WO2020132161A1

WO2020132161A1 - Compositions and methods for treating hearing loss

Info

Publication number: WO2020132161A1
Application number: PCT/US2019/067324
Authority: WO
Inventors: Jennifer Long; Federico Kalinec
Original assignee: The Regents Of The University Of California; United States Government Represented By The Department Of Veterans Affairs
Priority date: 2018-12-21
Filing date: 2019-12-19
Publication date: 2020-06-25

Abstract

Aspects of the disclosure provide methods and compositions involving extracellular vesicles that can be used to treat hearing loss. Extracellular vesicles are not immunogenic (they do not induce rejection or inflammatory responses), are easily incorporated by other cells, and lack of the limitations associated with the delivery of intact cells. Furthermore, it was discovered that exosome comprise or can be stimulated to comprise pro-resolution mediators and precursors that allow for the activation of endogenous hearing protection mechanisms rather than inhibition of natural defense processes. Accordingly, aspects of the disclosure relate to a method for treating, preventing, or delaying the onset of hearing loss, the method comprising administering a composition comprising extracellular vesicles to the ear of the subject.

Description

DESCRIPTION

COMPOSITIONS AND METHODS FOR TREATING HEARING LOSS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 62/783,426, filed December 21, 2018, and U.S. Provisional Application No. 62/931,296, filed November 6, 2019, the contents of which applications are incorporated into the present application in their entirety.

BACKGROUND

[0002] This work was supported by the U.S. Department of Veterans Affairs, and the Federal Government has certain rights in the invention.

1. Field of the Invention

[0003] The current disclosure relates to the field of molecular biology and therapeutic methods.

2. Description of Related Art

[0004] Hearing loss, induced by drugs (Drug-Related Hearing Loss - DRHL), noise (NRHL), or the normal process of aging (ARHL aka presbycusis), affect millions of people worldwide, causing social isolation, impaired communication with family, friends and coworkers, lost productivity, decreased self-esteem, depression, and cognitive decline. With an aging population their compounding socioeconomic impact is set to become even more significant. In spite of the importance of this problem, to date, there are no therapeutic strategies to prevent or alleviate DRHL, NRHL or ARHL.

[0005] The auditory organ, the organ of Corti (OC), localize inside the anatomical structure known as the cochlea, within the inner ear. The cochlea is a common site of inflammation, and the auditory sensory cells (hair cells) in the OC are very sensitive to inflammatory injury. There is a growing consensus in the hearing research field that cochlear inflammation is the common underlying cause of hearing loss induced by ototoxic drugs, noise, and aging, as well as by other agents such as viral infections, mitochondrial dysfunction, autoimmune inner ear disorders and cochlear surgery. The current approach to deal with these conditions in the clinic, in every case with inconsistent and sometimes negative outcomes, is the use of anti-inflammatory drugs (e.g., glucocorticoids) and/or antioxidant agents (e.g., inhibitors of NOX3). In fact, all the drugs currently on clinical trials in the USA for prevention of hearing loss are either anti-inflammatory drugs or antioxidants. However, inflammation is a life-saving protective response to overcome infection and/or injury and oxidative mediators are essential second messengers in innate and adaptive immune responses, and interfering with these processes may prevent the activation of natural defense mechanisms and usually have unintended negative consequences. Therefore, there is a need in the art for therapeutic approaches that overcome the limitations of current therapeutic approaches.

SUMMARY OF THE INVENTION

[0006] Aspects of the present disclosure overcome a major deficiency in the art by providing methods and compositions involving extracellular vesicles that can be used to treat hearing loss. Extracellular vesicles are not immunogenic (they do not induce rejection or inflammatory responses), are easily incorporated by other cells, and lack of the limitations associated with the delivery of intact cells. Furthermore, it was discovered that extracellular vesicles comprise or can be stimulated to comprise pro-resolution mediators and precursors that allow for the activation of endogenous hearing protection mechanisms rather than inhibition of natural defense processes. Accordingly, aspects of the disclosure relate to a method for treating, preventing, or delaying the onset of hearing loss, the method comprising administering a composition comprising extracellular vesicles to the ear of the subject.

[0007] Further aspects relate to a composition comprising extracellular vesicles , wherein the extracellular vesicles comprise one or more pro-resolving mediators. In some embodiments, the extracellular vesicles comprise one or more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, and neuromodulators. In some embodiments, the composition is formulated for administration by injection through the round window membrane and/or inside the cochlear scala tympani. In some embodiments, the composition further comprises additional therapeutic agents. In some embodiments, the additional therapeutic agents comprises aspirin, anti-inflammatory and/or anti-oxidant drugs. In some embodiments, the composition further comprises a pharmaceutical carrier. In some embodiments, the pharmaceutical carrier is a carrier or excipient described herein.

[0008] Further aspects of the disclosure relates to a method for making extracellular vesicles comprising isolating extracellular vesicles from auditory cells. In some embodiments, the auditory cells comprise human cells. In some embodiments, the auditory cells comprise HEI-OC1 cells. [0009] In some embodiments, the method comprises filtration of the extracellular vesicles, wherein the filtration comprises a 0.45 pm filter. In some embodiments, the filtration is performed more than once, such as at least 2, 3, 4, 5, or 6 times.

[0010] In some embodiments, the extracellular vesicles are 100-800nm. In some embodiments, the extracellular vesicles are at least or at most 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 nm (or any range derivable therein).

[0011] In some embodiments, the concentration of the extracellular vesicles is lxlO⁸- lxlO¹⁰ extracellular vesicles/mL. In some embodiments, the concentration of the extracellular vesicles is at least, at most, about, or exactly lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, lxlO⁸, 2xl0⁸, 3xl0⁸, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, lxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, 9xl0⁹, lxlO¹⁰, 2xl0¹⁰, 3xl0¹⁰, 4xl0¹⁰, 5xl0¹⁰, 6xl0¹⁰, 7xl0¹⁰, 8xl0¹⁰, or 9xl0¹⁰ (or any derivable range therein) exosomes/mL.

[0012] In some embodiments, the method further comprises inclubating a composition comprising the extracellular vesicles and a therapeutic agent. In some embodiments, the method further comprises sonication of the composition comprising the extracellular vesicles and the therapeutic agent. In some embodiments, the method further comprises washing the extracellular vesicles to remove excess therapeutic agent from the composition.

[0013] In some embodiments, the method further comprises freezing the extracellular vesicles. In some embodiments, the method further comprises storing the extracellular vesicles. In some embodiments, the extracellular vesicles are frozen and stored at a temperature of -30°C or lower. In some embodiments, the extracellular vesicles are frozen and stored at a temperature of 20, 10, 0, -10, -20, -30, -40, -50, -60, -70, or -80°C (or any derivable range therein). In some embodiments, the extracellular vesicles are stored for at least 2 months. In some embodiments, the extracellular vesicles are strored for at least, at most, about, or exactly 1, 2, 3, or 4 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years (or any derivable range therein).

[0014] In some embodiments, the hearing loss comprises drug-related, noise-related, and/or age-related hearing loss. In some embodiments, the hearing loss is due to a viral infection, mitochondrial dysfunction, an autoimmune inner ear disorder, or by physical disruption of the ear, such as by cochlear surgery.

[0015] The extracellular vesicles may be synthetically made or isolated from cells. In some embodiments, the extracellular vesicles are isolated from mesenchymal stem cells (MSCs). In related embodiments, the extracellular vesicles are isolated from adipose-derived MSCs. In further embodiments, the extracellular vesicles are isolated from a cell/cell type described herein. In some embodiments, the extracellular vesicles are isolated from mammalian cells. In some embodiments, the extracellular vesicles are isolated from human cells. In some embodiments, the extracellular vesicles are isolated from human, mouse, pig, goat, rabbit, guinea pig, horse, dog, or rat cells. In some embodiments, the extracellular vesicles are isolated from cells that comprise one or more of the following biomarkers: CD90+, CD105+, and CD73+.

[0016] In some embodiments, the cells are cultured in exosome-depleted medium or medium depleted of extracellular vesicles. In some embodiments, the cells are cultured in medium that is completely free of extracellular vesicles. In some embodiments, the cells are cultured in medium that contains less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pg, ng, or pg (or any derivable range therein) of extracellular vesicles per 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g, mg, or pg of cells (or any derivable range therein).

[0017] In some embodiments, the cells are contacted with one or more inducers, precursors of, or pro-resolving agents prior to, during, and/or after extracellular vesicle isolation. In some embodiments, the inducers, precursors of, or pro-resolving agents are selected from one or more of glucocorticoids like dexamethasone and prednisolone, anti inflammatory agents like curcumin, precursors of pro-resolving mediators like docosahexaenoic acid, arachidonic acid, and eicosapentaenoic acid, co-factors such as aspirin, pro-resolving mediators like lipoxins, resolvins, protectins, maresins, ANXA1, galectins, and others already known or to be discovered.

[0018] In some embodiments, the subject has been administered and/or has been prescribed a chemotherapeutic agent. In some embodiments, the subject has been administered and/or prescribed an ototoxic compound. In some embodiments, the subject has been administered and/or prescribed an ototoxic chemotherapeutic agent. In some embodiments, the ototoxic compound comprises cisplatin or aminoglycoside. In some embodiments, the subject has been or will be exposed to significant noise exposure. In some embodiments, the subject has been or will be exposed to at least, at most, about, or exactly 70, 75, 80, 90, 95, 100, 110, 120, 130, 140, or 150 decibel (dB) (or any derivable range therein) of noise exposure. In some embodimens, the noise exposure has taken place at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24 hours or days (or any derivable range therein) prior to administration. In some embodiments, the noise exposure will take place at a time point of at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24 hours or days (or any derivable range therein) after administration. In some embodiments, the noise exposure occurred or will occur for at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 20, 30, 40, 50, or 60 minutes or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,

9, 9.5, 10, 12, 14, 16, 18, 20, 22, or 24 hours or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 14, 16, 18, 20, 22, or 24 days (or any derivable range therein). In some embodiments, the subject has been diagnosed with and/or suffers from early partial hearing loss. In some embodiments, the subject has hearing loss of at least, at most, about, or exactly 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 dB (or any derivable range therein). The hearing loss may correspond to a dB at a frequency of at least, at most, about, or exactly 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7500, 8000, or 9000 Hertz (Hz), or any range derivable therein. In some embodiments, the subject is 40 years old or older. In some embodiments, the subject is at least, at most, about, or exactly 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old (or any derivable range therein). In some embodiments, the subject is genetically predisposed to hearing loss. In some embodiments, the subject has previously been treated for hearing loss. In some embodiments, the previous treatment was unsuccessful. In some embodiments, the previous treatment restored less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 2.5, or 1% hearing loss (or any range derivable therein).

[0019] In some embodiments, the composition is administered by injection into the inner ear. In some embodiments, the composition is administered by intracochlear delivery. In some embodiments, the composition is injected through the round window membrane. In some embodiments, the composition is injected through the round window membrane and inside the cochlear scala tympani.

[0020] In some embodiments, the composition comprises 0.5-3 ng extracellular vesicles per 0.1 pi of solution. In some embodiments, the composition comprises at least, at most, about, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,

4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,

6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,

8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 pg, ng, or pg extracellular vesicles (or any range derivable therein). In some embodiments, the extracellular vesicles are in a solution or suspension or at least, at most, about, or exactly 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nL, pL, or mL (or any derivable range therein) of solution or suspension. In some embodiments, the dose administered is at most, about, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,

1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,

3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,

5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,

7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,

9.7, 9.8, 9.9, 10.0 pg, ng, or pg extracellular vesicles (or any range derivable therein). In some embodiments, the subject may be given at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, or 100 doses (or any derivable range therein). In some embodiments, the subject may be given at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, or 100 doses (or any derivable range therein) per time period, wherein the time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or 1, 2, 3, 4, 5, or 6 months (or any derivable range therein). In some embodiments, the doses may be administered at least, at most, about, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days or months (or any derivable range therein) apart from each other.

[0021] In some embodiments, the composition is cell-free. In some embodiments, the composition is further defined as a pro-resolution composition. In some embodiments, the extracellular vesicles and/or composition comprises one or more pro-resolving mediators. In some embodiments, the extracellular vesicles and/or composition comprises one or more of ANXA1, Galectins 1, Galectins 3, Lipoxin A4, Lipoxin B4, Resolvin Dl, and Resolvin E7. In some embodiments, the extracellular vesicles comprise exogenously added lipoxin A4 and/or resolving Dl. In some embodiments, the extracellular vesicles and/or composition comprises one or more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, neuromodulators, NO gas, PUS gas, polyunsaturated fatty acids (PUFA), and CO gas. In some embodiments, the one ore more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, neuromodulators, NO gas, PhS gas, polyunsaturated fatty acids (PUFA), and CO gas is exogenously added. In some embodiments, the one ore more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, neuromodulators, NO gas, PhS gas, polyunsaturated fatty acids (PUFA), and CO gas is endogenous to the isolated exosome. In some embodiments, the PUFA comprises one or more of arachidonic, eicosapentaenoic, docosahexanoic, and linoleic acid. In some embodiments, the PUFA is exogenously added PUFA. In some embodiments, the pro-resolving mediator is one or more of ANXA1, Lipoxin A4, Lipoxin B4, Resolvin Dl, and Resolvin E7. In some embodiments, the pro-resolving mediator is one or more of lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, and neuromodulators. NO gas, FbS gas, and CO gas. In some embodiments, the pro-resolving mediator is one or more pro-resolving mediators described herein. In some embodiments, one or more pro-resolving mediators described herein is excluded from the composition and/or extracellular vesicle. In some embodiments, the pro-resolving mediator is contained in the interior of the extracellular vesicle. In some embodiments, the pro-resolving mediator is outside of the extracellular vesicle, in the composition.

[0022] Methods may involve administering a composition containing (or a composition comprising) about, at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,

2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,

4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,

6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3,

8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5,

12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110,

115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205,

210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300,

305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395,

400, 410, 420, 425, 430, 440, 445, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540,

550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700,

710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860,

870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 nanograms (ng), micrograms (meg), milligrams (mg), or grams of extracellular vesicles, or any range derivable therein. The above numerical values may also be the dosage that is administered to the patient based on the patient’s weight, expressed as ng/kg, mg/kg, or g/kg, and any range derivable from those values.

[0023] Alternatively, the composition may have a concentration of extracellular vesicles that are 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,

3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,

5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,

7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,

9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,

15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,

250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340,

345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450,

460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610,

620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 ng/ml, pg/ml, mg/ml, or g/ml, or any range derivable therein.

[0024] In some embodiments, the composition may have at least, at most, about, or exactly, about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500, or 1000 fold more or less nucleic acid content than a naturally derived extracellular vesicleor than an extracellular vesicle isolated from a mammal.

[0025] The composition may be administered to (or taken by) the patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, or any range derivable therein, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or any range derivable therein. It is specifically contemplated that the composition may be administered once daily, twice daily, three times daily, four times daily, five times daily, or six times daily (or any range derivable therein) and/or as needed to the patient. Alternatively, the composition may be administered every 2, 4, 6, 8, 12 or 24 hours (or any range derivable therein) to or by the patient. In some embodiments, the patient is administered the composition for a certain period of time or with a certain number of doses after experiencing symptoms of a demyelinating disorder.

[0026] In some embodiments of the methods and compositions of the disclosure, the extracellular vesicles are freshly isolated, meaning they have not been frozen. In some embodiments, the extracellular vesicles of the compositions and methods were isolated from cells at a time period of at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or 2, 3, 4, 5, or 6 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (or any derivable range therein). In some embodiments, the extracellular vesicles have previously been frozen. The frozen extracellular vesicles may be thawed and used in the methods and compsoitions of the disclosure. In some embodiments, the exosome composition has been size fractionated or the method further comprises size fractionation of a composition comprising isolated exosomes. In some embodiments, the extracellular vesicles have been stored after isolation for a time period of at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or 2, 3, 4, 5, or 6 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (or any derivable range therein).

[0027] In certain embodiments, the isolated extracellular vesicles may include one type or at least two, three, four, five, six, seven, eight, nine, ten or more different types of extracellular vesicles (or any range derivable therein). The type of extracellular vesicles may be characterized by their compositions, for example, the types of lipids, proteins and/or nucleic acids of interest or effects.

[0028] In a certain embodiment, the composition may be an autologous composition or the cells may be obtained from the same patient to be treated. Particularly, cells from a human subject may be harvested and cultured, and induced, stimulated or engineered to secrete an effective extracellular vesicle-containing composition according to certain aspects of the invention. The extracellular vesicle-containing composition may be then administered in a pharmaceutical composition to the same human donor.

[0029] In this particular embodiment, all the advantages of the autologous donation can apply. The skilled person will choose the nature and identity of the donor tissue or cells for extracellular vesicle production depending on the use and as is expedient. Here, it may be necessary to consider the criteria and the advantages relevant for the decision to use autologous donation, and/or the choice of donor tissue/cells. [0030] In another embodiment, the composition may be allogeneic, that is to that the say donor organism that provides extracellular vesicle-producing cells and recipient organism to be treated are the same species but different individuals.

[0031] In an alternative embodiment, the composition may be xenogeneic. This means that it is taken from an organism of a different species. For this purpose, cells are taken from a donor organism, for example an animal such as a, cow, pig, rat or yeast, and are induced, stimulated or engineered to produce an effective extracellular vesicle-containing composition, which is administered in a pharmaceutical composition to the individual to be treated which belongs to a different species, for example a human.

[0032] In another embodiment, the composition may be obtained from autologous, allogeneic, or xenogeneic cells that have been preserved ex vivo and/or cultured in vitro.

[0033] The cells for producing extracellular vesicles may be obtained from a subject that is relatively young, for example, at an age that is at most one tenths, one fifths, one third, or half of the subject’s expected life span. For example, the cells may be obtained from a human that is at most, less than or about one, two, three, four, five, six, seven, eight, nine, ten, 11, 12 months, or 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 years old, or any age or range derivable therein. In a particular aspect, the extracellular vesicles may be obtained from a human that is less than one year old or less than 18 years old. In a particular aspect, the extracellular vesicles may be obtained from a human that is between 18 and 50 years old. The human may be the same patient that is to be treated.

[0034] Furthermore, in some aspects, the isolated extracellular vesicles or nanovesicles (e.g., the artificially engineered extracellular vesicles from in vitro reconstitution) or compositions comprising the extracellular vesicles may contain endogenous extracellular vesicles or may be loaded with externally added therapeutic agents, such as nucleic acids, molecular compounds, or protein molecules. In some embodiments, the therapeutic agent comprises an anti-inflammatory agent. In some embodiments, the therapeutic agent comprises a non-steroidal anti-inflammatory agent (NSAID). In some embodiments, the therapeutic agent comprises aspirin. In some embodiments, the therapeutic agent comprises dexamethasone. In some embodiments, the nanovescicle is a liposome. The nucleic acids may be DNA or RNA, such as siRNA, miRNA, or mRNA.

[0035] In other aspects, the nanovesicles may be prepared from in vitro reconstitution of lipids. In other aspects, the nanovesicles may be loaded with one or more of the pro-resolving mediators described herein. The nanovesicles may have a diameter of at least, about, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 nm or any range derivable therein. In a particular aspect, the exosome or extracellular vesicles may have a diameter of about 40 to about 100 nm. As used herein,“substantially similar” refers to at least 50, 55, 60, 65, 70, 75, 80, 90, 95, 99 or 100% identical or any range derivable therein. In some embodiments, the composition comprises exosomes wherein at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% may have a diameter of at least, about, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,

950, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 nm or any range derivable therein. In some embodiments, at least 80% of the extracellular vesicles are 25-150 nm. In some embodiments, at least 80, 85, 90, 95, 96, 97, 98, or 99% of the extracellular vesicles are 25-150 nm, 50-150 nm, 50-140 nm, or 50-100 nm. In some embodiments, at least 80% of the extracellular vesicles are 150 nm - 10 mhi. In some embodiments, at least 80, 85, 90, 95, 96, 97, 98, or 99% of the extracellular vesicles are 150-1000 nm, 150-500 nm, 150-250 nm, or 150-200 nm.

[0036] In some embodiments, the methods comprise altering the extracellular vesicle surface to reduce potential inflammation caused by the extracellular vesicles. This can be done, for example, by stripping the surface of extracellular vesicles and adding back certain proteins. Stripping can be done by methods known in the art, and kits for performing such methods are commercially available ( e.g . from System Biosciences, XPEP kits for Mass Spec, XPEP100A- 1). In some embodiments, the extracellular vesicles and/or lipid nanovesicles have a modified extracellular vesicle surface that reduces or eliminates an inflammation response when administered to a patient. In some embodiments, the extracellular vesicles and/or nanovesicles are non-inflammatory or exhibit a low amount of inflammation that is easily tolerated by the patient.

[0037] Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well. For example, any of the disclosed methods of administration may be used to treat any of the disclosed hearing loss disorders.

[0038] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0040] FIG. 1: Flowchart of the experiments described in Examples 2-3.

[0041] FIG. 2 (A-D): Surgical procedure for intracochlear delivery of drug through the RWM in guinea pigs. Through the hole in the bulla, the RWM is visible and accessible (C, arrow). D. The procedure is easier in humans because the RWM is located right behind the tympanic membrane, and it can be performed with minimal preparatives.

[0042] FIG. 3: Example of NanoSight™ measurements. Fresh and 4 month-old (stored at -30°C) EVs samples were diluted 1:5 in PBS. EVs values (left and center) were automatically corrected by PBS data (right). EVs with diameters of >500 nm may represent aggregates.

[0043] FIG. 4: EVs attached to the hair bundle of cells of the bullfrog sacculus (picture obtained by Dr. Bechara Kachar).

[0044] FIG. 5A-C: Counting and sizing EVs - A: Computer-generated CSD vs. particle size for two independent samples (SI and S2) in the whole size range. Values for SI and S2 are already corrected by bin-by-bin background subtraction of the values obtained from matched CM+ED-FBS and PBS+0.1% BSA (See FIG. 9). Note that the TS-400 and TS-900 cartridges have an overlapping region. B: Bar graphic depicting the total number of particles in the S-EVs fraction (average of SI and S2 = (3.3+0.1)xl0⁹ particles/mL), the total background ((1.30+0.04)xl0⁸ particles/mL for CM+ED-FBS plus (1.35+0.08)xl0⁸ particles/mL for PBS+0.1% BSA = (2.65+0.12)xl0⁸ particles/mL), and average of adjusted values ((3.3+0.1)xl0⁹ - (2.65+0.12)xl0⁸ = (3.0+0.2)xl0⁹ particles/mL). C: Bar graphic depicting the total number of particles in the L-EVs fraction (average of SI and S2 = (1.39+0.07)xl0⁸ particles/mL), the total background ((4.8+0.2)xl0⁵ particles/mL for CM+ED- FBS plus (5.3+0.4)xl0⁶ particles/mL for PBS+0.1% BSA = (5.8+0.6)xl0⁶ particles/mL), and average of adjusted values ((1.39+0.69)xl0⁸ - (5.8+0.6)xl0⁶ = (1.3+0.7)xl0⁸ particles/mL). Note that in all panels the scale of the Y-axis is logarithmic. [0045] FIG. 6: Proteomic analysis - Characterization of proteins from HEI-OC1 EVs by cellular localization.

[0046] FIG. 7: Proteomic analysis - Characterization of proteins from HEI-OC1 EVs by associated biological process.

[0047] FIG. 8: Proteomic analysis - Characterization of proteins from HEI-OC1 EVs by molecular function.

[0048] FIG. 9: Computer-generated CSD vs. particle size for culture media+exosome- depleted FBS (CM+EDFBS) and PBS+0.1% BSA in the whole size range. The values for each point of CM+ED-FBS plus those corresponding to PBS+0.1% BSA were considered “Background” and subtracted from the measured values for HEI-OC1 EVs.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0049] Current therapies for treating hearing loss aim to prevent natural defense mechanisms. The current disclosure describes compositions and methods that can be used to activate endogenous hearing protection mechanisms by molecular mediators released by cells inside extracellular vesicles. Acute inflammation is an essential biological defense mechanism aimed at restoring normal function in the face of injury or illness. Until recently, it was considered that tissues automatically reset to homeostasis when pro-inflammatory signals dissipate. More recent studies have demonstrated that acute inflammation is terminated by a highly controlled and coordinated active process, the resolution phase of inflammation, regulated by endogenous signaling pathways driven by specialized molecules that: 1) switch from production of pro-inflammatory mediators to pro-resolution mediators; 2) turn off pro- inflammatory signaling pathways; 3) induce apoptosis of previously recruited inflammatory cells; 4) stimulate the clearance of apoptotic cells by phagocytes and; 5) reinstate homeostatic conditions. The current disclosure is based, at least in part, on promoting inflammatory resolution by delivery of pro-resolving mediators generated by mammalian cells.

I. Definitions

[0050] “Exosomes” or“extracellular vesicles” are nanovesicles released from a variety of different cells. These small vesicles may be derived from large multivesicular endosomes and secreted into the extracellular milieu. The precise mechanisms of exosome release/shedding remain unclear. They appear to form by invagination and budding from the limiting membrane of late endosomes, resulting in vesicles that contain cytosol and that expose the extracellular domain of membrane -bound cellular proteins on their surface. Using electron microscopy, studies have shown fusion profiles of multivesicular endosomes with the plasma membrane, leading to the secretion of the internal vesicles into the extracellular environment. In some embodiments, extracellular vesicles of a smaller size, such as less than 150 nm are referred to as exosomes.

[0051] The term“therapeutic agent” is art-recognized and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as“drugs”, are described in well-known literature references such as the Merck Index, the Physicians Desk Reference, and The Pharmacological Basis of Therapeutics, and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances which affect the structure or function of the body; nucleic acids such as miRNAs, siRNAs, and antisense molecules; or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.

[0052] The term“therapeutic effect” is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and/or conditions in an animal or human. The phrase “therapeutically-effective amount” means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The therapeutically effective amount of such substance will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. For example, certain compositions in some aspects may be administered in a sufficient amount to produce a at a reasonable benefit/risk ratio applicable to such treatment.

[0053] “About” and“approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.

[0054] Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term“about” or“approximately” can be inferred when not expressly stated.

[0055] The term“human-derived” as used herein, refers to extracellular vesicles or cells derived from a cell of human origin.

[0056] As used herein the terms“encode” or“encoding” with reference to a nucleic acid are used to make the invention readily understandable by the skilled artisan; however, these terms may be used interchangeably with“comprise” or“comprising” respectively.

[0057] As used herein the specification,“a” or“an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word“comprising”, the words“a” or “an” may mean one or more than one.

[0058] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” As used herein “another” may mean at least a second or more.

[0059] Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0060] Throughout this application, the term“effective” or“effective amount” is used to indicate that the compounds are administered at an amount sufficient to treat a condition in a subject in need thereof.

[0061] As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Although it is contemplated that any embodiment described using the terms comprising, having, including or containing (or variations thereof) may also be implemented with the term “consisting essentially of’ or“consisting of’. The term“consisting essentially of’ may include elements such as alternative carriers, but specifically excludes any active ingredients other than the actives specifically identified in the claim.

II. Extracellular vesicles

[0062] In certain aspects of the invention, extracellular vesicles may be prepared and used as a novel therapeutic modality for improvement and treatment of hearing loss. [0063] Extracellular vesicles are small membrane vesicles of endocytic origin that are secreted by many cell types. For example, extracellular vesicles may have a diameter of about 40 to about 100 nm. They may be formed by inward budding of the late endosome leading to the formation of vesicle-containing multivesicular bodies (MVB) which then fuse with the plasma membrane to release extracellular vesicles into the extracellular environment. Though their exact composition and content depends on cell type and disease state, extracellular vesicles all share certain characteristics.

A. Pro-Resolving Mediators

[0064] Aspects of the disclosure relate to compositions and methods comprising extracellular vesicles, wherein the extracellular vesiclescomprise pro-resolving mediators. Specialized pro-resolving mediators not only work in inflammatory responses, but they also have important functions in host defense, pain, organ protection and tissue remodeling.

[0065] Embodiments include compositions comprising and/or extracellular vesicle comprising one or more of the following pro-resolving mediators.

[0066] Exemplary pro-resolving mediators include lipoxins, (lipoxygenase interaction products; LX), which are eicosanoids generated in vivo from arachidonic acid; resolvins (resolution phase interaction products), which are co-3 essential fatty acids derivatives with powerful multilevel anti-inflammatory and pro-resolving properties; protectins, which control the magnitude and duration of inflammation in animal models, fight bacterial and viral infections, and can increase animal survival; maresins (macrophage mediators in resolving inflammation), which are anti-inflammatory and pro-resolving lipid mediators generated by macrophages from DHA; annexin A1 (ANXA1) and peptides derived therefrom (e.g. peptides from the N-terminus), which is a powerful anti-inflammatory and pro-resolving protein; galectins, which are a family of glycan-binding proteins; adenosine; NO, H2S and CO, nitric oxide, hydrogen sulfide and carbon monoxide, which are gaseous substances that can act as signaling molecules; neuromodulators, which control immune function and anti-inflammatory responses via a vagus nerve-mediated reflex; aspirin, which is a potent inhibitor of cyclo oxygenases (COX) and lipoxygenases (LOX), interfering with the synthesis of pro- inflammatory mediators and promotes resolution mechanisms; co-3 essential fatty acids.

[0067] Exemplary lipoxins include positional isomers LXA4 and LXB4, and aspirin- triggered LX (ATL). Exemplary resolvins include D-series resolvins (RvD - e.g. RvDl, RvD2, RvD3, and RvD5), such as those generated from docosahexaenoic acid (DHA; 22:6co-3), or E- series resolvins (RvE), such as those in which the biosynthesis is initiated from eicosapentaenoic acid (EPA; 20:5co-3), aspirin-triggered D-resolvins (ATR-D), which is generated from the oxidation of DHA by aspirin, T-series resolvins (RvT), which are generated from docosapentaenoic acid (DPA; 22:5co-3). Exemplary protectins include protectin-Dl and protectin-D2. PD1, originally identified in neural tissues (murine brain cells and human microglial cells), is also known as neuroprotectin. Exemplary maresins include MaRl, MaR2 and MaR3. Also included in the methods and compositions as pro-resolving mediators are sulfido-conjugates of maresins, protectins and D-resolvins. Exemplary galectins include Gal- 1, Gal-2, Gal-3, Gal-4, Gal-5, Gal-6, Gal-7, Gal-8, Gal-9, Gal-10, Gal-11, Gal-12, Gal-13, Gal- 14, and Gal- 15. Exemplary neuromodulators include acetylcholine and netrin-1 and their receptors, such as a7nAChR.

[0068] Further embodiments include exclusion of one or more pro-resolving mediators described herein.

B. Vesicle preparation and sources

[0069] In certain aspects, the extracellular vesicles may be purified by ultracentrifugation in a sucrose gradient, then identified by the presence of marker proteins such as Alix, CD81, CD63, and TSG101 (or enrichment of tetraspanins and heat shock protein 70). Furthermore, extracellular vesicles can be isolated in vivo from malignant effusions and normal body fluids such as urine, blood, saliva, breast milk and cerebrospinal fluid. In some other aspects, extracellular vesicles can be isolated using serial centrifugation and ultracentrifugation with sucrose density gradients, or other methods like ultrafiltration membranes and gel filtration, using polymers such as PEGs, magnetic beads coated with monoclonal antibodies specific for antigens contained within or on the surface of the vesicle, high-performance liquid chromatography (HPLC), or commercially available extracellular vesicles isolation kits like miRCURY, Invitrogen® Total Exosome, ExoQuick-TC™, or others to be developed.

[0070] The cargo on the interior of the extracellular vesicle is protected from degradation by proteases and RNases while the vesicle is in the interstitial space, and retains bioactivity once taken up by a recipient cell. In this way, they facilitate the transfer of interactive signaling and enzymatic activities that would otherwise be restricted to individual cells based on gene expression.

[0071] According to certain aspects, the extracellular vesicles and compositions can be produced using various preparations of cells. For example, the extracellular vesicle-producing cells may be cultured with one or more inducers, precursors of, or pro-resolving agents prior to, during, and/or after vesicle isolation. In some embodiments, the inducers, precursors of, or pro-resolving agents are selected from one or more of glucocorticoids like dexamethasone and prednisolone, anti-inflammatory agents like curcumin, precursors of pro-resolving mediators like docosahexaenoic acid, arachidonic acid, and eicosapentaenoic acid, co-factors such as aspirin, pro-resolving mediators like lipoxins, resolvins, protectins, maresins, ANXA1, galectins, and others already known or to be discovered.

[0072] The extracellular vesicles may be isolated from a variety of cell types. In some embodiments, the extracellular vesicles are isolated from stem cells, such as mammalian stem cells, human stem cells, and or induced pluripotent stem cells (iPSCs). In some embodiments the extracellular vesicles are isolated from established cell lines from human, mouse, or other species. In some embodiments, the cells are derived from human bone marrow, cord blood, PBMCs, or from adipose tissue. In some embodiments, the extracellular vesicles are derived from mesenchymal stem cells (MSCs). In some embodiments, the MSCs are adipose-derived MSCs.

[0073] Furthermore, the cells may be autologous are non- autologous with respect to the patient to be treated. In some embodiments, the cells are allogeneic or xenogeneic.

[0074] Extracellular vesicles may be used fresh (shortly after loading them with the cargo of pro-resolution agents and additional drugs), or after storage for different periods (days to months). This could be important for commercialization and therapy, since extracellular vesicles loaded with different molecules (targeting different inflammatory processes) could be available immediately to the practitioners to be used with the patients before the effects of the inflammatory reactions result in major damage of the auditory organ. For instance, soldiers in the field affected by noise (e.g., explosions) could be receiving the treatment in less than 24 hours.

[0075] The extracellular vesicles produced or released by cells may be isolated and/or purified using several techniques. These include filtration, centrifugation, ion- chromatography, or concentration, either alone or in combinations. An exemplary purification method comprises a step of density gradient centrifugation. Another exemplary method comprises a step of ultrafiltration, either alone or coupled to a centrifugation step. Suitable purification methods have been described in WO99/03499, WO00/44389 and WO01/82958, which are incorporated therein by reference.

[0076] Selective purification or enrichment of physiologically active subpopulations of extracellular vesicles may be achieved via several procedures. In certain embodiments, effective extracellular vesicles may be concentrated to an enriched sample via use of specific surface protein markers and related separation techniques. In other embodiments, effective extracellular vesicles may be harvested from enriched primary cells cultures identified as capable of producing the effective extracellular vesicles. In further embodiments, based on screening procedures used to identify candidate effective exosome species, other extracellular vesicles may be fabricated using molecular engineering strategies designed to selectively produce extracellular vesicles containing the target (i.e., postulated) therapeutic molecular species.

[0077] In certain embodiments, the extracellular vesicles or vesicles may be loaded with therapeutic agents such as inducers, precursors of, or pro-resolving agents, nucleic acid molecules or other small molecules. The methods may include, but are not limited to:

(a) Incubation. In this method, isolated extracellular vesicles are incubated at different temperatures and variable times (usually 37°C or room temperature (~22°C) and 2 hours) with the molecules of interest to be incorporated as cargo. After incubation, the mixture is centrifuged for 1 h at 100,000xg, and the drug-loaded extracellular vesicles are collected, washed, and resuspended in the vehicle. This method is further described in Sun et al, Molecular Therapy vol. 18 no. 9, 606-1614 sep. 2010 and Yang et al., Pharm Res (2015) 32:2003-2014 DOI 10.1007/sl l095-014-1593-y, which are herein incorporated by reference for all purposes.

(b) Electroporation. By this method, a number of holes are made in cells/extracellular vesicles by briefly shocking them with an electric field of 100-200 V/cm. The DNA/RNA can enter the cells/extracellular vesicles through the holes made by the electric field.

(c) Lipofection. The method commonly called transfection and can be used to transform cells/extracellular vesicles with DNA/RNA via vesicles containing the desired genetic constructs. The vesicles fuse with the cell membrane (similar to how two oil spots at the top of a broth will fuse) and the contents of the vesicles and the cells are combined. There are a number of transfection kits in the market, ready for use, e.g. DeliverX siRNA Transfection Kit (cat. No. DX0002) from Panomics, FuGENE® HD Transfection Reagent (Cat. no. 04709691001) from Roche and LIPOFECTAMINE™ 2000 (Cat. No. 11668-027) from Invitrogen.

(d) Transformation using heat shock. Chilling cells/extracellular vesicles in the presence of divalent cations such as Ca²⁺ (in CaCh) makes their membranes become permeable to RNA or DNA plasmids or fragments. Cells or extracellular vesicles are incubated with the DNA and then briefly heat shocked (42° C. for 30-120 seconds), which causes the DNA to enter the cell. This method may work well for condensed circular plasmid DNAs and may work for exosomal or lipid nanovesicle constituents. [0078] The above methods describe briefly how production and delivery of modified extracellular vesicles can be achieved to transfer RNA and DNA to recipient cells. Extracellular vesicles can be engineered to contain RNA/DNA or modified to contain the gene of interest and may be isolated and shifted to the recipient cells, to affect their biological function or survival. Consequently, the extracellular vesicles may dispose their content into the cytoplasm of the target cells, which in turn leads to translation of mRNA to specific proteins in the target cell. Further, extracellular vesicles are capable of carrying and transferring small coding and non-coding RNA such as microRNA and siRNA that may regulate translation of a specific gene.

III. Pharmaceutical compositions

[0079] In certain aspects, the compositions or agents for use in the methods are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the invention may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as the ear) or systemic delivery, in solid, semi- solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the invention also contemplate local administration of the compositions by intracochlear delivery. In some embodiments, the compositions are administered by injection into the inner ear, injection through the round window membrane, and/or inside the cochlear scala tympani.

[0080] Suitable carriers for intracochlear delivery include artificial cochlear perilymph, artificial cochlear endolymph, and saline solutions. Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0081] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0082] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0083] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active agent, such as an isolated exosome or extracellular vesicle, a related lipid nanovesicle, or an exosome or nanovesicle loaded with therapeutic agents or diagnostic agents. In other embodiments, the active agent may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non limiting examples of a derivable range from the numbers listed herein, a range of about 5 microgram/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered.

[0084] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. The compositions of the disclosure may comprise glycerol, liquid polyethylene glycols, and mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0085] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain less, than, equal to, or more than 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0086] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-fungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0087] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0088] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intracochlear, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol is between about 0.01 ml and 0.5 ml.

[0089] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term“unit dose” or“dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired. [0090] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment ( e.g ., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

IV. Kits

[0091] Some embodiments concern kits, such as kits for preparing and/or delivering exosomes or extracellular vesicles. For example, a kit may comprise one or more pharmaceutical compositions as described herein and optionally instructions for their use. Kits may also comprise one or more devices for accomplishing administration of such compositions. For example, a subject kit may comprise a pharmaceutical composition and catheter for accomplishing direct administration of the composition to a patient having or at risk for hearing loss. In other embodiments, a subject kit may comprise pre-filled ampoules of isolated extracellular vesicles, optionally formulated as a pharmaceutical, or lyophilized, for use with a delivery device.

[0092] Kits may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a composition which includes an antibody that is effective for therapeutic or non-therapeutic applications, such as described above. The label on the container may indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above. In some embodiments, kits will comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

V. Examples

[0093] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1: Treatment of Drug-Related Hearing Loss in a Mouse Model.

[0094] Drug-, noise-, and age-related hearing loss (DRHL, NRHL, and ARHL), together, affect 1 in 3 Americans, yet no FDA-approved clinical strategy exists to prevent or ameliorate any of them. Exosomes are cell-secreted vesicles carrying potent molecular mediators; exosomes released by Mesenchymal Stem Cells (MSCs) are particularly powerful. It is predicted that intracochlear delivery of MSC exosomes can be a novel and effective therapy for preventing or ameliorating DRHL, NRHL, and ARHL.

[0095] Recent studies strongly suggest the involvement of chronic inflammation in the genesis and progression of DHRL, NRHL, and ARHL. However, the most commonly used anti-inflammatory therapeutic approaches, glucocorticoids and antioxidants, produce inconsistent results. Thus, an alternative research paradigm is desperately needed. MSC secrete exosomes carrying mixtures of RNA, proteins, and lipids. This complexity confers power to alter multiple signaling pathways simultaneously, producing results that last much longer than the initial exposure and making this approach more advantageous than traditional pharmaceuticals. In addition, it has been shown that MSC release mediators that facilitate the resolution of inflammatory responses. Thus, MSC-derived mediators may be the key for protecting the delicate sensory cells of the inner ear by activating endogenous hearing defense mechanisms. Implanting MSC directly within a functional cochlea is not desirable; due to the confined bony space and precise geographic patterning of the sensory cells it may worsen or completely destroy hearing ability. Instead, supplying the soluble products from the cells may accomplish their beneficial actions without the risks of cell implantation. If so, the cell-free products (in the form of exosomes) could be injected into patient's inner ears with a minimally invasive procedure. Specific patients include those requiring ototoxic chemotherapy drugs and patients with significant noise exposure showing early partial hearing loss. For those persons at risk of deafness, this low-risk procedure could enhance normal repair mechanisms and halt the otherwise inevitable progression of hearing loss.

[0096] Applicants contemplate a cell-derived therapy aiming to facilitate rapid, safe and complete resolution of inflammation as a new strategy for preventing DRHL, NRHL and ARHL. This strategy employs exosomes derived from MSC to supply pro-resolution signals to the cochlea in a mouse ototoxicity model. Exosomes contain pro-resolution precursors, intact mediator molecules, and assorted pRNA. They are internalized in target cells, transferring the molecules from MSC. Exosomes therefore provide stem cell signals without any direct cell administration. MSC-exosomes can be delivered into cochlea via a simple, direct path through the round window membrane to protect the hearing organ.

[0097] The traditional approach to hearing protection is pharmaceutical delivery to the inner ear, but drugs lack the complex signaling ability to restore natural homeostasis. Alternative cell-based approaches aim to reproduce the neural patterning of the cochlea responsible for hearing. That concept requires significant development before translating to humans, and even then would only apply to profound hearing loss. Although derived from mesenchymal stem cells, Applicant’s cell-free approach is the most readily translated to humans. It is novel, feasible, and potentially revolutionary for hearing preservation.

[0098] The auditory organ, the organ of Corti (OC), is located inside the anatomical structure known as the cochlea, within the inner ear. The cochlea is a common site of inflammation. The auditory sensory cells (hair cells) in the OC are very sensitive to inflammatory injury, while supporting cell types are active in the entire sequence of inflammation and resolution. In addition to ototoxic drugs, noise, and aging, cochlear inflammation can be induced by viral infections, mitochondrial dysfunction, autoimmune inner ear disorders and by cochlear surgery, among other causes contributing to the overall pathogenesis of cochlear injury and hearing loss.

[0099] Acute inflammation is an essential biological defense mechanism aimed at restoring normal function in the face of injury or illness. Until recently, it was considered that tissues automatically reset to homeostasis when pro-inflammatory signals dissipate. However, it was found that acute inflammation is terminated by an active process, regulated by endogenous signaling pathways driven by specialized molecules that: 1) switch from production of pro-inflammatory mediators to pro-resolution mediators; 2) turn off pro- inflammatory signaling pathways; 3) induce apoptosis of previously recruited inflammatory cells; 4) stimulate the clearance of apoptotic cells by phagocytes and; 5) reinstate homeostatic conditions.

[0100] It is contemplated that inflammatory resolution may be improved by intracochlear delivery of pro-resolving mediators generated by MSCs.

[0101] Pro-resolving mediators are molecules of diverse origin that work in inflammatory responses and also have important functions in host defense, pain, organ protection and tissue remodeling. To date, the most important pro-resolution mediators described in the literature are: i) Lipoxins, eicosanoids generated in vivo from arachidonic acid; ii) Resolvins generated from docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or docosapentaenoic acid (DPA); iii) Protectins and Maresins, also derived from DHA; iv) Annexin A1 (ANXA1), a powerful glucocorticoid-regulated protein; v) Galectins, a family of glycan-binding proteins vi) Adenosine, an ATP metabolite; vii) Neuromodulators, like acetylcholine and netrin-1, and viii) NO, H2S and CO, gaseous substances.

[0102] Looking at the inner ear of guinea pigs, the inventors localized ANXA1 in several cell populations lining the cochlear scala media, particularly stored inside lipid droplets (LDs) in supportive Hensen cells of the OC. ALX/FPR2, the highly-conserved receptor for ANXA1, Lipoxins A4 and B4, Resolvins D1 and El as well as for aspirin-triggered lipoxins and resolvins, was also found expressed in cells throughout the guinea pig cochlea, being particularly abundant in hair cells and supportive cells. The massive release of ANXA1 from Hensen cells induced by glucocorticoids (GC) could be important for both stopping leukocyte migration into the cochlea and for facilitating the clearance of apoptotic hair cells by inducing the transformation of supporting cells in the organ of Corti to non-professional macrophages. Additional studies identified more than 300 LDs-associated proteins, and showed that dexamethasone treatment induced fast fusion of LDs, followed by their disintegration and generation of new LDs.

[0103] Importantly, although low concentrations of lipoxins were detected in these studies, no evidence was found of its glucocorticoid-induced release from any OC cell population. This result suggests the possibility that the inconsistent results obtained with GC therapies in the clinic could be associated with the absence (or low concentration of) other pro resolution mediators. Thus, the action of ANXA1 alone would be not enough for a fast and safe resolution of inflammatory responses, and the contribution of other pro-resolution mediators via MSC exosomes could be crucial for a complete protection of the OC.

[0104] MSC can be isolated from human lipoaspirate donated for research in accordance with local IRB-approved protocol. MSC phenotypic confirmation can be performed by flow cytometry using the MSC markers CD90, CD 105, and CD73. Cell multipotency can be confirmed by directed differentiation to osteogenic and adipogenic phenotypes. Cells can be cultured in exosome-depleted medium, either untreated (Control) or exposed to dexamethasone (DEXA, 100 mM) to promote secretion of pro-resolution mediator ANXA1. High-purity exosome fractions can be obtained following the protocol of Chiou & Amsel, and confirmed by particle counting and flow cytometry using exosome markers tetraspanins CD9, CD63 and CD81. The presence of pro-resolution proteins (ANXA1 and galectins) can be investigated by Western blotting. Quantitative proteomics and lipidomics of exosomes can then be performed. [0105] The inventors can next determine whether intracochlear delivery of exosomes generated by human MSC prevent or ameliorate hearing loss induced by the ototoxic drug cisplatin in C57BL/6J mice.

[0106] The inventors can validate the hypothesis with proof-of-concept studies of drug ototoxicity in a mouse model susceptible to hearing loss (C57BL/6J). Experiments with ototoxic drugs are acute and provide information that can be used later in noise and aging experiments performed in the context of more extended studies. Cisplatin and aminoglycoside antibiotics are the most clinically relevant ototoxic drugs. Because mice are resistant to treatments with aminoglycosides, cisplatin can be used to model drug-induced hearing loss. It can be investigated whether intracochlear delivery of the molecular products generated by MSC (either untreated or stimulated with DEXA) through the round window membrane (RWM) significantly diminish the toxic effects of cisplatin on auditory function. A standard hearing test to measure sound-evoked brainstem potentials can be administered to both ears of all animals three times: at baseline before cisplatin; after cisplatin before surgery; and 7 days after surgery before euthanasia. Control animals can undergo identical procedures, with injection of culture medium that was not exposed to cells.

[0107] Ten mice can be used for each group for this exploratory study. This sample size has 80% power to detect a 20% difference of quantitative hearing between groups based on typical evoked brainstem potentials. Estimated exosome dose for this exploratory study is 1 ng of exosomes in a delivery volume of 0.1 pi, for ten-fold the concentration of exosomes that was reported to produce biological activity in a cell culture. Unilateral injection can be used to identify any hearing impact of the injection procedure itself relative to the contralateral cisplatin-affected ear. After euthanasia, temporal bones will be harvested for histology, electron microscopy, and molecular analysis.

[0108] If successful, these experiments can provide strong evidence that precursors and/or mediators of inflammatory resolution delivered into the mammalian cochlea can significantly enhance the natural protective mechanisms of the inner ear.

[0109] It is contemplated that these experiments can confirm that MSCs contain precursors of lipoxins, resolvins, protectins and maresins, the lipid mediators responsible for the resolution phase of inflammatory responses, as well as intact pro-resolution mediators (e.g., proteins ANXA1 and galectins). These experiments can better identify the precise contents under two conditions. It is also contemplated that these experiments can demonstrate that intracochlear delivery of the molecular products generated by MSCs prevents or ameliorate hearing loss induced by ototoxic drugs in C57BL/6J mice. Example 2: Intracochlear Delivery of Stem Cells’ Exosomes as a Therapy for Age- Related Hearing Loss

[0110] It is contemplated that molecular mediators of inflammatory resolution— lipids, proteins and miRNA— released by human mesenchymal stem cells (MSCs) inside exosomes would be able to enhance natural defense mechanisms in the inner ear protecting it against Age-Related Hearing Loss (ARHL).

[0111] ARHL is the most common form of acquired deafness, affecting millions of people worldwide. Although ARHL is clearly associated to a combination of environmental and genetic factors, recent studies indicate that the state of chronic inflammation in the elderly known as‘inflammaging’ (a consequence of immune- senescence, the ageing of the immune system) should be a key contributor to this auditory dysfunction. Consequently, several laboratories around the world are working in the development of anti-inflammatory strategies aimed at reducing ARHL. Inflammation, however, is a beneficial host reaction aimed at protecting individuals from infections and tissue injury, and suppressing it may prevent the activation of natural defense mechanisms with unintended negative consequences. Rather than preventing activation of natural defense mechanisms, the inventors propose a novel therapeutic approach based on activation of endogenous hearing protection mechanisms by lipids, proteins and miRNA released by MSCs inside exosomes that facilitate the rapid, safe and complete resolution of inflammatory processes. Whereas implanting MSCs directly within a functional cochlea is not desirable due to the confined bony space and precise cellular patterning of the auditory cells, cell-free products (exosomes) injected into patient's cochleae through the round window membrane (RWM) could accomplish their beneficial actions without the risks of cell implantation. Thus, the cell-free approach described herein could be the origin of a novel, feasible, and potentially revolutionary clinical therapy for hearing preservation in the elderly.

[0112] To test the efficacy of the proposed therapeutic strategy, the following experiments can be performed.

[0113] MSCs, isolated from human lipoaspirate can be used. MSC phenotypic confirmation can be performed by flow cytometry using the MSC markers CD90, CD 105, and CD73. Cell multipotency can be confirmed by directed differentiation to osteogenic and adipogenic phenotypes. High-purity exosome fractions can be obtained from MSCs in culture, either untreated (Control) or exposed to DHA (100 pM/24 h), by density-gradient ultracentrifugation following the protocol of Chiou & Amsel, and confirmed by light scattering (NanoSight™, Malvern Instr.), atomic force microscopy (Bruker Instr.), and flow cytometry (FACS Arialll®, BD Biosciences) using exosome markers tetraspanins CD9, CD63 and CD81 . Quantitative proteomics and lipidomics of exosomes can be performed. Similar experiments can be performed with exosomes isolated from multipotent mouse stem cells (available from ATCC), which can be used in control experiments in animal studies.

[0114] To test the effects of the therapy under two different ARHL mechanisms, Proof- of-concept studies can then be performed in the most widely used mouse models of ARHL. The CBA strain has a slow, progressive hearing loss starting near 18 months after birth. The C57 strain, which possesses a mutation in the Ahl gene that leads to deficiencies in the cadherin-23 protein, have an accelerated ARHL that starts in the higher frequencies (basal turn) by 3 month of age and progress to lower frequencies (apical turn), rendering near deaf animals at 7 month of age. Mice from both genders— 2 groups, control (injected with artificial perilymph, no exosomes) and experimental with 10 animals per group, total 20 mice per strain— can receive a total of 2 injections separated by a 2 months-period, and they can then be euthanized 2 months after the 2^nd treatment (FIG. 1). However, C57 animals can be 2 months- old and CBA 16 months-old at the time of the first injection, just before the onset of ARHL in each strain. MSC exosomes can be delivered directly inside the cochlear scala tympani through the RWM, a well-known surgical approach in animals as well as accessible to clinical application (FIG. 2). The intracochlear delivery can be performed only in the right ear, using the left as a control for the procedure. Standard hearing test to measure sound-evoked brainstem potentials (ABR) and distortion products (DPOAE) can be administered to both ears of all animals before surgery and periodically along the study. These experiments can be performed with exosomes from human and mouse stem cells. After euthanasia, temporal bones can be harvested for histology, electron microscopy, and molecular analysis. During these last studies, special attention can be dedicated to detect signals of synaptopathy and the potential protective effect of exosomes therapy on this pathology. This exploratory study will confirm the feasibility of using MSC exosomes as natural enhancers of physiological protective mechanisms against ARHL.

Example 3: Intracochlear Delivery of Stem Cells’ Exosomes for Preventing Noise- Related Hearing Loss

[0115] Millions of people worldwide are exposed to harmful levels of noise daily in their work and leisure environment. According to recent global estimates released by the World Health Organization (World Health Statistics 2012), there are 360 million individuals (over 5% of the world’s population) with disabling hearing loss, with a significant proportion associated with NRHL. In the USA, a 2011-2012 CDC study suggested that as many as 40 million adults have some type of hearing loss from exposure to loud noise (nidcd.nih.gov/health/noise- induced-hearing-loss). In addition, based on data from 2005-2006, it has been estimated that as many as 17 percent of teens (ages 12 to 19) have features of their hearing test suggestive of NIHL in one or both ears. Besides traditional hazardous exposure to occupational noise in industrial and military settings, many recreational activities exceed recommended sound levels. For instance, Action on Hearing Loss has issued a serious warning that approximately two- thirds of 18- to 30-year olds in England are exposed to dangerously high-intensity sounds (> 85 dB) which can cause hearing damage, through personal listening devices. The decreased hearing sensitivity associated with NRHL interferes with social interactions, leading to withdrawal, depression, inability to work and increased economic hardship for the patients and their families. Currently, hearing aids and cochlear implants are the only, and unsatisfactory, management strategies available for NRHL. It is therefore crucial to develop therapies that can prevent or at least alleviate NRHL.

[0116] This example proposes a therapeutic strategy that enhances the natural pro resolution mechanisms in the cochlea, by delivering pro-resolution mediators released by human MSCs inside exosomes directly into the cochlear scala tympani. The achievement of these goals may lead to the development of a novel therapy to facilitate the rapid, safe and complete resolution of cochlear inflammatory responses as a new strategy for preventing NRHL. Since this cell-free approach may be readily translated to humans, it may be potentially revolutionary for the prevention and treatment of NRHL.

[0117] Most, if not all, of the current clinical trials related to prevention or treatment of NRHL involve anti-inflammatory or antioxidant agents. However, inflammation is a life saving protective response to overcome infection and/or injury and ROS/RNS are essential second messengers in innate and adaptive immune responses, and interfering with these processes may prevent the activation of natural defense mechanisms and have unintended negative consequences. In contrast, rather than looking for ways to prevent inner ear inflammation, the methods in this example provide a therapeutic strategy for strengthening the mechanisms associated with the resolution of the inflammatory response in order to diminish, and perhaps eliminate, its negative side effects on the hearing organ while preserving its natural protective role.

[0118] MSCs, isolated from human lipoaspirate can be used. MSC phenotypic confirmation can be performed by flow cytometry using the MSC markers CD90, CD 105, and CD73. Cell multipotency can be confirmed by directed differentiation to osteogenic and adipogenic phenotypes. High-purity exosome fractions can be obtained from MSCs in culture, either untreated (Control) or exposed to DHA (100 pM/24 h), by density-gradient ultracentrifugation following the protocol of Chiou & Amsel, and confirmed by light scattering (NanoSight™, Malvern Instr.), atomic force microscopy (Bruker Instr.), and flow cytometry (FACS Arialll®, BD Biosciences) using exosome markers tetraspanins CD9, CD63 and CD81 . Quantitative proteomics and lipidomics of exosomes can be performed. Similar experiments can be performed with exosomes isolated from multipotent mouse stem cells (available from ATCC), which can be used in control experiments in animal studies.

[0119] Proof-of-concept studies can be performed following the protocol of Sanz L, et al. (Front Aging Neurosci. 2015;7:7) in two mouse models with different susceptibilities to NRHL. The CBA/Ca strain is more resistant to NRHL than the C57BL/6, which possess a mutation in the Ahl gene that leads to deficiencies in the cadherin-23 protein. A total of 48 young animals (4-8 weeks old) of both sexes of each strain can be used in these experiments. They can be divided in 4 groups: 1) control (no exposed, n=12), 2) exposed to 105 dB swept- sine sound stimuli in the range of 2-20 kHz for 30 minutes (n=12), 3) exposed to 105 dB swept- sine sound stimuli in the range of 9-13 kHz for 30 minutes (n=12), and 4) exposed to 120 dB swept-sine sound stimuli in the range of 2-20 kHz for 30 minutes (n=12). This sample size has 80% power to detect a 20% difference of noise-induced hearing damage between groups. Exosome’ s injection can be performed 24 hours after sound exposure, during the early onset of inflammatory responses. Estimated exosome dose for this exploratory study can be set at 1 ng of exosomes in a delivery volume of 0.1 pi, for ten-fold the concentration of exosomes that was reported to produce biological activity in a cell culture. MSC exosomes will be delivered directly inside the cochlear scala tympani through the RWM, a well-known surgical approach in animals as well as accessible to clinical application (FIG. 2). In humans, the RWM is located directly behind the tympanic membrane (FIG. 2 (D)), and minimal surgical procedures would be necessary to deliver exosomes into the cochlea. Unilateral injection can be used to identify any hearing impact of the injection procedure itself relative to the contralateral noise-affected ear. Animals can be euthanized 2, 14 and 28 days after noise exposure. After euthanasia, temporal bones can be harvested for histology, electron microscopy, and molecular analysis. During these last studies, special attention will be dedicated to detect signals of synaptopathy and the potential protective effect of exosomes therapy on this pathology. These experiments can be performed with exosomes isolated from human or mouse stem cells. Example 4: Inflammatory Resolution and the Prevention of Drug- and Noise-Related Hearing Loss

[0120] Isolation, countins and sizing of EVs: High-purity EVs fractions from auditory HEI-OC1 cells in culture were obtained in the inventors’ laboratory using the commercially available EVs isolation kit ExoEasy™ (Qiagen, USA). In the protocol the inventors used 0.45 pm filters, since the inventors were not sure whether exosomes were better nanocarriers than small ectosomes. EVs size was characterized by dynamic light scattering (DLS) with a Zetasizer (Malvern Instr., USA), and by nanoparticle tracking with a NanoSight™ NS300 (Malvern Instr., USA) (FIG. 3). Although EVs’ size and concentration varied among samples, typical values were in the ranges 100-800 nm and 9xl0⁸-3xl0⁹ EVs/mL, respectively. In subsequent experiments the inventors performed a second filtration (0.45 pm filters) to eliminate aggregates, the number of EVs was normalized to a value of 5xl0⁸ EVs/mL, and only freshly isolated EVs were used.

[0121] SEM is one of the best techniques for morphological control of EVs. The results using this technique with freshly isolated EVs and samples stored for 2 and 4 months at both - 30°C and -80°C suggest minimal effects of storage on EVs morphology and integrity. Moreover, the inventors investigated the feasibility of detecting EVs in the inner ear using SEM and, as showed in FIG. 4, they can be clearly identified.

[0122] — Protein profiling of EVs: Quantitative proteomics of HEI-OC1 EVs, either freshly obtained or stored for up to 4 month at -30°C, was performed using deuterium-labeled standards. The inventors found that EVs from HEI-OC1 naturally expressed abundant copies of the pro-resolution mediators ANXA1 and Galectins 1 and 3. A very preliminary profile analysis determined that, among the most abundant EVs proteins, 445 were cytoplasmic, 73 localized in the plasma membrane, 66 in the cell surface, and 78 in the nucleus. Investigating their primary function, the inventors found that 328 had catalytic activity, 236 bound proteins, 50 bound DNA, 43 bound RNA, and 13 possessed strong antioxidant activity; likewise, 242 were associated primarily with cell organization and biogenesis, 156 with cell metabolism, 91 with cell differentiation, 43 with regulation of biological processes, 30 with cell communication, and 20 with cell defense. Comparison with the proteomic profile of whole HEI-OC1 cells indicated, as expected, that EVs concentrate some proteins while others were below the limit of detection. No significant differences between fresh and stored EVs were detected. The significance of these results is still under investigation. [0123] — Loading of EVs with pharmacological drugs: The inventors investigated the loading of fresh EVs with aspirin (ASP) and dexamethasone (DEXA). For these experiments the inventors used, in addition to pure drugs (Sigma-Aldrich), deuterium labeled ASP (04- ASP) and DEXA (D4-DEXA) (Clearsynth Canada Inc.) as standards for quantification. The inventors found that HEI-OC1 EVs were easily loaded with the drugs by simple co-incubation at room temperature for 1 hour, followed by a second EVs purification step with ExoEasy™ columns to wash away the unloaded drug. The loading could be further improved with 5 minutes sonication. No significant differences in drug loading capacity between fresh and stored EVs were detected.

Example 5: Extracellular vesicles from Auditory Cells as Nanocarriers for Antiinflammatory Drugs and Pro-resolving Mediators

[0124] Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. The inventors propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to cochlear cells. The present study provides evidence that extracellular vesicles from auditory HEI-OC1 cells incorporate anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. Extracellular vesicles generated by HEI-OC1 cells were divided by size in two fractions, small (<150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin Dl, and the polyunsaturated fatty acids arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with polyunsaturated fatty acids and the large ones with aspirin, LXA4 and resolvin Dl. Importantly, this data indicates that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large extracellular vesicles from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

[0125] Drug-, noise-, and age-related hearing loss (DRHL, NRHL and ARHL, respectively) are intimately associated with inflammatory responses in the inner ear (Kaur et al. 2016, Lowthian et al. 2016, Kalinec et al. 2017, Keithley 2018). Inflammation, a normal biological reactions aimed at restoring tissue and organ functionality and homeostasis, is usually divided in two phases: initiation and resolution (Kumar et al. 2014). The initiation of inflammation is characterized by the up-regulation of pro-inflammatory mediators such as leukotrienes, prostaglandins, and thromboxanes. When the inflammatory response peaks, the resolution phase starts. Inflammatory resolution is an active process achieved mostly by the action of specialized protein and lipid pro-resolving mediators (Perretti 2015, Perretti et al. 2015, Kalinec et al. 2017). The inventors have recently proposed that stimulation of pro resolving pathways associated with cochlear inflammatory processes could be an important new therapeutic approach for preventing or ameliorating DRHL, NRHL, and ARHL (Kalinec et al. 2017), with pro-resolving mediators accelerating the return to homeostasis by modifying the immune response rather than by inhibiting inflammation (Dalli and Serhan 2019). The successful implementation of this clinical strategy, however, has several challenges. It requires, for instance, identifying safe and efficient ways to deliver a combination of pro-resolving mediators into the cochlea, without compromising their pharmacokinetics and therapeutic efficacy, with minimal adverse effects to the host. Thus, this study was aimed at evaluating whether extracellular vesicles (EVs) from auditory HEI-OC1 cells could be adequate carriers for these agents.

[0126] Recent studies have shown that packaging drugs into nanoscale synthetic particles can improve pharmacokinetic efficiency and therapeutic efficacy (Li et al. 2017, Hao and Li 2019). Nonetheless, the use of these drug-loaded artificial nanoparticles has several disadvantages. Lor example, microparticles are usually toxic, they are not stable in biological environments, assembling them is usually expensive, and they are not well suited to carry several different drugs simultaneously (Tang et al. 2012). EVs, in contrast, are naturally adapted for conveying molecular products from cells that generate and/or store them to cells that need them. After dexamethasone treatment, for example, Hensen cells in the guinea pig organ of Corti accumulate Annexin Al in their cytoplasm and release it to the external milieu inside EVs (Kalinec et al. 2009). EVs have demonstrated promise as a natural delivery system for combinations of small molecules, proteins, oligonucleotides, and pharmacological drugs (Robbins and Morelli 2014, Armstrong and Stevens 2018).

[01271 EVs have unique advantages as carriers to deliver drugs, such as their ability to overcome natural biological barriers and their intrinsic cell targeting properties. Besides, since EVs are formed from cellular membranes, they are not toxic and they can be easily manipulated for drug-packaging without restrictions associated with the physicochemical properties of drugs. Furthermore, since EVs do not self-replicate, they lack endogenous tumor- formation potential. Importantly, EVs are known to be effective for gene and drug delivery, and their surface and cargo can be engineered to target specific cell types and to deliver specific components (Marcus and Leonard 2013). Moreover, the toxicity of some pharmacological drugs can be reduced by encapsulating them into EVs (Tang et al. 2012). Finally, EVs can be produced in a scaled manner more easily than other therapeutics (Lai et al. 2019), and because of their small size and scarce presence of membrane histocompatibility molecules, they carry a reduced possibility for immune rejection (Reis et al. 2016). Not less important, intracochlear delivery of EVs loaded with adeno-associated virus has been already successfully used for rescuing hearing in a mouse model for hereditary deafness (Gyorgy et al. 2017).

[0128] EVs are released by cells as self-contained vesicles encapsulating a small portion of the parent cell cytoplasm. Cells naturally produce a diverse spectrum of EVs, spanning from small vesicles of about 50 nm diameter, to large vesicles up to 10 pm diameter (Mathieu et al. 2019). In the 1980’s the smallest EVs, between 50 nm to 150 nm in diameter, were called ‘exosomes’, and this term rapidly became the most frequently used in the EV field (Tkach et al. 2017); the bigger EVs are known as‘ectosomes’ or microvesicles. This categorization is also associated with the specific origin of the vesicles, with exosomes deriving from the fusion of multivesicular endosomes with the plasma membrane and microvesicles shedding directly from the plasma membrane (Colombo et al. 2014). Independently of their origin, EVs consist of a lipid bilayer, with integral and surface proteins, and containing in the core cytoplasmic proteins, lipids, RNAs (including messenger RNA and microRNAs), DNA, and metabolites (Pinheiro et al. 2018). They are usually enriched in proteins involved in vesicle genesis and trafficking, signal transduction, cytoskeleton organization, antigen presentation and transport, and vesicle targeting to acceptor cells or to the extracellular matrix (Robbins and Morelli 2014). Typically, they also contain proteins belonging to the tetraspanin protein family such as CD9, CD63 and CD81. The lipid components of EVs include ceramide (sometimes used to differentiate them from lysosomes), cholesterol, sphingolipids, and phosphoglycerides with long and saturated fatty-acyl chains (Skotland et al. 2017). Importantly, it has been reported that EVs naturally contain lipid mediators such as arachidonic and 12-hydroxyeicosatetraenoic (12-HETE) acids, leukotriene B4, leukotriene C4, and prostaglandins E2 and J2 (PGE2 and 15d-PGJ2, respectively), as well as lipid-related proteins (Record 2018). The outer surface of EVs, in turn, is rich in mannose, polylactosamine, alpha-2,6 sialic acid, N-linked glycans, lectins, and galectins (Laulagnier et al. 2004, Yanez-Mo et al. 2015). Nonetheless, each type of EV has a unique molecular signature that depends on the parent cell lineage and status (for example, healthy or pathological), and the stimulus that elicited their generation and release.

[01291 There is no actual proof yet that small EV s have a function different to that of larger

EVs (Tkach et al. 2017, Vagner et al. 2019), although there is agreement in that these different EV populations may have different composition even when released by the same cell type (Mathivanan et al. 2010, Bobrie et al. 2012, Romancino et al. 2013, Zaborowski et al. 2015, Kowal et al. 2016, Xu et al. 2016, Vagner et al. 2019). A recent‘consensus’ publication, co authored by several prominent scientists in the field of EVs, stated that“EVs in the small size range likely represent vesicles heterogeneous in origin” with unknown portions of exosomes and ectosomes (Mateescu et al. 2017). The definition of larger EVs is even less precise, with these vesicles comprising a wide range of membrane-enclosed entities. Indeed, it is not yet clear how to divide EVs into their relevant subtypes, or even how many functionally distinct subtypes there are (Mateescu et al. 2017). In addition, increasing evidence suggests that different classes of EVs, and different populations within each class, may harbor unique molecular cargos and have specific functions (Kowal et al. 2016, Willms et al. 2016, Jeppesen et al. 2019), clearly indicating the limitations of the size-based classification. Thus, looking for a better characterization EVs have been divided by size into small and large (Tkach et al. 2017, Vagner et al. 2019). A similar approach was followed in the present study, with HEI-OC1 EVs divided into two fractions: small EVs (S-EVs, <150 nm in diameter, mostly exosomes), and large EVs (L-EVs, >150 nm in diameter, mostly microvesicles).

[0130] In summary, endogenously produced EVs are an attractive drug delivery system mainly because their small size, low immunogenicity, absence of toxic effects, and stability in biological environments (Akao et al. 2011, Fuhrmann et al. 2015, Wong et al. 2016). In addition, EVs can be loaded with a designed combination of therapeutic agents and engineered to target specific cell types (Marcus and Leonard 2013). Although the actual suitability of EVs for intracochlear delivery of anti-inflammatory and pro-resolving agents, as well as its true potential for preventing or alleviating DRHL, NRHL, and ARHL, will require additional research, confirming them as a possible safe and efficient nanocarrier for these agents is a crucial initial step. In the present study the inventors investigate the production of EVs by auditory HEI-OC1 cells, characterize them by proteomics and targeted lipidomics, and evaluate their capability to incorporate simultaneously anti-inflammatory and pro-resolving agents. These results support the idea that EVs from HEI-OC1 cells could, indeed, be used as nanocarriers for the delivery of drugs and molecular mediators aimed at facilitating the resolution of inflammatory processes.

A. Materials and Methods

1. HEI-OC1 cells

[0131] Immortomouse™-derived HEI-OC1 cells were grown in polystyrene cell culture dishes (CellStar™, Greiner Bio-One, NC, USA) using DMEM (Gibco, MD, USA) supplemented with 10% FBS (HyClone, Thermo Fisher Sci, USA), at 33°C and 5% C02 as previously described (Kalinec et al. 2003). At approximately 80% confluence, cells were washed with PBS and fresh cell culture media supplemented with 10% exosome-depleted FBS (Cat. #A2720801, Thermo Fisher Sci., USA) was added. After a further 24 hours under the same incubation conditions the cell culture media was removed and processed for EVs isolation and characterization.

2. EVs isolation and physical characterization

[0132] EVs were isolated from the cell culture media using the commercially available exoEasy™ isolation kit (Qiagen) following the manufacturer-suggested procedure. Two cell culture dishes of 100 mm diameter were used per condition, yielding 16 mF (8 mF/dish) of medium; this medium was pre-filtered to exclude particles larger than 800 nm, and the filtrate loaded onto the exoEasy™ spin columns (4 mL per column, total 4 columns per condition). After centrifugation (500xg, 5 min) to remove the residual liquid, 400 pL of elution buffer was added to each column, and them centrifuged again for 5 min at 500xg to collect the eluate. Isolated EVs samples (400 pL each in elution buffer per column) were pooled, PBS was added to obtain a final volume of 200 mL, and then this EVs suspension was filtered by tangential flow with a Sartorious Vivaflow 50 device with a 200 nm pore size (Cat. #VF05P7. Sartorious GmbH, Gottingen, Germany). This device allows the recovery of two fractions: the retained one (L-EVs) with particles of diameter greater than 200 nm, and the filtrate (S-EVs) with particles smaller than 200 nm diameter. The L-EVs fraction was further ultra-filtered and concentrated using Sartorious Vivaspin 2 with 200 nm pore size (Cat. #VS0271), and the second filtrate incorporated to the S-EVs fraction. Next, this combined S-EVs fraction was further ultra-filtered and concentrated using Sartorious Vivaspin 2 with 100 kDa pore size (Cat. #VS0241). Using this procedure, the original EVs obtained from HEI-OCl cells were separated by size into two fractions, S-EVs (diameter < 150 nm) and L-EVs (diameter > 150 nm).

[0133] The counting and sizing of EVs in the L-EVs and S-EVs fractions was accomplished with the Microfluidic Resistive Pulse Sensing (MRPS™) technique using a Spectradyne nCS l™ instrument (Spectradyne LLC, Torrance, CA) (Cleland et al. 2016, Grabarek et al. 2019). This method is based on monitoring transient changes in electric current, also known as the Coulter principle, caused by particles passing through a narrow orifice (Song et al. 2017). When a nanoparticle passes through the constriction, it blocks some of the electrical sensing current, increasing the electrical resistance of the constriction by an amount proportional to the nanoparticle volume. Monitoring the electrical resistance as a function of time thus yields a number of short pulses, each corresponding to the passage of a nanoparticle, with the pulse amplitude yielding the nanoparticle volume, and the duration corresponding to the particle dwell time and thus the particle velocity. As the particles are entrained in the suspending fluid, the pulse duration yields the volumetric flow rate. One can thus obtain measurements of the concentration of nanoparticles as a function of nanoparticle size, directly from the temporal record of electrical resistance. This is a well-established technique, with the Coulter counter being the most commonly-used automated instrument for particle analysis (Vaclavek et al. 2019). At present, there are two commercially- available RPS instruments for counting and sizing EVs, the qNano (Izon Science Ltd., New Zealand) and the nCS l (Spectradyne LLC, Torrance, CA) (Vaclavek et al. 2019). The qNano instrument expands RPS technique to a broader range of particle diameters by using an elastic pore that can be stretched, an approach known as Tunable Resistive Pulse Sensing (TRPS) (Weatherall and Willmott 2015). This instrument, however, requires user-dependent manual settings that reduce data reproducibility (Tkach et al. 2017). Rather than a single elastic pore, the MRPS™ technique in the nCS l instrument uses disposable poly dimethyl siloxane cartridges, each with a constriction of a particular size in a microfluidic channel acting as a sensing gate (Cleland et al. 2016).

[0134] In the present study TS-400 cartridges were used for S-EVs (range 65 - 400 nm particle size) and TS-900 cartridges for L-EVs (range 130 - 900 nm particle size) (Cleland et al. 2016, Grabarek et al. 2019). Samples were first diluted with PBS (1: 10) and then supplemented with 0.1% BSA as recommended by the instrument manufacturer, and counting and sizing determined as the average of triplicate measurements on two independent samples. Since the instrument does not discriminate between EVs generated by HEI-OC1 cells from those contributed by the FBS used in cell culture or the abundant silicate particles present in BSA solutions, the values for the number of particles per diameter were background-corrected by subtracting bin-by-bin the numbers obtained in matched control samples of both culture media containing exosome-depleted FBS (CM+ED-FBS) and PBS+0.1% BSA. Particle concentration is reported in the form of a concentration spectral density (CSD), which corresponds to the density of particles per mL of solution per nm of particle diameter (particles/mL.nm). The use of CSD allows the comparison of measurements made with different cartridges, since the CSD is independent of the cartridge parameters and bin widths.

3. Proteomic studies

[0135] Bottom-up proteomics was performed using well-established protocols as recently described (Kalinec et al., 2019). Briefly, peptide samples were desalted using a modified version of Rappsilber's protocol (Rappsilber et al. 2007) and fractionated via high pH reverse phase chromatography (Agilent Poroshell 120). The fractions were then analyzed by in-line nanobore reversed phase chromatography coupled to nanospray ionization on a hybrid quadrupole-Orbitrap mass spectrometer (nLC-MS/MS; QE-Plus, Thermo Fisher Scientific) (Capri and Whitelegge 2017). The data was processed using Proteome Discoverer 2.2 (Thermo Fisher Scientific, USA), which provides measurements of relative abundance for the identified peptides, and mined using mouse protein databases (Kanehisa and Goto 2000, Kanehisa et al. 2014).

4. Loading of EVs and Targeted Lipidomic Analysis

[0136] HEI-OC1 EVs were loaded with 10 mM aspirin (ASP) (Catalog No. A5376, Sigma-Aldrich, St. Louis, Missouri, USA), arachidonic (AA), eicosapentaenoic (EPA), docosahexaenoic (DHA), and linoleic (LA) acids, (Sigma-Aldrich, St. Louis, MO), lipoxin A4 (LXA₄) and resolvin D1 (RvDl) (CAS No. 89663-86-5 and CAS No. 872993-05-0, respectively, Cayman Chemical, Ann Arbor, MI), alone or combined. Loading was performed by co-incubation for lh at 25°C with sonication for 5 minutes (Kalinec et al. 2019), a procedure favored by the hydrophobic nature of the molecules selected as cargo (Armstrong et al. 2017). These particular molecules were chosen because they are either pro-resolving mediators (ASP, LXA4, RvDl) or precursors of pro-resolving mediators (AA, EPA, DHA, and LA). Unloaded drug was removed by tangential flow filtration followed by ultrafiltration and concentration as already described in subsection 2.2. This procedure increases more than 20-fold the PBS washing volume, rendering a more thorough wash-out of any unincorporated drug than the second purification step with exoEasy™ columns performed in the inventors’ previous study (Kalinec et al. 2019). [0137] For confirming ASP incorporation by S-EVs and L-EVs, dried samples were treated with /V,Obis(trimethylsilyl)trifluoroacetamide reagent (Pierce, 50 pL of reagent, 60°C, 60 min), and aliquots (1 pL) of the solution were analyzed by GC/MS (Thermo Q Exactive GC) for detection of the trimethylsilyl ASP derivative. Peak areas from the reconstructed ion chromatograms (m/z 195.04622-195.04818) for the fragment ion corresponding to the loss of a methyl and acetyl group from the molecular ion (observed m/z 195.04713, calculated 195.04720 for CgHnOsSi) were compared to those obtained from known amounts of ASP treated in the same way.

[0138] For targeted lipidomics, S-EVs and L-EVs were divided in 10 groups: S-Control: S-EVs untreated; S-EVs #2: S-EVs incubated with 10 mM ASP; S-EVs #3: S-EVs incubated with 10 mM LXA4 + 10 mM RvDl; S-EVs #4: S-EVs incubated with 10 mM AA+EPA+DHA+LA; S-EVs #5: S-EVs incubated with 10 mM AA+EPA+DHA+LA+ASP; L- Control: L-EVs untreated; L-EVs #2: L-EVs incubated with 10 mM ASP; L-EVs #3: L-EVs incubated with 10 mM LXA4+RvDl; L-EVs #4: L-EVs incubated with 10 mM AA+EPA+DHA+LA; L-EVs #5: L-EVs incubated with 10 mM AA+EPA+DHA+LA+ASP. Samples of each of these groups were sent to the UC San Diego Lipidomic Core for fatty acid and eicosanoid analysis (found on the world wide web at ucsd-lipidmaps.org). As previously described (Eguchi et al. 2016), samples were supplemented with 26 deuterated internal standards and brought to a volume of 1 ml with PBS containing 10 % methanol. They were then partially purified by solid-phase extraction on Strata-X columns (Phenomenex, Torrance, CA) following the procedure outlined by the manufacturer. The columns were eluted with methanol (1 ml), the eluent was dried under vacuum and redissolved in 50 pL of buffer A (water/acetonitrile/acetic acid, 60:40:0.02 (v/v/v) and immediately used for LC-MS analysis. Eicosanoids were analyzed as previously described (Quehenberger et al. 2010, Quehenberger et al. 2011). Briefly, eicosanoids were separated by reverse phase chromatography using a 1.7- mM 2.1 x 100-mm BEH Shield Column (Waters, Milford, MA) and an Acquity UPLC system (Waters, Milford, MA). The column was equilibrated with buffer A and 5 pi of sample was injected via the autosampler. Samples were eluted with a step gradient to 100 % buffer B consisting of acetonitrile/isopropanol = 50:50 (v/v). The liquid chromatography effluent was interfaced with a mass spectrometer, and mass spectral analysis was performed on an AB SCIEX 6500 QTrap mass spectrometer equipped with an IonDrive Turbo V source (AB SCIEX, Framingham, MA). Eicosanoids and polyunsaturated fatty acids (PUFA) were measured using multiple reaction monitoring (MRM) transitions with the instrument operating in the negative ion mode (Wang et al. 2014). Collisional activation of the eicosanoid precursor ions was achieved with nitrogen as the collision gas, and eicosanoids were identified by matching their MRM signals and chromatographic retention times with those of pure identical standards. Detailed instrument settings are summarized elsewhere (Quehenberger et al. 2018). Eicosanoids were quantified by the stable isotope dilution method. Briefly, identical amounts of deuterated internal standards were added to each sample and to all the primary standards used to generate standard curves. To calculate the amounts of eicosanoids in a sample, ratios of peak areas between endogenous eicosanoids and matching deuterated internal eicosanoids were calculated. Ratios were converted to absolute amounts by linear regression analysis of standard curves generated under identical conditions.

B. Results

1. Counting and sizing EVs

[0139] The first goal of the present study was to evaluate the number of S-EVs and L-EVs generated by HEI-OC1 cells. Since the nanotracking technique used in previous study is dependent on the optical properties of the particles, which vary with their size, the inventors switched to a different technique, Microfluidic Resistive Pulse Sensing (MRPS).

[0140] The data provided by the nCSl MRPS instrument, using TS-400 and TS-900 cartridges, is summarized in FIG. 5. The number of particles vs. diameter of particles curves corresponding to S-EVs and L-EVs from two independent samples (#1 and #2) are depicted in FIG. 5 (A). Each point of these curves represents the average of three measurements per sample, and they are already adjusted by bin-by-bin background subtraction of the values obtained from matched CM+ED-FBS and PBS+0.1% BSA control samples (confidence intervals are depicted by the diameter of the graphic points corresponding to every value; for background values see FIG. 9). The data shows that the number of EVs varies in inverse proportion to their size, with a maximum of around lxlO⁷ and a minimum of about lxlO⁵ particles per mL for S-EVs, and a range of around lxlO⁶ to lxlO³ particles per mL for L-EVs (FIG. 5 (A)). When the areas under the size distribution plots were summed, and background subtracted, the S-EVs samples contained an adjusted average of (3.0±0.2)xl0⁹ particles/mL, corresponding to a total of (3.3+0.1)xl0⁹ particles/mL minus (2.65+0.08)xl0⁸ particles/mL for background (FIG. 5 (B)). L-EVs samples, in turn, contained an adjusted average of (1.3+0.7)xl0⁸ particles/mL, as a consequence of a total value of 1.39+0.69)xl0⁸ particles/mL in experimental samples and (5.8+0.4)xl0⁶ particles/mL for background (FIG. 5 (C)). Thus, in contrast to the results using the nanotracking technique (Kalinec et al. 2019), currentt data indicates that HEI-OC1 cells generate more S-EVs than L-EVs. Other information apparent from these figures is that the fractions are not absolutely“pure”, since the s-EVs one still contains a certain number of particles with diameters >150 nm and, vice-versa, the L-EVs fraction contains vesicles with diameters smaller than 150 nm (FIG. 5 (A)), .

[0141] Interestingly, using the formulas V=4/3 (p r³) and S=4 p r² and assuming the EVs are perfectly spherical, the calculated total volumes (S-EVs=3.1xl0¹⁴ nm³; L-EVs=6.4x 10¹⁴ nm³) and surface (membrane) areas (S-EVs=1.8xl0¹³ nm²; L-EVs=1.2xl0¹³ nm²) of the vesicles present in each fraction are of the same order of magnitude.

2. Protein profiling of HEI-OC1 EVs

[0142] Next, the inventors wonder whether S-EVs and L-EVs shared a similar protein profile or, instead, they contain some unique protein markers that could be used for identification or classification. To investigate this issue, the inventors performed bottom-up proteomics.

[0143] A total of 620 bona fide EVs proteins were detected in these proteomic studies, 489 of them in the S-EVs fraction and 131 in the L-EVs fraction (Supplemental Tables 1 and 2). From them, 381 (61.5%) were unique for S-EVs, 23 (3.7%) were specific for the L-EVs, and 108 (17.4%) were found in both fractions. Interestingly, 86.7% (26 out of 30) of the proteins listed in the ExoCarta exosome database as those more frequently identified in exosomes (Keerthikumar et al. 2016) were detected in S-EVs; in contrast, only 15 out of these 30 (50%) were detected in the L-EVs fraction. CD63, CD81, and PDCD6IP (aka Alix), all considered exosome biomarkers, were identified only in S-EVs (Table 1). More importantly, the protein mediators of inflammatory resolution Annexin Al (ANXA1) and Galectins 1 and 3 (Gal-1 and Gal-3) were only detected in the S-EVs fraction too (Supplemental Tables 1 and 2). On the other hand, only one cytokine (CSF1, colony stimulating factor 1) was detected in HEI-OC1 EVs, but it was present in both fractions S-EVs and L-EVs.

Table 1 - Expression in S-EVs and L-EVs fractions of the proteins more frequently identified in exosomes. Twenty-six out of the 30 (86.7%) proteins more frequently identified in exosomes were found in S-EVs, but only fifteen (50%) of them were found in L-EVs.

[0144] Except for the difference in the total number of proteins in each fraction, the distribution profiles by cellular origin, biological and molecular function were quite similar in S- EVs and L-EVs (FIGS. 6, 7, and 8). Most of the proteins detected in both fractions originated from cell membranes, cytoplasm, nucleus, or cytosol, with minor percentages of extracellular origin or from organoids (mitochondria, endoplasmic reticulum) and the cytoskeleton (FIG. 6). They were mostly involved in metabolism or regulation of other biological processes, as well as in response to stimuli, cell organization and biogenesis, and transport (FIG. 7). The most common function was molecular binding, either to other proteins, RNA, metal ions or nucleotides, but proteins with catalytic or structural function were also significantly represented (FIG. 8). In every case, the proteins identified as“Others” include all those belonging to groups with less than 5% of the total.

[0145] In summary, the inventors found that the S-EVs fraction contains a larger number and greater diversity of proteins than the L-EVs, but the similarities observed in their distribution profiles (FIGS. 6, 7, and 8) suggest that this difference could be associated with the involvement of a more efficient cellular mechanism of protein sorting and/or loading into S-EVs from a single pool of molecules rather than the existence of two different pools, one for S-EVs and other for L-EVs. The presence of ANXA1, Gal-1, and Gal-3 makes S-EVs more attractive as potential nanocarriers in pro-resolving therapies.

3. EVs’ Loading and Target Lipidomic Analysis

[0146] The absence of toxic effects and ability to incorporate as cargo the drugs and molecules of interest are crucial requirements for a useful drug carrier. As already mentioned, EVs are not toxic, lack endogenous tumor- formation potential, and they show very low immunogenicity. In addition, they can be easily loaded with pharmacological agents using simple procedures. Therefore, the inventors decided to investigate the loading of S-EVs and L- EVs from HEI-OC1 cells with ASP, the eicosanoids LXA4, RvDl, and the PUFA AA, DHA, EPA, and LA, all of them recognized anti-inflammatory and pro-resolving agents. While ASP incorporation was evaluated by GC/MS, eicosanoids were identified and quantified by targeted lipidomics using LC/MS/MS-MRM. Importantly, in addition to revealing the identity and concentration of around 150 eicosanoids, PUFA, and related compounds, the lipidomic profiles revealed the presence and amounts of endogenous pro-inflammatory components that could counter the pro-resolving effects of the cargo.

[0147] Co-incubation of HEI-OC1 EVs (10 mL, with lxlO⁸ S-EVs and L-EVs per mL) with ASP (10 mM) resulted in the incorporation of 6.9+0.1 pg/mL (-38 mM) of ASP in S-EVs samples, and 61.8+0.6 pg/mL (-0.34 mM) ASP in L-EVs samples. These results suggest that EVs, particularly L-EVs could be loaded with pharmacologically effective quantities of this drug.

[0148] Of the 150 components of the lipidomic profiles, only 19 were detected in untreated (Control) samples, with 4 of them detected only in S-Control, 6 only in L-Control, and 9 found in both fractions (Table 2 and Supplemental Table 3). In addition, while free AA, DHA, and EPA were found in untreated EVs, LXA4, ATL-LXA4, Resolvins, Maresins, and Protectins were not detected (Table 2 and Supplemental Table 3). Table 2

Metabolites Unique to S-Evs

Metabolites Unique to U-Evs

Metabolites Detected in S-Evs and U-Evs

[0149] As shown in Table 3 both, S-EVs #3 and #4 and L-EVs #3 and #4, were able to simultaneously incorporate AA, EPA, and DHA in amounts between 5-6 nmol per mL of suspension (about 1.5-2.0 ng/mL).

Table 3: Lipidomic analysis - Incorporation of fatty acids and pro-resolving mediators by HEI-

OC1 EVs.

S-Control: S-EVs untreated; S-EVs #2: S-EVs incubated with 10 mM ASP; S-EVs #3: S-EVs incubated with 10 mM LXA4 + 10 mM RvDl; S-EVs #4: S-EVs incubated with 10 mM AA+EPA+DHA+LA; S-EVs #5: S-EVs incubated with 10 mM AA+EPA+DHA+LA+ASP; L-Control: L-EVs untreated; L-EVs #2: L-EVs incubated with 10 mM ASP; L-EVs #3: L- EVs incubated with 10 mM LXA4+RvDl; L-EVs #4: L-EVs incubated with 10 mM AA+EPA+DHA+LA; L-EVs #5: L-EVs incubated with 10 mM AA+EPA+DHA+LA+ASP. Results are expressed in pmol/mL. ND=Not Detected. [0150] Importantly, in EVs loaded with these PUFA, all 19 metabolites originally detected in S -Control and L-Control samples (Table 2) showed an increased concentration in both fractions. Moreover, 50 other metabolites previously undetected were found in these EVs, making a total of 69 eicosanoids identified in the samples (Supplemental Table 3). Inflammatory agents were not detected in Control and ASP-loaded EVs, but those loaded with PUFAs generate several prostaglandins (e.g., PGD₂(189.18 pmol/mL), PGE2 (48.94 pmol/mL), and PGF2a (15.64 pmol/mL)), leukotriene B4 (LTB4, 3.84 pmol/mL), and thromboxane B2 (TXB2, 1.18 pmol/mL), among others (Supplemental Table 3). The generation of PUFA derivatives suggests the existence of an active biosynthetic mechanism within HEI-OC1 EVs. LXA4 and AT-LXA4 (ASP triggered-LXA4) were also detected in these groups in amounts varying roughly between 3 - 10 pmol/mL (about 0.35 ng/mL). Since the limit of detection of the lipidomic experiments was around 0.002 pmol/mL, the measured values represent increases of at least 3 to 4 orders of magnitude (1,000-fold to 10,000-fold) relative to the control conditions. As expected, S-EVs and L-EVs also incorporated LXA4 and RvDl, although in lower amounts than their precursors AA, EPA, and DHA. It should be noted, however, that the amounts of LXA4 and RvDl incorporated by HEI-OC1 EVs ranged from 15 to 210 pmol/mL (roughly 5 to 70 pg/mL), six orders of magnitude higher than the normal concentrations of these pro-resolving mediators in human serum (30 to 120 pg/mL (Colas et al.)). Curiously, AT- LXA4 was not found in ASP-loaded EVs (S-EVs #2 and #5, and L-EVs #2, and #5), but was detected in EVs loaded with LXA4 (S-EVs #3 and L-EVs#3) and A A, its fatty acid precursor (S-EVs #4 and #5, and L-EVs #4, and #5).

C. Discussion

[0151] The present study provides evidence that auditory HEI-OC1 cells generate abundant S-EVs and L-EVs, with the number of particles varying in inverse proportion to their size. The data provided herein demonstrates that HEI-OC1 EVs have theability to incorporate molecules and drugs as cargo. The proteomic studies detected a differential presence of selected proteins in S-EVs and L-EVs. In particular, S-EVs contained a larger variety of proteins, including the pro-resolving mediators ANXA1, Gal-1 and Gal-3. Lipidomic studies, in turn, identified eicosanoid that were present in one of the EVs fractions and not in the other. Most importantly, the inventors confirmed that particles from both fractions, S-EVs and L- EVs, can be loaded with anti-inflammatory drugs, PUFA, and pro-resolving mediators, either alone or combined. These results support to the idea that HEI-OC1 EVs could be advantageously used as nanocarriers for delivery of drugs and molecular mediators aimed at facilitating the resolution of inflammatory processes. In particular, they could potentially be useful as vehicles for the intracochlear delivery of pro-resolving agents aimed at preventing or alleviating DRHL, NRHL, and ARHL.

Counting and sizing EVs

[0152] The present results indicate that HEI-OC1 cells secrete near two orders of magnitude more S-EVs than L-EVs, with the number of EVs decreasing monotonically with their increase in diameter (FIG. 5A). Since the instrument used for the measurements counts individual particles and estimates their diameter, arithmetic calculations from the data (assuming that all the EVs were perfect spheres) indicated that the total volume and total membrane area of the vesicles present in each fraction were of the same order of magnitude, suggesting that the drug-loading capability of both fractions should be roughly similar.

[0153] The abundance of S-EVs is in contradiction to the data the inventors reported in a recent publication, where the counting and sizing of HEI-OC1 EVs was based on light scattering technology (Kalinec et al. 2019). The distribution of EVs by size as estimated with that technique showed a low concentration of particles with diameters below 200 nm, suggesting that HEI-OC1 EVs consisted mostly of L-EVs. However, as already discussed in that publication, although currently considered as reliable, light- scattering techniques are not exempt of problems (Witwer et al. 2013, Koritzinsky et al. 2017, Grabarek et al. 2019). In particular, particles with an optical index close to that of the suspension medium or with high curvature radius (small diameter) scatter much less light than those with a significantly different index or larger diameter. This sensitivity dependence makes small biological particles essentially undetectable (Cleland et al. 2016, Grabarek et al. 2019), a fact that could explain the difference between the preliminary results and those reported here.

[0154] In the present study HEI-OC1 EVs were counted and sized with an instrument (Spectradyne nCS 1™) based on a completely different technique, Microfluidic Resistive Pulse Sensing (MRPS™) (Cleland et al. 2016, Grabarek et al. 2019). MRPS is based on monitoring transient changes in electric current, also known as the Coulter principle, caused by particles passing through a narrow orifice (Song et al. 2017). Using the nCS l for counting and sizing EVs has some important advantages. The nCS l is capable of measuring the size distribution of EVs with diameters ranging from 35 nm up to 10 micrometers over concentrations ranging from 10⁷ to 10¹² particles/mL, and statistically significant data sets can be acquired rapidly (minutes). Because the nCS l uses electrical sensing, not optical detection, measurements are independent of the material properties of the particles. Importantly, the small sample volume required for analysis (3 pL) is set by the size of the analyte reservoir in the disposable cartridge. Moreover, the measurement does not rely on user-adjustable parameters, rendering more reproducible results. Although not completely free of limitations (analysis of particles with diameters <55 nm with this technique is still complicated, and the use of different cartridges makes cumbersome the evaluation of samples with a big range of particle’s diameters), MRPS is probably the best currently available technique for analyzing particles in the nanometer range (Grabarek et al. 2019).

2. Protein profiles of S-EVs and L-EVs

[0155] These results show that S-EVs and L-EVs do not contain the same repertoire of proteins. In fact, only 17.4% (108 out of the 620) proteins identified as belonging to HEI-OC1 EVs are common to both fractions, whereas 61.5% (381 out of 620) are unique to S-EVs and 3.7% (23 out of 620) were found only in L-EVs. As shown in Table 1, 86.7% of the proteins more frequently found in exosomes were present in S-EVs from HEI-OC1 cells, whereas only 50% were also present in the L-EVs fraction. Two proteins (SDCBP, an adapter protein involved in exosome biogenesis, and FASN, a fatty acid synthase) frequently identified in exosomes and previously reported in HEI-OC1 EVs (Kalinec et al. 2019) were not detected in the present study. Interestingly, the protein most frequently found in exosomes from other cell populations, tetraspanin CD9, was also absent in EVs from HEI-OC1 cells. This confirmed preliminary results (Kalinec et al. 2019), and those of others, suggesting that not all S-EVs have the same tetraspanin profile (Vagner et al. 2019). For instance, CD9 and CD63 were variably detected in S-EVs and L-EVs, whereas CD81 was exclusively detected in S-EVs. However, only the co-localization of CD81 with CD63 qualify an S-EV as an exosome (Kowal et al. 2016, Tkach et al. 2017). Thus, the inventors can conclude that S-EVs samples probably contain classical exosomes since CD63 and CD81 were abundant in this fraction (Table 1).

[0156] Other differences between current and earlier preliminary results was the finding of ANXA1 and ANXA5 in S-EVs (Supplemental Tables 1 & 2). The inventors commented in the previous work the oddity that“two of the most common annexins found in exosomes (ANXA1 and ANXA5) were either found at low levels or not appeared at all” in the samples (Kalinec et al. 2019). The fact that both of them were now found only in the S-EVs fraction suggests that they were probably present in preliminary samples, but in non-detectable levels. The presence of ANXA1, in particular, provides important support to the idea of using HEI- OC1 EVs as nanocarriers of drugs and molecular mediators aimed at the resolution of inflammatory responses in the cochlea. ANXA1 (Annexin Al) is a potent anti-inflammatory and pro-resolving protein (see (Kalinec et al. 2017) and references therein). Many of the cellular and molecular processes associated with the anti-inflammatory properties of glucocorticoids are modulated by ANXA1, and it is considered an important modulator of both the innate and adaptive immune systems.

[0157] Interestingly, the authors of a recent publication (Jeppesen et al. 2019) proposed that ANXA1 would be a specific marker of microvesicles shed from the plasma membrane, and ANXA5 a component of apoptotic vesicles. The subcellular distribution of ANXA1 is unusual (see, for instance, (Buckingham and Flower 2017)); it is abundant in the cytoplasm of some cell populations, but a small proportion is also found on the external surface of the plasma membrane or attached to their inner leaflet. Sometimes, even a single membrane pool of ANXA1 has been detected in particular cells. In guinea pig Hensen cells (Kalinec et al. 2009) and HEI-OC1 cells ANXA1 was found in the cytoplasm, with membrane localization observed only after stimulation with glucocorticoids; in contrast, Jeppesen et al. localized ANXAlonly in the plasma membrane of DKO-1 and Gli36 human cancer cells, which they used in the reported study. Therefore, while Jeppesen et al. detected only membrane -bound ANXA1 in microvesicles from human cancer cells, cytoplasmic ANXA1 could be abundant in exosomes from auditory HEI-OC1 cells. Likewise, although ANXA5 is prominent in apoptotic vesicles, its presence in exosomes or microvesicles from some cell populations cannot be overlooked. [0158] The inventors also confirmed the presence in S-EVs of Gal-1 and Gal-3 (Supplemental Tables 1 & 2), a family of glycan-binding proteins that regulate the initiation, amplification and resolution of acute and chronic inflammatory responses (see (Kalinec et al. 2017) and references therein). Gal-1 has been associated with a range of anti-inflammatory effects on various cells types, whereas Gal-3 enhances the phagocytic capabilities of neutrophils, a property that may in part account for its protective role in infections. On the other hand, the only pro -inflammatory cytokine detected in HEI-OC1 EVs, the Colony Stimulating Factor 1 (CSF-1), was found in both fractions. CSF-1 is known to be involved in the proliferation, differentiation, and survival of monocytes and macrophages, and it has been implicated in promoting tissue repair following injury (Zhang et al. 2012). Recently, however, it was proposed that CSF-1 has a dual role as pro-inflammatory and anti inflammatory/regulatory cytokine dependent on the particular immune response (Bhattacharya et al. 2015).

3. Lipids in S-EVs and L-EVs

[0159] LXA4, AT-LXA4, Resolvins, Maresins, and Protectins, in contrast to free AA, DHA, and EPA, were not detected in S -Control and L-Control (Table 2 and Supplemental Table 3). On the other hand, 4 eicosanoids unique to S-EVs and 6 unique to L-EVs were detected in Control samples, while another 9 were common to both fractions (Table 2). Those unique to S-Control were the HydroxyEicosaTetraEnoic acids 8-HETE, 15-HETE, and 8- HETrE, which derive from AA, and 16-HDoHE (HydroxyDocosaHexaEnoic acid), an autoxidation product of DHA (Poorani et al. 2016) (for a complete list of eicosanoid abbreviations see Supplemental Table 3, page 3). These products of PUFAs are pre-dominantly pro-inflammatory, but they are also intermediaries in the generation of lipoxins and epi- lipoxins and enhance the generation of nitric oxide (NO), another pro-resolving mediator (Kalinec et al. 2017). Interestingly, two pro-inflammatory eicosanoids, prostaglandin D2 (PGD2) and 13,14-dihydro- 15-keto-PGE2 ( dhk-PGE2 ), were detected in L-Control but not in S-Control (Table 2). In mammalian organs, large amounts of PGD₂ are found only in mast cells and the brain; in fact, PGD2 is the most abundant prostaglandin in the brain and the one that changes the most under pathological conditions (Figueiredo-Pereira et al. 2014). While prostaglandin E2 (PGE2) and 15-deoxy-A12, 14-prostaglandin J2 (15-d-PGJ2) are frequently found in EVs (Subra et al. 2010), to the inventors’ knowledge the literature only reports the presence of PGD2 in EVs in one study performed on exosomes from RBL-2H3 cells, a mast cell model (Subra et al. 2010). Other eicosanoids only detected in L-Control were 15-oxoETE (oxo EicosaTetraEnoic acid, an oxylipin produced by oxidation of 15-HETE), 14(15)-EpETE (a cytochrome P450 (CYP)-metabolite of EPA), 9(10)-EpOME and 12(13)-EpOME (products of the metabolism of linoleic acid by CYP enzymes). Just like all those detected in both fractions, these metabolites participate in pro-inflammatory and pro-resolving pathways, suggesting they are part of a delicate physiological balance.

[0160] An important piece of information provided by the lipidomic studies described in this example was the significant increase in the variety and amount of lipid metabolites present in EVs loaded with AA, EPA, and DHA (see below). More than 50 metabolites undetected in S-Control or L-Control were found in these EVs, making a total of 69 eicosanoids identified in the inventors’ samples (Supplemental Table 3). For instance, in the absence of exogenously added PUFAs few pro-inflammatory eicosanoids were detected, and those that were detected were present at trace levels. In contrast, prostaglandins PGD2, dhk-PGE2, PGF2a, PGE2, PGE1, PGD1, PGE3, PGD3, 15kPGF2 a, 15k PGE2, dhk-PGD2, llb PGE2, thromboxane B2 (TxB2), and other pro-inflammatory agents were detected in both S-EVs and L-EVs loaded with PUFAs (Supplemental Table 3). The data suggest that all pathways, including those mediated by COX (cyclooxygenase), 5-LOX (lipooxygenase), 12-15 LOX and CYP (cytochrome P450), are endogenously present in the HEI-OC1 EVs, and are sufficiently active to produce abundant molecular products when supplied with substrates. It should be emphasized that, since EVs were loaded exogenously in the absence of cells, the inventors can confidently assume that all these metabolites were generated in the EVs, suggesting the existence in EVs of an active metabolic mechanism.

[0161] Most of these AA, EPA and DHA metabolites are part of pathways that give rise to potent pro-resolving mediators but also to pro-inflammation agents. Many of the prostaglandins, including the highly induced PGD2, are considered pro-inflammatory. In general, under steady-state physiological conditions, one would expect a balance between pro- and anti-inflammatory molecules so that homeostasis is preserved. When this balance is altered, depending on the side to which it is tilted, either there will be suppression or exaggeration of inflammation. These results suggest that incorporation of PUFAs as cargo induces the generation of pro-inflammatory agents in the HEI-OC1 EVs, which could interfere with the goal of accelerate the resolution of inflammatory processes. Thus, if HEI-OC1 EVs are used as nanocarriers, their cargo should be carefully tailored to ensure that it will promote the return to the normal balance. 4. Loading of S-EVs and L-EVs

[0162] Incorporation of dexamethasone by HEI-OC1 EVs was already reported in the preliminary study (Kalinec et al. 2019), and here the inventors present proof that they can be loaded with ASP, LXA4, RvDl, and the eicosanoid precursors AA, EPA, and DHA. These results support the idea that HEI-OC1 EVs could be ideal vehicles for intracochlear delivery of drugs and molecular mediators aimed at facilitating the resolution of cochlear inflammatory processes.

[0163] The amount of ASP incorporated by HEI-OC1 EVs was surprisingly high, with significantly more loading by L-EVs (61.8+0.6 pg/mL) than by S-EVs (6.9+0.1 pg/mL). Since the volume of L-EVs per mL is only about twice the volume of S-EVs per mL (6.4x 10¹⁴ nm³ vs. 3.1xl0¹⁴ nm³), the inventors speculate that could be some structural problem limiting ASP incorporation in S-EVs. For instance, the different loading of ASP by S-EVs and L-EVS could be related to differences in the curvature of the EVs membrane in one and other case. It has been shown that ASP interacts with membrane lipids, being incorporated first to the external layer and translocating to the internal by flip-flop before being internalized. During this process, the physical properties of the full structure, including their thickness, bending elasticity, and permeability, are affected (Zhou and Raphael 2005, Sharma et al. 2017). These changes could be more significant in small EVs, hindering the incorporation of ASP.

10164] ASP, in addition to its proven anti-inflammatory, pro-resolving, and anti-oxidant properties, is the only drug to date that has showed beneficial effects for the mitigation of sensorineural hearing loss in clinical trials (see (Kalinec et al. 2017) and references therein). ASP not only blocks the biosynthesis of prostaglandins, but also stimulates the endogenous production of pro-resolving mediators, such as ASP-triggered lipoxins (AT-LXs) and resolvins (AT-RVs), which promote the resolution of inflammation by stimulating phagocytosis of cellular debris and counter-regulate proinflammatory cytokines without being immunosuppressive (Serhan 2014). ASP-triggered pro-resolving mediators are generated by the activity of ASP-acetylated cyclo-oxygenase on PUFA substrates, including AA, EPA, and DHA (Claria and Serhan 1995, Claria et al. 1996, Serhan et al. 2002, Dalli et al. 2013, Serhan 2014). In addition to exhibiting similar anti-inflammatory and pro-resolving characteristics of native mediators, the ASP-triggered forms (R epimers) resist rapid inactivation by oxido- reductases and have longer in vivo half-lives (Serhan 2014). Intriguingly, present results indicate that incorporation of ASP as cargo did not produce detectable amounts of AT-LXA4 in HEI-OC1 EVs, in contrast to the significant amounts found in EVs loaded with AA, EPA and DHA (Table 3: compare groups S-EVs #2 and L-EVs #2 with S-EVs #4 and #5, and L- EVs #4 and #5). These results present a puzzle: how is an ASP-triggered mediator produced in the absence of ASP, and why the generation of an ASP-triggered mediator is not affected by the presence of ASP? Since ATLs are generated from PUFA by ASP-acetylated COX-2, the inventors can speculate that minimum amounts of acetylated COX-2 could be naturally present in EVs and the process triggered by an excess of substrate even in absence of ASP. Conversely, if no PUFAs are present, even incorporation of abundant ASP will not trigger the production of ATLs because of the absence of substrate. However, without specific data, this is only guesswork and, clearly, finding answers to these questions requires further investigation.

[0165] Whereas the incorporation of lipid precursors and pro-resolving mediators by HEI- OC1 EVs was not unexpected given their hydrophobic nature, the amounts were surprisingly high. All these agents were found in concentrations roughly six to seven orders of magnitude higher than the normal values present in human serum (micrograms/mL in HEI-OC1 EVs vs. picograms/mL in human serum (Colas et al.)). Therefore, delivery of very small volumes of EVs suspension would be enough to reach clinically significant amounts of these pro-resolving mediators in specific organs and tissues. This is particularly important for their intracochlear delivery, given the small size of the auditory organ. Other very important result is the demonstration that EVs can be loaded simultaneously with different precursors and drugs, and that this can be accomplished by simple co-incubation of the EVs with the different agents. As abundantly described in the literature, EVs have been loaded with different molecules either endogenously or exogenously. For instance, mesenchymal and tumor cells incubated with chemotherapeutic drugs, subsequently produced EVs loaded with these drugs (Tang et al. 2012, Pascucci et al. 2014). The main disadvantage of this endogenous approach, however, is that drug incorporation into the cells and into the EVs depends on the particular cells and the mechanisms involved in molecular sorting into EVs, which largely remain to be elucidated. The exogenous approach, which involves loading of EVs after their isolation as described here, is simpler, but not exempt of potential complications. For example, incorporation of hydrophilic agents may be problematic, but even in these situations high loading efficiencies can be obtained with sonication, extrusion or following saponin treatments (Vader et al. 2016).

5. S-EVs or L-EVs?

[0166] As known from the literature (Mathivanan et al. 2010, Bobrie et al. 2012, Romancino et al. 2013, Zaborowski et al. 2015, Kowal et al. 2016, Xu et al. 2016, Vagner et al. 2019) and confirmed by the results, S-EVs and L-EVs have different composition and, probably, different functions. Since the inventors are interested in using them as nanocarriers of pre-defined molecular cargoes to play specific functions, the inventors wonder whether one of these fractions could be more qualified than the other for this purpose or, alternatively, if not only small EVs but also larger EVs could be exploited for the therapeutic goal.

[0167] A first important parameter, the potential amount of cargo to be incorporated by one fraction or the other, does not provide any clue. As reported in Results, the total volume and surface (membrane) area of the vesicles present in each fraction are of the same order of magnitude, suggesting that they should be able to incorporate similar amounts of exogenous cargo. However, the already discussed issue of ASP incorporation indicates that this is not necessarily true.

[0168] Proteomic results suggest that HEI-OC1 S-EVs, but not L-EVs, contain ANXA1, Gal-1 and Gal-3, known regulators and mediators of inflammation resolution (Kalinec et al. 2017). In addition, they contain many unique proteins that are probably part of the proteome of the parent cells and could perhaps be associated with the function and/or protection of the hearing organ (Kowal et al. 2016, Kalinec et al. 2019). However, dexamethasone can be incorporated as cargo in any EV, and it is already known that this glucocorticoid induces the release of ANXA1 (Kalinec et al. 2009), galectins and other proteins (Kalinec et al, unpublished) from Hensen cells of the organ of Corti. Thus, the differences in the proteome of S-EVs and L-EVs could be compensated, at least partially, by the judicious choice of their cargo to include the right molecules and/or pharmacological agents.

[0169] These results showed only small differences in the targeted lipidome between S- Control and L-Control. Most importantly, they showed a huge increase in the variety and amount of lipid metabolites present in EVs of both fractions loaded with AA, EPA, and DHA, erasing any difference between S-Control and L-Control (Supplemental Table 3). Therefore, since these precursors of the pro-resolving lipid mediators lipoxins, resolvins, protectins and maresins should definitely be part of the cargo to be delivered into the cochlea, the lipidome of S-EVs and L-EVs does not provide any definitive hint to select one fraction over the other to be used as nanocarriers for the intracochlear delivery of anti-inflammatory and pro-resolving agents.

D. Conclusions

[0170] In the present study the inventors provide evidence that auditory HEI-OC1 cells generate abundant extracellular vesicles, and that they can be loaded simultaneously with several anti-inflammatory drugs and pro-resolving agents in amounts significantly higher levels than those normally required for clinical significance. Proteomic and lipidomic studies detected a differential distribution of selected proteins and lipids between small (S-EVs) and large (L-EVS) vesicles. For instance, the S-EVs fraction contains a larger number and diversity of proteins than the L-EVs that could be associated with the involvement of a more efficient cellular mechanism of protein sorting and/or loading. In particular, S-EVs contain the pro resolving protein mediators ANXA1, Gal-1 and Gal-3 as well as a variety of other molecules that were not found in the larger vesicles. Most importantly, the inventors confirmed that extracellular vesicles from both fractions can be loaded with anti-inflammatory drugs and pro resolving mediators, either alone or mixed, making possible the generation of particles with cargoes containing a cocktail of molecules aimed at accelerating inflammation resolution and improving the organ response to inflammation damage. In contrast, the inventors found that incorporation of PUFAs as cargo induces the generation of pro-inflammatory agents, which could interfere with the resolution of inflammatory processes. Altogether, these results provide support to the idea that EVs from auditory HEI-OC1 cells could be useful nanocarriers for the delivery of anti-inflammatory drugs and pro-resolving mediators, but their cargo should be carefully tailored to ensure that it will indeed promote the successful resolution of inflammatory processes.

E. Supplemental Tables

Supplemental Table 1

Supplemental Table 2

Supplemental Table 3

Method

Extraction

Amount of sample Used = 450 uL

lOOuL internal standard mix added to each sample

EIC were purified by SPE:

Strata-x polymeric reverse phase columns (8B-S100-UBJ Phenomenex)

Dissolved in 50uL Buffer A (63%H20, 37%ACN, 0.02%Acedic Acid)

Analysis

Analysis by RP-UPLC/MS

Chromatography: ACQUITY UPLC System, Waters

Column: Waters BEH-Shield 2.1x100mm 1.7uM

Injection volume: 10 uL

Analysis: Mass spectrometer, Sciex 6500 Qtrap

Software Packages: Analyst; Multiquant;

Quantification: Full Standard Mix

Metabolites included in the Comprehensive Eicosanoid Panel

6k PGFla, TxB2, PGF2a, PGE2, PGD2, l ib PGF2a, TXB1 PGFla, PGE1, PGD1, dl7 6k PGFla, TXB3, PGF3a, PGE3, PGD3, dihomo PGF2a, dihomo PGE2, dihomo PGD2, dihomo PGJ2, dihomo 15d PGD2, 6k PGE1, 6,15 dk-,dh- PGFla, 15k PGFla, 15k PGF2a, 15k PGE2, 15k PGD2, dh PGF2a, dhk PGF2a, dhk PGE2, dhk PGD2, bicyclo PGE2, l ib dhk PGF2a,

19oh PGF2a, 20oh PGF2a, 19oh PGE2, 20oh PGE2, 2,3 dinor l ib PGF2a, tetranor-PGFM, tetranor-PGEM, tetranor 12-HETE, l ib PGE2, PGK2, 12-HHTrE, 11-HETE, 11-HEPE, 13 HDoHE, PGA2, PGB2, 15d PGA2, PGJ2, 15d PGD2, 15d PGJ2, 5-iso PGF2a VI, 8-iso PGF2a III, 9-HETE, 9-HEPE, 8 HDoHE, 16 HDoHE, 20 HDoHE, LTB4, 20oh LTB4, 20cooh LTB4, 5,6-diHETE, 6t LTB4, 12epi LTB4, 6t,12epi LTB4, 12oxo LTB4, LTC4, LTD4, LTE4, l it LTC4, l it LTD4, l it LTE4, 5-HETE, 5-HEPE, 7 HDoHE, 4 HDoHE, 9-HOTrE, 5-HETrE,

5.15-diHETE, 6R-LXA4, 6S-LXA4, 15R-LXA4, LXA5, LXB4, Resolvin El, Resolvin D1 7,17 dHDPA, 15t-Protectin Dl, PDX, 8,15-diHETE, 15-HETE, 15-HEPE, 17 HDoHE, 13- HODE, 13-HOTrE, 13-HOTrE(y), 15-HETrE, 8-HETE, 8-HEPE, 10 HDoHE, 8-HETrE, 14,15 LTC4, 14,15 LTD4, 14,15 LTE4, 12-HETE, 12-HEPE, 14 HDoHE, 11 HDoHE, 9-HODE, HXA3, HXB3, 5-oxoETE, 12-oxoETE, 15-oxoETE, 9-oxoODE, 13-oxoODE, 15 oxoEDE, 20- HETE, 19-HETE, 18-HETE, 17-HETE, 16-HETE, 18-HEPE, 5,6-EET, 8,9-EET, 11,12-EET,

14.15-EET, 14(15) EpETE, 17(18) EpETE, 16(17) EpDPE, 19(20) EpDPE, 19,20 DiHDPA, 9,10 EpOME, 12,13 EpOME, 5,6-diHETrE, 8,9-diHETrE, 11,12-diHETrE, 14,15-diHETrE, 9,10 diHOME, 12,13 diHOME, 20cooh AA, 17k DPA, 2,3 dinor TXB2, 1 ld-TXB2, 2,3 dinor

8-iso PGF2a, 2,3 dinor-6k PGFla, PGK1, 8-iso PGF3a, 8-iso-15k PGF2b, 9-Nitrooleate, 10- Nitrooleate, tetranor-PGDM, 7(R) Maresin-1, Resolvin D2, Resolvin D3, and Resolvin D5.

1 1 1

[0171] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

1. A method for treating or preventing hearing loss in a subject, the method comprising administering a composition comprising extracellular vesicles to the ear of the subject.

2. The method of claim 1, wherein the hearing loss comprises drug-related, noise-related, and/or age-related hearing loss.

3. The method of claim 1 or 2, wherein the extracellular vesicles are isolated from cells.

4. The method of claim 3, wherein the extracellular vesicles are isolated from mesenchymal stem cells (MSCs).

5. The method of claim 4, wherein the MSCs are adipose-derived MSCs.

6. The method of any one of claims 3-5, wherein the cells are cultured in exosome- depleted medium.

7. The method of any one of claims 3-6, wherein the cells were contacted with one or more of inducers, precursors of, or pro-resolving agents prior to isolation of the exosomes, wherein the one or more inducers, precursors of, or pro-resolving agents are selected from one or more of dexamethasone, prednisolone, curcumin, docosahexaenoic acid, arachidonic acid, eicosapentaenoic acid, aspirin, a lipoxin, a resolvin, a protectin, a maresin, ANXA1, and a galectin.

8. The method of any one of claims 3-7, wherein the cells comprise one or more of the following biomarkers: CD90+, CD105+, and CD73+.

9. The method of any one of claims 1-8, wherein the subject has been administered or has been prescribed a chemotherapeutic agent.

10. The method of claim 9, wherein the chemotherapeutic agent is ototoxic.

11. The method of any one of claims 1-10, wherein the subject has been or will be exposed to significant noise exposure.

12. The method of any one of claims 1-11, wherein the subject is one that suffers from early partial hearing loss.

13. The method of any one of claims 1-12, wherein the subject has been administered or is prescribed an ototoxic compound.

14. The method of claim 13, wherein the ototoxic drug comprises cisplatin or aminoglycoside.

15. The method of any one of claims 1-14, wherein the subject is 40 years old or older.

16. The method of any one of claims 1-15, wherein the subject has previously been treated for hearing loss.

17. The method of any one of claims 1-16, wherein the composition is administered by injection into the inner ear.

18. The method of any one of claims 1-17, wherein the composition is administered by intracochlear delivery.

19. The method of claim 18, wherein the composition is injected through the round window membrane.

20. The method of any one of claims 1-19, wherein the composition comprises 0.5-3 ng extracellular vesicles per 0.1 pi.

21. The method of any one of claims 1-20, wherein the extracellular vesicles are administered to the subject in multiple doses that are at least one day apart.

22. The method of any one of claims 1-21, wherein the composition is administered by injection through the round window membrane and inside the cochlear scala tympani.

23. The method of any one of claims 1-22, wherein the composition is cell-free.

24. The method of any one of claims 1-23, wherein the extracellular vesicles comprise pro resolving mediators.

25. The method of any one of claims 1-24, wherein the extracellular vesicles comprise one or more of ANXA1, Galectins 1, Galectins 3, Lipoxin A4, Lipoxin B4, Resolvin Dl, and Resolvin E7.

26. The method of claim 25, wherein one or more of ANXA1, Galectins 1, Galectins 3, Lipoxin A4, Lipoxin B4, Resolvin Dl, and Resolvin E7 is exogenously added or is endogenous to the isolated exosome.

27. The method of any one of claims 1-26, wherein the extracellular vesicles comprise one or more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, neuromodulators, NO gas, PUS gas, polyunsaturated fatty acids (PUFA), and CO gas.

28. The method of claim 27, wherein the one ore more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, neuromodulators, NO gas, PUS gas, polyunsaturated fatty acids (PUFA), and CO gas is exogenously added or is endogenous to the isolated exosome.

29. The method of claim 27 or 28, wherein the PUFA comprises one or more of arachidonic, eicosapentaenoic, docosahexanoic, and linoleic acid.

30. The method of any one of claims 1-29, wherein the composition is further defined as a pro-resolution composition.

31. The method of any one of claims 1-30, wherein the exosome comprise one or more exogenously added molecules.

32. The method of claim 1, wherein the exogenously added molecule comprises a drug.

33. The method of claim 32, wherein the drug comprises an anti-inflammatory drug.

34. The method of claim 33, wherien the drug comprises aspirin.

35. The method of any one of claims 1-30, wherein the extracellular vesicles are freshly isolated.

36. The method of any one of claims 1-30, wherein the extracellular vesicles have been fractioned by size.

37. The method of claim 36, wherein at least 80% of the extracellular vesicles are 25-150 nm.

38. The method of claim 36, wherein at least 80% of the extracellular vesicles are 150 nm- 10 pm.

39. The method of any one of claims 1-34 or 36-38, wherein the extracellular vesicles have previously been frozen.

40. A composition comprising exosomes, wherein the extracellular vesicles comprise one or more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, neuromodulators, NO gas, FbS gas, polyunsaturated fatty acids (PUFA), and CO gas.

41. The composition of claim 1 wherein the one or more lipoxins, resolvins, protectins, maresins, ANXA1, galectins, adenosine, neuromodulators, NO gas, FbS gas, polyunsaturated fatty acids, and CO gas is exogenously added or is endogenous to the isolated exosome.

42. The composition of claim 40 or 41, wherein the PUFA comprises one or more of arachidonic, eicosapentaenoic, docosahexanoic, and linoleic acid.

43. The composition of any one of claims 40-42, wherein the composition comprises 0.5-3 ng extracellular vesicles per 0.1 pi.

44. The composition of claim 40 or 43, wherein the composition is formulated for administration by injection through the round window membrane and inside the cochlear scala tympani.

45. The composition of any one of claims 40-44, wherein the composition is cell-free.

46. The composition of any one of claims 40-45, wherein the extracellular vesicles comprise pro-resolving mediators.

47. The composition of any one of claims 40-46, wherein the extracellular vesicles comprise one or more of ANXA1, Galectins 1, Galectins 3, Lipoxin A4, Lipoxin B4, Resolvin

Dl, and Resolvin E7.

48. The composition of claim 47, wherein the extracellular vesicles comprise exogenously added lipoxin A4 and/or resolvin Dl.

49. The composition of any one of claims 40-48, wherein the composition further comprises an additional therapeutic agent.

50. The composition of claim 49, wherein the additional therapeutic agent comprises aspirin, dexamethasone, or a combination thereof.

51. The composition of any one of claims 40-50, wherein the composition further comprises a pharmaceutical carrier.

52. A method for making extracellular vesicles comprising isolating extracellular vesicles from auditory cells.

53. The method of claim 52, wherein the auditory cells comprise human cells.

54. The method of claim 52 or 53, wherein the auditory cells comprise HEI-OC1 cells.

55. The method of any one of claims 52-54, wherein the method comprises filtration of the exosomes, wherein the filtration comprises a 0.45 pm filter.

56. The method of claim 55, wherein the filtration is performed more than once.

57. The method of any one of claims 52-56, wherien the extracellular vesicles are fractionated by size.

58. The method of any one of claims 52-57, wherein the extracellular vesicles are 100- 800nm.

59. The method of any one of claims 52-56, wherein at least 80% of the extracellular vesicles are 25-150 nm.

60. The method of any one of claims 52-56, wherein at least 80% of the extracellular vesicles are 150 nm-10 pm.

61. The method of any one of claims 52-60, wherein the concentration of the extracellular vesicles is Ixl0⁸-lxl0¹⁰ exosomes/mL.

62. The method of any one of claims 52-61, wherein the method further comprises incubating a composition comprising the extracellular vesicles and a therapeutic agent.

63. The method of claim 62, wherein the method further comprises sonication of the composition comprising the extracellular vesicles and the therapeutic agent.

64. The method of claim 62 or 63, wherein the method further comprises washing the extracellular vesicles to remove excess therapeutic agent from the composition.

65. The method of any one of claims 62-64, wherein the therapeutic agent comprises aspirin or dexamethasone.

66. The method of any one of claims 52-65, wherein the method further comprises freezing the exosomes.

67. The method of claim 66, wherein the extracellular vesicles are frozen and stored at a temperature of -30°C or lower.

68. The method of claim 66, wherein the extracellular vesicles are stored for at least 2 months.