Methods for producing extracellular vesicles, compositions and methods of use thereof

Abstract

Methods for producing extracellular vesicles (EVs) are provided by culturing keratinocytes in a culture medium containing a ROCK inhibitor and harvesting EVs secreted by the keratinocytes. In some embodiments, the EVs comprise or consist of exosomes. Typically, keratinocyte proliferation and / or secretion of EVs is increased in the presence of a ROCK inhibitor compared to its absence. EVs produced according to the methods of the present disclosure and pharmaceutical compositions formed therefrom are also provided. The pharmaceutical compositions may contain an effective amount of EVs to be useful, for example, for nutritional and therapeutic applications (e.g., for improving skin, treating skin-related diseases or disorders, or enhancing recovery from injury).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 63 / 270,875, filed October 22, 2021, and U.S. Patent Application No. 63 / 333,854, filed April 22, 2022, which are specifically incorporated by reference herein in their entireties.

[0002] FIELD OF THEINVENTION The field of the invention relates generally to compositions and methods for culturing cells, harvesting extracellular vesicles, and compositions and methods of use thereof. [Background technology]

[0003] 2. Background of the Invention Extracellular vesicles (EVs) are secreted lipid membranes that have the ability to regulate cellular functions by exchanging biological components between different cells (Nasiri et al., Stem Cell Research & Therapy volume 11, Article number: 421 (2020)). Skin cells (e.g., keratinocytes, fibroblasts, melanocytes, and inflammatory cells) can secrete different types of EVs depending on their biological status. These vesicles can affect physiological properties and pathological processes of the skin (e.g., pigmentation, cutaneous immunity, and wound healing). As keratinocytes constitute the majority of cells in the skin, secreted EVs from these cells can alter the pathophysiological behavior of other skin cells.

[0004] The properties of EVs as biological carriers have potential for various skin therapeutic purposes including repair, regeneration, and rejuvenation (Basu et al., Expert Opin Biol Ther. 16(4):489-506 (2016)). The rapid degradation of primary human keratinocytes in culture limits their utility in drug discovery research, as well as a source of nutritional supplements and therapeutic use in regenerative medicine. To make these therapeutic approaches accessible to patients, good manufacturing practice (GMP) standard protocols are required to ensure the quality of the EVs used (Chen et al., Tzu-Chi Med J., 32(2):113 (2019), Lener et al., J Extracellular Vesicles, 4(1):30087 (2015)). It is therefore an object of the present invention to provide improved methods for culturing keratinocytes and producing and harvesting EVs therefrom, compositions comprising EVs, and methods of use thereof. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nasiri et al., Stem Cell Research & Therapy volume 11, Article number: 421 (2020) [Non-Patent Document 2] Basu et al., Expert Opin Biol Ther. 16(4):489-506 (2016) [Non-Patent Document 3] Chen et al., Tzu-Chi Med J., 32(2):113 (2019) [Non-Patent Document 4] Lener et al., J Extracellular Vesicles, 4(1):30087 (2015) Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention Methods for producing extracellular vesicles (EVs) are provided by culturing keratinocytes in a culture medium containing a ROCK inhibitor and harvesting EVs secreted by the keratinocytes. In some embodiments, the EVs comprise or consist of exosomes.

[0007] Typically, the proliferation of the keratinocytes and / or secretion of EVs is increased in the presence of the ROCK inhibitor compared to its absence. An exemplary ROCK inhibitor is Y-27632. In some embodiments, the cells are also cultured with an inhibitor of TGFβ signaling. An exemplary inhibitor of TGFβ signaling is A83-01.

[0008] In a preferred embodiment, the keratinocytes are primary keratinocytes.

[0009] Also provided are EVs produced according to the methods of the present disclosure, and pharmaceutical compositions formed therefrom. The pharmaceutical compositions may contain an effective amount of EVs, for example, to serve nutritional and therapeutic applications (e.g., to improve skin, treat skin-related diseases or disorders, or enhance recovery from injury). And such therapeutic and non-therapeutic methods and uses are also provided. In some embodiments, the compositions are used to treat or prevent dry skin, irritation, stress, allergy, infection, and / or fever / sweating. For example, in some embodiments, the methods include administering EVs or compositions thereof to a subject in need thereof to treat or prevent atopic dermatitis. In some embodiments, the compositions are administered in an effective amount to reduce or prevent one or more symptoms and / or biological or physiological indicators of atopic dermatitis.

[0010] In some embodiments, the exosomes may increase expression of type I collagen (COL1A1) and / or elastin; may reduce expression of thymic stromal lymphopoietin (TSLP), Th2, eosinophil-recruiting chemokines, inflammatory cytokines (e.g., IL-33 and IL-25), or any combination thereof, in cells with which the exosomes are contacted. [Brief description of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figures 1A-1H are 4x (Figures 1A, 1C, 1E, 1G) and 10x (Figures 1B, 1D, 1F, 1H) photomicrographs showing keratinocytes cultured in keratinocyte medium alone on days 2 (Figures 1A, 1B), 3 (Figures 1C, 1D), and 7 (Figures 1E and 1F), and after addition of Y-27632 on day 9 (Figures 1G and 1H).

[0012] [Diagram 2] Figures 2A-2F are 4x (Figures 2A, 2C, 2E) and 10x (Figures 2B, 2D, 2F) photomicrographs showing keratinocytes cultured alone (Figures 2A, 2B), with Y-27632 (Figures 2C, 2D), and with Y-27632 + A83-01 (Figures 2E, 2F) on day 17.

[0013] [Diagram 3] Figures 3A-3F are 4x (Figures 3A, 3C, 3E) and 10x (Figures 3B, 3D, 3F) photomicrographs of keratinocytes cultured alone (Figures 3A, 3B), with Y (Figures 3C, 3D), and with Y-27632 + A83-01 (Figures 3E, 3F) on day 20.

[0014] [Figure 4] FIG. 4 is a bar graph showing the concentration of extracellular vesicle (EV) particles (×10 particles / ml) in medium alone, control keratinocytes, keratinocytes cultured with Y-27632 (Y), and keratinocytes cultured with Y-27632 + A83-01 (A).

[0015] [Diagram 5] FIG. 5 is a bar graph showing particle weight (ratio) of control keratinocytes, keratinocytes cultured with Y-27632 (Y), and keratinocytes cultured with Y-27632 + A83-01 (A).

[0016] [Figure 6] Figure 6 is a bar graph showing gene expression levels of type I collagen (COL1A1) and elastin in human fibroblasts 72 hours after addition of 1,000 exosomes / cell. Relative expression levels (genes / actin): The expression level of each gene was calculated using the amount of β-actin as a control.

[0017] [Figure 7-1] Figure 7A is a flow chart of the experimental protocol for the analysis of the effect of extracellular vesicles (EVs) secreted from cultured keratinocytes on gene expression in an in vitro model of atopic dermatitis. Figure 7B is a bar graph showing gene expression of TSLP, IL-25, and IL-33 in untreated control epidermal keratinocytes compared to epidermal keratinocytes treated with EVs prepared by regular keratinocyte culture or long-term keratinocyte culture. [Figure 7-2] Figure 7A is a flow chart of the experimental protocol for the analysis of the effect of extracellular vesicles (EVs) secreted from cultured keratinocytes on gene expression in an in vitro model of atopic dermatitis. Figure 7B is a bar graph showing gene expression of TSLP, IL-25, and IL-33 in untreated control epidermal keratinocytes compared to epidermal keratinocytes treated with EVs prepared by regular keratinocyte culture or long-term keratinocyte culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention I. Definition As used herein, the term "carrier" or "excipient" refers to an inactive ingredient, organic or inorganic, natural or synthetic, in a formulation with which one or more active ingredients are combined.

[0019] As used herein, the term "pharmacologically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0020] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water and emulsions (e.g., oil / water or water / oil emulsions), as well as various types of wetting agents.

[0021] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to alleviate one or more symptoms of the disorder, disease, or condition being treated, or to otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, and the route of administration and pharmacokinetics of the agent being administered.

[0022] As used herein, the term "prevention" or "preventing" means administering a composition to a subject or system at risk for or predisposed to one or more symptoms caused by a disease or disorder, to cause the arrest of a particular symptom of said disease or disorder, the reduction or prevention of one or more symptoms of said disease or disorder, the reduction of the severity of said disease or disorder, the complete elimination of said disease or disorder, or the stabilization or delay of the onset or progression of said disease or disorder.

[0023] As used herein, the terms "subject", "individual" and "patient" refer to any individual who is a target of treatment using the compositions of the present disclosure. The subject may be a vertebrate, e.g., a mammal. Thus, the subject may be a human. The subject may be symptomatic or asymptomatic. The terms do not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. The subject may include a control subject or a test subject.

[0024] As used herein, "substantially altered" means at least, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100% or more altered compared to a control.

[0025] As used herein, the terms "purified," "isolated," and the like refer to the isolation of a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) of other components that are normally associated with the molecule or compound in its natural environment.

[0026] As used herein, the term "antibody" refers to a natural or synthetic antibody that binds a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, fragments or polymers of those immunoglobulin molecules, and humanized or humanized versions of immunoglobulin molecules that bind a target antigen are also encompassed within the term "antibody."

[0027] As used herein, "treatment" refers to the medical management of a patient with the intent of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at removing the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the occurrence of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific treatment aimed at improving the associated disease, pathological condition, or disorder.

[0028] The term "inhibit" or "reduce" means to decrease, prevent or suppress a particular characteristic (e.g., an activity, a response, a condition, a disease, or other biological parameter). It is understood that this is typically relative to some standard or expected value, i.e., it is relative, but it is not always necessary for the standard or relative value to be referred to. "Inhibit" or "reduce" can also mean to prevent or suppress the synthesis, expression or function of a protein compared to a standard or control. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. Inhibition can also include, for example, a 10% reduction in the activity, response, condition, disease, or other biological parameter compared to a natural or control level. Thus, the reduction may be about 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%, The reduction may be 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% or any amount of reduction therebetween.

[0029] As used herein, "primary cells" refer to non-immortalized cells that are obtained from a living organism or tissue source.

[0030] As used herein, "prolonging the viability" of a cell (eg, a primary cell) refers to extending the period during which said cell is capable of normal growth and / or survival.

[0031] As used herein, "senescence" refers to the point at which mitosis (cell division) can no longer occur.

[0032] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually set forth herein.

[0033] Use of the term "about" is intended to describe values ​​that are either above or below the stated value in a range of approximately ±10%; in other forms, values ​​can range between values ​​that are either above or below the stated value in a range of approximately ±5%; in other forms, values ​​can range between values ​​that are either above or below the stated value in a range of approximately ±2%; in other forms, values ​​can range between values ​​that are either above or below the stated value in a range of approximately ±1%. The above ranges are intended to be clear by context, and no further limitation is implied.

[0034] Ranges may be expressed herein as "about" one particular value and / or to "about" another particular value. When such a range is expressed, the range from the one particular value and / or to the other particular value is also considered to be specifically contemplated and disclosed, unless the context specifically dictates otherwise. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another specifically contemplated embodiment that should be considered disclosed, unless the context specifically dictates otherwise. It is further understood that the endpoints of each of the above ranges are significant both relative to the other endpoint and independently of the other endpoint, unless the context specifically dictates otherwise. It should be understood that all individual values ​​and subranges of values ​​included within the explicitly disclosed ranges are also to be considered to be specifically contemplated and disclosed, unless the context specifically dictates otherwise. Finally, it should be understood that all ranges refer to both the ranges described and to a collection of individual numbers from a first endpoint (inclusive) to a second endpoint (inclusive). In the latter case, it should be understood that any of the individual numbers can be selected as one form of the amount, value, or characteristic to which the range refers. In this way, a range describes a set of numbers or values ​​from a first endpoint (inclusive) to a second endpoint (inclusive), from which a single member of the set (i.e., a single numerical value) can be selected as the amount, value, or characteristic to which the range refers. The above applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[0035] Any compound disclosed herein is intended to be specifically disclosed herein and should be considered to be specifically disclosed herein.Furthermore, any subrange that may be specified within this disclosure is intended to be specifically disclosed herein and should be considered to be specifically disclosed herein.As a result, it is specifically contemplated that any compound, or subgroup of compounds, may be either specifically included or excluded for use, or included or excluded in a list of compounds.

[0036] Disclosed are the components to be used to prepare the compositions of the present disclosure, and the compositions themselves to be used within the methods disclosed herein. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, when a particular polypeptide is disclosed and discussed, and many modifications that can be made to many polypeptides are discussed, each and every combination and permutation of the polypeptides, and possible modifications, are specifically contemplated, unless specifically indicated to the contrary. Thus, when a class of molecules A, B, and C is disclosed, as is a class of molecules D, E, and F, and an example of a combination, A-D, is disclosed, each is individually and specifically contemplated, even if each is not individually described. That is, the combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, subgroups AE, BF, and CE are considered to be disclosed.This concept applies to all aspects of this application, including but not limited to the steps in the method of making and using the disclosed compositions.Thus, when there are various additional steps to be performed, it is understood that each of these additional steps can be performed in any specific embodiment or combination of embodiments of the disclosed method.

[0037] II. Methods for culturing keratinocytes Keratinocytes are cells found in the epidermis that produce keratin. Keratinocytes constitute about 90% of epidermal cells. Keratinocytes are produced by keratinocyte stem cells in the basal layer of the epidermis. It has been discovered that small molecule signaling inhibitors are useful for maintaining various regenerative functions of primary human keratinocytes, including regeneration of the epithelium of the skin, reconstitution of a functional epidermal barrier, and growth factor productivity that induces the production of extracellular vesicles for skin regeneration. Importantly, these culture conditions allow primary human keratinocytes to maintain the production of extracellular vesicles. Thus, methods are disclosed for producing, harvesting, and using EVs from long-term cultured keratinocytes. Such EVs can be used in a variety of applications, including, but not limited to, nutritional supplementation and therapeutic intervention (e.g., acellular skin regeneration and / or disease treatment, as well as a research-based platform to promote keratinocyte-based drug discovery for the above treatments). Specifically provided are cells obtained according to the culture methods of the present disclosure, as well as pharmaceutical compositions thereof and methods of their therapeutic and non-therapeutic use for the treatment of skin diseases and conditions, e.g., as described in further and greater detail elsewhere herein with respect to EVs.

[0038] The results of the following experiments show that Y-27632 (a Rock inhibitor) alone, and the combination of Y-27632 and A83-01 (a TGFβ signaling inhibitor) can enhance long-term culture of keratinocytes and increase the accumulation of EVs. Thus, the culture methods of the present disclosure typically include a Rock inhibitor and optionally one or more additional small molecules, including but not limited to a TGFβ inhibitor. In some embodiments, a TGFβ signaling inhibitor is not included.

[0039] The keratinocytes are typically cultured in tissue culture medium containing a ROCK inhibitor and, optionally, one or more additional small molecules. The tissue culture medium may be a sterile liquid medium for long-term serum-free culture of human epidermal keratinocytes (e.g., EpiLife TMThe medium may be Keratinocyte Growth Medium (Ready-to-Use) (Promocell; C20011), HuMedia-KG2 (KK-2150S) (Kurabou), and KGM-Gold TM Keratinocyte Growth Medium BulletKit TM (LONZA)).

[0040] The keratinocytes are cultured in the presence of the inhibitor and / or other inhibitor(s) for a period of time sufficient to increase the proliferation of the cells and / or increase the number of extracellular vesicles that can be collected, compared to untreated cells. In some embodiments, the keratinocytes are cultured in the presence of the inhibitor(s) for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, at least 20 days, at least 40 days, at least 60 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, at least 350 days, at least 400 days, at least 450 days, or at least 500 days. Typically, the cells are cultured with the inhibitor(s) for 14 days or longer.

[0041] In some embodiments, the cells are cultured without the inhibitor(s) for a period of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, at least 20 days) before the inhibitor(s) are added.

[0042] In the following experiments, the cells were cultured for 4-8 days until the cells were subconfluent (i.e., day 9), then inhibitor(s) were added, the cells were subsequently cultured for 7-14 days (i.e., day 10), and then EV collection medium (serum-free) was added for 2 days for collection of EVs. Typically, the EV collection medium does not contain inhibitors.

[0043] The cells most typically begin as primary keratinocytes. As used herein, a "primary keratinocyte" is a keratinocyte that has been isolated from a tissue and grown in culture, but has not been immortalized. In some embodiments, the primary keratinocyte is obtained by tissue biopsy. In some instances, the tissue biopsy is taken from the skin (e.g., skin and / or mucosal squamous epithelium). In some embodiments, the primary keratinocyte is a foreskin keratinocyte, a vaginal keratinocyte, a cervical keratinocyte, an oral keratinocyte, or a skin keratinocyte.

[0044] Typically, primary keratinocytes cultured according to the method of the present disclosure can be considered and referred to as long-term cultured or reprogrammed keratinocytes.Cells treated according to the method of the present disclosure can show the typical characteristics of normal primary keratinocytes, including having normal karyotype and intact DNA damage response.Furthermore, primary keratinocytes cultured long-term or reprogrammed by exposure to ROCK inhibitors can retain the ability to differentiate into stratified epithelium upon removal of the ROCK inhibitor.

[0045] Thus, in some embodiments, keratinocytes cultured long-term or reprogrammed with ROCK inhibitors are functionally equivalent or improved compared to normal cells. In some embodiments, they have normal karyotypes, intact DNA damage responses, and / or can form stratified epithelia in organotypic culture. In some embodiments, the long-term cultured or reprogrammed keratinocytes show upregulated telomerase mRNA levels and have shortened but stable telomeres. Myc mRNA levels can also be increased in keratinocytes cultured long-term or reprogrammed with ROCK inhibitors.

[0046] In some embodiments, the primary keratinocytes are cultured in the presence of a ROCK inhibitor and / or other inhibitor(s) for a sufficient time to allow long-term culture or reprogramming of the primary keratinocytes, if desired, and further cultured in the absence of the ROCK inhibitor and / or other inhibitor(s).

[0047] In some embodiments, the cultured keratinocytes may differentiate to form an organotypic tissue equivalent. In some embodiments, the organotypic tissue equivalent comprises primary keratinocytes that have been cultured in the presence of a ROCK inhibitor to increase proliferation of these cells, but the cells have not yet been immortalized. Thus, organotypic tissue equivalents having primary keratinocytes that have been cultured in the presence of a ROCK inhibitor for a period of time sufficient to increase proliferation and / or reprogramming of the primary keratinocytes are also provided.

[0048] Typically, the keratinocytes are cultured in the presence of at least a ROCK inhibitor. Rho-associated kinases (also known and / or referred to herein as ROCK, Rock, Rho-associated coiled-coil kinase, and Rho kinase) include ROCK1 (also referred to as ROKβ or p160ROCK) and ROCK2 (also referred to as ROKα). ROCK proteins are serine-threonine kinases that interact with Rho GTPases.

[0049] Treatment of primary keratinocytes with a ROCK inhibitor can result in the immortalization of these cells. See, for example, U.S. Patent Application Publication No. 2011 / 0243903, which is specifically incorporated herein by reference in its entirety. In some embodiments, the method of the present disclosure does not immortalize the primary keratinocytes as described in U.S. Patent Application Publication No. 2011 / 0243903.

[0050] A. ROCK Inhibitors ROCK inhibitors are proteins, nucleic acids, small molecules, antibodies or other agents that reduce or prevent the expression of ROCK or downregulate ROCK activity (e.g., its kinase activity).Thus, ROCK inhibitors include, but are not limited to, small molecules, antibodies, antisense compounds and negative regulators of ROCK.ROCK inhibitors include inhibitors of ROCK-1, ROCK-2 or both.

[0051] The ROCK inhibitor may also be a negative regulator of ROCK, such as, but not limited to, small GTP-binding proteins, such as Gem, RhoE and Rad. In another example, the ROCK inhibitor is an antibody that specifically binds ROCK1 or ROCK2 or both isoforms.

[0052] In another example, the ROCK inhibitor is an antisense compound. In general, the principle behind antisense technology is that antisense compounds hybridize to target nucleic acids, resulting in modulation of gene expression activity, or function (e.g., transcription, translation, or splicing). Modulation of gene expression can be achieved, for example, by target RNA degradation or occupancy-based inhibition. An example of modulation of target RNA function by degradation is RNase H-based degradation of target RNA upon hybridization with a DNA-like antisense compound (e.g., an antisense oligonucleotide). Antisense oligonucleotides can also be used to modulate gene expression, such as splicing, by occupancy-based inhibition (e.g., by blocking access to splice sites).

[0053] Antisense compounds include, but are not limited to, antisense oligonucleotides, siRNAs, miRNAs, shRNAs, and ribozymes. Antisense compounds can specifically target ROCK nucleic acids.

[0054] Each of the above antisense compounds provides sequence-specific target gene regulation. This sequence specificity makes antisense compounds an effective tool for selective modulation of target nucleic acid of interest. In some embodiments, the target nucleic acid is human ROCK1 (e.g., Genbank Accession No. NM_005406) ​​and / or human ROCK2 (Genbank Accession No. NM_004850). However, other known ROCK sequences can be used to design antisense compounds. Methods for designing, preparing and using antisense compounds that specifically target ROCK are within the capabilities of those skilled in the art. Examples of ROCK antisense oligonucleotides are described in U.S. Patent Application Publication No. 2004 / 0115641.

[0055] Antisense compounds that specifically target ROCK1 or ROCK2 can be prepared by designing a compound that is complementary to ROCK1 or ROCK2 nucleotide sequence. Antisense compounds that target ROCK1 or ROCK2 do not need to be 100% complementary to ROCK1 or ROCK2 to specifically hybridize to and regulate the expression of target genes. For example, the antisense compound, or the antisense strand of the compound if it is a double-stranded compound, can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% complementary to the selected ROCK1 or ROCK2 nucleic acid sequence. Methods for screening antisense compounds for specificity are well known in the art (see, for example, U.S. Patent Application Publication No. 2003 / 0228689). Antisense compounds can include one or more modifications to enhance nuclease resistance and / or increase the activity of the compound. Modified antisense compounds include those containing modified internucleoside linkages, modified sugar moieties and / or modified nucleosides.

[0056] Preferably, the ROCK inhibitor is a small molecule.Exemplary small molecule ROCK inhibitors include Y-27632 (US Pat. No. 4,997,834, which is specifically incorporated herein by reference in its entirety) and fasudil (also known as HA 1077; Asano et al., J. Pharmacol. Exp. Ther. 241:1033-1040, 1987, which is specifically incorporated herein by reference in its entirety).These inhibitors bind to kinase domain and inhibit ROCK enzyme activity. Other small molecules that have been reported to specifically inhibit ROCK include H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine, Ikenoya et al., J. Neurochem. 81:9, 2002; Sasaki et al., Pharmacol. Ther. 93:225, 2002); N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea (Takami et al., Bioorg. Med. Chem. 12:2115, 2004); and 3-(4-pyridyl)-1H-indole (Yarrow et al., Chem. Biol. 12:385, 2005), GSK269962A (Axon medchem), and fasudil hydrochloride (Tocris Bioscience).

[0057] Additional small molecule Rho kinase inhibitors include those described in PCT Publication Nos. WO 03 / 059913, WO 03 / 064397, WO 05 / 003101, WO 04 / 112719, WO 03 / 062225 and WO 03 / 062227; U.S. Patent Nos. 7,217,722 and 7,199,147; and U.S. Patent Application Publication Nos. 2003 / 0220357, 2006 / 0241127, 2005 / 0182040 and 2005 / 0197328, each of which is specifically incorporated by reference herein in its entirety.

[0058] In another embodiment, the ROCK inhibitor is a negative regulator of ROCK activity.The negative regulator of ROCK activation includes small GTP-binding proteins (such as Gem, RhoE and Rad), which can attenuate ROCK activity.The autoinhibitory activity of ROCK has also been demonstrated by reducing kinase activity with its carboxy terminus and its kinase domain interaction.

[0059] In another embodiment, the ROCK inhibitor can be an antibody that specifically binds ROCK1 or ROCK2 or both isoforms. In one example, the antibody specifically binds ROCK1 (e.g., human ROCK1) or ROCK2 (e.g., human ROCK2). By way of example and not limitation, an antibody specific for a ROCK protein can interfere with the binding of ROCK to Rho or other binding partners, or the antibody can directly disrupt the kinase activity of ROCK.

[0060] In a particularly preferred embodiment, the ROCK inhibitor is Y-27632. Y-27632, also known as (+ / -)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, is a small molecule inhibitor that specifically inhibits the activity of Rho-associated kinase. Y-27632 is disclosed in U.S. Pat. No. 4,997,834 and PCT Publication No. WO 98 / 06433. In some embodiments, when the ROCK inhibitor is Y-27632, the effective amount of the ROCK inhibitor is about 1 to about 100 μM, or about 5 to about 25 μM, or about 10 μM.

[0061] B. Other Inhibitors The culture method of the present disclosure may include one or more additional inhibitors, such as inhibitors of TGF-β / Smad signaling. For example, the following experimental results also show that combining A83-01 and Y-27632 together appears to be better than no small molecule inhibitor for culturing keratinocytes and generating extracellular vesicles, but not as good as Y-27632 alone. A83-01 is a potent selective inhibitor of TGF-βRs ALK4, 5, and 7, which are part of the TGF-β / Smad signaling pathway. Thus, in some embodiments, the cells are cultured with a protein, nucleic acid, small molecule, antibody, or other agent that reduces or prevents or otherwise downregulates the expression of molecules in the TGF-β / Smad signaling pathway. Thus, inhibitors of TGF-β / Smad signaling include, but are not limited to, small molecules, antibodies, antisense compounds, and negative regulators of TGF-β / Smad signaling molecules. Antibodies, antisense compounds and negative regulators can be designed to target TGF-β signaling molecules (eg, ALK4, 5, and / or 7) following the same strategies discussed above for ROCK inhibitors.

[0062] Exemplary small molecule inhibitors of TGF-β / Smad signaling include A83-01, SB431542, LDN-193189, galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox (E-616452), LDN-193189 2HCl, K02288, BIBF-0775, TP0427736 HCl, LDN-214117, SD-208, bactosertib (TEW-7197), ML347, LDN-212854, DMH1, dorsomorphin (compound C), 2HCl, pirfenidone (S-7701), sulfasalazine (NSC 667219), AUDA, PD 169316, TA-02, ITD-1, LY 3200882, alantolactone, halofuginone, SIS3 HCl, dorsomorphin (compound C), and hesperetin.

[0063] Other examples include, but are not limited to, 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-ylamine (SD-208), 3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all from Merck) and SB431542 (Sigma Aldrich).Preferred examples include A-83-01. Typically, for example, the inhibitor, A83-01, is used at a concentration of about 1 to about 10 μM, or about 0.1 to about 10 μM, or about 0.5 μM.

[0064] Inhibitors are also described in WO 2020 / 080550, WO 2017 / 119512, U.S. Patent No. 10,961,507, and U.S. Patent Application No. 17 / 285,038, each of which is specifically incorporated by reference in its entirety.

[0065] III. Extracellular vesicles A cell-free composition comprising extracellular vesicles (EVs) and its method of use are provided. The EVs can be a part of a heterogeneous mixture of factors (e.g., conditioned medium) or a fraction isolated therefrom. In other embodiments, the EVs, or one or more subtypes thereof, are isolated or otherwise collected from conditioned medium. The EVs, or one or more subtypes thereof, can be suspended in a pharma- ceutically acceptable composition (e.g., carrier or matrix or depot) prior to administration to the subject.

[0066] A. Extracellular vesicles The compositions of the present disclosure may typically be or include extracellular vesicles derived from primary cultured keratinocytes, or isolated or fractionated subtypes or other cell types derived therefrom. Extracellular vesicles are lipid bilayer bounded particles that are naturally released from cells and, unlike cells, cannot replicate. EVs range in diameter from sizes approaching the smallest physically possible unilamellar liposomes (approximately 20-30 nanometers) to 10 microns or larger, although the vast majority of EVs are smaller than 200 nm.

[0067] A variety of EV subtypes have been proposed, including ectosomes, microvesicles (MVs), microparticles, exosomes, oncosomes, apoptotic bodies (ABs), tunneling nanotubes (TNTs), and others (Yanez-Mo, et al., J Extracell Vesicles. 4: 27066 (2015) doi:10.3402 / jev.v4.27066. PMC 4433489). These EV subtypes are defined by various, often overlapping, definitions, mostly based on biogenesis (cellular pathway, cellular or tissue entity, state of origin) (Thery, et al., J Extracell Vesicles. 7 (1): 1535750 (2018). doi:10.1080 / 20013078.2018.1535750, which is specifically incorporated by reference herein in its entirety). However, EV subtypes can also be defined by size, constituent molecules, function, or isolation method. As discussed in Thery et al., subtypes of EVs can be defined by: a) physical characteristics of EVs (e.g., size ("small EVs" (sEVs) and "medium / large EVs" (m / lEVs), ranges are defined (e.g., <100 nm or <200 nm [small], or >200 nm [large and / or medium], respectively) or density (low, medium, high, ranges are defined for each); b) biochemical composition (e.g., CD63+ / CD81+- EVs, annexin A5 staining EVs); or c) Description of state or cell of origin (podocyte EVs, hypoxic EVs, large oncosomes, apoptotic bodies).

[0068] Thus, in some embodiments, the composition is or comprises one or more EV subtypes defined according to (a), (b), or (c) as discussed above.

[0069] In some embodiments, the vesicles are or include exosomes (which may also be referred to or include "small EVs", "sEVs", etc.). Exosomes have surface proteins that facilitate endocytosis, and they have the ability to deliver macromolecules. Also, when exosomes are obtained from the same individual to whom they are delivered, the exosomes are immune tolerant.

[0070] Exosomes are vesicles with sizes of 30-150 nm, often 40-100 nm, that are observed in most cell types. Exosomes are often similar to MVs, but with an important difference: instead of originating directly from the plasma membrane, they are generated by inward budding into multivesicular bodies (MVBs). The formation of exosomes involves three distinct steps: (1) the formation of endocytic vesicles from the plasma membrane, (2) the inward budding of the endosomal vesicle membrane resulting in MVBs composed of intraluminal vesicles (ILVs), and (3) the fusion of these MVBs with the plasma membrane, which releases the vesicle contents (known as exosomes).

[0071] Exosomes have a lipid bilayer with an average thickness of about 5 nm (see, e.g., Li, Theranostics, 7(3):789-804 (2017) doi: 10.7150 / thno.18133). The lipid components of exosomes include ceramides (sometimes used to distinguish exosomes from lysosomes), cholesterol, sphingolipids, and phosphoglycerides with long and saturated fatty-acyl chains. The outer surface of exosomes is typically rich in saccharide chains (e.g., mannose, polylactosamine, α-2,6 sialic acid, and N-linked glycans).

[0072] Many exosomes contain proteins such as platelet-derived growth factor receptor, lactadherin, transmembrane and lysosomal-associated membrane protein-2B, membrane trafficking and fusion proteins (annexins, flotillins, GTPases, heat shock proteins, tetraspanins, etc.), proteins involved in multivesicular body biogenesis, as well as lipid-associated proteins and phospholipases. Thus, these characteristic proteins serve as good biomarkers for the isolation and quantification of exosomes. Another important cargo that exosomes can carry is nucleic acid, including coding and non-coding ribonucleic acid (RNA), such as deoxynucleic acid (DNA), messenger RNA (mRNA) and microRNA (miRNA).

[0073] In some embodiments, the vesicles include or are one or more alternative extracellular vesicles (e.g., ABs, MVs, TNTs, or others discussed herein or elsewhere).

[0074] ABs are heterogeneous in size and originate from the plasma membrane. They can be released from all cell types and are approximately 1-5 μm in size.

[0075] MVs, with sizes ranging from 20 nm to 1 μm, are formed due to blebbing involving the incorporation of cytosolic proteins. In contrast to ABs, MVs are uniform in shape. They arise from the plasma membrane and are observed in most cell types.

[0076] TNTs are thin (e.g., 50-700 nm) and up to 100 μm long actin-containing tubes that form from the plasma membrane.

[0077] In some embodiments, the EVs are between about 20 nm and about 500 nm, In some embodiments, the EVs are between about 20 nm and about 250 nm or 200 nm or 150 nm or 100 nm.

[0078] B. Methods for Producing Extracellular Vesicles 1. Cell source for production Extracellular vesicles As used herein, EVs (including AB, MV, exosomes and TNT) refer to lipid vesicles typically formed by cells or tissues.Generally, EVs can be isolated directly from tissues, cells and fluids, including cultured and uncultured tissues, cells or fluids, and fluids derived from or conditioned by cultured cells (e.g., conditioned medium) from subjects.For example, exosomes are present in physiological fluids (e.g., plasma, lymph, malignant pleural effusion, amniotic fluid, milk, semen, saliva and urine) and are secreted into the medium of cultured cells.

[0079] The disclosed EVs are typically formed from cultured primary keratinocytes as disclosed herein.

[0080] Methods for isolating extracellular vesicles directly from tissues, cells, and fluids, including cultured and uncultured tissues, cells, or fluids, as well as fluids derived from or conditioned by cultured cells (e.g., conditioned medium), from a subject are known in the art. For example, Li, Thernaostics, 7(3):789-804 (2017) doi: 10.7150 / thno.18133, Ha, et al., Acta Pharmaceutica Sinica B, 6(4):287-296 (2016) doi: 10.1016 / j.apsb.2016.02.001, Skotland, et al., Progress in Lipid Research, 66:30-41 (2017) doi: 10.1016 / j.plipres.2017.03.001, Phinney and Pittenger, Stem Cells, 35:851-858 (2017) doi: 10.1002 / stem.2575, each of which is specifically incorporated by reference and describes isolating extracellular vesicles, particularly exosomes.

[0081] The disclosed EVs are typically collected from cultured primary cells or subsequent cell types derived therefrom.In some embodiments, the vesicles are isolated from primary cells isolated from the subject to be treated.The advantage of using EVs that can be isolated from natural sources includes avoiding the immunogenicity that may be associated with artificially produced lipid vesicles.

[0082] The EVs can also be collected from cell lines or tissues. The EVs of the present disclosure are most typically collected from keratinocytes cultured as described herein. In some embodiments, the medium of the cultured keratinocytes is replaced before collecting the EVs. Such medium can be described as collection medium and can be the same or different from the culture medium. The collection medium can, but need not, contain one or more inhibitors used to culture the cells.

[0083] 2. How to collect extracellular vesicles Extracellular vesicles (including exosomes) can be isolated using differential centrifugation, buoyant density gradient centrifugation, filtration, high performance liquid chromatography, and immunoaffinity capture.

[0084] For example, one of the most common isolation techniques for isolating exosomes from cell cultures is differential centrifugation, whereby large particles and cell debris in the culture medium are separated using centrifugal forces between 200 and 100,000 × g, and the exosomes are separated from the supernatant by sedimenting the exosomes at approximately 100,000 × g. However, purity can be improved by centrifuging the sample using buoyant density gradient centrifugation on sucrose or Optiprep. Tangential flow filtration combined with deuterium / sucrose-based density gradient ultracentrifugation has been used to isolate therapeutic exosomes for clinical trials.

[0085] Ultrafiltration and high performance liquid chromatography (HPLC) are further methods to isolate EVs based on their size differences. EVs prepared by HPLC are highly purified.

[0086] Hydrostatic diafiltration has been used to isolate extracellular vesicles from urine.

[0087] Other common techniques for EV collection involve positive and / or negative selection using affinity-based methodologies. Antibodies can be immobilized in different media conditions and combined with magnetic beads, chromatography matrices, plates, and microfluidic devices for separation. For example, antibodies against exosome-associated antigens (e.g., cluster of differentiation (CD) molecules, CD63, CD81, CD82, CD9, epithelial cell adhesion molecule (EpCAM), and Ras-associated protein (Rab5)) can be used for affinity-based separation of exosomes. Non-exosomal vesicles carrying these or different antigens can also be isolated in a similar manner.

[0088] Microfluidic-based devices have also been used to rapidly and efficiently isolate EVs (e.g., exosomes), taking advantage of both the physical and biochemical properties of exosomes at the microscale. In addition to size, density, and immunoaffinity, sorting techniques (e.g., acoustic, electrophoretic, and electromagnetic manipulation) can be implemented.

[0089] Methods for characterizing EVs, including exosomes, are also known in the art. Exosomes can be characterized based on their size, protein content, and lipid content. Exosomes are spherical structures with sizes between 40-100 nm, much smaller compared to other systems (e.g., microvesicles with a size range of 100-500 nm). Several methods can be used to characterize EVs, including flow cytometry, nanoparticle tracking analysis, dynamic light scattering, Western blotting, mass spectrometry, and microscopy techniques. EVs can also be characterized and scored based on their protein composition. For example, integrins and tetraspanins are two of the most abundant proteins found in exosomes. Other protein markers include TSG101, ALG-2 interacting protein X (ALIX), flotillin 1, and cell adhesion molecules. Like proteins, lipids are major constituents of EVs and can be utilized to characterize them.

[0090] C. Pharmaceutical Compositions Pharmaceutical compositions comprising the EVs and / or cells are also provided, which may be administered parenterally (intramuscularly (IM), intraperitoneally (IP), intravenously (IV), subcutaneous injection (SubQ), subdermal), transdermally (passively or using iontophoresis or electroporation), or by any other suitable means, and may be formulated in dosage forms suitable for each administration route.

[0091] In some embodiments, the compositions are administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective for delivery of the composition to targeted cells.

[0092] In a preferred embodiment, the composition is administered locally, e.g., by injection directly at or near the site to be treated. Typically, local injection results in a greater increase in the local concentration of the composition than can be achieved by systemic administration.

[0093] In some embodiments, the composition is delivered locally to the appropriate cells by using a catheter or syringe.Other means of delivering such compositions locally to cells include using an infusion pump (e.g., Alza Corporation, Palo Alto, Calif.) or incorporating the composition into a polymeric implant (e.g., see P. Johnson and JG Lloyd-Jones, eds., Drug Delivery Systems: Fundamentals and Techniques (Chichester, England: Ellis Horwood Ltd., 1988 ISBN-10: 0895735806), which can provide sustained release of material in the immediate area of ​​the implant.

[0094] The EV composition can be provided to the cells either directly (e.g., by contacting it with or with the cells) or indirectly (e.g., through the action of any biological process). For example, the vesicles can be formulated in a physiologically acceptable carrier and injected into tissues or fluids surrounding the cells.

[0095] Exemplary dosages for in vivo methods are discussed in the following experiments. As further tests are conducted, information regarding dosage levels suitable for treating various conditions in various patients will become clear, and those skilled in the art can ascertain appropriate administration, taking into account the recipient's treatment status, age, and general health. The dosage selected depends on the desired therapeutic effect, the route of administration, and the desired duration of treatment.

[0096] Generally, for local injection or infusion, the dosage may be low. In general, the total amount of active agent administered to an individual using the vesicles of the present disclosure may be smaller than the amount of the relevant active agent that must be administered for the same desired or intended effect, and / or may exhibit reduced toxicity.

[0097] In a preferred embodiment, the compositions are administered in an aqueous solution by parenteral injection (eg, intramuscular, intraperitoneal, intravenous, subcutaneous, subdermal, etc.).

[0098] The formulations may be in the form of suspensions or emulsions. In general, pharmaceutical compositions are provided that contain an effective amount of one or more active agents, and optionally contain pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions may contain diluents, sterile water, buffered salt solutions of various buffer contents at various pH and ionic strengths (e.g., Tris-HCl, acetate, phosphate); and, optionally, additives (e.g., detergents and solubilizers (e.g., TWEEN® 20, TWEEN® 80 (also known as polysorbate 20 or 80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), as well as preservatives (e.g., thimerosal, benzyl alcohol) and bulking substances (e.g., lactose, The non-aqueous solvent or vehicle may include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). The formulation may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0099] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations can include penetration enhancers. Chemical enhancers and physical methods, including electroporation and microneedles, can work with this method. Typically, penetration enhancers are selected so as not to destroy and / or eliminate the biological activity of the EVs.

[0100] D.How to use Methods of using the compositions of the present disclosure are also provided. In some embodiments, the methods include administering to a subject in need of contacting or administering cells an effective amount of a composition comprising extracellular vesicles.

[0101] Resident skin cells (e.g., keratinocytes, fibroblasts, melanocytes, and inflammatory cells) can secrete various types of EVs depending on their biological state (Nasiri et al., “Shedding light on the role of keratinocyte-derived extracellular vesicles on skin-homing cells”, Stem Cell Research & Therapy, volume 11, Article number: 421 (2020), which is specifically incorporated by reference herein in its entirety). These vesicles can affect physiological properties and pathological processes of the skin (e.g., pigmentation, cutaneous immunity, and wound healing). As keratinocytes constitute the majority of skin cells, EVs secreted from these cells can alter the pathophysiological behavior of other skin cells. For example, keratinocyte EVs have been shown to harbor various biomolecules including DNA, miRNA, mRNA, and proteins. They are believed to promote crosstalk between keratinocytes and melanocytes, and between keratinocytes and immune cells, modulating cell proliferation, migration, and angiogenesis during homeostasis and wound healing. Therefore, it is believed that keratinocyte EVs could be used for nutritional supplementation and therapeutic approaches. For example, the physiological function of keratinocyte-derived exosomes in regulating melanocyte proteins is also well established and may provide therapeutic approaches for hypo- and hyperpigmentation disorders. In addition, keratinocyte-derived exosomes may function as intercellular messengers and immunomodulators through interactions with APCs, which may provide therapeutic approaches through the reduction of immune responses.

[0102] In some embodiments, exosomes / EVs produced according to the methods of the present disclosure may increase the expression of type I collagen (COL1A1) and / or elastin in cells contacted with the exosomes. Such cells may include, but are not limited to, fibroblasts. Collagen and elastin are the main fibers that form the extracellular matrix. See, for example, Mehta-Ambalal, J Cutan Aesthet Surg. 2016 Jul-Sep; 9(3): 145-151. doi: 10.4103 / 0974-2077.191645. Both are formed by fibroblasts. Collagen is responsible for tensile strength and elastin provides elasticity to the skin. The production and density of both decrease as a function of age, resulting in sagging and wrinkling. Wounding alters the quantity and quality of these fibers. Thus, exosomes produced according to the methods of the present disclosure may be used to manage cosmetic conditions such as skin aging and scarring.

[0103] Additionally or alternatively, specific additional therapeutic molecules such as genetic material, proteins, or even inhibitor drugs can be engineered into EVs and delivered to target abnormal cells, i.e., fibroblasts, melanocytes, or inflammatory cells, to improve their biological activity, for example, for the treatment of skin disorders such as dyspigmentation, autoimmune diseases such as psoriasis, chronic wounds, etc. Methods for loading drugs into preformed vesicles, including exosomes, are known in the art and are reviewed in Ha et al., Acta Pharmaceutica Sinica B, 6(4):287-296 (2016) doi: 10.1016 / j.apsb.2016.02.001 and discussed in Yang et al., J Control Release, 243:160-171 (2016). doi: 10.1016 / j.jconrel.2016.10.008 (each of which is specifically incorporated by reference).

[0104] Briefly, small molecules have been loaded by mixing and incubation, and for example, via complexation with surface elements. Proteins and peptides have been loaded by incubation with or without permeabilizers (e.g., saponin), via freeze-thaw cycling, sonication, and extrusion procedures. Nucleic acids have been loaded by chemical transfection and electroporation. See also Table 2 in Ha et al., Acta Pharmaceutica Sinica B, 6(4):287-296 (2016) doi: 10.1016 / j.apsb.2016.02.001, and references cited therein.

[0105] Thus, in some embodiments, the compositions of the present disclosure are administered in an effective amount to a subject in need thereof to contact cells or to have a biochemical or physiological effect on one or more cell types of the skin (e.g., keratinocytes, fibroblasts, melanocytes, inflammatory cells, etc.). In some embodiments, the compositions of the present disclosure are administered in an effective amount to a subject in need thereof to have, for example, a nutritional or therapeutic effect. In some embodiments, the compositions are administered topically, for example, by contact with the skin of the subject. Exemplary non-limiting diseases include skin disorders (e.g., pigmentation disorders), autoimmune diseases such as psoriasis, chronic wounds, atopic dermatitis, etc., as well as others mentioned herein and elsewhere.

[0106] In some embodiments, the composition is used to treat or prevent skin, irritation, stress, allergy, infection and / or skin heat / sweat. For example, the following experiment shows that keratinocyte EVs prepared according to the method of the present disclosure inhibited TSLP, IL-25, and IL-33, which are factors highly related to the induction of the pathogenesis of atopic dermatitis. Atopic dermatitis (also known as eczema) is a condition characterized by red and itchy skin. It is common in children, but can occur at any age. Atopic dermatitis lasts for a long time (is chronic), tends to flare periodically, and can be accompanied by asthma or hay fever. Atopic dermatitis symptoms vary widely between individuals and may include: dry skin; itching, which may be particularly worse at night; red to brownish-gray patches, especially on the hands, feet, ankles, wrists, neck, upper chest, eyelids, inside the crooks of the elbows and knees, and in young children, on the face and scalp; small raised bumps, which may leak fluid and become scabbed over if scratched; thickened, cracked, scaly skin; and rough, sensitive, swollen skin from scratching.

[0107] In some embodiments, compositions of the present disclosure (e.g., EVs prepared by the disclosed culture methods and / or compositions formed therefrom) are more efficacious than corresponding compositions prepared according to traditional (e.g., non-long term, non-reprogramming methods). In some embodiments, the traditional methods do not involve culturing the keratinocytes with a ROCK inhibitor and / or an inhibitor of TGFβ signaling. Thus, in some embodiments, the traditional culture methods do not involve culturing the keratinocytes with a ROCK inhibitor and / or an inhibitor of TGFβ signaling.

[0108] For example, the following experiment shows that the inhibitory effect of EVs prepared according to the long-term culture method disclosed herein is much stronger than that of EVs prepared according to traditional keratinocyte culture methods. Thus, in some embodiments, the skin disease or disorder to be treated is atopic dermatitis. In some embodiments, EVs prepared according to the methodology of the present disclosure reduce or prevent one or more symptoms or biochemical or physiological indicators of atopic dermatitis. The biochemical and physiological indicators may include, but are not limited to, thymic stromal lymphopoietin (TSLP), Th2, eosinophil-recruiting chemokines, inflammatory cytokines (e.g., IL-33 and IL-25), and combinations thereof. Thus, in some embodiments, EVs prepared according to the methodology of the present disclosure reduce or prevent one or more symptoms or biochemical or physiological indicators of atopic dermatitis to a greater extent than EVs prepared according to traditional (e.g., non-long-term) culture methods.

[0109] In some embodiments, the EVs may be administered as part of a heterogeneous mixture of factors (e.g., conditioned medium or a fraction isolated therefrom). In some embodiments, the EVs, or more of its more subtypes, are isolated or otherwise collected from conditioned medium. The EVs, or one or more of its subtypes, may be suspended in a pharma- ceutically acceptable composition (e.g., a carrier or matrix or depot) prior to administration to the subject.

[0110] EVs may have the versatility and ability to rapidly interact with multiple cell types in distant regions to modulate cellular responses (Zhang et al., Cell Prolif., 49:3-13 (2016)). Thus, local or regional administration to the site of interest or adjacent thereto is preferred, although systemic administration is also contemplated.

[0111] The administration frequency of the treatment method can be, for example, once, twice, three times, four times or more times every day, every week, every two weeks, or every month.In some embodiments, the composition is administered to the subject once every day, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, every ten days, every eleven days, every twelfth day, every thirteenth day, every fourteenth day, every fifteenth day, every sixteenth day, every seventeenth day, every eighteenth day, every nineteenth day, every twenty days, every twenty-first day, every twenty-second day, every twenty-third day, every twenty-fourth day, every twenty-fifth day, every twenty-sixth day, every twenty-seventh day, every twenty-eighth day, every twenty-ninth day, every thirty-first day, or every thirty-first day.In some embodiments, the administration frequency is once, twice or three times every week, or once, twice or three times every two weeks, or once, twice or three times every four weeks. In some embodiments, the composition is administered to a subject one to three times, preferably two times, per week.

[0112] In some embodiments, the effect of the compositions and methods of the present disclosure on a subject is compared to a control. For example, the effect of the composition on a particular symptom, pharmacological, or physiological indicator (including those mentioned above and elsewhere herein) can be compared to an untreated subject, or to the condition of the subject before treatment. In some embodiments, the symptom, pharmacological, or physiological indicator is measured in the subject before treatment, and measured again one or more times after treatment is initiated. In some embodiments, the control is a reference level or an average determined based on the measurement of the symptom, pharmacological, or physiological indicator in one or more subjects (e.g., healthy subjects) that do not have the disease or condition to be treated. In some embodiments, the effect of treatment is compared to a conventional treatment known in the art (e.g., one of those discussed herein).

[0113] E.Kit Also disclosed is a dosage unit that includes the composition of the present disclosure in a pharma- ceutically acceptable carrier for, for example, transport and storage and / or administration. The components of the kit may be individually packaged and may be sterile. In some embodiments, the pharma-ceutically acceptable carrier that includes an effective amount of the composition is transported and stored in a sterile vial. The sterile vial may include enough composition for one or more doses. The composition may be transported and stored in a volume suitable for administration or may be provided in a concentration that is diluted before administration. In another embodiment, the pharma-ceutically acceptable carrier that includes the drug may be transported and stored in a syringe.

[0114] Kits are provided that include syringes of various volumes or containers with deformable sides (e.g., plastic containers or containers with plastic sides) that can be crushed to force the liquid composition out of the opening. The size and design of the syringes depend on the route of administration. Any of the kits can include instructions for use.

[0115] The present invention can be further understood by the following numbered paragraphs: 1. A method for producing extracellular vesicles (EVs), the method comprising culturing keratinocytes in a culture medium containing a ROCK inhibitor, and harvesting EVs secreted by the keratinocytes.

[0116] 2. The method of paragraph 1, wherein the ROCK inhibitor is Y-27632.

[0117] 3. The method of paragraph 2, wherein the Y-27632 is at a concentration of about 1 μM to about 100 μM, or about 5 μM to about 25 μM, or about 10 μM.

[0118] 4. The method of any one of paragraphs 1 to 3, wherein the cells are cultured with an inhibitor of TGFβ signaling.

[0119] 5. The method of paragraph 4, wherein the inhibitor of TGFβ signaling is A83-01.

[0120] 6. The method of paragraph 5, wherein the A83-01 is at a concentration of about 1 μM to about 10 μM, or about 0.1 μM to about 10 μM, or about 0.5 μM.

[0121] 7. The method of any one of paragraphs 1 to 6, wherein proliferation and / or secretion of EVs is increased in the presence of the ROCK inhibitor compared to its absence.

[0122] 8. The method of any one of paragraphs 1-7, wherein the cells are cultured in the ROCK inhibitor, and optionally an inhibitor of TGFβ signaling, for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days; or about 5 days to about 25 days, or any subrange or integer number of days therebetween, optionally for about 7 days to about 22 days, about 5 days to about 25 days, or about 10 days to about 20 days, or about 12 days to about 17 days; or about 13 days, 14 days, or 15 days.

[0123] 9. The method of any one of paragraphs 1 to 8, wherein the EVs comprise or consist of exosomes.

[0124] 10. Extracellular vesicles (EVs) produced according to the method described in any one of paragraphs 1 to 9.

[0125] 11. A pharmaceutical composition comprising an effective amount of the EVs described in paragraph 10.

[0126] 12. A therapeutic or non-therapeutic method of treating a subject, said method comprising administering to said subject a pharmaceutical composition described in paragraph 11.

[0127] 13. The method of paragraph 12, wherein the subject has a skin disease or disorder or injury.

[0128] 14. A therapeutic or non-therapeutic method for improving the skin of a subject in need thereof, said method comprising administering to said subject a pharmaceutical composition described in paragraph 11.

[0129] 15. A therapeutic or non-therapeutic method for reducing or preventing skin aging and scarring of said skin, said method comprising administering to said subject a pharmaceutical composition described in paragraph 11.

[0130] 16. The method of any one of paragraphs 12 to 15, comprising contacting skin and / or cells thereof with the pharmaceutical composition.

[0131] 17. The method of any one of paragraphs 12 to 16, wherein the pharmaceutical composition increases expression of type I collagen (COL1A1) in cells of the subject.

[0132] 18. The method described in any of paragraphs 12 to 17, wherein the pharmaceutical composition increases expression of elastin in cells of the subject.

[0133] 19. The method of any one of paragraphs 16 to 18, wherein the cells are fibroblasts.

[0134] 20. The method of paragraph 19, wherein the fibroblasts are dermal fibroblasts.

[0135] 21. A method for treating atopic dermatitis, said method comprising administering to the subject a pharmaceutical composition described in paragraph 11.

[0136] 22. The method of paragraph 21, comprising contacting the skin and / or cells thereof with the pharmaceutical composition.

[0137] 23. The method of paragraph 22, wherein the subject's skin is dry, scaly, rough, sensitive, swollen, red, bumpy, or a combination thereof.

[0138] 24. The method of any one of paragraphs 21 to 23, wherein the pharmaceutical composition reduces expression of thymic stromal lymphopoietin (TSLP), Th2, eosinophil-recruiting chemokines, inflammatory cytokines (e.g., IL-33 and IL-25), or a combination thereof, in cells of the subject.

[0139] 25. The method of paragraph 24, wherein the pharmaceutical composition reduces expression of TSLP, IL-25, IL-33, or a combination thereof in cells of the subject.

[0140] 26. The method of any one of paragraphs 16 to 18, wherein the cells are keratinocytes.

[0141] 27. The method of paragraph 26, wherein the keratinocytes are epidermal keratinocytes.

[0142] 28. The method of any one of paragraphs 12 to 27, wherein the EVs are more effective than EVs prepared according to non-long-term or non-reprogramming keratinocyte culture methods.

[0143] 29. The method of paragraph 28, wherein the non-long-term or non-reprogramming keratinocyte culture method does not involve culturing the keratinocytes with a ROCK inhibitor.

[0144] 30. The method of paragraphs 28 or 29, wherein the non-long-term or non-reprogramming keratinocyte culture method does not involve culturing the keratinocytes with an inhibitor of TGFβ signaling. EXAMPLES

[0145] Working Example Example 1: Rock inhibitors enhance secretion of extracellular vesicles during long-term culture of keratinocytes material and method "Y" refers to Y-27632 (Rock inhibitor). "A" refers to A83-01 (TGFβ signaling inhibitor). "KC" refers to keratinocyte cells.

[0146] Human primary keratinocytes were cultured in EpiLife with or without supplementation of Y or Y+A according to the following schedule: TM The cells were cultured in medium containing 10 μM Y-27632 (Wako) and 0.5 μM A-83-01 (Wako) and analyzed for extracellular vesicle secretion. The collection medium was serum-free EpiLife® without inhibitors. TM Therefore, depending on the experiment or stage of the experiment, human epidermal keratinocytes were cultured in EpiLife® culture medium with or without small molecule inhibitors, i.e., 10 μM Y-27632 (Wako) alone or 10 μM Y-27632 + 0.5 μM A-83-01 (Wako). TM The cells were cultured in serum-free medium. [Table 1]

[0147] result Long-term culture of keratinocytes was performed by adding low molecular weight compounds, Y and Y+A.

[0148] The amount of EVs secreted from normal KCs (control EpiLife culture only) was compared with the amount of EVs secreted from KCs cultured long-term with low molecular weight compounds Y and YA.

[0149] The results showed that incubation with low molecular weight compound Y increased cell proliferation and EVs secretion. Untreated cells showed slightly advanced cell differentiation. Y-treated cells showed good cell growth and good morphology. YA-treated cells showed poor condition and poor adhesion.

[0150] By culturing with Y, cell proliferation was enhanced compared to normal cells cultured without the addition of Y. Compared with normal KC-EVs, the amount of secreted EVs was increased by culturing with low molecular weight compounds Y and YA. However, cell morphology was best for low molecular weight compound Y, and cell condition was poor in YA culture.

[0151] The results are shown in Table 2 (below) and illustrated in Figures 1A-5.

[0152] Inhibitor-treated keratinocytes (Y alone or Y+A) secreted significantly more exosomes / EVs compared to the original culture of keratinocytes without inhibitors (see, for example, Figures 4 and 5). Exosomes / EVs from inhibitor-treated keratinocytes were CD9- and CD63-positive. Nanoparticle tracking system nanosight showed that the particles were approximately 100 nm in diameter, which is consistent with the particles being exosomes or small EVs. [Table 2]

[0153] Example 2: Exosomes from cultured keratinocytes increase the expression of collagen and elastin material and method Exosomes / EVs were harvested from keratinocytes treated with 10μM Y-27632 for 14 days, and the culture medium was replaced with the harvested culture supernatant. The culture supernatant was filtered to remove cell debris, and then ultracentrifuged to harvest and purify the exosomes / EVs. The purified exosomes / EVs were resuspended in PBS(-) and the particle number was counted by Nanosight.

[0154] Culture of human dermal fibroblasts: Human dermal fibroblasts (adult, normal, cryopreserved)<NHDF-c Adult> : C-12302 Funakoshi p2 was cultured in HFDM-1 medium (Funakoshi 2102P05) until p10 (passage 10) (70% confluent, 6 cm cell culture dish).

[0155] Keratinocyte exosomes / EVs were added to human fibroblasts (cultured in glucose-supplemented DMEM) at 1,000 exosomes / well.

[0156] After 72 hours, fibroblasts were harvested, mRNA was prepared from the cells with Qiagen's RNeasy Mini kit, cDNA was synthesized, and expression levels of type I collagen (COL1A1) (ThermoFisher Assay ID: Hs00164004_m1) and elastin (ThermoFisher Assay ID: Dr03073243_g1) were quantified by quantitative PCR with Taqman probes (catalog number: 4331182).

[0157] result The results are shown in Figure 6 and demonstrate that exosome particles secreted by low molecular weight compound-treated human keratinocytes induce the expression of type I collagen and elastin genes in human fibroblasts. These results are consistent with the skin beautifying effect of exosomes derived from low molecular weight compound-treated keratinocytes.

[0158] Example 3: Exosomes derived from cultured keratinocytes inhibit factors that induce atopic dermatitis material and method An in vitro model of atopic dermatitis was developed, which is illustrated in Figure 7 A. Epidermal keratinocytes were cultured with 10 μM IL-10, TNFα, and IFNγ for 24 hours to induce inflammation.

[0159] Long-term keratinocytes were prepared by treating keratinocytes with 10μM Y-27632 for 14 days. Exosomes / EVs were prepared by culturing normal keratinocytes (EpiLife only) and long-term keratinocytes (EpiLife only) for 48 hours and collecting the supernatant. The culture supernatant was filtered to remove cell debris and then ultracentrifuged to collect and purify the exosomes / EVs. The purified exosomes / EVs were resuspended in PBS(-) and particle numbers were counted with a Nanosight.

[0160] Keratinocyte exosomes / EVs were added to epidermal keratinocytes at a concentration of 100 particles / cell for 48 h. Gene expression of TSLP, IL-25, and IL-33 was analyzed by qPCR.

[0161] result Barrier disruption and keratinocyte injury, which stimulates thymic stromal lymphopoietin (TSLP), a Th2 and eosinophil-recruiting chemokine, together with IL-33 and IL-25 released from keratinocytes, are key players in the pathogenesis of atopic dermatitis. See, for example, Rerknimitr et al., Inflamm Regen. 37:14, doi:10.1186 / s41232-017-0044-7 (2017).

[0162] An in vitro model was developed and used to test the effect of keratinocyte exosomes / EVs on the pathogenesis of atopic dermatitis. The results (shown in Figure 7B) indicate that keratinocyte exosomes / EVs prepared using long-term culture significantly inhibited TSLP, IL-25, and IL-33, which are highly relevant to the induction of atopic dermatitis pathogenesis. The inhibitory effect of keratinocyte exosomes / EVs prepared by long-term culture was much stronger than that of exosomes / EVs prepared from conventionally cultured keratinocytes. These results support the conclusion that exosomes / EVs harvested from reprogrammed keratinocytes could be an effective treatment for atopic dermatitis.

[0163] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the materials from which they are derived are specifically incorporated by reference.

[0164] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method for producing extracellular vesicles (EVs), the method comprising culturing keratinocytes in a culture medium containing a ROCK inhibitor, and harvesting EVs secreted by the keratinocytes.

2. The method of claim 1, wherein the ROCK inhibitor is Y-27632.

3. 3. The method of claim 2, wherein the Y-27632 is at a concentration of about 1 μM to about 100 μM, or about 5 μM to about 25 μM, or about 10 μM.

4. The method of any one of claims 1 to 3, wherein the cells are cultured with an inhibitor of TGFβ signaling.

5. 5. The method of claim 4, wherein the inhibitor of TGFβ signaling is A83-01.

6. 6. The method of claim 5, wherein the A83-01 is at a concentration of about 1 μM to about 10 μM, or about 0.1 μM to about 10 μM, or about 0.5 μM.

7. The method of claim 1, wherein the proliferation and / or secretion of EVs is increased in the presence of the ROCK inhibitor compared to its absence.

8. 2. The method of claim 1, wherein the cells are cultured in the ROCK inhibitor, and optionally the inhibitor of TGFβ signaling, for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days; or about 5 days to about 25 days, or any subrange or integer number of days therebetween, optionally from about 7 days to about 22 days, from about 5 days to about 25 days, or from about 10 days to about 20 days, or from about 12 days to about 17 days; or for about 13 days, 14 days, or 15 days.

9. The method of claim 1, wherein the EVs comprise or consist of exosomes.

10. Extracellular vesicles (EVs) produced according to the method of claim 1.

11. A pharmaceutical composition comprising an effective amount of the EVs of claim 10.

12. 12. The pharmaceutical composition of claim 11 for use in a therapeutic or non-therapeutic method of treating a subject.

13. The pharmaceutical composition of claim 12, wherein the subject has a skin disease or disorder or injury.

14. 12. The pharmaceutical composition of claim 11 for use in a therapeutic or non-therapeutic method of improving the skin of a subject in need thereof.

15. 12. A pharmaceutical composition according to claim 11 for use in a therapeutic or non-therapeutic method of reducing or preventing skin ageing and skin scarring.

16. A pharmaceutical composition described in any one of claims 12 to 15, wherein the method includes contacting the skin and / or cells thereof with the pharmaceutical composition.

17. The pharmaceutical composition according to any one of claims 12 to 15, wherein the pharmaceutical composition increases the expression of type I collagen (COL1A1) in cells of the subject.

18. The pharmaceutical composition according to any one of claims 12 to 15, wherein the pharmaceutical composition increases the expression of elastin in cells of the subject.

19. 17. The pharmaceutical composition of claim 16, wherein the cells are fibroblasts.

20. 20. The pharmaceutical composition of claim 19, wherein the fibroblasts are dermal fibroblasts.

21. 12. The pharmaceutical composition of claim 11 for use in a method for treating atopic dermatitis.

22. The pharmaceutical composition of claim 21, wherein the method comprises contacting the skin and / or cells thereof with the pharmaceutical composition.

23. 23. The pharmaceutical composition of claim 22, wherein the skin of the subject is dry, scaly, rough, sensitive, swollen, red, bumpy, or a combination thereof.

24. 24. The pharmaceutical composition of any one of claims 21 to 23, wherein the pharmaceutical composition reduces expression of thymic stromal lymphopoietin (TSLP), Th2, eosinophil-recruiting chemokines, inflammatory cytokines (e.g., IL-33 and IL-25), or a combination thereof, in cells of the subject.

25. 25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition reduces the expression of TSLP, IL-25, IL-33, or a combination thereof in cells of the subject.

26. The pharmaceutical composition of claim 16 , wherein the cells are keratinocytes.

27. 27. The pharmaceutical composition of claim 26, wherein the keratinocytes are epidermal keratinocytes.

28. The pharmaceutical composition of any one of claims 12 to 15 and 21 to 23, wherein the EV's are more effective than EV's prepared according to non-long-term or non-reprogramming keratinocyte culture methods.

29. 29. The pharmaceutical composition of claim 28, wherein the non-long-term or non-reprogramming keratinocyte culture method does not involve culturing the keratinocytes with a ROCK inhibitor.

30. 29. The pharmaceutical composition of claim 28, wherein the non-long-term or non-reprogramming keratinocyte culture method does not involve culturing the keratinocytes with an inhibitor of TGFβ signaling.

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