials and Methods

[0186] Production of naive Extracellular Vesicles (MSC-EVs).

[0187] Extracellular vesicles (EVs) were produced using human mesenchymal stromal cells (MSCs) derived from umbilical cord tissue (UCT).

[0188] Approximately 40 cm of UCT was transported to the production lab and processed depending on the arrival time on the same day or on the next day (maximum 24 hrs from the C-section). The cord slices were transferred to cryovials and cryopreserved with Via Freeze (Cytiva, VF_30001) electric-controlled rate freezer. UCT cryovials were thawed and the MSCs were enzymatically extracted from the slices of the umbilical cord using different enzymes (company proprietary information). Digested slices were immediately placed into 2D culture plates to initiate the expansion of MSCs. Defined medium guarantees very high homogeneity of the MSCs and EVs with no risk of contamination with unknown EVs, and proteins present in sera, platelet lysates or other undefined components of culture media. During primary growth, the MSCs attached and started to proliferate forming colonies. After the 1st passage, cells continued proliferating as a typical monolayer culture of MSCs adherent to plastic (synthetic coating, company proprietary information). Cell viability below 80% at any passage terminated the production process. Cell detachment was achieved by incubation with a combination of enzymes (company proprietary information). After centrifugation, the cell pellet was resuspended in DPBS with Ca / Mg and was ready for cell count and viability test. After cell harvesting the MSCs are cryopreserved (company proprietary information). Cell phenotypes are tested twice, after 2D expansion and after 3D expansion, using a standard CD markers panel described for identification of MSCs in ATMPs manufacturing (BD Stemflow™, Human MSC Analysis Kit).

[0189] 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. Bioreactor, beads and culture media are proprietary information. This approach allowed very effective EVs production with multiple harvests and an efficient large-scale manufacturing process. Supernatants containing MSC-EVs were collected, and fresh medium was replaced in the bioreactor every 24 hrs using a peristaltic pump and semi-automated module of the MSC-EVs production. Bags with harvested supernatants were stored at 4°C-8°C until pooling in the next production step.

[0190] Pooled supernatants (from all harvests qualified for MSC-EVs production) were subsequently processed using a semi-automated module of the MSC-EVs purification. Particle concentration and size were measured using nanoparticle tracking analysis (NTA) as a critical in-process quality control test (IPC) in a pooled bag. In addition, a sample of pooled medium was taken for mycoplasma testing and a sample of cell supernatant was collected for viral testing.

[0191] Quantitative analysis of EVs-related proteins i.e. Annexin V was performed using ELISA and measured as a ratio to the particle concentration. Proteomics batch identity is performed using MACSPlex (FACS) and mass spectrometry. The latter method is used for R&D characteristics of selected EVs batches only.

[0192] Quantification of non-EVs proteins i.e. albumin was performed with ELISA, measured in correlation to the particles concentration.

[0193] EVs are tested for activity using macrophage polarisation assay (RAW cells assay). The assay, measures the ability of EVs to inhibit the acquisition of the Ml phenotype in LPS stimulated murine RAW 264.7 macrophage cell line.

[0194] Production of naive EVs derived from cytokine-primed MSCs.

[0195] Cytokine primed EVs were produced using a similar protocol of naive EVs described above except on Day 4 the cells were cultured with cytokines cocktail (TNFa 25ng / ml, ILip 25ng / ml, IL6 20ng / ml) containing media for 24 hours and washed one time with production media to remove the cytokines before adding the media for EVs production. All the QC parameters were documented and compared with naive EVs.

[0196] EV concentration and characterization.

[0197] Tangential flow filtration (TFF) was performed using cassettes with a lOOkDa molecular weight cut-off membrane. TFF cassette purity (nanoparticles free) was tested using NTA. lOOkDa TFF membrane filtration allows the impurities to pass the membrane into the permeate (waste). Of note, the defined culture medium components were smaller than lOOkDa and could not be accumulated in the retentate during FTT filtration. EVs were larger than lOOkDa and could be concentrated in the retentate. An additional single wash step was performed after pre-concentration using saline.

[0198] After washing the EVs were re-concentrated by TFF filtration and NTA was performed to confirm a target concentration of 1.2E+11 particles / mL used in this project. Subsequently, EVs were removed from the TFF cassette and aliquoted. The vials with EVs were transferred into cryo-boxes and frozen. EVs were ready to use immediately after thawing.

[0199] The EVs batch was tested using nanoparticle tracking (NTA) ZetaView®, PMX220 TWIN (Particle Metrics). The mean particle size was 106 nm (normal distribution) and the concentration was 1.2E+11 particles / mL.

[0200] Analysis of surface markers on EVs

[0201] Surface markers of naive or cytokines-primed EVs were analysed using MACSplex EVs kit by Miltenyi Biotec, (catalogue number 130-108-813). EVs surface markers were analysed using kit manufacturers protocol. Briefly, EVs were incubated with capture beads, which are conjugated with antibodies targeting different vesicle surface markers, followed by washing to remove unbound particles. Beads were incubated with detection antibodies conjugated with fluorophores to label vesiclebound markers, this was followed by washing to remove free antibody. Samples were analysed on a flow cytometer; each bead population has a distinct fluorescence signature, allowing simultaneous analysis of multiple surface markers. Results were expressed in Median Fluorescence Intensity (MFI) and data is normalised and expressed in fold change of relative quantification of total fluorescence (here normalised to CD9, CD63, and CD81 expression).

[0202] BAK-induced dry eye disease animal model and experimental set up.

[0203] 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). The following experimental groups were defined:

[0204] 1. 1st generation extracellular vesicles (EXOB-OOl) - blinded group

[0205] 2. Extracellular vesicles derived from cytokines-primed MSCs - blinded group

[0206] 3. saline (placebo) - blinded group

[0207] 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.

[0208] Dose: one drop (lOpL) was instilled in the conjunctival sac 3 times per day.

[0209] • Single administration volume app. lOpL and EVs dose 1.2E+9 particles / administration (eye).

[0210] • Total dose (48 administrations per animal / 24 per eye): 480uL and EVs per mouse.

[0211] • Cumulative dose per eye: 2.9E+10

[0212] • Cumulative dose per animal: 5.8E+10

[0213] EVs administration : topical (eye drops) in the conjunctival sac, 3 times per day for a period of 8 days (total 24 per eye).

[0214] Animals' well-being was monitored during the entire experiment. Disease activity was monitored by biomicroscopy.

[0215] 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 at 5 mg / kg subcutaneously.

[0216] 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 following multiple injections was observed and the EVs product is considered as save with no risk of addition harm to the animals.

[0217] Corneal damage quantification

[0218] 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).

[0219] Tear secretion quantification

[0220] 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.

[0221] Leukocyte infiltration quantification.

[0222] Leukocyte infiltration was quantified using CD45 staining.

[0223] 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.

[0224] Lymph-angiogenesis quantification.

[0225] Lymph-angiogenesis was quantified by CD31 and LYVE1 staining.

[0226] 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 three rinses in PBS. Corneas were flat mounted on glass slides using Vectashield mounting medium. Positive signal was quantified using fluorescent microscope.

[0227] Results Expression of surface markers on EVs

[0228] Surface markers on naive EVs and cytokine primed EVs were quantified using MACSplex EVs kit as described in materials and methods. We observed higher expressed signal of CD44 and CD29 surface markers compared to other markers in all the batches of naive EVs produced. 18 different batches of naive EVs were analysed and we observed 5.16- and 4.46-fold change respectively in CD44 and CD29 markers expression when normalised with CD9, CD63, and CD81 expression (table 1). For cytokines-primed EVs the fold change for CD44 and CD29 observed was increased to 18.10 and 12.61 respectively. Only selected markers from total 37 markers analysed are represented here, which show values above certain threshold.

[0229] Table 1: average fold change values of selected markers expressed on EVs when normalized with expression of CD9-CD63 and CD9 beads.

[0230] Study timeline and Dry Eye Disease induction

[0231] The study timeline is represented in Figure 1. 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 2).

[0232] Tear secretion quantification

[0233] 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 EV EXOB-OOl) 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 3).

[0234] CD45 staining (leukocyte infiltration)

[0235] Markers of leukocyte infiltration indicating the inflammation have been considered on whole cornea samples to evaluate the efficacy of the treatment Compounds (Figure 4A). Mice treated with Compound 1 (MSC-derived EVs) 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 4B). This confirms immunomodulatory and anti-inflammatory activity of MSC-derived EVs.

[0236] CD31 and LYVE1 Staining (lymph-angiogenesis)

[0237] Corneal blood and lymphatic vessels were marked with CD31 and LYVE1, respectively (Figure 5A). 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.

[0238] The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use in the treatment of an ocular surface disease, wherein said EVs are administered to the eye of a patient suffering from said ocular surface disease, wherein said EVs are MSC (Mesenchymal Stem Cells) derived EVs, preferably umbilical cord MSC- derived EVs, wherein said EVs are associated with albumin and Annexin V, and wherein said EVs are positive for CD44 and CD29.

2. Isolated MSC-derived EVs or a pharmaceutical composition comprising an effective amount of said EVs for use according to claim 1, wherein said EVs are further positive for one or more markers chosen from CD105, CD9, CD63, CD81, SSEA4, HLA1, CD49e or MSCP.

3. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the previous claims, wherein said EVs express CD105, CD29, CD9, CD63, CD81, SSEA4, HLA1, MSCP, CD49e and CD44.

4. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to claim 3, wherein the respective expression level of CD44 and CD29 is between 2.5- and 5-fold higher than the level of expression of CD105, CD49e or MSCP when normalized with CD9, CD63 and CD81 expression.

5. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the previous claims wherein the ratio between EV-associated Annexin and EV- associated albumin is between 5 and 222 pg Annexin V per g albumin.

6. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the previous claims, wherein said EVs have a mean particle size of below 120 nm, such as between 95 and 110 nm.

7. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the previous claims, wherein said MSCs are primed with one or more cytokines.

8. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the previous claims, wherein said MSCs are primed with one or more cytokines chosen from TNFa, ILip or IL6.

9. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to claims 7 or8, wherein the levels of expression of CD29 and CD44 in said primed EVs are higher than in said naive EVs, preferably at least 2 times higher.

10. Isolated Extracellular Vesicles (EVs) or a pharmaceutical composition comprising an effective amount of said EVs for use according to any of the previous claims, wherein said ocular surface disease is Dry Eye Disorder, Blepharitis, Neurotrophic keratitis, Ocular rosacea, or Meibomian gland dysfunction, preferably Dry Eye Disorder.

11. Isolated MSC-derived EVs or the pharmaceutical composition for use according to any of the previous claims wherein said EVs are administered at a dose of between 1.2 x 107and 1.2 x 1012EVs / eye.

12. Isolated MSC-derived EVs or the pharmaceutical composition for use according to any of the previous claims, wherein said EVs or composition is to be administered one time, repeatedly, or continuously.

13. Isolated MSC-derived EVs or the pharmaceutical composition for use according to claim 11, wherein said administration is repeated, preferably with at least 1 to 24 hours between each administration, more preferably once or twice a day.

14. Isolated MSC-derived EVs or the pharmaceutical composition for use according to any of the previous claims, wherein said isolated MSC-derived EVs are autologous to the patient, or wherein the isolated MSC-derived EVs are allogeneic to the patient.

15. Isolated MSC-derived EVs or the pharmaceutical composition for use according to any of the previous claims, wherein said patient has received at least one prior treatment, said prior treatment being chosen from eye drops or artificial tears, corticosteroid therapy, NSAID therapy, anti-VEGF therapy, or surgical corneal transplantation.

16. Isolated MSC-derived EVs or the pharmaceutical composition for use according to any of the previous claims, wherein said EVs when administered induce tear and / or mucin secretion in the eye or part of said eye.

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