WO2025151822A1

WO2025151822A1 - Mesenchymal stem cell-derived exosome drug delivery for dry eye disease and other disorders

Info

Publication number: WO2025151822A1
Application number: PCT/US2025/011244
Authority: WO
Inventors: Carl Randall HARRELL
Original assignee: MAM Holdings of West Florida LLC
Current assignee: MAM Holdings of West Florida LLC
Priority date: 2024-01-12
Filing date: 2025-01-10
Publication date: 2025-07-17
Anticipated expiration: 2026-07-12

Abstract

The present invention relates generally to compositions, formulations, and methods for immunotherapy and drug delivery, including the treatment of eye diseases such as dry eye disease (DED). In particular, the present invention relates to methods of producing exosomes from mesenchymal stem cells (MSC-Exos) and, optionally, loading said exosomes with one or more bioactive substances. The exosomes may be loaded using electroporation with one or more bioactive substances such as proteins, miRNAs, and/or siRNA. In specific embodiments, the exosomes, and/or one or more biological compounds derived from the exosomes, may be provided to an individual in need thereof, including as part of one or more compositions containing mesenchymal stem cells (MSCs). The individual in need thereof may be an individual having a medical disorder such as DED, immune disorders, cancer, and other disorders. Therefore, the compositions containing MSCs can be used for topical application to the eye. The compositions can contain MSCs, MSC-Exos, one or more MSC-derived biological compounds (e.g., growth factors, proteins, etc.).

Description

MESENCHYMAL STEM CELL-DERIVED EXOSOME DRUG DELIVERY FOR DRY EYE DISEASE AND OTHER DISORDERS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made without government support or grants awarded by the National Institutes of Health. The government has no rights in the invention.

FIELD OF THE DISCLOSURE

The present invention relates generally to methods and compositions for immunotherapy and drug delivery. In particular, the present invention relates to methods of producing exosomes from mesenchymal stem cells and, optionally, loading said exosomes with one or more bioactive substances. Various embodiments of the disclosure relate to methods and compositions for the prevention, management, and treatment of various ophthalmic diseases, ocular injuries, and other disorders, including dry eye disease (DED). In certain embodiments, therapeutic compositions may include, for example, mesenchymal stem cells, mesenchymal stem cell-derived exosomes, and/or one or more biological molecules derived from the mesenchymal stem cells and/or mesenchymal stem cell-derived exosomes, including, for instance, macromolecular proteins, nucleic acids, growth factors, drugs, treatment compounds, and various immunoregulatory biomolecules.

BACKGROUND

A primary function of the mammalian cornea and its surrounding structures is to moisten the eye. When the lubricated state of the eye is impaired, the subject may suffer from a condition known as dry eye disease (DED). Dry eye disease, also known as keratoconjunctivitis sicca or dysfunctional tear syndrome, is a common, multifactorial disease of the lacrimal system and ocular surface characterized by a deficiency in quality and/or quantity of the tear fluid. Dehydration of moisture from the eye of the subject gives rise to various discomforts related to ocular dryness as well as burning and scratching sensations. An even more serious consequence of a dry eye condition is the loss of visual acuity, which if not corrected, may result in permanent damage. In fact, dry eye disease may act to degrade the exposed ocular surface and may cause a complete breakdown of corneal tissues. In an extreme case, this may necessitate a corneal transplant. In advanced cases, decreased tear secretion or altered tear composition leads to tear fdm instability/imbalance which, in DED patients, can result in the abnormally rapid breakup of the tear film. The lives of people with DED are negatively impacted due to consistent pain, redness and/or dryness of the eyes. Typically, artificial tears and over-the counter eye drops are used by patients to sooth eye irritation and to lubricate the eyes. The majority of these treatment options provide minimal relief for limited duration and require several daily reapplications.

These treatment options provide varying degrees of relief with limited capacity to modify the underlying disease state. Human amniotic membrane (HAM) has been used efficaciously to treat specific eye surface injuries and maladies. However, the use of HAM often involves the skills of a physician and additional expense to patients. Additionally, these procedures usually impose severe vision impairment during treatment as the amniotic membrane is non-transparent. Ultimately, the benefits of the procedure last only as long as the membrane is in place, so the procedure is not particularly useful for chronic conditions such as dry eye, dry eye discomfort, and tear hyperosmolarity, which is an important step in the development, progression, and aggravation of dry eye discomfort.

Mesenchymal stem cells (“MSC” or “MSCs”) are self-renewable, multipotent, and multifunctional stem cells that regulate innate and/or adaptive immune responses in various human tissues. MSCs may originate from different sources (e.g., bone marrow, amniotic fluid, placental tissue, spinal cord, umbilical cord blood, umbilical cord tissue, adipose tissue, etc.) and contain a variety of biological compounds (e.g., carbohydrates, proteins and peptides, lipids, lactate, pyruvate, electrolytes, enzymes, hormones, and various growth factors). With low immunogenicity, multi-directional differentiation ability, in particular homing ability, and immunosuppressive properties, MSCs have significant therapeutic potential in alleviating various diseases (e.g., ophthalmic diseases, ocular diseases, autoimmune diseases, specific cancers, cardiovascular diseases, nervous diseases, hematopoietic diseases, and the like). Exosomes derived from mesenchymal stem cells (“MSC-Exos”) are nano-sized extracellular vesicles enriched with biological compounds and/or bioactive molecules (e.g., microRNAs, enzymes, cytokines, chemokines, immunomodulatory, trophic, growth factors, and the like) that regulate survival, phenotype, and/or function of various cells (e.g., immune cells, malignant cells, tumor-infiltrated cells, and the like). Due to their nano-sized dimensions and bilayer lipid envelope, MSC-Exos can bypass biological barriers and may serve as carriers to deliver bioactive substances, biological compounds, and/or biological precursors (e.g., drugs, chemotherapeutics, and the like) directly into one or more cells, including, for instance, normal cells, malignant cells, tumor cells, and the like. A lipid bilayer maintains the integrity of exosomes and stabilizes biological activities. Protein modification on the surface enhances the recognition and targeting ability of exosomes. MSC-Exos have many unique characteristics, such as small size, low immunogenicity, long-circulating half-life, good penetration, and good biocompatibility. MSC-Exos can be used, for instance, as drug carriers and/or as carriers to deliver RNA, protein, and/or molecular drugs to specific parts of the body (e.g., eye tissues) to achieve targeted therapy.

In view of the foregoing, there is a significant need for methods, systems, compositions, and formulations for the production and use of exosomes, including, for instance, MSC-sourced exosomes and/or MSC-derived exosomes. In particular, there is a need for providing clinical uses of exosomes for the prevention, management, and/or treatment of various eye diseases (e.g., DED), injuries, and disorders, and that are affordable, readily accessible, and easy to use for both clinician and patient.

SUMMARY

It is to be understood that both the following summary and the detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Neither the summary nor the description that follows is intended to define or limit the scope of the invention to the particular features mentioned in the summary or in the description.

In certain embodiments, the disclosed embodiments may include one or more of the features described herein.

Embodiments of the present disclosure are directed towards method, systems, and compositions for the production and use of exosomes, including exosomes derived from mesenchymal stem cells (“MSC” or “MSCs”), referred to herein as “MSC-Exos,” optionally wherein the exosomes are loaded with one or more bioactive substances. In particular embodiments, the disclosure concerns systems, methods, and compositions for production of exosomes to be used as a treatment (e.g., for one or more eye disorders such as dry eye disease (DED)) or as part of a treatment, including as a bioactive substance such as a drug, treatment compound, therapeutic, chemotherapeutic, and/or as a delivery to an individual in need thereof. Further embodiments are directed towards using one or more types of MSCs, MSC-Exos, and/or biological compounds derived from MSCs and/or MSC-Exos for preventing, managing, and/or treating various ophthalmic and ocular conditions and/or diseases (e.g., dry eye disease (DED), including severe DED).

In at least one embodiment, the present disclosure includes a composition for delivering target specific exosomes (e.g., MSC-Exos) to one or more cells, including one or more cells of the eye. In embodiments, the composition comprises, in addition to one or more exosomes (e.g., MSC- Exos), a biological compound, a bioactive substance, a plasmid, and the like. In other embodiments, the exosome (e.g., MSC-Exos) is isolated from autologous cells of a subject, from a cell line, from a primary cell culture, and/or from a mesenchymal stem cell. In other embodiments, the at least one plasmid is an RNA plasmid, a DNA plasmid, or any combination thereof.

Also disclosed, in some embodiments, are methods of treating any medical disorder (e.g., DED) for which the exosomes (e.g., MSC-Exos), optionally loaded with one or more bioactive substances, would be therapeutic. In some embodiments, the MSC-Exos, optionally loaded with one or more biological compounds and/or bioactive substances (e.g., one or more compounds derived from MSC-Exos), can both treat a disease or a condition in an individual and protect against toxicities associated with other treatments for said disease or condition administered to the individual. In certain embodiments, exosomes (e.g., MSC-Exos) are produced from particular cells using multiple agents in the production method of the exosomes. Such exosomes may be produced from particular cells, including at least stem cells, and for example, MSCs. The MSCs may be derived from any suitable tissue, but in a specific case they are derived from umbilical cord tissue and/or amniotic fluid. Such MSC-Exos may be modified to harbor one or more biological compounds and/or bioactive substances, and in some cases, the exosomes are electroporated to be made to harbor the aforementioned one or more biological compounds and/or bioactive substances.

In some embodiments, the umbilical MSCs are from cord tissue, bone marrow, adipose tissue, dental tissue, placental tissue, amniotic fluid, synovial fluid, peripheral blood, Wharton's Jelly, umbilical cord blood, skin tissue, liver tissue, lung tissue, blood vessels, salivary glands, skeletal muscle, mammary gland, or a mixture thereof. In some embodiments, the MSCs are from umbilical cord tissue or amniotic fluid. In some embodiments, the one or more biological compounds and/or bioactive substances is miRNA, siRNA, shRNA, protein, peptides, drug, lipids, DNA, RNA, or a combination thereof. In some embodiments, the one or more biological compounds and/or bioactive substances is protein, peptides, drugs, and/or lipids, and wherein the concentration of the protein, peptides, drugs, and/or lipids is between 1 pg/mL and 1000 mg/mL. In some embodiments, the protein comprises an antibody or antibody fragment. In some embodiments, the one or more biological compounds and/or bioactive substances is miRNA, a nucleic acid therapeutic, and/or a protein therapeutic. Notably, synovial fluid is a clear, thick liquid that acts as a lubricant and cushion in joints. It helps to reduce friction between bones and provides nutrients to the cartilage in the joint. It is produced and maintained by the synovium, which is the soft tissue lining the joint capsule.

In at least one embodiment, compositions are disclosed (referred to herein as “MSC Compositions”) that comprise one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos-derived biological compounds. The MSC Compounds can, for instance, include one or more topical solutions. In at least one embodiment, the MSC Compounds can be used to treat one or more diseases, including one or more eye disorders e.g., DED). The MSC Compounds can further favor development of tolerogenic and/or regulatory phenotypes in activated monocytes and lymphocytes, indicating its potential for therapeutic use in various diseases, including one or more of the eye diseases described herein (e.g., DED, as well as other diseases, such as various cancers).

In at least one embodiment, a method for prevention and treatment of a disease (e.g., one or more eye conditions and/or diseases, such as DED) is disclosed, including altering the response of endogenous immune cells in the subject provided, comprising administering to the subject an effective amount of one or more MSC Compositions, thereby altering the response of endogenous immune cells (e.g., dendritic cells, macrophages, natural killer cells, T cells, and the like) in the subject. Tn embodiments, administration of an effective amount of MSC Compositions improves one or more symptoms of one or more eye diseases in the subject. In some embodiments, MSC Compositions may be administered in combination with one or more biological compounds, including, for instance, proteins, nucleic acids, growth factors, cytokines, antimicrobial agents, analgesic agents, local anesthetic agents, anti-inflammatory agents, anti-oxidant agents, immunosuppressant agents, anti-allergenic agents, enzyme cofactors, essential nutrients, cells, and combinations thereof.

In some embodiments, the MSC Compositions are pharmaceutical compositions that may be formulated in various formulations, including, for example, as one or more liquid solutions. At least one such solution is suitable for administration to the eyes of a subject as eye drops. Other formulations (e.g., gels, solids) are possible. As stated herein, the MSC Compositions may comprise one or more types of MSC-Exos (e.g., exosomes generated ex vivo from mesenchymal stem cells, wherein the mesenchymal stem cells may be, for instance, placental tissue-derived mesenchymal stem cells). Such exosomes may be used as a delivery vehicle for one or more MSC- sourced and/or MSC-Exos-sourced biological molecules (e.g., anti-tumorigenic miRNAs, messenger RNAs (mRNAs), enzymes, cytokines, chemokines, growth factors, immunomodulatory factors, small-molecule drugs, proteins, and combinations thereof). The MSC Compositions may further comprise one or more pharmaceutically acceptable excipients. The MSC Compositions may also comprise one or more agents selected from the group consisting of adjuvants, antioxidants, anti-inflammatory agents, growth factors, neuroprotective agents, antimicrobial agents, local anesthetics, and combinations thereof.

In at least one example, the MSC Compositions may be formulated as a formulation for topical application to the eye for the treatment DED and/or other eye diseases. Further conditions, diseases, and/or injuries that may be treated include Sjogren’s syndrome, cataracts, burns, and injuries to the eye tissues. The aforementioned composition may, in some instances, contain a human amniotic fluid formulation. The MSC Compositions may be applied directly to the eye(s), preferably as a liquid ocular solution, much like a common liquid eye drops, lubricant, or gel. The MSC Compositions can alleviate or prevent at least one symptom of a number of ocular injuries and diseases, including DED, dry eye discomfort, Sjogren’s syndrome, and burns or injuries, corneal neovascular disorders, corneal opacities (including corneal haze), prolonged redness and inflammation of the eye(s), and the like.

In the specific, non-limiting example of DED, decreased tear secretion or altered tear composition can lead to tear film instability/imbalance which, in DED patients, may result in the abnormally rapid breakup of the tear film after blinking. Numerous structural changes in epithelial cells and mucin-producing goblet cells develop as a consequence of exposition of these cells to the hyperosmolar tears. Tear hyperosmolarity causes and/or induces oxidative stress, disruption of DNA repair systems, and DNA damage, particularly in the cells of the ocular surface and lacrimal system. This can result in, for instance, cell apoptosis. An injury of the lacrimal glands may result in decreased tear secretion, enabling the creation of a positive feedback loop that leads to DED progression and aggravation. Eye drops containing MSC Compositions can treat DED and/or one or more symptoms thereof, including restoring tear homeostasis at the corneal surface. Such drops can therefore break the aforementioned positive feedback loop and relieve eye pain, irritation, discomfort, and vision disturbance in DED patients. In at least one embodiment, the MSC Compositions may be formulated as a hypotonic solution enriched with osmoprotectants, which may help support tear stability and assist in relieving eye dryness in DED patients.

In at least one embodiment, the MSC Compositions may include a pharmaceutically accepted carrier, and may be administered using a standard eye dropper apparatus. The MSC Compositions can contain over 300 human growth factors, and may be devoid of amniotic stem cells and elements of micronized membrane or chorion particles. The dilution ratio of the MSC Compositions may be dependent on the severity of the disorder or injury. For example, early to moderate DED or chronic redness, surface inflammation and, intraocular inflammation may be best treated with a low concentration, whereas Sjogren’s syndrome, severe DED, a corneal neovascular disorder, or corneal opacity will typically utilize a higher concentration of MSC Compositions. Daily applications of the MSC Compositions can deliver a sustainable level of beneficial growth factors.

Methods for treating or preventing an ocular disease, disorder, or injury of the eye using one or more of the described MSC Compositions are disclosed. In some embodiments, the aforementioned compositions are administered with a pharmaceutically acceptable carrier. In some embodiments, such compositions are administered as a solution, suspension, ointment, or gel, with or without an implant. In some embodiments, the disorders associated with the eye that are suitable for treatment include DED, dry eye discomfort, ocular burns, tears or injury to the eye or associated structures, corneal neovascular disorders, corneal opacities (including corneal haze), ocular blast injuries, eye infections, eye surgeries, drug-induced eye conditions, and prolonged redness and inflammation of the eye. In some embodiments, the disorders to be treated include amoebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, onchorcercal keratitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, corneal dystrophic diseases, Fuchs’ endothelial dystrophy, Sjogren’s syndrome, Stevens-Johnson syndrome, autoimmune dry eye diseases, environmental dry eye diseases, corneal neovascularization diseases, post-corneal transplant rejection prophylaxis and treatment, autoimmune uveitis, infectious uveitis, anterior uveitis, posterior uveitis (including toxoplasmosis), pan-uveitis, an inflammatory disease of the vitreous or retina, endophthalmitis prophylaxis and treatment, macular edema, macular degeneration, age related macular degeneration, proliferative and non-proliferative diabetic retinopathy, hypertensive retinopathy, an autoimmune disease of the retina, primary and metastatic intraocular melanoma, other intraocular metastatic tumors, open angle glaucoma, closed angle glaucoma, pigmentary glaucoma, and combinations thereof. Other disorders include injury, burns, or abrasion of the cornea, cataracts and age related degeneration of the eye or vision associated therewith.

Methods for treating, or preventing a disease, disorder, or injury of the eye using one or more MSC Compositions in combination with one or more therapeutic, prophylactic or diagnostic agents are also described. In some embodiments, one or more MSC Compositions is administered prior to, in conjunction with, subsequent to, or alternation with treatment with one or more therapeutic, prophylactic or diagnostic agents. In some embodiments, the one or more therapeutic, prophylactic or diagnostic agents are selected from the group consisting of an anti-glaucoma agent, an anti-angiogenesis agent, an anti-infective agent, an anti-inflammatory agent, an analgesic agent, a local anesthetic, a growth factor, an immunosuppressant agent, an anti-allergic agent, an antioxidant, a cytokine, and combinations thereof. In some embodiments, the one or more diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, contrast media.

These and further and other objects and features of the invention are apparent in the disclosure, which includes the above and ongoing written specification, as well as the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable a person skilled in the pertinent art to make and use these embodiments and others that will be apparent to those skilled in the art. The invention will be more particularly described in conjunction with the following drawings wherein:

Figure 1 shows various molecular and cellular effects that occur in dry eye disease (DED), according to at least one embodiment of the disclosure.

Figure 2 shows various ways that mesenchymal stem cells (MSCs) can modulate the phenotype and/or function of immune cells that play a pathogenic role in the development and progression of severe DED, according to at least one embodiment of the disclosure.

Figure 3 shows various effects of MSC-sourced and/or MSC-derived exosomes (MSC- Exos) in suppressing eye inflammation and other DED symptoms, according to at least one embodiment of the disclosure.

Figure 4 shows various effects of immune cells, including myeloid-derived suppressor cells (MDSCs), in the suppression of detrimental immune responses in the inflamed eyes of DED patients, according to at least one embodiment of the disclosure.

Figure 5 shows various therapeutic effects of MSC-Exos on the modulation and/or suppression of T-cell driven inflammation (e.g., eye inflammation in DED patients), according to at least one embodiment of the disclosure.

Figure 6 shows various immunosuppressive effects of MSC-Exos-sourced and/or MSC- Exos-derived miRNAs (e.g., miRNA-125b), according to at least one embodiment of the disclosure.

Figure 7 shows various effects of MSC-Exos, and/or compositions, formulations, and/or treatments containing such MSC-Exos (e.g., MSC Compositions), on eye tissues and DED, according to at least one embodiment of the disclosure.

Reference to various embodiments does not limit the scope of the invention. Figures represented herein are not limitations to the various embodiments according to the invention and are presented for exemplary illustration of the invention. DETAILED DESCRIPTION

The present invention is more fully described below with reference to the accompanying figures. The following description is exemplary in that several embodiments are described (e.g., by use of the terms “preferably,” “for example,” or “in one embodiment”); however, such should not be viewed as limiting or as setting forth the only embodiments of the present invention, as the invention encompasses other embodiments not specifically recited in this description, including alternatives, modifications, and equivalents within the spirit and scope of the invention. Further, the use of the terms “invention,” “present invention,” “embodiment,” and similar terms throughout the description are used broadly and not intended to mean that the invention requires, or is limited to, any particular aspect being described or that such description is the only manner in which the invention may be made or used. Additionally, the invention may be described in the context of specific applications; however, the invention may be used in a variety of applications not specifically described.

The embodiment(s) described, and references in the specification to “one embodiment,” “an embodiment,” “an example embodiment,” etc., indicate that the embodiment(s) described may include a particular feature, structure, or characteristic. Such phrases are not necessarily referring to the same embodiment. When a particular feature, structure, or characteristic is described in connection with an embodiment, persons skilled in the art may affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

In the several figures, like reference numerals may be used for like elements having like functions even in different drawings. The embodiments described, and their detailed construction and elements, are merely provided to assist in a comprehensive understanding of the invention. Thus, it is apparent that the present invention can be carried out in a variety of ways and does not require any of the specific features described herein. Also, well-known functions or constructions are not described in detail since they would obscure the invention with unnecessary detail. Any signal arrows in the drawings/figures should be considered only as exemplary, and not limiting, unless otherwise specifically noted. Further, the description is not to be taken in a limiting sense, but is made merely for the purpose of illustrating the general principles of the invention, since the scope of the invention is best defined by the appended claims. It will be understood that, although the terms “first,” “second,” etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Purely as a non-limiting example, a first element could be termed a second element, and, similarly, a second element could be termed a first element, without departing from the scope of example embodiments. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. As used herein, “at least one of A, B, and C” indicates A or B or C or any combination thereof. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be noted that, in some alternative implementations, the functions and/or acts noted may occur out of the order as represented in at least one of the several figures. Purely as a non-limiting example, two figures shown in succession may in fact be executed substantially concurrently or may sometimes be executed in the reverse order, depending upon the functionality and/or acts described or depicted.

As used herein, ranges are used herein in shorthand, so as to avoid having to list and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range.

Unless indicated to the contrary, numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims, each numerical parameter should be constmed in light of the number of significant digits and ordinary rounding approaches.

The words “comprise,” “comprises,” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including,” and “or” should all be constmed to be inclusive, unless such a constmction is clearly prohibited from the context. The terms “comprising” or “including” are intended to include embodiments encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include embodiments encompassed by the term “consisting of.” Although having distinct meanings, the terms “comprising,” “having,” “containing,” and “consisting of’ may be replaced with one another throughout the description of the invention. Conditional language, such as, among others, “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment.

Terms such as, among others, “about,” “approximately,” “approaching,” or “substantially,” mean within an acceptable error for a particular value or numeric indication as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. The aforementioned terms, when used with reference to a particular non-zero value or numeric indication, are intended to mean plus or minus 10% of that referenced numeric indication. As an example, the term “about 4” would include a range of 3.6 to 4.4. All numbers expressing dimensions, velocity, and so forth used in the specification are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

“Typically” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

Wherever the phrase “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.

Definitions

The following is a non-exhaustive and non-limiting list of terms used herein and their respective definitions.

The terms “agent” or “active agent,” which are used interchangeably herein, refer to a physiologically or pharmacologically active substance that acts locally and/or systemically in a subject’s body. An “agent” or “active agent” is a compound or substance that is administered to an individual for the treatment (e.g., therapeutic agent, cancer therapeutic agent, and the like), prevention (e.g., prophylactic agent), or diagnosis (e.g., diagnostic agent) of a disease or disorder. Such agents may also include therapeutics that prevent or alleviate symptoms, such as, for instance, symptoms associated with one or more eye disorders or treatments for such disorders. “Ophthalmic drug” or “ophthalmic active agent,” as used herein, refers to an agent that is administered to a patient to alleviate, delay onset of, and/or prevent one or more symptoms of a disease or disorder of the eye, or a diagnostic agent useful for imaging or otherwise assessing the eye.

The term “administering” or “administration” refers to providing or giving a subject one or more agents and/or formulations, such as one or more MSC Compositions, either alone or in conjunction with any other compound and/or agent (including, e.g., prophylactic or therapeutic agents), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as, e.g., subcutaneous, subdermal, intramuscular, intradermal, intraperitoneal, intracerebroventricular, intraosseous, intratumoral, intraprostatic, and intravenous), transdermal, intranasal, oral, vaginal, rectal, and inhalation.

The term “amniotic factor” generally refers to one or more compounds naturally present in the amniotic fluid. These include, for example, carbohydrates, proteins and peptides (e.g., enzymes, hormones), lipids, metabolic substrates and products (e.g., lactate, pyruvate), and electrolytes.

The term “antigen” refers to a compound, composition, and/or substance that can stimulate the production of antibodies or an immune response in a subject, including compositions that are injected or absorbed into a subject. An “antigen” may react with the products of specific humoral and/or cellular immunity, including, for example, those induced by heterologous antigens.

The term “biocompatible” or “biologically compatible,” as used herein, generally refers to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient or subject.

The term “biodegradable polymer,” as used herein, generally refers to a polymer that will degrade or erode by enzymatic action and/or hydrolysis under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. The degradation time is a function of polymer composition, morphology, such as porosity, particle dimensions, and environment.

The term “cancer” refers to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. A malignant cancer is one in which a group of tumor cells display one or more of uncontrolled growth (e.g., division beyond normal limits), invasion (e.g., intrusion on and destruction of adjacent tissues), and/or metastasis (e.g., spread to other locations in the subject’s body via lymph or blood). As used herein, the terms “metastasis” or “metastasize” refer to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are similar to those in the original tumor (i.e., the tumor at the primary site of tumor growth). A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A “tumor” refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but some, e.g., leukemia, and some blood cancers, do not necessarily form tumors. For those cancers that form tumors, the terms “cancer,” “cancer cell,” “tumor,” and “tumor cell” are used interchangeably. The amount of a tumor in a given subject is the “tumor burden,” which can be measured as the number, volume, and/or weight of the tumor.

The term “combination therapy” refers to the administration of different compounds, agents, and/or individual therapies in a sequential and/or simultaneous manner. Individual elements of a “combination therapy” may be administered at different times and/or by different routes, but act in combination to provide a beneficial effect on the subject.

The term “compound” refers to a substance formed from one or more chemical elements, arranged together in any proportion or structural arrangement. The one or more chemical elements may be either naturally occurring and/or non-naturally occurring. As used herein, the term “biological compound” refers to a compound of biological origin and/or having one or more effects on a subject’s local and/or systemic biological functions. Accordingly, “compounds” or “biological compounds” include, as non-limiting examples, various proteins (e.g., growth factors, hormones, enzymes), nucleic acids, and pharmaceutical products (e.g., drugs, prodrugs). The term “drug” generally refers to a medicine or other substance that has a physiological effect when introduced into a subject. The term “prodrug” generally refers to a biologically and/or chemically inactive compound that can be metabolized by a subject to produce a drug.

The terms “decrease,” “lower,” “lessen,” “reduce,” and “abate,” which are used interchangeably herein, refer generally to the ability of a compound, formulation, or therapy (including those disclosed herein) to produce, elicit, and/or cause a lesser physiological response (e.g., downstream effects) compared to the response caused by a respective control compound, formulation, or therapy. A “decrease” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that is, for instance, 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

The term “dendritic cell” refers to a type of specialized antigen-presenting cell (“APC”) involved in innate and/or adaptive immunity. Dendritic cells may also be referred to herein as “DC” or “DCs.” Dendritic cells may be present in the tumor microenvironment, and these are referred to as “tumor-associated dendritic cells” (“tDC” or “tDCs”).

The terms “effective amount” or “therapeutically effective amount,” which are used interchangeably herein, refer to the amount of an agent (e.g., including one or more MSC Compositions described herein) that is sufficient to effect beneficial or desired therapeutic result, including clinical results. An “effective amount” may vary depending upon one or more of: the subject and disease condition being treated, the sex, weight and age of the subject, the severity of the disease condition, the manner of administration, the ability of one or more formulations to elicit a desired response in the subject, and the like. The beneficial therapeutic effect can include, but is not limited to, enablement of diagnostic determinations; prevention of disease or tumor formation; amelioration of a disease, symptom, disorder, and/or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, and/or pathological condition; and generally counteracting a disease, symptom, disorder, and/or pathological condition. The term “effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient or individual), including an amount effective to alleviate, delay onset of, and/or prevent one or more symptoms, particularly of a disease or disorder of the eye. When a therapeutic amount is indicated, the precise amount of one or more formulations described in the present disclosure to be administered can be determined by a physician, based on, for instance, considerations such as individual differences in age, weight, extent of the disease or disorder, and/or condition of the subject (individual).

The terms “enhance,” “induce,” “induction,” and “increase,” which are used interchangeably herein, refer generally to the ability of a compound, formulation, or therapy (including those disclosed herein) to produce, elicit, and/or cause a greater physiological response (e.g., downstream effects) compared to the response caused by a respective control compound, formulation, or therapy. An “enhanced” or “increased” amount is typically a “statistically significant” amount, and may include an increase that is, for instance, 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

The term “growth factor” refers to any compound (e.g., one or more groups of proteins or hormones) that stimulate cellular growth. Generally, growth factors play an important role in promoting cellular differentiation and cell division, and they occur in a wide range of organisms, including humans.

The term “immune cell” refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of antibody-dependent cell-mediated cytotoxicity (ADCC), and/or induction of complementdependent cytotoxicity (CDC)).

The terms “immunologic,” “immunological,” or “immune” response, which are used interchangeably herein, refer to the development of a beneficial humoral (i.e., antibody-mediated) and/or a cellular (e.g., mediated by immune cells, such as antigen-specific T cells, or their secretion products) response directed against an antigen and/or immunogen in a specific subject. Such a response can be an active response induced by administration of an antigen and/or immunogen, or a passive response induced by administration of antibodies or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II major histocompatibility complex (MHC) molecules to activate antigen-specific CD4+ healer T cells and/or cos+ cytotoxic T cells. The response may also involve, for instance, activation of monocytes, macrophages, natural killer (NK) cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, and/or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (e.g, CD4+ T cells) or cytotoxic T lymphocyte (CTL) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an antigen and/or immunogen can be distinguished by, for example, separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

The term “implant,” as generally used herein, refers to a polymeric device or element that is structured, sized, or otherwise configured to be implanted, preferably by injection or surgical implantation, in a specific region of the body so as to provide therapeutic benefit by releasing one or more therapeutic, prophylactic or diagnostic agents over an extended period of time at the site of implantation. For example, intraocular implants are polymeric devices or elements that are structured, sized, or otherwise configured to be placed in the eye, preferably by injection or surgical implantation, and to treat one or more diseases or disorders of the eye by releasing one or more therapeutic, prophylactic or diagnostic agents over an extended period. Intraocular implants are generally biocompatible with physiological conditions of an eye and do not cause adverse side effects. Generally, intraocular implants may be placed in an eye without disrupting vision of the eye.

The term “ionizing radiation” refers to radiation, traveling as a particle or electromagnetic wave, that carries sufficient energy to detach electrons from atoms or molecules, thereby ionizing an atom or a molecule. Generally, ionizing radiation is made up of energetic subatomic particles, ions, or atoms moving at high speeds and electromagnetic waves on the high-energy end of the electromagnetic spectrum. Radiation has been demonstrated to induce adaptive immune responses to mediate tumor regression. In addition, the induction of type I interferons (“IFNs”) by radiation is essential for the function of CD8+ T cells. Radiation induces cell stress and causes excess deoxyribonucleic acid (DNA) breaks, indicating that the nucleic acid-sensing pathway likely accounts for the induction of type I IFNs upon radiation. Type I IFN responses in DCs dictate the efficacy of antitumor radiation. In contrast, chemotherapeutic agents and anti-human epidermal growth factor receptor 2 (HER2) antibody treatments have been demonstrated to depend on a distinct immune mechanism to trigger adaptive immune responses. In general, therapeutic radiation-mediated antitumor immunity depends on a proper cytosolic DNA sensing pathway. In at least one embodiment, one or more agents, formulations, and/or methods (e.g., including one or more types of MSCs, either alone or in conjunction with one or more other agents) described herein is administered in combination with radiation therapy.

The term “macrophage” refers to a type of white blood cell of the immune system that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and the like. These phagocytes include various subtypes (e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia, and others), but all are part of the mononuclear phagocyte system. Besides phagocytosis, macrophages play a critical role in both innate and adaptive immunity by recruiting other endogenous immune cells (e.g., lymphocytes). For example, they are important as antigen presenters to T cells. In humans, dysfunctional macrophages can cause severe diseases (e.g., chronic granulomatous disease) that result in frequent infections. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of various compounds (e.g., cytokines). Macrophages that encourage inflammation may be termed “Ml macrophages” because they have the so-called “Ml phenotype,” whereas those that decrease inflammation and encourage tissue repair may be termed “M2 macrophages” because they have the so-called “M2 phenotype.”

The term “mean particle size,” as used herein, generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a non- spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering.

The term “microparticle,” as used herein, generally refers to a particle having a diameter, such as an average diameter, from about 1 micron to about 100 microns, preferably from about 1 micron to about 50 microns, more preferably from about 1 to about 30 microns. The microparticles can have any shape. Microparticles having a spherical shape are generally referred to as “microspheres.”

The term “molecular weight” generally refers to the relative average chain length of a bulk polymer or protein, unless otherwise specified. In practice, molecular weights can be estimated or characterized using various methods including, for example, gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (MW), as opposed to the number-average molecular weight (MN). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

The term “nanoparticle,” as used herein, generally refers to a particle having a diameter, such as an average diameter, from about 10 nanometers (nm) up to but not including about 1 micron, preferably from 100 nm to about 1 micron. The particles can have any shape. Nanoparticles having a spherical shape are generally referred to as “nanospheres.”

The term “parenteral administration” refers to a type of administration by any method other than through the digestive tract or non-invasive topical or regional routes. As a non-limiting example, parenteral administration may include administration to a subject via intravenous, intradermal, intraperitoneal, intrapleural, intratracheal, intraarticular, intrathecal, intramuscular, subcutaneous, subjunctival, injection, and/or infusion.

The term “peptide” refers to a polymer of amino acid residues. The amino acid residues may be naturally occurring and/or non-naturally occurring. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein. The terms apply to, for instance, amino acid polymers of one or more amino acid residues, an artificial chemical mimetic of a corresponding naturally occurring amino acid, naturally occurring amino acid polymers, and non-naturally occurring amino acid polymers.

The term “pharmaceutically acceptable,” as used herein, refers to compounds, carriers, excipients, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The terms “subject,” “individual,” or “patient,” which are used interchangeably herein, refer to a vertebrate, such as a mammal (e.g., a human). Mammals include, but are not limited to, murines (e.g., mice), simians, humans, farm animals, sport animals, and pets. In at least one embodiment, the subject is a non-human mammal, such as a monkey or other non -human primate, mouse, rat, rabbit, guinea pig, pig, goat, sheep, dog, cat, horse, or cow. In at least one example, the subject has a tumor, such as a cancer, that can be treated using one or more agents, formulations, and/or methods (e.g., including one or more MSC Compositions, either alone or in conjunction with one or more other agents) disclosed herein. In at least an additional example, the subject is a laboratory animal/organism, such as, for example, a mouse, rabbit, guinea pig, or rat. In at least a further example, a subject includes, for instance, farm animals, domestic animals and/or pets (e.g., cats, dogs). In at least a still further example, a subject is a human patient that has one or more eye disorders, has been diagnosed with an eye disorder, and/or is at risk of having an eye disorder. A “patient” can specifically refer to a subject that has been diagnosed with a particular disease, condition, and/or indication that can be treated with refers to a subject that has been diagnosed with a particular indication that can be treated with one or more agents, formulations, and/or methods (e.g., including one or more MSC Compositions, either alone or in conjunction with one or more other agents) disclosed herein.

The term “topical administration” refers to a type of non-invasive administration to the skin, orifices, and/or mucosa of a subject. Topical administrations can be administered locally; that is, they are capable of providing a local effect in the region of application without systemic exposure. Topical formulations can, however, provide one or more systemic effects via, e.g., adsorption into the blood stream of the individual. Routes of topical administration include, but are not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, pulmonary administration, and rectal administration.

The terms “treating,” “treatment,” and “therapy” refer, either individually or in any combination, to any success or indicia of success in the attenuation or amelioration of an injury, disease, symptom, disorder, pathology, and/or condition, and/or pathological condition, including any objective or subjective parameter such as, for instance, abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, and/or prolonging the length of survival. Treatment does not necessarily indicate complete eradication or cure of the injury, disease, symptom, disorder, pathology, and/or condition, and/or pathological condition, or any associated symptom(s) thereof. The treatment may be assessed by one or more objective or subjective parameters, including, for example, the results of a physical examination, blood and other clinical tests (e.g., imaging), and the like. In at least one example, treatment with the disclosed one or more agents, formulations, and/or methods (e.g., including one or more MSC Compositions, either alone or in conjunction with one or more other agents) results in a clinical improvement in one or more eye diseases in a subject.

Further, unless otherwise noted, technical terms are generally used according to conventional usage. Aspects of the disclosed methods employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and/or cell biology, many of which are described below solely for the purpose of illustration. Such techniques are explained fully in technical literature sources. General definitions of common terms in the aforementioned fields, including, for instance, molecular biology, may be found in references such as, e.g., Krebs et al., Lewin ’s Genes X, Jones & Bartlett Learning (2009) (ISBN 0763766321); Redei, Encyclopedic Dictionary of Genetics, Genomics, Proteomics and Informatics (3rd ed.), Springer (2008) (ISBN: 1402067532); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons (updated July 2008) (ISBN: 047150338X); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology (2nd ed.), Wiley-Interscience (1989) (ISBN 0471514705); Glover, et al., DNA Cloning: A Practical Approach, Vol. 1-11, Oxford University Press (1985) (ISBN 0199634777); Anand et al., Techniques for the Analysis of Complex Genomes, Academic Press (1992) (ISBN 0120576201); Hames et al., Transcription and Translation: A Practical Approach, Oxford University Press (1984) (ISBN 0904147525); Perbal et al ., A Practical Guide to Molecular Cloning (2nd ed ), Wiley-Interscience (1988) (ISBN 0471850713); Kendrew et al., Encyclopedia of Molecular Biology, Wiley -Blackwall (1994) (ISBN 0632021829); Meyers et al., Molecular Biology and Biotechnology: A Comprehensive Desk Reference, Wiley-VCH (1996) (ISBN 047118571X); Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988) (ISBN 0879693746); Coligan et al., Current Protocols in Immunology, Current Protocols (2002) (ISBN 0471522767); Annual Review of Immunology, articles and/or monographs in scientific journals (e.g., Advances in Immunology), and other similar references.

Dry eve disease (PED)

Dry eye disease (DED), also known as dry eye syndrome or keratoconjunctivitis sicca, is an inflammatory condition characterized by, for instance, a lack of sufficient moisture or lubrication on the surface of the eye. Severe DED represents as an advanced form of DED characterized by, for instance, significant and persistent symptoms that greatly impact the quality of life and visual function of the affected individual. Such DED is a chronic condition in which the eyes are unable to produce enough tears or maintain a healthy tear film, leading to various symptoms, including, for example, severe dryness, discomfort, and potential damage to the ocular surface. Patients suffering from severe DED often experience severe and constant eye discomfort, and/or a constant burning or stinging sensation in the eyes. Further symptoms frequently observed in DED patients include, for example, dryness, grittiness, scratchiness, soreness, irritation, burning, watering, foreign body sensation, and eye fatigue. Additionally, the eyes may appear persistently red or bloodshot due to inflammation and irritation caused by the lack of sufficient lubrication. The eyes may feel extremely dry, gritty, or sandy. Severe dry eye disease can also cause vision disturbances, such as blurred or fluctuating vision.

The lack of sufficient tear production and lubrication can lead to chronic inflammation. Prolonged and severe dryness of the ocular surface can lead to comeal damage, including the development of corneal erosions, ulcers, or thinning. Turning now to Figure 1, various molecular and cellular effects that occur in DED are shown. Injured epithelial cells (e.g., injured corneal and conjunctival epithelial cells) 102 can, as shown at block 104, release damaged associated molecular patterns (DAMPs) and alarmins. These molecules can, as shown at block 106, activate professional antigen presenting cells (e.g., dendritic cells (DCs) and macrophages). Such antigen presenting cells can, as shown at block 108, produce various inflammatory cytokines (e.g., tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1 )) and can, as shown at block 110, induce increased expression of E and P selectins on endothelial cells (ECs), enabling massive influx of circulating leukocytes in inflamed eyes of DED patients.

Among recruited inflammatory immune cells, effector, interferon gamma (IFN-y), and IL- 17-producing CD4+ Thl and Th 17 lymphocytes can play important pathogenic roles in the development and progression of severe DED. Specifically, Thl cell-sourced IFN-y, shown at block 112, may induce the increased synthesis of inflammatory mediators (e.g., nitric oxide (NO), reactive oxygens species (ROS), TNF-a, IL-10, IL-6, IL-12, IL-23), shown at block 114. Such increased synthesis may favor the generation of an inflammatory phenotype in eye-infiltrated monocytes/macrophages, shown at block 116. Additionally, Thl7 cell-derived IL-17, shown at block 118, can promote the production of ROS, NO, TNF-a, and IL-1 P in neutrophils, shown at block 120, and may enhance neutrophil extracellular trap (NET) formation, shown at block 122, which can importantly contribute to the progression of ongoing eye inflammation, shown at block 124. In addition to CD4+ T helper cells, CD8+ cytotoxic lymphocytes (CTLs), shown at block 126, also infiltrate lacrimal glands of patients with severe DED. CTLs can produce TNF-a, perforins, and granzymes, shown at block 128, which may induce apoptosis of secretory cells in lacrimal glands, shown at block 130, thereby impairing tear production and exacerbating the dryness and discomfort of the eyes, shown at block 132. Further, chronic T cell-driven inflammation, shown at block 134, can lead to the damage of corneal and conjunctival epithelial cells, shown at block 136. These cells form the protective barrier of the eye and play a crucial role in maintaining the health of the ocular surface. The damage to these cells can result in compromised barrier function, increased evaporation of tears, and further exacerbation of DED- related symptoms, all shown at block 138.

Conventional treatments like tear supplementation, lubricating eye drops, and punctal plugs may be used to alleviate symptoms and promote ocular surface healing in DED patients. However, modulation of immune cells’ phenotype and function is a key aspect of managing severe DED and improving the overall ocular health of affected individuals. Therefore, targeted treatment approaches that are based on an understanding of the molecular mechanisms which orchestrate detrimental immune responses in the eyes of patients with severe DED are essential. Antiinflammatory medications, such as corticosteroids or immunomodulatory agents, may suppress immune cell-driven eye inflammation and might alleviate DED-related symptoms. Although corticosteroids and immunosuppressive drugs can be effective in managing severe dry eye disease, their long-term use can have detrimental effects, including an increased susceptibility to microbial pathogens and an impaired ability for the repair and regeneration of injured ocular surface. Moreover, long-term steroid use may induce development of glaucoma, cataracts, and corneal thinning in the eyes of DED patients. Finally, these immunomodulatory drugs can interact with other medications, leading to potential drug interactions and side effects.

It should be noted that eye drops which are presently used in the treatment of severe DED do not contain growth factors and are not able to promote repair and regeneration of injured cells in the lacrimal glands or ocular surface of DED patients. Also, the bioavailability and long-term effects of eye drops are generally low since the well -developed protective mechanisms of the eye ensure their rapid clearance from the pre-comeal space. Accordingly, there is an urgent need for the therapeutic use of new immunomodulatory agents which will be able to concurrently attenuate on-going eye inflammation without impairing protective immune response and to promote regeneration of injured epithelial barriers and lacrimal glands in the eyes of patients with severe DED.

Dry eye disease is often classified into two primary subtypes: aqueous tear-deficient dry eye (ADDE), which can be characterized by the inefficiency or inability of the lacrimal glands to produce tears, and evaporative dry eye (EDE), which is typically attributed to excessive evaporation of the tear fluid. ADDE may have an autoimmune origin or else can be attributed to a compromise in the LFU integrity. EDE is the more common form of dry eye disease and is frequently associated with meibomian gland dysfunction (MGD). MGD is often characterized by the modification or reduction of tear fluid lipids; as a result, the integrity and quality of the tear fluid may be compromised. Although dry eye disease has traditionally been classified into these two subtypes, there is considerable overlap between them. As such, dry eye disease is most often characterized as a “hybrid” or “mixed” form of these two subtypes, where each subtype adopts some clinical features of the other, initiating and exacerbating its pathology.

The multifactorial nature of dry eye disease involves several inter-related underlying pathologies, including the loss of homeostasis, chronic eye inflammation, and instability and hyperosmolarity of the tears. This can lead to neurosensory dysfunction and visual disturbance. Moreover, these detrimental events can create a “pathological loop” that promotes the progression and aggravation of dry eye disease. DED is usually manifested by dryness, grittiness, scratchiness, soreness, irritation, burning, watering, foreign body sensations, eye fatigue, and/or reduced functional visual acuity. Since significantly impaired performance of vision-dependent daily activities diminishes the quality of life of dry eye disease patients, a better understanding of the pathological steps in dry eye disease pathogenesis is of crucial importance for appropriate dry eye disease treatment.

Mesenchymal stem cells-derived exosomes in ophthalmology

Mesenchymal stem cells (“MSC” or “MSCs”) are self-renewable adult stem cells which are able to differentiate into corneal epithelial cells under specific culture conditions. Additionally, MSC secrete a large number of growth factors that support the viability of injured cells and produce immunomodulatory proteins which can regulate the phenotype and/or function of immune cells that participate in the development and progression of dry eye disease (DED). Many MSC-derived bioactive factors are contained in MSC-sourced exosomes (“MSC-Exos”), extracellular vesicles which, due to their nano-sized dimension and lipid envelope, can easily bypass all biological barriers to reach the target epithelial and/or immune cells in the eyes and lacrimal system of DED patients without affecting neighboring parenchymal cells and, therefore, without causing any severe side effects. Due to their enormous differentiation potential and immunosuppressive characteristics, MSCs, MSC-Exos, and/or MSC-Exos-derived biological compounds (e.g., the bioactive factors referred to above herein and described further below herein) are new remedies in regenerative ophthalmology. Accordingly, in at least one embodiment of the present disclosure, compositions containing MSCs, MSC-Exos, and/or MSC-Exos-derived biological compounds (referred to herein as “MSC Compositions”), which can, for instance, include one or more topical solutions, are set forth in further detail herein.

MSCs may, under specific culture conditions, differentiate in the cells of all three germ layers. Multi -lineage differentiation potential of MSCs could be a consequence of their complex development origin. During embryogenesis, different subpopulations of MSCs originate from different precursor cells, including epithelial-to-mesenchymal transition-derived cells, Soxl+ neuroepithelial cells, lateral plate mesoderm-derived mesoangioblast cells from the embryonic dorsal aorta, and blood-vessel-derived precursor cells.

MSCs reside in almost all postnatal tissues from where MSC can be isolated, propagated in vitro, and used in cell-based therapies of degenerative and inflammatory diseases. For clinical use, MSCs can be most frequently derived from bone marrow (BM), umbilical cord (UC), amniotic fluid (AF), and adipose tissue (AT). Specific functional properties of BM-derived MSC (“BM- MSC” or “BM-MSCs”) which favor their clinical application include, for instance, rapid proliferation in vitro, genomic stability after long-term cultivation, and the capacity for the increased production of immunosuppressive cytokines. Although BM-MSC have enormous therapeutic potential, harvesting of BM is an invasive procedure and, therefore, UC, AF, and AT can be used as alternative tissue sources for the isolation of MSCs. Collection of UC-derived MSC (“UC-MSC” or “UC-MSCs”) is noninvasive, painless, and safe. UC-MSC share similar functional properties with BM-MSC, but have a higher capacity for exosome (Exos) production. AF, obtained through amniocentesis, serves as an important source of AF-derived MSC (“AF-MSC” or “AF- MSCs”). AF-MSCs can produce large amount of neurotrophins and have a high therapeutic potential in the repair and regeneration of injured neural cells. Lastly, AT-derived MSC (“AT- MSC” or “AT-MSCs”), easily derived from patients’ AT, are usually used for autologous transplantation of MSC. AT-MSC have a high proliferation capacity and potent immunoregulatory properties.

Under specific culture conditions, BM-MSC and AT-MSC may differentiate into corneal epithelial cells. For instance, one week of exposure to hormonal epidermal medium (SHEM) or standard MSC cultured Dulbecco's Modified Eagle Medium (DMEM) supplemented with all- trans-retinoic acid (ATRA), may be sufficient to cause both BM-MSC and AT-MSC to differentiate into corneal epithelial cells.

Higher expression of epithelial markers (e.g., cytokeratin (CK)12, CK3, CK19, E- cadherin) and lower expression of mesenchymal markers (e.g., Vim, snail and alpha smooth muscle actin (a-SMA)) can occur in BM-MSC and AT-MSC which were cultured in SHEM (MSC^SHEM) or ATRA-supplemented medium (MSC^ATRA) than in BM-MSC and AT-MSC that grew under standard culture conditions (MSC^DMEM). Down-regulation or suppression of the Wnt/p-catenin signaling pathway is crucially responsible for BM-MSC and AT-MSC differentiation towards corneal epithelial cells. Importantly, human corneal epithelial cells (HCE) that were co-cultured with MSC^SHEM or MSC^ATRA can have an increased proliferation rate and an improved capacity for wound healing than HCE which grew with MSC^DMEM. The fact that MSC^SHEM or MSC^ATRA may better guide HCE-driven wound healing than MSC^DMEM indicates that SHEM or ATRA not only increases expression of pro-epithelial genes in MSC, but can also induce enhanced secretion of MSC-derived bioactive factors, which improve the viability and proliferation rate of injured HCE. From 720 different proteins which were detected in BM-MSC and AT-MSC-sourced secretome, around 122 proteins participate in the proliferation and differentiation of comeal epithelial cells. Specific proteins such as, for instance, TGF-P receptor type-1, TGF-P receptor type-2, Ras-related C3 botulinum toxin substrate 1, and/or Ras-related C3 botulinum toxin substrate 2 derived from UC-MSC can be responsible for MSC-mediated regulation of epithelial cell proliferation. These molecules activate Jun-N-terminal kinase (JNK) and p38 mitogen activated kinase in HCE, which can elicit signaling pathways that improve their proliferation and migration, which may contribute to the enhanced healing of corneal wounds.

MSCs from all tissue sources are potent immunoregulatory cells that produce a large number of immunomodulatory factors (e.g., IL-10, TGF-0, growth related oncogene (GRO), indoleamine 2,3 dioxygenase (IDO), nitric oxide (NO), interleukin 1 receptor antagonist (IL-IRa), prostaglandin E2 (PGE2)), which can alter the phenotype and/or function of all immune cells that play a pathogenic role in the development and progression of DED. For instance, by suppressing the Jak-Stat signaling pathway in T cells, MSC-sourced TGF-0 can induce G1 cell cycle arrest and prevent the proliferation of these cells. MSC-derived IDO can promote expansion of immunosuppressive T regulatory cells (Tregs) and prevent their conversion in inflammatory Thl7 lymphocytes.

Tregs are regulatory T cells (also referred to as “suppressor T cells”) that are generally immunosuppressive and can, for instance, help to prevent autoimmune diseases. Tregs can express several biomarkers, such as, for example, CD4 and forkhead box P3 (FOXP3). FOXP3 (also referred to as “scurfin”) is a protein that assists in regulation of regulatory pathways, including, for example, development of Tregs. Thus, the aforementioned CD4+ FOXP3+ T regulatory cells are positive for (i.e., express) both CD4 and FOXP3.

MSC-sourced NO, in an autocrine manner, can increase IDO expression in MSC and significantly enhance their immunosuppressive properties. Additionally, MSC-derived PGE2 can attenuate the proliferation of activated T cells and prevent the conversion of naive CD4+T cells in effector Thl and Thl7 cells by suppressing IL-2 production in T lymphocytes. Moreover, MSC- sourced PGE2 can stimulate the generation of an immunoregulatory tolerogenic phenotype in DC and induce expansion of alternatively activated macrophages, contributing to the creation of an immunosuppressive microenvironment in inflamed tissues in which MSC are transplanted. Similar to PGE2, MSC-derived IL- 10 and TGF-P can prevent the generation of inflammatory Thl and Thl7 cells by inhibiting the maturation of DC and by inducing the generation of alternatively activated (M2) phenotype in macrophages. Therefore, attenuated expression of co-stimulatory molecules (e.g., CD80 and CD86) and suppressed production of pro-Thl and pro-Thl7 cytokines (e.g., IL-12, IL-ip, IL-6, IL-23) can be observed in MSC-primed DC and macrophages. In addition to T cells, DC, and macrophages, MSC are also able to efficiently inhibit proliferation and cytotoxicity of NK cells. MSC-derived TGF-0 and NO can suppress the expansion of activated NK cells, while MSC-sourced IDO and PGE2 can generate the immunosuppressive and regulatory phenotype in NK cells. MSC-derived IL-10 can also down- regulate expression of pro-apoptotic and toxic molecules (e.g., perforins and granzymes) and inhibit the production of inflammatory and cytotoxic cytokines (e.g., TNF-a and IFN-y) in NK cells, significantly reducing their cytotoxic potential.

Juxtacrine communication (e.g., direct cell-to-cell interaction between immune cells and MSC) may also be involved in MSC-dependent suppression of detrimental immunity. MSC can express pro-apoptotic molecules (e.g., programmed death-ligand (PDL)-l, PDL-2, Fas ligand (FasL)), which bind to PD and Fas receptors on the membranes of activated T and NK cells and can induce their apoptosis in a caspase-3-dependent manner.

MSCs isolated from human and murine lacrimal glands possess potent regenerative and immunomodulatory properties and can be used as therapeutic agents for the treatment of severe DED. By producing various growth and immunoregulatory factors, MSCs can suppress detrimental immune responses and promote repair and/or regeneration of injured and inflamed eyes. The regenerative potential of MSCs is based on their ability to differentiate into the cells of all three germ layers. For instance, MSCs grown under specific culture conditions can differentiate into neural, epithelial and acinar-like cells. Additionally, as shown in Figure 2, MSCs are capable of modulating the phenotype and/or function of all immune cells that play a pathogenic role in the development and progression of severe DED. For example, MSCs 202 can, as shown at block 204, induce the alternative activation of macrophages, induce the generation of a tolerogenic phenotype in DCs, and attenuate NO, TNF-a, IL-ip, and/or ROS production in eye-infiltrated neutrophils. Such mechanisms can suppress ongoing eye inflammation, as shown at block 206. Moreover, MSCs 202 can, as shown at block 208, inhibit the expression of co-stimulatory molecules and suppress the synthesis of pro-Thl and Thl7 cytokines (e.g., IL-12, IFN-y, IL-ip, IL-6, IL-12) in macrophages and DCs, resulting in the alleviation of their antigen-presenting properties, as shown at block 210. Accordingly, MSCs 202 can, as shown at block 212, impede the expansion of Thl and Thl7 cells and prevent the generation of Thl and/or Thl7 cell-driven eye inflammation. Although MSCs have enormous therapeutic potential, several side effects caused by engrafted MSCs can limit their present clinical use in DED treatment. Although MSCs do not highly express major histocompatibility class (MHC) II molecules, these stem cells are not immune privileged cells and, accordingly, a detrimental immune response can be elicited upon transplantation of allogeneic MSCs. The recipient’s immune system may recognize foreign MHC class I and II molecules on the membranes of engrafted MSCs, which can result in the rejection of transplanted cells and in the generation of immune cell-driven inflammation. An additional potential side effect of MSCs’ transplantation is their unwanted differentiation. In certain cases, spontaneous differentiation of MSCs in chondrocytes and osteocytes can compromise tissue structure, integrity and function. Although rare, there have been reports of MSCs contributing to tumor formation or promoting the growth of existing tumors. This risk is associated with the potential for MSCs to differentiate into different cell types, including those involved in cancer development. MSCs can release various pro-angiogenic factors (e.g., angiopoietin, vascular endothelial growth factor (VEGF), IL-6) which may, under specific circumstances, promote neoangiogenesis in the tumor microenvironment, enabling dissemination of malignant cells.

Since MSC-dependent beneficial effects mainly rely on the biological activity of MSC- derived growth, pro-angiogenic, and immunomodulatory factors, injection of the MSC-derived secretome is a novel approach for the treatment of inflammatory diseases that can overcome all potential safety issues associated with the transplantation of MSCs. The majority of MSC-sourced bioactive factors are contained within MSC-sourced and/or MSC-derived exosomes (“MSC- Exos”), which are nano-sized extracellular vesicles (EVs) that are abundantly present in the MSC- sourced secretome. MSC-Exos represent homogeneous EVs which display surface markers of their parental cells (e.g., CD9, CD63, CD81, and/or CD44), and a consistent size and shape. MSC-Exos can be characterized by their small size (e.g., 30-150 nm), rounded or cup-shaped morphology, and lipid bilayer membrane, all of which can collectively enable their important roles in paracrine intercellular communication. The outer membrane of MSC-Exos is composed of phospholipids, cholesterol, and glycolipids. Due to small size and lipid envelope, MSC-Exos can easily bypass all biological barriers in the body and deliver their cargo directly into the target cells. MSC-Exos can contain a variety of bioactive molecules, including proteins (e.g., growth factors, immunoregulatory molecules, cytokines, chemokines), lipids, nucleic acids (e.g., messenger RNA (mRNA) and microRNAs (miRNAs)), one or more of which can affect the viability, proliferation, phenotype, and/or function of parenchymal and immune cells in injured and inflamed tissues (e.g., one or more tissues of the eye in DED patients).

Indeed, MSC-Exos can have numerous beneficial effects in the treatment of severe inflammatory eye diseases, including DED, suggesting their potential therapeutic use in clinical settings. Various molecular and cellular mechanisms, which will be described herein, are responsible for the trophic, anti-inflammatory, immunoregulatory, and/or regenerative properties of MSC-Exos in the treatment of severe DED.

With a greater understanding of these molecular and cellular mechanisms, methods and systems of producing MSC-Exos can enable the development of targeted treatments for DED patients, including, for instance, the various compositions and formulations described herein (e.g., the topical administration of eye-drops containing MSCs, MSC-Exos, and/or MSC-Exos-derived biological compounds).

MSC-Exos-based attenuation of innate immune cell-driven eye inflammation in DED

MSC-Exos can attenuate cell-driven eye inflammation in DED processes. For instance, in murine models, benzalkonium chloride (BAC)-induced DED can be used to examine the therapeutic efficiency of MSC-Exos in suppressing eye inflammation. Such DED can be induced by, for example, topical administration of specific amounts of BAC (e.g., 0.2% BAC). Mice can be divided into experimental and control groups to receive either MSC-Exos, one or more types of commercial eye drops (e.g., 0.1% pranoprofen), and/or one or more buffers as a control (e.g., phosphate-buffered saline (PBS)). Such agents can be given various times a day (e.g., three times a day) for a specific period (e.g., seven days). In such models, MSC-Exos can suppress ongoing eye inflammation in a dose-dependent manner. Specifically, MSC-Exos can trigger ocular surface epithelial repair in BAC-treated mice (e.g., mice that received 50 mg/mL of MSC-Exos). Further, MSC-Exos can improve tear film stability and prevented inflammation-induced apoptosis of corneal epithelial cells (CECs). BAC may cause a large release of alarmins from injured CECs. These alarmins can activate the NLRP3 inflammasome in eye-infiltrated neutrophils and macrophages, which may result in the increased production of inflammatory cytokines (e.g., IL- ip, IL-6, IL-la, and TNF-a). Elevated concentrations of these mediators can result in the enhanced recruitment of circulating leukocytes in the inflamed eyes of such BAC-treated animals. Lower levels of various inflammatory cytokines (e.g., IL-10, IL-6, IL-1 a, TNF-a, IFN-y) may be observed in serum samples of MSC-Exos-treated mice than in mice from control groups, indicating the suppressive effects of MSC-Exos on systemic immune responses. Importantly, MSC-Exos can enhance the production of immunosuppressive IL- 10, which has the capacity to suppress the generation of an inflammatory phenotype in eye-infiltrated neutrophils and monocytes. Accordingly, reduced expression of NLRP3, IL-10, and/or IL-18 may be observed in conjunctival tissue samples of MSC-Exos+B AC-treated mice compared to PBS+B AC -treated animals, indicating that MSC-Exos-dependent suppression of the NLRP3 inflammasome in eye-infiltrated immune cells can be primarily responsible for the beneficial effects of MSC-Exos in DED treatment. Importantly, MSC-Exos-based attenuation of NLRP3 -driven inflammation may result in the suppression of caspase 3. MSC-Exos-dependent inhibition of caspase-3 -driven apoptosis can prevent the increased loss of BAC-injured CECs and enable the enhanced regeneration of the ocular surface epithelial barrier.

By suppressing activation of the NLRP3 inflammasome, MSC-Exos can induce generation of alternatively activated (M2) phenotype in eye-infiltrated macrophages. M2 macrophages interact with other eye-infiltrated anti-inflammatory cells (e.g., tolerogenic DCs and forkhead box P3 (FoxP3)-expressing CD4+CD25+T regulatory cells (Tregs)) and can create an immunosuppressive microenvironment in inflamed eyes, crucially contributing to the attenuation of eye inflammation and to the alleviation of DED -related symptoms. Tolerogenic DCs do not optimally express MHC class II and co- stimulatory molecules and, instead of pro-Thl and pro- Thl7 cytokines (e.g., IL-12, IL-23, IL-10, IL-6), produce IL-10 and indoleamine 2,3 -dioxygenase (IDO), which promote the generation and/or expansion of immunosuppressive Tregs in inflamed tissues. MSC-derived IL-10 can suppress the maturation of DCs and promote their differentiation in tolerogenic DCs. IDO, produced by MSCs and tolerogenic DCs, metabolizes tryptophan (TRP) and generates kynurenine (KYN), which enhances the expression of lineage-defining FoxP3 transcription factor in naive CD4+ T lymphocytes, enabling the expansion of immunosuppressive CD4+CD25+FoxP3+Tregs in inflamed eyes.

Turning now to Figure 3, various effects of MSC-Exos in suppressing eye inflammation and other DED symptoms are shown. First, treatment with MSC-Exos 302 can, as shown at block 304, provide various effects, such as (1) triggering ocular surface epithelial repair, (2) improving tear film stability, and (3) preventing inflammation-induced apoptosis of corneal epithelial cells (CECs). Second, treatment with MSC-Exos 302 can, as shown at block 306, result in decreased levels of various inflammatory cytokines (e.g., IL-ip, IL-6, IL-la, and TNF-a). Third, treatment with MSC-Exos 302 can, as shown at block 308, enhance the production of immunosuppressive IL- 10, which, as shown at block 310, can suppress the generation of an inflammatory phenotype in eye-infiltrated neutrophils and monocytes. Fourth, treatment with MSC-Exos 302 can, as shown at block 312, result in reduced expression of NLRP3, IL-ip, and/or IL-18 (e.g., in one or more eye tissues, such as, for instance, conjunctival tissues), indicating that the MSC-Exos-dependent suppression of the NLRP3 inflammasome in eye-infiltrated immune cells can be primarily responsible for the beneficial effects of MSC-Exos in DED treatment. Finally, treatment with MSC-Exos 302 can, as shown at block 314, result in attenuation of NLRP3 -driven inflammation, which may result in the suppression of caspase-3, as shown at block 316. Suppression of caspase 3 can, as shown at block 318, result in the inhibition of caspase-3 -driven apoptosis, which can (1) prevent the loss of CECs and (2) enable enhanced regeneration of the ocular surface epithelial barrier, both shown at block 320.

In addition to M2 macrophages, tolerogenic DCs, and Tregs, myeloid-derived suppressor cells (MDSCs) can play an important role in the suppression of detrimental immune responses in the inflamed eyes of DED patients, as shown in Figure 4. Specifically, MDSCs 402 can produce anti-inflammatory cytokines (e.g., TGF-P and IL-10), as shown at block 404. MDSCs 402 can also, as shown at block 406, increase the production and/or expression of immunoregulatory factors (e.g., arginase-1 and NO) by, for example, MDSC-derived IL-6. Such immunoregulatory factors can then inhibit the proliferation of activated Thl and Thl7 cells, as shown at block 408. Further, MDSC-derived NO 410 may, as shown at block 412, inhibit the activity of cyclin- dependent kinases and promote the apoptosis of inflammatory T cells (e.g., by triggering the activation of caspase-3). Such effects can result in the suppression of cell cycle arrest, as shown at block 414. Additionally, MDSC-sourced NO 410 can, as shown at block 416, downregulate the expression of IL-2, which is crucially responsible for the proliferation of activated T cells. Further, MDSC-produced arginase 1 418 can, as shown at block 420, metabolize the amino acid arginine, thereby depleting the arginine. Through such depletion, MDSCs can, as shown at block 422, inhibit the expansion of effector Thl and Thl 7 cells. Moreover, MDSC-derived arginase 1 418 can, as shown at block 424, divert the metabolism of arginine towards the production of polyamines and proline, which are involved in tissue repair. In this way, MDSCs can, as shown at block 426, promote the regeneration of the injured ocular surface barrier.

In murine models of pSS, murine olfactory ecto-mesenchymal stem cell-derived exosomes (OE-MSC-Exos) can enhance the immunosuppressive properties of eye-infdtrated MDSCs and attenuate DED-related symptoms in experimental animals. OE-MSC-Exos, which can be intravenously infused (e.g., 100 micrograms) on specific days (e.g., days 18 and 25) after disease induction, can significantly increase the production of arginase- 1 and NO and downregulate the expression of MHC class II and co-stimulatory molecules (e.g., CD40, CD80, CD86) in MDSCs, which resulted in the suppression of T cell-driven eye inflammation. OE-MSC-Exo-derived S100A4, a member of the SI 00 calcium -binding protein family and ligand of toll like receptor (TLR)-4, may be responsible for the immunosuppressive effects of OE-MSC-Exos. Through the activation of TLR-4 signaling, OE-MSC-Exos can modulate the Jak2/Stat3 axis in MDSCs, enhancing the production of IL-6. MDSC-derived IL-6, in turn, in an autocrine, paracrine, and endocrine manner, may promote the expression of arginase-1 and NO in eye-infiltrated MDSCs, crucially contributing to the suppression of Thl and Thl7 cell-driven eye injury and inflammation in DED.

Therapeutic potential of MSC-Exos in the modulation and/or suppression of T-cell driven eye inflammation

MSC-Exos-dependent polarization of T cells can also contribute to the beneficial effects of MSC-Exos in the treatment of DED.

Ongoing eye inflammation and antigen-dependent priming of T cell receptors can result in the phosphorylation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR) in resting Tregs. Activation of Akt/mTOR pathways can alter the immunoregulatory phenotype of Tregs and induces their reprogramming into a pro-inflammatory Thl7-like phenotype, which may be characterized by the enhanced production of inflammatory cytokines, particularly IL- 17 and IL-22. IDO, derived from tolerogenic DCs, can attenuate the concentration of TRP in the inflamed microenvironment. Low levels of TRP may induce the activation of general control nonderepressible 2 (GCN2) kinase, which inhibits Akt/mTOR2 signaling in Tregs. Accordingly, tolerogenic DCs, in an IDO-dependent manner, can induce the generation of FoxP3 -expressing Tregs and prevent their transdifferentiation in inflammatory Thl 7 cells. By delivering IL-10, MSC-Exos can prevent maturation and induce the generation of a tolerogenic phenotype in DCs. Moreover, MSC-Exos are enriched with IDO and, therefore, in an IDO-dependent manner, may promote the expansion of immunosuppressive Tregs in inflamed eyes of DED patients.

In line with these findings, MSC-Exos can attenuate DED-related symptoms via, for instance, MSC-Exos-dependent enhancement of Treg-driven immunosuppression of inflammatory Thl and Thl7 cells. For example, MSC-Exos can be isolated from human labial glands (LG-MSC- Exos), whose immunoregulatory effects can be evaluated in vivo (e.g., in mice models) and in vitro by analyzing MSC-Exos-dependent changes of mononuclear cells (MNCs). Such MNCs can previously be isolated from the blood of patients suffering with various diseases (e.g., primary Sjogren's syndrome (pSS)). A hallmark characteristic of this autoimmune disorder is dryness of the eyes due to immune cell-mediated destruction of lacrimal glands. Thl and Thl7 cells, in an IFN-y and IL-17-dependent manner, can cause apoptosis of epithelial and acinar cells and induce a potent systemic inflammatory response, stimulating the production of auto-antibodies against self-antigens of lacrimal glands. An increased number of effector Thl and Th 17 lymphocytes and the reduced presence of immunosuppressive Tregs may create a vicious inflammatory cycle in the eyes of pSS patients, which results in the development of chronic eye inflammation. The systemic, intravenous infusion of LG-MSC-Exos (e.g., 50 pg/mouse, 3 times a week for 2 weeks) may significantly increase saliva flow rate in experimental animals. Importantly, the number and area of lymphocyte infiltration foci can be remarkably reduced in the salivary glands of LG-MSC-Exos- treated animals compared to control PBS-treated animals. LG-MSC-Exos can further decrease serum levels of Thl7-related inflammatory cytokines (e.g., IL-6 and IL- 17), increase serum levels of immunosuppressive TGF-P, downregulate the presence of Thl7 cells, and promote the expansion of Tregs in experimental mice. MSC-Exos-sourced IL- 10 can induce the generation of tolerogenic DCs which, in turn, interact with naive CD4+T cells and induce their differentiation in FoxP3+Tregs, enabling creation of an immunosuppressive environment in the inflamed eyes of patients with various eye diseases (e.g., pSS patients). Additionally, MSC-Exos, in a TGF-P dependent manner, can prevent the proliferation and expansion of inflammatory Th 17 cells. MSC- Exos-derived TGF-P can suppress the activation of the Jak-Stat signaling pathway in IL-17- producing Thl7 cells, causing G0/G1 cell cycle arrest. In this way, LG-MSC-Exos can increase the Treg:Thl 7 ratio in inflamed eyes, which may result in the attenuation of ongoing inflammation and alleviate DED-related symptoms.

Similar findings can be observed in vitro, specifically with respect to flow cytometry analysis of pSS patients’ MNCs, which can be cultured with LG-MSC-Exos (e.g., for 72 hours). A significantly increased percentage of CD4+CD25+FoxP3+ Tregs and a reduced percentage of Thl7 lymphocytes may be observed in the population of pSS patients’ MNCs that were exposed to LG-MSC-Exos. Moreover, LG-MSC-Exos can alter the secretory profile of T cells. The downregulated production of inflammatory cytokines (e.g., IL- 17, IL-6, TNF-a, IL-6) and the increased production of immunosuppressive IL-10 and/or TGF- can be observed in LG-MSC- Exos-primed pSS patients’ MNCs, confirming the therapeutic potential of LG-MSC-Exos in the attenuation of T cell-driven eye inflammation.

Almost identical findings can be obtained through isolation of MSC-Exos from the umbilical cord (UC-MSC-Exos). For instance, UC-MSC-Exos can suppress the proliferation of pSS patients’ Thl7 cells by inducing G0/G1 cell cycle arrest and inducing the expansion of Tregs by enhancing expression of FoxP3 in naive CD4+T cells. A UC-MSC-Exos-dependent increase in pSS patients’ Tregs:Thl7 ratio can be accompanied by, for instance, a downregulated production of various inflammatory cytokines (e.g., IFN-y, TNF-a, IL-6, IL-17A, and IL-17F) and with an upregulated secretion of immunosuppressive TGF-0 and IL-10 in MSC-Exos-primed T cells, further confirming the therapeutic potential of UC-MSC-Exos in the attenuation of T cell-driven eye inflammation.

Based on these promising results, various clinical trials should investigate the therapeutic potential of UC-MSC-Exos in the alleviation of DED-related symptoms in patients suffering from various diseases. For instance, a clinical trial for patients suffering from ocular graft versus disease (oGVHD) is currently recruiting. According to the study protocol, oGVHD patients will receive artificial tears for 14 days to normalize the baseline, and, afterwards, UC-MSC-Exo eye drops (10 pg/drop, four times a day) will be administered for two weeks. Changes in the ocular surface disease index, conjunctiva redness scores, tear secretion, tear break time, ocular surface staining, best corrected visual acuity, and tear meniscus height will be determined during the follow-up of 12 weeks. The first results of this trial are expected in the next two years. Turning now to Figure 5, various therapeutic effects of MSC-Exos on the modulation and/or suppression of T-cell driven inflammation (e.g., eye inflammation in DED patients) are shown. First, MSC-Exos 302 can, as shown at block 502, deliver IL- 10, which can prevent maturation and induce the generation of a tolerogenic phenotype in DCs, shown at block 504. Moreover, MSC-Exos are enriched with IDO and, therefore, in an IDO-dependent manner 506, may promote the expansion of immunosuppressive Tregs in inflamed eyes of DED patients, as shown at block 508. Further, treatment with MSC-Exos 302 can, as shown at block 510, reduce the number and/or area of lymphocyte infiltration foci. MSC-Exos 302 can also, as shown at block 512, further decrease serum levels of Thl7-related inflammatory cytokines (e.g, IL-6 and IL-17), increase serum levels of immunosuppressive TGF-P, downregulate the presence of Thl7 cells, and promote the expansion of Tregs. MSC-Exos-sourced IL-10514 can, as shown at block 516, induce the generation of tolerogenic DCs. Such DCs can interact with naive CD4+T cells 518 and, as shown at block 520, induce their differentiation in FoxP3+Tregs, enabling creation of an immunosuppressive environment in one or more eye tissues of patients with various eye diseases (e.g, DED), as shown at block 522. Additionally, MSC-Exos, in a TGF-P dependent manner 524, can prevent the proliferation and expansion of inflammatory Thl7 cells, as shown at block 526. MSC-Exos-derived TGF-P 528 can, as shown at block 530, suppress the activation of the Jak-Stat signaling pathway in IL-17-producing Thl7 cells. Such suppression can, as shown at block 532, cause G0/G1 cell cycle arrest. MSC-Exos can also, as shown at block 534, increase the Treg:Thl7 ratio in inflamed eyes, which may result in the attenuation of ongoing inflammation and alleviate DED-related symptoms, as shown at block 536. Further, MSC-Exos can alter the secretory profile of T cells by, for instance, (1) downregulating production of inflammatory cytokines (e.g, IFN-y, IL-6, IL-17, IL-17A, IL-17F, TNF-a), and (2) increasing production and/or secretion of immunosuppressive IL- 10 and/or TGF-P, both shown at block 538. Such effects further validate the therapeutic potential of MSC-Exos in the attenuation of T cell-driven eye inflammation.

Therapeutic potential of MSC-Exos in the modulation and/or suppression of B cell- driven eye inflammation

MSC-Exos are enriched with MSC-derived miRNAs, small non-coding RNA molecules that can regulate gene expression by affecting the stability of mRNAs and by impairing protein synthesis in eye-infiltrated immune cells, modulating their phenotype and/or function. For example, MSC-sourced miRNA-125b may be responsible for the immunosuppressive effects of MSC-Exos in the attenuation of DED-related symptoms. In murine models, LG-MSC-Exos (e.g., at 50 qg/mouse) can be intravenously injected (e.g., 3 times per week for 2 weeks) in mice with spontaneous pSS. Such a treatment can suppress detrimental inflammatory responses in inflamed lacrimal and salivary glands of pSS mice, completely alleviating DED-related symptoms. Further, LG-MSC-Exos may significantly reduce the total number of auto-antibody -producing CD 19- CD138+ plasma cells in the spleens of experimental animals. Similarly, a remarkably reduced percentage of CD19+CD20-CD27+CD38+ plasma cells can be observed in pSS patients’ MNCs that are co-cultured with LG-MSC-Exos compared to PBS-treated MNCs. Importantly, mRNA profiling of B-lymphocytes activated in the presence or absence of LG-MSC-Exos revealed that LG-MSC-Exo-sourced miRNA-125b can be mainly responsible for the modulation of autoantibody production and for the attenuation of B cell-driven autoimmune response in pSS.

The transcript of PR domain zinc finger protein 1 (PRDM1) gene can be the main intranuclear target of MSC-derived miRNA-125b. MSC-sourced miRNA-125b may recognize specific sequences in the mRNA called miRNA recognition elements (MREs) or binding sites. The binding between the MSC-sourced miRNA- 125b and PRDM1 mRNA can be mediated by the RNA-induced silencing complex (RISC) and Argonaute proteins. The binding of the MSC-derived miRNA- 125b to targeted PRDM1 mRNA can trigger its degradation by promoting the recruitment and activation of the exonuclease complex. Additionally, MSC-derived miRNA-125b can prevent the interaction between PRDM1 mRNA and ribosomes, impairing the synthesis of PRDM1 protein in plasma cells. PRDM1 protein is a transcription factor that has been identified as a key regulator of B cell differentiation into antibody-producing plasma cells. Additionally, PRDM1 can regulate the expression of various genes involved in immune responses and inflammation. For example, it can modulate the production of pro-inflammatory cytokines in lacrimal gland-infiltrated B cells (e.g., IL-6 and TNF-a), importantly contributing to the inflammatory processes in DED. PRDMl can also influence the expression of B cell-recruiting chemokines (e.g., CXCL13) and adhesion molecules (e.g., E and P selectins), which are involved in the recruitment and activation of B cells at the ocular surface. Furthermore, PRDMl can interact with the STAT-3 transcription factor, which regulates the development of B cells from their progenitors in bone marrow. Therefore, by inhibiting synthesis of PRDMl, MSC-derived miRNA-125b can regulate the development, recruitment, and/or activation of B cells and impair their differentiation in auto-antibody-secreting plasma cells, thereby attenuating detrimental B cell-driven immune responses in the inflamed eyes of DED patients.

Turning now to Figure 6, various immunosuppressive effects of MSC-Exos-sourced and/or MSC-Exos-derived miRNAs (e.g., miRNA-125b) are shown. Such miRNAs 602 may, as shown at block 604, result in (1) a reduction in the number of auto-antibody-producing plasma cells, (2) a concomitant reduction in the amount of auto-antibody production, and (3) an attenuation of B cell-driven autoimmune responses. MSC-Exos-sourced miRNAs (e.g., miRNA- 125b) can, as shown at block 606, bind to targeted PR domain zinc finger protein 1 (PRDM1) mRNA. Such binding can, as shown at block 608, result in the degradation of PRDM1 mRNA by promoting the recruitment and activation of the exonuclease complex. Additionally, MSC-Exos- sourced miRNAs (e.g., miRNA-125b) can, as shown at block 610, prevent and/or reduce the interaction between PRDM1 mRNA and ribosomes, thereby impairing the synthesis of PRDM1 protein in plasma cells, as shown at block 612. PRDM1 protein is a transcription factor that has been identified as a key regulator of B cell differentiation into antibody -producing plasma cells. Additionally, PRDM1 can (1) regulate the expression of various genes involved in immune responses and inflammation, (2) influence the expression of B cell-recruiting chemokines (e.g., CXCL13) and adhesion molecules (e.g., E and P selectins), and (3) interact with the STAT-3 transcription factor, which regulates the development of B cells from their progenitors in bone marrow. Therefore, by inhibiting synthesis of PRDM1, MSC-Exos-sourced miRNAs (e.g., miRNA-125b) can, as shown at block 614, regulate the development, recruitment, and/or activation of B cells and impair their differentiation in auto-antibody-secreting plasma cells, thereby attenuating detrimental B cell-driven immune responses in the inflamed eyes of DED patients, as shown at block 616.

MSC-Exos based therapy and treatments for DED

Since MSC-Exos may deliver their cargo directly into the target cells, these extracellular vesicles may be used as vehicles for the transport of bioactive molecules. For instance, eye treatments and eye drops containing MSC-Exos can be formulated, including eye drops containing ascorbic acid (AA) and MSC-Exos (mExo@AA). These eye drops can be prepared based on the capacity of AA to reduce ROS production of eye-infiltrated immune cells and MSC-Exos to deliver their cargo directly in target cells. In vitro, MSC-Exos containing eye drops can improve the viability and migration of injured CECs, attenuate the production of inflammatory cytokines (e.g., IL-1 and IL-6), and induce the generation of an immunosuppressive phenotype in macrophages. Such eye drops can, at least in murine models, further inhibit ROS production in eye-infiltrated immune cells, attenuate detrimental immune response in inflamed eyes, enhance repair and regeneration of the ocular surface barrier, and efficiently restore tear production in experimental animals. Importantly, side effects may be minimized or non-existent after topical application of various eye drops containing MSC-Exos (e.g., mExo@AA), indicating their potential clinical use.

Despite the fact that results from pre-clinical studies indicate the therapeutic potential of MSC-Exos in the modulation of immune cell-driven eye inflammation and DED treatment, it should be noted that several challenges still limit their clinical application. There is a lack of standardized protocols for the isolation and characterization of MSC-Exos. Different isolation methods and techniques can yield MSC-Exos with varying purity, size, and content. Establishing standardized procedures, including those described herein, for isolation and characterization is essential to ensure consistent and reproducible results. Also, MSC-Exos has to be produced in large quantities for clinical applications. However, current methods for their production and purification are often time-consuming, expensive, and yield low quantities. Developing scalable and cost-effective production methods, including those described herein, is crucial to meet the demands of clinical use.

Turning now to Figure 7, various effects of MSC-Exos, and/or compositions, formulations, and/or treatments containing such MSC-Exos (e.g., MSC Compositions), on eye tissues and DED are shown. Specifically, MSC-Exos-containing eye treatments e.g., any of the compositions, formulations, and/or treatments described herein, such as any MSC Composition) 702 can, as shown at block 704, reduce ROS production of eye-infiltrated immune cells. Additionally, MSC-Exos-containing eye treatments 702 can, as shown at block 706, (1) improve the viability and migration of injured CECs, (2) attenuate the production of inflammatory cytokines (e.g., IL-ip and IL-6), and (3) induce the generation of an immunosuppressive phenotype in macrophages. Further, MSC-Exos-containing eye treatments 702 can, as shown at block 708, (1) attenuate detrimental immune responses in inflamed eyes of DED patients, (2) enhance repair and regeneration of the ocular surface barrier, and (3) restore tear production in the eyes of DED patients.

Although MSCs that reside in different organs share large numbers of morphological and functional characteristics, MSCs are not homogenous cell populations. Their phenotypic and/or functional properties are affected by the tissue microenvironment to which the MSCs are exposed. MSCs that are primed with the inflammatory cytokines can obtain an immunosuppressive phenotype and secrete large amounts of immunoregulatory factors. On the contrary, MSCs that are exposed to low concentrations of inflammatory cytokines can develop a pro-inflammatory phenotype and could aggravate ongoing inflammation. Therefore, the contents of MSC-Exos, isolated from diverse tissue sources, can vary depending on the phenotype, function, and/or tissue source of their parental MSCs. This heterogeneity can lead to inconsistent therapeutic effects and challenges in defining a specific set of functional characteristics for clinical use. Moreover, MSC- Exos are sensitive to environmental conditions, such as temperature, freeze-thaw cycles, and storage duration. Maintaining the stability and integrity of exosomes during storage and transportation is critical for their clinical application. Identifying and standardizing specific markers or cargo profiles associated with therapeutic efficacy and developing appropriate storage conditions and techniques, such as cryopreservation, is necessary to preserve the functional properties and therapeutic efficacy of MSC-Exos.

Finally, bearing in mind that MSC-Exos are enriched with large number of different bioactive proteins and miRNAs, the safety profile of each MSC-Exos-based treatment (e.g., eye drops) should be thoroughly evaluated in clinical trials before these immunoregulatory therapeutic agents are offered as new remedies for the treatment of severe DED.

Systems and methods of producing exosomes, including MSC-Exos

As described above herein, systems and methods of producing target-specific exosomes e.g., MSC-Exos, and the like) are disclosed. Thus, the term “exosomes” includes, but is not limited to, MSC-Exos. In specific cases, the present disclosure concerns a manufacturing practice to produce exosomes. In some embodiments, the exosomes are MSC-derived and produced under particular conditions in combination with being produced from particular cells. In some embodiments, MSCs are from umbilical cord tissue or amniotic fluid, but they may be sourced from and/or derived from any source including, but not limited to, bone marrow, adipose tissue, dental tissue, placental tissue, and/or any other tissue disclosed herein.

Any step in the process may have a particular media, duration of time, presence of one or more particular gases at specific concentrations, presence or absence of movement (such as rotation), and a combination thereof, for example. In particular aspects, the cells are incubated (e.g., in a cell culture reactor or flask) with media for a particular amount of time, in some cases. This is followed by washing and collection of the cells and exosomes secreted from the cells. The collection of the exosomes (that may be referred to herein as “harvesting”) may include one step or multiple steps; in cases when the collection of the exosomes occurs more than once, there may or may not be an interval of time by which the exosomes are collected, such as at least, at most, or about 12, 18, 24, 36, 48, 60, 72 hours, or more, or any range or value derivable therein, between collections. The media in which the cells and exosomes are collected may be of a particular kind, and in specific steps when the cells and exosomes are collected the media lacks platelet lysate (PLT-free). In specific cases, the cells are cultured over the course of about 22 hours, and then cells are washed and exosomes secreted from the cells are collected approximately every 48 hours in the EC media-PLT-free (the EC media-PLT free may or may not comprise alpha MEM media supplemented with 2mM of GLUTAMAX™ (synthetic reagent similar to L-glutamine and that comprises L-alanyl-L-glutamine dipeptide)). These sequential steps may be repeated, such as repeated for a total of 2, 3, 4, or more times.

In specific aspects, the suspension of cells and exosomes are harvested from the system under conditions in which the exosomes produced from the cells consistently have the same, or substantially the same, markers and physiology. Thus, in specific cases at different times of harvesting, the exosomes are the same or substantially the same, at least as judged by the majority of their exosomes having one or more of the same expression markers.

In particular aspects, the process to produce the exosomes occurs in a cell culture reactor, although in alternative cases it does not. In particular aspects, the process to produce the exosomes occurs in flasks. In specific aspects, part or all of the process occurs in a cell culture reactor having controllable conditions that in specific cases may be automated. Although the cell culture reactor may be of any kind, in specific aspects the cell culture reactor comprises a hollow fiber system that may or may not comprise one or more pathways. The multiple porous microchannels comprise inner surfaces suitable for adherence of cells or suitable for modification such that cells may adhere to them, in particular aspects. Alternative cell culture reactor systems include the WAVE CELL CULTURE REACTOR™ (GE Healthcare) or the G-REX® system (Wilson Wolf), as non-limiting examples.

In certain aspects, the hollow fiber cell culture reactor may be a functionally closed (or semi-closed) system designed for a large-scale cell culture of adherent or non-adherent cells. The system allows the cells to grow (expand in number) in a dynamic environment allowing the continuous perfusion of medium that under suitable conditions mimics particular in vivo intravascular and extravascular compartments in at least some cell culture reactors. That is, in specific cases, an intravascular compartment is configured to mimic the intravascular region of the blood system and/or an extravascular compartment is configured to mimic the extravascular hematopoietic system. The hollow fiber system in specific cases comprises hundreds or thousands of semi-permeable pores for the culture of desired cells, including adherent cells. Membranes may make up the inner walls of the porous microchannels and allow the exchange of gas and/or nutrients with a homogenous approach, maximizing the growth rate of the cells in a short time. In particular aspects, the process is specifically designed to be suitable for growth of MSCs and to allow for the collection of the exosomes secreted by the cells (e.g., MSC-Exos) in a customized method.

Components of the cell culture reactor system can comprise vessels and/or compartments for introducing media and/or cells to the system, vessels and/or compartments for expanding the cells (and thereby produce exosomes from the expanding/expanded cells), and vessels and/or compartments for harvesting the cells, the conditioned media comprising the exosomes, and so forth. Non-limiting examples of compartments for any part of the system include a cell inlet bag, media bag, harvest bag, and waste bag, in specific aspects. The cell culture reactor system can utilize thousands of semi-permeable porous microchannels onto which the cells are adherent, either naturally or because the porous microchannels in the system have been manipulated to allow for adherence of the desired cells. In specific aspects, the system also comprises a gas regulator (that may be referred to as a “gas transfer module”) that stabilizes desired gas concentrations in the media. Such a gas regulator allows for, if desired, continual infusion of one or more gases into the cell culture reactor. In specific aspects, the process to produce the desired exosomes utilizes well- defined concentrations of CO2 (for example, about 5%), O2 (for example, about 20%), and nitrogen (for example, the conditions are nitrogen balanced).

Appropriate steps are taken to prepare the system prior to loading of the cells, such as, for instance, preparation of the physical components of the system to facilitate expansion of the cells. The system may be closed or may be semi-closed (which, as used herein, refers to some steps during the production of exosomes requiring the opening of the system and the exposure of the sample to the air). Prior to subjecting the cells to be expanded to the system, the cell culture reactor may be subjected to one or more components and/or one or more conditions to facilitate adherence of cells to the cell culture reactor. Cell media may be loaded into the system prior to loading of the cells. For adherent cell production, cells attach and proliferate on the inner surface of each fiber. Suspended cells can be flushed, leaving the adherent cell production for expansion. Automated cell feeding and waste removal may be part of the system, in specific aspects. In at least some cases, sampling of cells/conditioned media from the system may be provided for without or with interruption of the process. In particular aspects, after cell expansion, the adherent cells are released from the hollow fiber walls into suspension, and the suspension including cells and exosomes secreted therefrom are collected.

The exosomes (e.g., MSC-Exos) may be separated by any suitable tools, methods, and/or processes from the supernatant and cells once the cells are harvested from the process, including from the system. In some cases, there are multiple harvests from the process, and the supernatant, cells, and exosomes from the process may be pooled prior to any further separation or modification steps. In certain cases, exosomes from multiple harvests are processed separately and combined later. In some embodiments, the exosomes are enriched or concentrated following the production process. As one non-limiting example, the exosomes are separated from cells, cell fragments, and/or larger or smaller vesicles through physical and/or chemical tools, methods, and/or processes. In specific cases, the exosomes are concentrated through one or more centrifugations, one or more filtrations (such as ultrafiltration and/or diafiltration), one or more of immunoisolation, chemical precipitation, size exclusion chromatography, microfluidics, or a combination thereof. Different centrifugation steps may occur at different speeds, and/or different filtration steps may occur at different sizes. In some embodiments, exosomes are enriched or concentrated from the medium of cultured MSCs using differential ultracentrifugation. In some embodiments, differential ultracentrifugation comprises the following steps: (1) the supernatant is centrifuged at 2000 x g for 20 minutes, and the pellet comprising cells is discarded; (2) the supernatant is filtrated using at 0.2 pm filter; (3) the supernatant is centrifuged at 100000 x g for 240 minutes, and the pellet comprising exosomes and cell proteins is obtained and washed in PBS; and (4) the PBS-washed pellet comprising exosomes and cell proteins is centrifuged at 100000 x g for 70-180 minutes, and the pellet comprising exosomes is obtained.

Although differential ultracentrifugation provides reasonably pure exosomes, in some embodiments, an extra purification step is performed using, for instance, a sucrose cushion. Thus, in some embodiments, exosomes are enriched or concentrated from the medium of cultured MSCs using differential ultracentrifugation followed by filtration through a sucrose gradient. Using a sucrose cushion eliminates more contaminants, such as proteins nonspecifically associated with exosomes, or large protein aggregates, which are sedimented by centrifugation but do not float on a sucrose gradient. Therefore, in some embodiments, the recited differential ultracentrifugation steps further comprise the following steps: (5) resuspend partially purified exosome pellet in PBS total; (6) load Tris/sucrose/heavy water (D2O) solution at the bottom of a centrifuge tube, to make a cushion; (7) add the diluted exosomes gently above the sucrose cushion without disturbing the interface, and centrifuge 75 minutes at 100,000 x g at 4 °C; (8) with a 5-ml syringe fitted with an 18-G needle, collect ~3.5 ml of the Tris/sucrose/EhO cushion, which now contains exosomes, from the side of the tube; (9) transfer the exosomes to a fresh ultracentrifuge tube, dilute with PBS, and centrifuge 70 min at 100,000 x g, at 4 °C; and (10) resuspend the pellet in PBS.

Exosomes may be used immediately or substantially immediately, or they may be stored prior to use, for example at -80 °C or in liquid nitrogen. In some embodiments, the exosomes are concentrated prior to modification of any kind, whereas in other cases the exosomes are modified prior to concentration. The exosomes may be analyzed following the production process, following the concentration step, and/or during the process itself. Such analysis includes identifying one or more markers, identifying size, determining concentration, determining one or more specific activities for the exosomes (such as migration or immunosuppression, and/or anti-T cell activity), or a combination thereof. In some embodiments, high concentrations of exosomes are required for effective treatment of one or more diseases or disorders (e.g., one or more eye diseases, including, but not limited to, DED). In these cases, stem cells are induced to produce an increased amount of exosomes. Exemplary methods for induction of exosomes (“Exos”) from stem cells include treatment of the stem cells with cytokines, treatment with liposome stimulation using one or more stimulant liposomes such as neutral or cationic liposomes (Emam SE et al., Biol Pharm Bull. 2018;41(5):733-742), or other physical and/or biological methods previously described (Phan et al., J Extracell Vesicles. 2018; 7(1): 1522236). Generally, methods of isolating exosomes are known, including one or more of differential ultracentrifugation-based techniques, size-based techniques, immunoaffinity capture-based techniques, exosome precipitation, and microfluidics- based techniques (Li et al., Theranostics. 2017; 7(3): 789-804). In some embodiments, the MSC- Exos formulations comprise exogenous exosomes generated ex vivo from amniotic fluid MSCs, or derived from MSCs of other sources and/or tissues of origin.

Exosome modifications

In some embodiments, the exosomes comprise one or more certain characteristics or activities as a result of being produced from MSCs (including particular MSCs, such as from umbilical cord tissue). In other embodiments, the exosomes may be further modified. In particular cases, the exosomes are further modified to harbor and/or carry one or more bioactive substances, including any of the biological compounds described herein. In some cases, the MSCs are modified (e.g., transfected, transduced, electroporated, etc.), and modified exosomes are generated by the modified MSCs. In some cases, the exosomes themselves are modified (e.g., transfected, transduced, electroporated, etc.).

The modification of the exosomes may occur by any suitable method in the art, but in specific, non-limiting cases the exosomes are loaded with one or more bioactive substances by a vector, electroporation, transfection, using a cationic liposome transfection agent, or a combination thereof. Additionally, or alternatively, in some embodiments, MSCs are modified by any suitable method in the art, but in specific, non-limiting cases the MSCs are loaded with one or more bioactive substances by a vector, electroporation, transfection, using a cationic liposome transfection agent, or a combination thereof, and exosomes comprising the one or more bioactive substances are generated from the modified MSCs. Notably, bioactive substances may also be referred to as “biological compounds,” “agents,” and/or “therapeutic agents” interchangeably throughout this specification.

The bioactive substance(s) loaded into the exosomes in particular aspects are exogenous with respect to the MSCs. They can be introduced into the exosomes by a number of different techniques. In particular aspects of the disclosure, the exosomes are loaded by electroporation or the use of a transfection reagent.

In specific embodiments, the exosomes are of a specific size such that their size determines the type of bioactive substances that they can carry. In particular cases, the exosomes are 20-500 nm in size, including 30-350, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 50- 400, 50-350, 50-300, 50-250, 50-200, 50-150, 50-100, 100-400, 100-350, 100-300, 100-250, 100-200, 200-400, 200-350, 200-300, 200-250, 250-400, 250-350, 250-300, 300-400, 300-350, or 350-400 nm in size, or any range or value derivable therein.

Exosome loading via vector, electroporation, transfection, and the like

In some examples, exosomes are modified by loading the MSCs or exosomes with one or more bioactive substances by a vector, electroporation, transfection using a cationic liposome transfection agent, for example, or a combination thereof.

I. Vectors

In one embodiment, exosomes may be loaded by transforming or transfecting the MSCs with a nucleic acid construct that expresses the bioactive substance(s), such that the bioactive substance(s) are present in the exosomes as the exosomes are produced from the cell. In another embodiment, exosomes may also be loaded by directly transforming or transfecting the exosomes with a nucleic acid construct that expresses the bioactive substance(s).

In some embodiments, the nucleic acid construct encoding the bioactive substance(s) is comprised in a vector. In some cases, the nucleic acid construct encoding the bioactive substance(s) is linked to a promoter and incorporated into an expression vector, which is taken up and expressed by cells. The vectors can be suitable for replication and, in some cases, integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers In some embodiments, a suitable vector is capable of crossing the blood-brain barrier.

In certain embodiments, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. A number of viral-based systems have been developed for gene transfer into mammalian cells. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including self-inactivating lentivirus vectors). For example, adenoviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. Thus, in some embodiments, the nucleic acid encoding the polypeptide sequences is introduced into cells using a recombinant vector such as a viral vector including, for example, a lentivirus, a retrovirus, gamma-retroviruses, an adeno-associated virus (AAV), a herpesvirus, and/or an adenovirus.

Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that influence binding or targeting to cells (including, for instance, components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as, for example, agents mediating nuclear localization); and components that influence expression of the polynucleotide.

In some embodiments, such components also might include markers, such as, for instance, detectable and/or selection markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as, for example, the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. A large variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell’s nucleus or cytoplasm.

Eukaryotic expression cassettes may be included in the vectors, and can particularly contain (in a 5'-to-3' direction) regulatory elements including a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, a transcriptional termination/polyadenylation sequence, post-transcriptional regulatory elements, and origins of replication.

2. Electroporation

In particular embodiments of the disclosure, the MSCs and/or exosomes (e.g., MSC-Exos) are loaded by electroporation. As used herein, “electroporation” refers to application of an electrical current or electrical field to facilitate entry of an agent of interest into cells, exosomes, or derivatives thereof. One of skill in the art will understand that any method and technique of electroporation is contemplated by the present disclosure. In some embodiments, an electroporation system may be controlled to create electric current and send it through a cell- or exosome-containing solution. In some embodiments, a static electroporation apparatus is used. In some embodiments, a flow electroporation apparatus is used. In specific embodiments, static or flow electroporation is used with parameters described herein.

The process of electroporation generally involves the formation of pores in a cell membrane, or in an exosome, by the application of electric field pulses across a liquid cell suspension containing cells or exosomes. The pulse induces a transmembrane potential that causes the reversible breakdown of the cellular membrane. This action results in the permeation or “pore formation” of the cell membrane, which allows introduction of bioactive substance(s) into the cells or exosomes. During the electroporation process, cells or exosomes are often suspended in a liquid media and then subjected to an electric field pulse. The medium may be electrolyte, nonelectrolyte, or a mixture of electrolytes and non-electrolytes.

The outcome of an electroporation process is largely controlled by the magnitude of the applied electrical field (EF) pulse and the duration of the pulse. Field strength is measured as the voltage delivered across an electrode gap and may be expressed as kV/cm. Field strength is critical to surpassing the electrical potential of the cell membrane to allow the temporary reversible permeation or pore formation to occur in the cell membrane, and the methods of the present disclosure are capable of subjecting the cells to a range of electric field strengths. Field strength is a function of several factors, including, but not limited to, voltage magnitude of an applied electrical pulse, duration of the electrical pulse, and conductivity of the sample being electroporated.

The conductivity of the sample is a function of parameters comprising an ionic composition of electroporation buffer, concentration of an agent to be loaded, cell or exosome density, temperature, and pressure. Ionic strength of an electroporation buffer has a direct effect on the resistance of the sample, which in turn affects the pulse length or time constant of the pulse. The size and concentration of an agent will have an effect on the electrical parameters used to transfect the cell. Smaller molecules (for example, siRNA or miRNA) may need higher voltages with microsecond pulse lengths, while larger molecules (for example, DNA and proteins) may need lower voltages with longer pulse lengths. Cell or exosome density can be related to cell size. Generally, smaller cell or exosome sizes require higher voltages while larger cell or exosome sizes require lower voltages for successful cell membrane permeation.

Pulse duration, or pulse length, is the duration of time the sample is exposed to an electrical pulse and is typically measured as time in microseconds to milliseconds ranges. The pulse length works indirectly with the field strength to increase pore formation and therefore the uptake of target molecules. Generally, an increase in voltage should be followed by an incremental decrease in pulse length. Decreasing the voltage, the reverse is true. In addition to pulse duration, electrical pulses can also be characterized by pulse number, pulse width, pulse shape, pulse pattern, and pulse polarity. Thus, in some embodiments, the first and second electrical pulses further comprise characteristics selected from the group consisting of pulse number, width, shape, pattern, polarity, and combinations thereof. Electroporation can be carried out as a single pulse or as multiple pulses as disclosed herein to achieve maximum transfection efficiencies. Pulse pattern can comprise a single pulse or multiple pulses, and a combined duration of the multiple pulses corresponds to the pulse duration. Pulse polarity can be positive or negative. Pulse width depends on the wave shape generated by a pulse generator of an electroporation system. Pulse shape, or wave form, generally falls into two categories, square wave or exponential decay wave. Square wave pulses rise quickly to a set voltage level and maintain this level during the duration of the set pulse length before quickly turning off. Exponential decay waves generate an electrical pulse by allowing a capacitor to completely discharge. A pulse is discharged into a sample, and the voltage rises rapidly to the peak voltage set then declines over time. The pulse width in an exponential decay wave system corresponds to the time constant and is characterized by the rate at which the pulsed energy or voltage is decayed to one-third (1/3) the original set voltage. The time constant is modified by adjusting the resistance and capacitance values in an exponential decay, and the calculation for the time is T = RC, where T is time, R is resistance of a sample, and C is capacitance of an electroporation system power supply. Thus, in some embodiments, the rate of exponential decay is a function of a resistance of the sample and the capacitance of a power supply used to effect electroporation.

The strength of the electric field applied to the suspension and the length of the pulse (the time that the electric field is applied to a cell suspension) varies according to the cell or exosome type. To create a pore in the outer membrane of a cell or exosome, the electric field must be applied for such a length of time and at such a voltage as to increase permeability of the membrane to allow the bioactive substance(s) to enter the cell or exosome. As long as the pulse magnitude is above a certain threshold level, an increase in either the magnitude or the duration of the pulse generally results in a greater accumulation of the bioactive substance(s) inside the cell or exosomes (e.g., MSC-Exos).

Each electrical pulse applied to a cell suspension can be characterized by a certain amount of energy, which is equal to the product of voltage on the electrodes, current through the buffer, and duration of the high voltage pulse. Electroporation parameters may be adjusted to optimize the strength of the applied electrical field and/or duration of exposure such that the pores formed in membranes by the electrical pulse reseal after a short period of time, during which bioactive substance(s) have a chance to enter into the cell or exosome (e.g., MSC-Exos).

Electroporation conditions may vary depending on the charge and size of the bioactive substance(s). Typical field strengths are in the range of 20 to 1000 V/cm or kV/cm, such as 20 to 100 V/cm or kV/cm. In some embodiments, field strengths are 0.01 to 10, 0.01 to 1, 0.1 to 10, 0.1 to 1, or 1 to 10 V/cm or kV/cm, or any value from 0.01 to 10 V/cm or kV/cm or range derivable therein. Field strength is a function of several factors, including, for instance, voltage magnitude of an applied electrical pulse, duration of the electrical pulse, and conductivity of the sample being electroporated.

A voltage in the range of 150 mV or V to 250 mV or V, particularly a voltage of 200 mV or V, may be used for loading exosomes (e.g., MSC-Exos) with bioactive substance(s) according to the present disclosure. In some embodiments, the voltage magnitude of the electrical pulses is at most or at least about 0.001 to 10,000, 0.01 to 10,000, 0.1 to 10,000, 1 to 10,000, 1 to 9,000, 1 to 8,000, 1 to 7,000, 1 to 6,000, 1 to 5,000, 1 to 4,000, 1 to 3,000, 1 to 2,000, or 1 to 1,000 mV or V, or any value from 0.001 to 10,000 mV or V or range derivable therein. In some embodiments, the voltage magnitude of the electrical pulses is between 0.001 and 10,000, 0.01 and 10,000, 0.1 and 10,000, 1 and 10,000, 1 and 9,000, 1 and 8,000, 1 and 7,000, 1 and 6,000, 1 and 5,000, 1 and 4,000, 1 and 3,000, 1 and 2,000, or 1 and 1,000 mV or V, or any value from 0.001 to 10,000 mV or V or range derivable therein.

In some embodiments, the conductivity of the sample is a function of parameters comprising an ionic composition of electroporation buffer, concentration of an agent to be loaded into the cells, cell density, temperature, and pressure. In some embodiments, the conductivity of the sample is at most or at least about 0.01 Siemens/meter to 10 Siemens/meter, 0.01 Siemens/meter to 1 Siemens/meter, 0.1 Siemens/meter to 10 Siemens/meter, 0.1 Siemens/meter to 1 Siemens/meter, 1 Siemens/meter to 10 Siemens/meter, or any value from 0.01 Siemens/meter to 10 Siemens/meter or range derivable therein. In some embodiments, the conductivity of the sample is between 0.01 Siemens/meter and 10 Siemens/meter, 0.01 Siemens/meter and 1 Siemens/meter, 0.1 Siemens/meter and 10 Siemens/meter, 0.1 Siemens/meter and 1 Siemens/meter, 1 Siemens/meter and 10 Siemens/meter, or any value from 0.01 Siemens/meter to 10 Siemens/meter or range derivable therein. In some embodiments, the conductivity of the sample is between 1.0 and 3.0 Siemens/meter, any value from 1.0 Siemens/meter to 3.0 Siemens/meter, or any range or value derivable therein.

The ionic composition of a buffer used for electroporation can vary depending on the cell type. For example, highly conductive buffers such as PBS (Phosphate Buffered Saline <30 ohms) and HBSS (Hepes Buffer <30 ohms) or standard culture media, which may contain serum, may be used. Other buffers include hypoosmolar buffers in which cells absorb water shortly before an electrical pulse, which can result in cell swelling and can lower the optimal permeation voltage while ensuring the membrane is more easily permeable. Cells requiring the use of high resistance buffers (>3000 ohms) may require preparation and washing of the cells to remove excess salt ions to reduce the chance of arcing and sample loss. Ionic strength of an electroporation buffer has a direct effect on the resistance of the sample, which in turn affects the pulse length or time constant of the pulse. The volume of liquid in contact with an electrode also has significant effect on sample resistance for ionic solutions, and the resistance of the sample is inversely proportional to the volume of solution and pH. As volume increases, resistance decreases, which increases the probability of arcing and sample loss, while lowering the volume increases the resistance and decreases arc potential.

The size and concentration of an agent will have an effect on the electrical parameters used to transfect the cell. Smaller molecules (for example, siRNA or miRNA) may need higher voltages with microsecond pulse lengths, while larger molecules (for example, DNA and proteins) may need lower voltages with longer pulse lengths. The concentration of a bioactive substance may be, may be at least, may be at most, or may be from 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 75, 100, 150, 200, 250, 300 to about 350, 400, 500, 1000, 1500, 2000, 3000, 4000, or 5000 pg/mL, mg/mL, or g/mL, or any value from 0.01 to 5000 pg/mL, mg/mL, or g/mL, or any range derivable therein. In further embodiments, the concentration of the bioactive substance is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 125, 150, 175, 200, 225, 250, 275, or 300 pg/mL, mg/mL, or g/mL, or any value from 1 to 300 pg/mL, mg/mL, or g/mL, or any range derivable therein. In certain embodiments, the concentration of the bioactive substance is at least 1 pg/mL, mg/mL, or g/mL. In some embodiments, concentration of the bioactive substance is between 1 pg/mL and 200 pg/mL, such as between 5 pg/mL and 100 pg/mL, any value from 5 pg/mL and 100 pg/mL, or any range derivable therein.

Cell density can be related to cell size. Generally, smaller cell sizes require higher voltages while larger cell sizes require lower voltages for successful cell membrane permeation. The temperature at which cells are maintained during electroporation can affect the efficiency of the electroporation. Samples pulsed at high voltage or exposed to multiple pulses and long pulse durations can cause sample heating, which can contribute to increased cell death and lower transfection efficiency. Maintaining the sample at a lower temperature can diminish the effects of overheating on cell viability and efficiency. In general, the standard pulse voltage used for cells at room temperature should be approximately doubled for electroporation at 4°C in order to effectively permeate the cell membrane.

Pulse width depends on the wave shape generated by a pulse generator of an electroporation system. Pulse shape, or wave form, generally falls into two categories, square wave or exponential decay wave. Square wave pulses rise quickly to a set voltage level and maintain this level during the duration of the set pulse length before quickly turning off. In some embodiments, the pulse generator generates a square wave pulse, and pulse width can be inputted directly. Exponential decay waves generate an electrical pulse by allowing a capacitor to completely discharge. A pulse is discharged into a sample, and the voltage rises rapidly to the peak voltage set then declines over time. In some embodiments, the pulse generator generates an exponential decay wave pulse, and the pulse width is a function of a rate of exponential decay. The pulse width in an exponential decay wave system corresponds to the time constant and is characterized by the rate at which the pulsed energy or voltage is decayed to one-third (1/3) the original set voltage. The time constant is modified by adjusting the resistance and capacitance values in an exponential decay, and the calculation for the time is T = RC, where T is time, R is resistance of a sample, and C is capacitance of an electroporation system power supply. Thus, in some embodiments, the rate of exponential decay is a function of a resistance of the sample and the capacitance of a power supply used to effect electroporation.

The resistance of a sample can be at most or at least 1 ohm to 10000 ohms, 1 ohm to 9000 ohms, 1 ohm to 8000 ohms, 1 ohm to 7000 ohms, 1 ohm to 6000 ohms, 1 ohm to 5000 ohms, 1 ohm to 4000 ohms, 1 ohm to 3000 ohms, 1 ohm to 2000 ohms, 1 ohm to 1000 ohms, 1 ohm to 900 ohms, 1 ohm to 800 ohms, 1 ohm to 700 ohms, 1 ohm to 600 ohms, 1 ohm to 500 ohms, 1 ohm to 400 ohms, 1 ohm to 300 ohms, 1 ohm to 200 ohms, 1 ohm to 100 ohms, 1 ohm to 90 ohms, 1 ohm to 80 ohms, 1 ohm to 70 ohms, 1 ohm to 60 ohms, 1 ohm to 50 ohms, 1 ohm to 40 ohms, 1 ohm to 30 ohms, 1 ohm to 20 ohms, 1 ohm to 10 ohms, or any value from 1 ohm to 10000 ohms, or any range derivable therein. In some embodiments, the resistance of the sample is between 1 ohm and 10000 ohms, 1 ohm and 9000 ohms, 1 ohm and 8000 ohms, 1 ohm and 7000 ohms, 1 ohm and 6000 ohms, 1 ohm and 5000 ohms, 1 ohm and 4000 ohms, 1 ohm and 3000 ohms, 1 ohm and 2000 ohms, 1 ohm and 1000 ohms, 1 ohm and 900 ohms, 1 ohm and 800 ohms, 1 ohm and 700 ohms, 1 ohm and 600 ohms, 1 ohm and 500 ohms, 1 ohm and 400 ohms, 1 ohm and 300 ohms, 1 ohm and 200 ohms, 1 ohm and 100 ohms, 1 ohm and 90 ohms, 1 ohm and 80 ohms, 1 ohm and 70 ohms, 1 ohm and 60 ohms, 1 ohm and 50 ohms, 1 ohm and 40 ohms, 1 ohm and 30 ohms, 1 ohm and 20 ohms, 1 ohm and 10 ohms, or any value from 1 ohm to 10000 ohms, or any range derivable therein. In some embodiments, the resistance of the sample is between 1 ohm and 1000 ohms, any value from 1 ohm to 1000 ohms, or any range derivable therein.

The bioactive substances may be any biological compound, agent, and/or therapeutic agent described herein, including, for instance, proteins and peptides (e.g., synthetic, natural, and mimetics, including antibodies or fragments thereof), oligonucleotides (e.g., anti-sense oligonucleotides, ribozymes, etc.), short nucleic acid sequences less than about 1000 nucleotides (e.g., double sense linear DNA, inhibitory RNA, siRNA, miRNA, anti-miRNA, shRNA, expression vectors, etc ), ribonucleoproteins, vectors, small molecules, lipids, carbohydrates, cytokines, hemobioactive substances, anti-cancer drugs, anti-inflammatory drugs, anti-fungal drugs, anti-viral drugs, anti-microbial drugs, thrombomodulating agents, immunomodulating agents, and the like.

In certain embodiments, the bioactive substance is miRNA, and the concentration of miRNA is between 1 pg/mL and 200 pg/mL, such as between 5 pg/mL and 100 pg/mL, any value from 5 pg/mL and 100 pg/mL, or any range derivable therein. In certain embodiments, the bioactive substance is siRNA, shRNA, and/or RNA, and the concentration of siRNA, shRNA, and/or RNA is between 1 pg/mL and 200 pg/mL, such as between 10 pg/mL and 50 pg/mL, any value from 10 pg/mL and 50 pg/mL, or any range derivable therein. In certain embodiments, the bioactive substance is DNA, the DNA is at least, at most, or about 1000 base pairs, and the concentration of DNA is between 1 pg/mL and 200 pg/mL, such as between 10 pg/mL and 100 pg/mL, any value from 10 pg/mL and 100 pg/mL, or any range derivable therein.

In certain embodiments, the bioactive substance is protein, peptides, lipids, and/or drugs, the protein, peptides, lipids, and the concentration of protein, peptides, lipids, and/or drugs is between 1 pg/mL and 1000 mg/mL, such as between 100 pg/mL and 3 mg/mL, any value from 100 pg/mL and 3 mg/mL, or any range derivable therein, certain embodiments, the bioactive substance is protein, peptides, lipids, and/or drugs, the protein, peptides, lipids, and the concentration of protein, peptides, lipids, and/or drugs is 1 pg/mL or mg/mL, 10 pg/mL or mg/mL, 20 pg/mL or mg/mL, 960 pg/mL or mg/mL, 970 pg/mL or mg/mL, 980 pg/mL or mg/mL, 990 pg/mL or mg/mL, 1000 pg/mL or mg/mL, or any range or value derivable therein.

In particular embodiments, the parameters for an electroporation pulse comprise a power between 100 to 240 VAC, with a frequency of between 50 to 60 Hz and a voltage of about 1500 V, with a limitation of 100 A. In some embodiments, all components for electroporation, including but not limited to buffers, exosomes and cuvettes or electrodes, should be kept at at least about 4°C. In some embodiments, the electroporation pulse is performed at at least about 25°C, and the electroporated exosomes are placed at at least about 4°C following electroporation, for example, immediately following electroporation.

Electroporation is capable of achieving loading, or transfection, efficiencies of bioactive substance(s) into cells or exosomes of greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% (or any range or value derivable therein). In some embodiments, a loading efficiency of bioactive substance(s) is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Transfection efficiency can be measured either by the percentage of the cells that express the product of the gene or the secretion level of the product expressed by the gene or by directly measuring concentration of the bioactive substance(s) in the exosomes using, for example, realtime quantitative PCR (RT-qPCR) or similar quantitative analyses.

3. Transfection

In particular embodiments of the disclosure, the MSCs and/or exosomes (e.g, MSC-Exos) are loaded by use of a transfection reagent. Particular transfection reagents for use in accordance with the present disclosure include, but are not limited to, cationic lipids and/or liposomes.

The use of lipid formulations is contemplated for the introduction of the bioactive substance(s) into MSCs and/or exosomes (e.g., MSC-Exos). In another embodiment, the bioactive substance(s) may be associated with a lipid. The bioactive substance(s) associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, and/or otherwise associated with a lipid. Lipid-, lipid/DNA-, lipid/expression vector-, or lipid/bioactive substance(s)- associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as, for instance, fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In at least one embodiment, the bioactive substance(s) may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The amount of liposomes used may vary upon the nature of the liposome as well as the entity to be transfected, for example, about 5 to about 20 pg vector DNA per 1 to 10 million cells may be contemplated.

Liposome-mediated bioactive substance(s) delivery and expression of bioactive substance(s) in vitro has generally been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of bioactive substance(s) in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980). In certain embodiments, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and/or a liposome.

In various embodiments, lipids suitable for use can be obtained from commercial sources. For example, lipofectamine can be obtained from Thermo Fisher Scientific, Waltham, Mass.; dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem- Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform can be used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al. (1991) Glycobiology 5: 505-510). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.

In specific embodiments, the exosomes (e.g., MSC-Exos) are able to be loaded with any type of bioactive substance(s). Non-limiting examples of suitable bioactive substance(s) include, but are not limited to, bioactive materials. Bioactive materials particularly suited to incorporation into exosomes include, but are not limited to, therapeutic and prophylactic agents. Non-limiting examples of bioactive materials include, but are not limited to, proteins and peptides (e.g., synthetic, natural, and mimetics, including antibodies or fragments thereof), oligonucleotides (e.g., anti-sense oligonucleotides, ribozymes, etc.), short nucleic acid sequences less than about 1000 nucleotides (e.g., double sense linear DNA, inhibitory RNA, siRNA, miRNA, anti-miRNA, shRNA, expression vectors, etc.), ribonucleoproteins, vectors, small molecules, lipids, carbohydrates, cytokines, hemobioactive substances, anti-cancer drugs, anti-inflammatory drugs, anti-fungal drugs, antiviral drugs, anti-microbial drugs, thrombomodulating agents, immunomodulating agents, and the like.

It is to be understood that other bioactive substance(s) can also be introduced into the exosomes (e.g., MSC-Exos). These bioactive substances of interest include, but are not limited to, smooth muscle inhibitors, anti-infective bioactive substances (e.g., antibiotics, antifungal agents, antibacterial agents, antiviral agents), chemotherapeutic/antineoplastic agents, and the like. The bioactive substance(s) may be bioactive substances for eye diseases (e.g., DED), cancer bioactive substances, bioactive substances for auto- or alloimmune disease, bioactive substances for microbial infection, bioactive substances for heart disease, bioactive substances for lung disease, bioactive substances for liver disease, bioactive substances for kidney disease, bioactive substances for neurological disease, or a combination thereof. For cancer bioactive substances, the bioactive substance(s) may be, for example, a drug, small molecule, antibody, inhibitory RNA targeting an oncogene, tumor suppressor protein, or any combination or mixture thereof.

In some embodiments, the exosomes (e.g., MSC-Exos) comprise one or more biological compounds, such as, for instance, short DNA sequences and/or one or more short RNA sequences. The one or more short RNA sequences may comprise inhibitory RNA, including miRNA, anti- miRNA, siRNA, shRNA, Morpholino oligomers, or any combination or mixture thereof. A microRNA (“miRNA” or “miR”) can refer to a small single- stranded non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression by basepairing with complementary sequences within mRNA molecules. Upon base-pairing between the miRNA and complementary mRNA molecule, the mRNA molecule is silenced by either cleavage of the mRNA strand into two pieces, destabilization of the mRNA through shortening of its poly(A) tail, and/or less efficient translation of the mRNA into proteins by ribosomes. Anti- miRNA (also known as “anti-miRNA oligonucleotide” or “AMO”) can refer to synthetically designed molecules used to neutralize miRNA function in cells. By controlling the miRNA that regulate mRNAs in cells, AMOs can be used as further regulation through, for example, a steric blocking mechanism as well as hybridization to miRNA. These interactions between miRNA and AMOs can be therapeutic in disorders in which miRNA over/under expression occurs or aberrations in miRNA lead to coding issues. Small interfering RNA (“siRNA” or “ “short interfering RNA” or “silencing RNA” can refer to a class of double-stranded RNA non-coding RNA molecules that operate in sequence-specific suppression of gene expression. siRNA interfere with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation of the mRNA into amino acids and then proteins. siRNA may be introduced into cells using an expression vector in which the siRNA sequence is modified to introduce a short loop between the two strands. The resulting transcript may be a short hairpin RNA (“shRNA” or “short hairpin RNA”), which can be processed into a functional siRNA by Dicer, an enzyme that cleaves double-stranded RNA into siRNA (and pre- microRNA into microRNA). Morpholino oligomers (“morpholino” or “phosphorodiamidate morpholino oligomer” or “PMO”) can refer to oligomer molecules containing DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos can sterically block access of other molecules to small specific sequences of the basepairing surfaces of RNA, thereby modifying gene expression. For example, Morpholinos can modify pre-mRNA splicing, block translation by interfering with progression of the ribosomal initiation complex from the 5’ cap to the start codon, or block other functional sites on RNA (z.e., blocking miRNA activity and maturation, blocking ribozyme activity, etc.) depending on the Morpholino’ s base sequence.

In some embodiments, the exosomes (e.g., MSC-Exos) comprise biological compounds that include, for instance, one or more antibodies or antibody fragments. The term “antibody” as referred to herein includes, for example, whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof. An antibody can refer to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). An antibody of use in at least one embodiment of the invention may be a monoclonal antibody or a polyclonal antibody and will preferably be a monoclonal antibody. An antibody of use in the invention may be a chimeric antibody, a CDR- grafted antibody, a nanobody, a human or humanized antibody or an antigen binding portion of any thereof. For the production of both monoclonal and polyclonal antibodies, the experimental animal is typically a non-human mammal such as a goat, rabbit, rat or mouse, but may also be raised in other species such as camelids. The term “antigen-binding portion” of an antibody can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Non-limiting examples of binding fragments encompassed within the term “antigen binding portion” of an antibody include a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, a Fd fragment, a Fv fragment, a dAb fragment, and an isolated complementarity determining region (CDR). Single chain antibodies such as scFv antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies. An antibody of use in at least one embodiment of the invention may be a human antibody or a humanized antibody.

In some embodiments, the exosomes (e.g., MSC-Exos) are loaded with one or more drugs, including drugs for treating one or more eye diseases e.g., DED), drugs for treating other diseases such as cancer e.g., one or more chemotherapies), or the like. A wide variety of chemotherapeutic substances may be used in accordance with the present embodiments. The term “chemotherapy” can refer to the use of drugs to treat cancer. A “chemotherapy” or “chemotherapeutic substance” is used to connote a compound or composition that is administered in the treatment of cancer. These bioactive substances or drugs can be categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, a bioactive substance may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Non-limiting examples of various types of biological compounds that can be delivered by exosomes (e.g., MSC-Exos) according to at least one embodiment of the invention are disclosed below.

In at least one embodiment, non-limiting examples of drugs or therapeutic compounds include, for instance, alkylating agents, such as thiotepa, procarbazine, and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as plicomycin and the enediyne antibiotics (e.g, calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6- mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, gemcitabine, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., taxol, paclitaxel, and docetaxel; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as transplatinum, cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; protease inhibitors, like bortezomib; kinase inhibitors, like palbociclib, ibrutinib, dasatinib, pp2, pazopanib, and gefitinib; checkpoint inhibitors, like nivolumab, pembrolizumab, and ipilimumab; colony stimulating factors, like pegfilgrastim and filgrastim; monoclonal antibodies, like bevacizumab, trastuzumab, and rituximab; immunomodulatory agents, like lenalidomide; navelbine; farnesyl-protein transferase inhibitors; pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations thereof.

In at least one embodiment, non-limiting examples of antimicrobial agents (that is, any natural or synthetic substance that kills or inhibits the growth of microorganisms or pathogens, such as bacteria, fungi, algae, or viruses) include, for instance, an antibiotic, an antifungal, an antiviral, and combinations thereof.

In at least one embodiment, non-limiting examples of antibiotics include, but are not limited to, aminoglycosides, ansamycins, carbacephem, carbapenems, cephalosporins (first, second, third, fourth, or fifth generation), glycopeptides, linocsamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quinolones/fluoroquinolones, sulfonamides, tetracyclines, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole, trimethoprim, and combinations thereof. Aminoglycosides can include, but are not limited to, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, and Spectinomycin. Ansamycins can include, but are not limited to, Geldanamycin, Herbimycin, and Rifaximin. Carbacephem can include, but is not limited to, Loracarbef. Carbapenems can include, but are not limited to, Ertapenem, Doripenem, Imipenem/Cilastatimn, and Meropenem. Cephalosporins can include, but are not limited to, Cefadroxil, Cefazolin, Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan, Cefotan, Cefamandole, Cefmetazole, Cefonicid, Loracarbef, Cefprozil, Cefzil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole. Glycopeptides can include, but are not limited to, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, and Oritavancin. Lincosamides can include, but are not limited to, Clindamycin and Lincomycin. Lipopeptides can include, but are not limited to, Daptomycin. Macrolides can include, but are not limited to, Azithromycin, Clarithromycin, Erythromycin, Roxithromycin, Telithromycin, Spiramycin, and Fidaxomicin. Monobactams can include, but are not limited to, Aztreonam. Nitrofurans can include, but are not limited to, Furazolidone and Nitrofurantoin. Oxazolidinones can include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Penicillins can include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Dicloxacillin, Flucloxxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate. Polypeptides can include, but are not limited to, Bacitracin, Colistin, and Polymyxin B. Quinol ones/fluoroquinolones can include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin. Sulfonamides can include, but are not limited to, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, and Sulfoamidochrysoidine. Tetracyclines can include, but are not limited to, Demeclocy cline, Doxycydine, Metacycline, Minocycline, Oxytetracycline, and Tetracycline. In some embodiments, the antibiotic is a macrolide. In some embodiment, the antibiotic is azithromycin.

Non-limiting examples of antibiotics also include, but are not limited to, antimicrobial proteins or peptides. The antimicrobial proteins or peptides can be of any class, including, but not limited to, the following classes: anionic peptides (e.g., dermicidin), linear cationic a-helical peptides (e.g, LL37), cationic peptides enriched for proline, arginine, phenylalanine, glycine, or tryptophan, anionic and cationic peptides that contain cysteine and form disulfide bonds (e.g., defensins), and combinations thereof. Defensins can include, but are not limited to, trans- defensins, cis-defensins, and related defensin-like proteins. Trans-defensins include, but are not limited to, a-defensins and b-defensins.

Non-limiting examples of antibiotics also include, but are not limited to, anti- mycobacterials, including, but not limited to, isoniazid, rifampin, streptomycin, rifabutin, ethambutol, pyrazinamide, ethionamide, aminosalicylic, and cycloserine. Non-limiting examples of antivirals include, but are not limited to, anti-herpes agents such as acyclovir, famciclovir, foscamet, ganciclovir, acyclovir, idoxuridine, sorivudine, trifluridine, valacyclovir and vidarabine; anti-retroviral agents such as ritonavir, didanosine, stavudine, zalcitabine, tenovovir and zidovudine; and other antiviral agents such as, but not limited to, amantadine, interferon-alpha, ribavirin, rimantadine, and combinations thereof.

Non-limiting examples of antifungals include, but are not limited to, polyene antifungals (e.g, amphotericin B, nystatin, natamycin, and the like), flucytosine, imidazoles (e.g., n- ticonazole, clotrimazole, econazole, ketoconazole, and the like), triazoles (e.g, itraconazole, fluconazole, and the like), griseofulvin, terconazole, butoconazole ciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifme, terbinafme, any other antifungal that can be lipid encapsulated or complexed, and combinations thereof.

In at least one embodiment, the exosomes (e.g, MSC-Exos) are loaded with biological compounds that include, for instance, one or more bioactive substances for the treatment of a disease (e.g, an eye diseases such as DED, an auto- or alloimmune disease, or the like). Nonlimiting examples of such disease therapies include, but are not limited to, anti-microbial agents (for example, antibiotics, antiviral agents and anti-fungal agents), anti-tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (for example, azathioprine, or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen or naproxen sodium), cytokines (for example, interleukin- 10 or transforming growth factor- beta), hormones (for example, estrogen), and/or a vaccine. Tn addition, immunosuppressive and/or tolerogenic agents including, but not limited to, calcineurin inhibitors (e.g., cyclosporin and tacrolimus); mTOR inhibitors (e.g., Rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic substances (e.g., Methotrexate, Treosulfan, Busulfan); irradiation; and/or chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors) can be administered.

In alternative embodiments, the exosomes (e.g., MSC-Exos) are not loaded with a therapeutic drug, but instead are loaded with one or more gene-modifying components, such as that comprise a CRISPR-Cas system, including a specific guide RNA and an endonuclease. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA- processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus. The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include, for example, a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains).

In some embodiments, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. Typically, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. The CRISPR system can induce double stranded breaks (DSBs) at the target site, followed by disruptions or alterations as discussed herein. In other embodiments, Cas9 variants, deemed “nickases,” are used to nick a single strand at the target site. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression. The target sequence may comprise any polynucleotide, such as DNA and/or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of the cell, such as, for instance, within an organelle of the cell.

In some embodiments, exosomes (e.g., MSC-Exos) loaded with one or more vectors can be introduced into cells to drive expression of one or more elements of the CRISPR system such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells via exosomes as proteins and/or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. The vector may comprise one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. A vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein.

Methods of using exosomes, including MSC-Exos, in therapies and/or treatments

In particular embodiments, and as described above herein, exosomes (e.g., MSC-Exos) are useful for the treatment of one or more medical conditions, including one or more eye disorders or diseases, such as, for instance, DED. The exosomes may be used for the systemic or local delivery of therapeutic compounds. The disclosure encompasses methods for delivering bioactive substances of interest using exosomes as a delivery vehicle. The present disclosure also includes methods of treating a patient in need of one or more bioactive substances comprising administering to the patient an effective amount of exosomes containing the bioactive substance(s). The exosomes of the disclosure may or may not be utilized directly after production. In some cases they are stored for later purpose. In any event, they may be utilized in therapeutic or preventative applications for a mammalian subject (e.g., human, dog, cat, horse, etc.), such as a patient. The individual may be in need of exosome-based therapy for a medical condition of any kind, including any eye disease, cancer, infections of any kind, any immune disorder, any tissue injury, any skin disorder, any wounds, any trauma, and/or any burns or injuries (e.g., eye tissue injuries), as non-limiting examples. Methods may be employed with respect to individuals who have tested positive for a medical condition, who have one or more symptoms of a medical condition, and/or who are deemed to be at risk for developing such a condition.

Individuals treated with the present exosome-based therapy, including, but not limited to, any of the compositions described herein containing MSC-Exos (described herein as “MSC Compositions”), may or may not have been treated for the particular medical condition prior to receiving the exosome-based therapy. In some embodiments, the patient has received at least 1, 2, 3, 4, 5, 6, 7, 8, or more prior treatments for a medical condition. The prior treatments may include a treatment or therapy described herein. In some embodiments, the prior treatments comprise conventional therapies, including, for instance, conventional eye treatment therapies, conventional chemotherapies, conventional radiotherapy, conventional antiviral therapies, conventional antiseptic and antibacterial therapies, conventional immunosuppressive therapies, conventional anti-inflammatory therapies, conventional burn treatment therapies, and the like. In some embodiments, the patient had received the prior therapy within 10, 20, 30, 40, 50, 60, 70, 80, or 90 days or hours of administration of the current compositions and exosomes of the disclosure. In some embodiments, the patient is one that has undergone prior therapy and has failed the prior treatment either because the prior treatment was not effective or because the prior treatment was deemed too toxic.

Exosomes (e.g., MSC-Exos) loaded with one or more bioactive substances as contemplated herein, and/or pharmaceutical compositions comprising the same, can be administered either alone or in any combination, and in at least some embodiments, together with a pharmaceutically acceptable carrier or excipient, and can be used for the prevention, treatment, or amelioration of one or more eye diseases (e.g., DED), cancer, immune disorders, heart disease, lung disease, microbial infections, tissue injuries, skin disorders, wounds, trauma, and/or burns or injuries (e.g., eye tissue injuries) of any kind.

Exosomes (e.g., MSC-Exos) loaded with one or more bioactive substances as contemplated herein, and/or pharmaceutical compositions comprising the same, can also be used for the mitigation of chemo- and radiotherapy-induced CNS toxicity, and the treatment of other chemotherapy or radiation-induced vital organ toxicities involving the heart, lung, kidney, gastrointestinal tract where regenerative or reparative properties are often needed.

Aspects of the disclosure include methods for treating, reversing, or ameliorating cognitive impairment in response to or providing neuroprotection against chemo- and radiotherapy-induced central nervous system (CNS) toxicity. In specific embodiments, exosomes (e.g., MSC-Exos, which may be derived from, for instance, umbilical cord tissue-derived MSCs (UC-Exos)) are useful for treating or reversing cognitive impairment in response to or providing neuroprotection against chemo- and radiotherapy-induced central nervous system (CNS) toxicity. Methods and compositions of the disclosure allow for generation of a large scale of activated exosomes from MSC-Exos, carrying bioactive substance(s), including at least miR, anti-miR, siRNA, and therapeutic drugs for treating or reversing cognitive impairment in response to or providing neuroprotection against chemo- and radiotherapy-induced central nervous system (CNS) toxicity.

In certain embodiments, the exosomes (e.g., MSC-Exos) are utilized for individuals in need of regeneration and/or reparation of tissue for any reason. The tissue in need of regeneration and/or reparation may be of any kind, but in specific embodiments the tissue is any of the tissues of the eye, soft tissue (e.g., fat, fibrous tissue such as tendons and/or ligaments), muscle (e.g., smooth muscle, skeletal muscle, and/or cardiac muscle), synovial tissue, blood vessels, lymph vessels, and/or nerves), brain, lung, spleen, liver, heart, kidney, pancreas, intestine, testis, or bone. For example, the individual may be in need of regeneration and/or reparation of one or more eye tissues due to the effect of one or more eye diseases and/or disorders (e.g., DED), As another non-limiting example, the individual may be in need of regeneration and/or reparation of heart, lung, kidney, and/or gastrointestinal tract tissue due to chemotherapy or radiation-induced vital organ toxicities. The individual may be in need of regeneration and/or reparation of soft tissue (e.g., fat, fibrous tissue such as tendons and/or ligaments), muscle (e.g., smooth muscle, skeletal muscle, and/or cardiac muscle), synovial tissue, blood vessels, lymph vessels, and/or nerves) due to inflammation, trauma (e.g., contusions, sprains, tendonitis, bursitis, stress injuries, strains), burns (e.g., thermal bums, chemical bums, electric burns, frostbite), or any combination thereof. In some embodiments, the tissue is in need of regeneration or repair due to toxicity due to burns (e.g., thermal burns, chemical burns, electric bums, frostbite) or trauma e.g., contusions, sprains, tendonitis, bursitis, stress injuries, strains), and/or due to toxicity due to a prior treatment for bums (e.g., thermal burns, chemical bums, electric bums, frostbite) or trauma (e.g., contusions, sprains, tendonitis, bursitis, stress injuries, strains). In particular embodiments, the exosomes (e.g., MSC- Exos) produced by methods encompassed herein are useful as regenerative and/or reparative therapies to target soft tissues and organs including the eyes, brain, lung, spleen, liver, heart, kidney, pancreas, intestine, testis, and bone, as examples of target tissues. The exosomes in such cases are therapeutic at least in part because they are suitable to migrate in the individual.

In certain embodiments, the exosomes (e.g., MSC-Exos, including, for instance, UC-Exos) are utilized for individuals in need of regeneration and/or reparation of skin for any reason. For example, the individual may be in need of regeneration and/or reparation of skin due to chemotherapy or radiation-induced vital organ toxicities. The individual may be in in need of regeneration and/or reparation of skin due to toxicity due to bums (e.g., thermal burns, chemical bums, electric burns, frostbite) or trauma (e.g., cutaneous wound damage, contusions, cuts, lacerations, gashes, tears, punctures, scrapes, abrasions, scratches, bites, stings, bruises, pressure injuries, crush injuries, incisions) and/or due to toxicity due to a prior treatment administered for bums (e.g., thermal bums, chemical bums, electric bums, frostbite) or trauma (e.g., cutaneous wound damage, contusions, cuts, lacerations, gashes, tears, punctures, scrapes, abrasions, scratches, bites, stings, bruises, pressure injuries, crush injuries, incisions). The individual may be in need of regeneration and/or reparation of skin due to a skin disorder. Non-limiting examples of skin disorders include inflammation, aging, skin cancer, acne, cold sores, blisters, seromas, hematomas, ulcers, carbuncles, warts, psoriasis, eczema, cellulitis, lupus, actinic keratosis, keratosis pilaris, shingles, hives, melasma, impetigo, sunburn, dermatitis, rosacea, thermal burns, chemical bums, electric burns, frostbite, cutaneous wound damage, contusions, cuts, lacerations, gashes, tears, punctures, scrapes, abrasions, scratches, bites, stings, bruises, pressure injuries, crush injuries, incisions, or any combination thereof. In certain embodiments, the exosomes (e.g., MSC-Exos) are utilized for individuals in need of wound healing (e.g., wound repair) for any reason, including, for instance, due to one or more eye disorders such as DED. For example, the individual may be in need of regeneration and/or reparation of wounded skin or tissue due to chemotherapy or radiation-induced vital organ toxicities. The individual may be in need of regeneration and/or reparation of wounded skin or tissue due to toxicity due to burns (e.g., thermal bums, chemical bums, electric burns, frostbite) or trauma (e.g., sprains, tendonitis, bursitis, stress injuries, strains, cutaneous wound damage, contusions, cuts, lacerations, gashes, tears, punctures, scrapes, abrasions, scratches, bites, stings, bruises, pressure injuries, crush injuries, incisions) and/or toxicity due to a prior treatment forburns (e.g., thermal burns, chemical burns, electric burns, frostbite) or trauma (e.g., sprains, tendonitis, bursitis, stress injuries, strains, cutaneous wound damage, contusions, cuts, lacerations, gashes, tears, punctures, scrapes, abrasions, scratches, bites, stings, bruises, pressure injuries, crush injuries, incisions). The individual may be in need of regeneration and/or reparation of wounded skin or tissue due to disease effects (e.g., the effects of DED), inflammation, aging, skin cancer, acne, cold sores, blisters, seromas, hematomas, ulcers, carbuncles, warts, psoriasis, eczema, cellulitis, lupus, actinic keratosis, keratosis pilaris, shingles, hives, melasma, impetigo, sunburn, dermatitis, rosacea, thermal bums, chemical bums, electric bums, frostbite, cutaneous wound damage, contusions, cuts, lacerations, gashes, tears, punctures, scrapes, abrasions, scratches, bites, stings, bruises, pressure injuries, crush injuries, incisions, sprains, tendonitis, bursitis, stress injuries, strains, or any combination thereof.

Aspects of the disclosure include methods for treatment of one or more diseases, including, for example, cancer. In some cases, the exosomes are useful for one or more cancers. In specific embodiments, exosomes derived from umbilical cord tissue-derived MSCs (UC-Exos) are useful for the treatment of cancer and for the systemic delivery of therapeutic compounds for the cancer. Methods and compositions of the disclosure allow for generation of a large scale of activated exosomes from UC-Exos, carrying bioactive substance(s), including at least miR, anti-miR, siRNA, and/or therapeutic drugs for the treatment of cancer.

In some embodiments, the present invention includes a composition for delivering target specific exosomes (e.g., MSC-Exos) to the cytoplasm of a tumor cell, wherein the exosomes modulate angiogenesis. In embodiments, the composition (e.g., any of the MSC Compositions described herein) comprises an exosome e.g., MSC-Exos), a bioactive substance, and/or a plasmid. In other embodiments, the exosome (e.g., MSC-Exos) is isolated from autologous cells of a subject, from a cell line, from a primary cell culture, and/or from a mesenchymal stem cell. In other embodiments, the at least one plasmid is an RNA plasmid, a DNA plasmid, or any combination thereof.

Cancers for which the present exosomes are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. In cases wherein the individual has cancer, the cancer may be primary, metastatic, resistant to therapy, and so forth. In specific cases, the present therapy is useful for individuals with cancers that have been clinically indicated to be subject to immune cell regulation, including multiple types of solid tumors (melanoma, colon, lung, breast, and head and neck cancers), for example. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, glioblastoma, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo- alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin’s disease; Hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS- related lymphoma; Waldenstrom’s macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

In some embodiments, cancers for which the present exosomes (e.g., MSC-Exos) are useful is glioblastoma multiforme (GBM). Adult glioblastoma is notoriously recalcitrant to most therapies, not only because its molecular, cellular and immune biology are unique compared with other cancers, but also because of the formidable delivery challenges imposed by the blood brain/blood tumor barriers (BBB/BTB). Consequently, there is an urgent need to identify anticancer therapeutics that specifically target GBMs, and to elucidate strategies for delivering these new agents across the BBB/BTB. In some cases, these exosomes home efficiently to human gliomas, overcoming the BBB/BTB.

In some embodiments, exosomes used to treat GBM are loaded with the anti-GMB miRNA miR-124. Validation studies proved that miR-124 is highly efficacious against all subtypes of glioma stem cells, functioning by down-regulating GBM-relevant targets, particularly F0XA2, and leading to apoptotic cell death. MiR-124 also enhances T-cell responses by inhibiting STAT- 3, a known mediator of immune suppression in GBM, further supporting its therapeutic potential. Recent work has also shown that miR-124 reverses neurodegeneration after brain injury, rendering miR-124 one of the first anti-glioma agents that may also mitigate neuro-toxicity.

Aspects of the disclosure include methods for treatment of immune disorders. In some cases, the exosomes (e.g., MSC-Exos) are useful for one or more immune disorders. In specific embodiments, exosomes derived from umbilical cord tissue-derived MSCs (UC-Exos) are useful for the treatment of immune disorders and for the systemic delivery of therapeutic compounds for the immune disorders. Methods and compositions of the disclosure allow for generation of a large scale of activated exosomes from UC-Exos, carrying bioactive substance(s), including at least miR, anti-miR, siRNA, and therapeutic drugs, for the treatment of immune disorders.

Immune disorders for which the present exosomes are useful include, but are not limited to, autoimmune or inflammatory disorders. Non-limiting examples of autoimmune or inflammatory disorders include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet’s disease, bullous pemphigoid, cardiomyopathy, celiac spate-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn’s disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves’ disease, Guillain- Barre, Hashimoto’s thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere’s disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (such as minimal change disease, focal glomerulosclerosis, or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud’s phenomenon, Reiter’s syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitides (such as polyarteritis nodosa, takayasu arteritis, temporal arteritis/giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, graft versus host disease (GVHD), and Wegener’s granulomatosis. Thus, some examples of an autoimmune disease that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosis, type I diabetes mellitus, Crohn’s disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subject can also have an allergic disorder such as asthma.

Aspects of the disclosure include methods for treatment of heart disease of any kind, including at least coronary artery disease, heart failure, cardiomyopathy, valvular heart disease, arrhythmia, genetic defects of the heart, and so forth. Aspects of the disclosure include methods for treatment of lung disease, such as pulmonary hypertension, asthma, bronchopulmonary dysplasia (BPD), allergy, cystic fibrosis, Chronic Obstructive Pulmonary Disease, idiopathic pulmonary fibrosis, acute respiratory distress syndrome (ARDS), pneumonia, pleural effusion, and so forth.

Aspects of the disclosure include methods for treatment of a microbial infection of any kind, including a pathogenic infection. The infection may be bacterial, viral, fungal, or protozoan. Examples of bacteria include, but are not limited to, Actinomyces, Bacillus, Bacteroides, Bordetella, Bartonella, Borrelia, Brucella, Campylobacter, Capnocytophaga, Chlamydia, Corynebacterium, Coxiella, Dermatophilus, Enterococcus, Ehrlichia, Escherichia, Francisella, Fusobacterium, Haemobartonella, Haemophilus, Helicobacter, Klebsiella, L-form bacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neisseria, Neorickettsia, Nocardia, Pasteurella, Peptococcus, Peptostreptococcus, Pneumococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea polypeptides, Salmonella, Shigella, Staphylococcus, group A streptococcus, group B streptococcus, Treponema, and Yersinia. Examples of fungi include, but are not limited to, Absidia, Acremonium, Altemaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida, Coccidioides, Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton, Exophiala, Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum, Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium, Phialophora, Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha. Examples of protozoa include, but are not limited to, Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium. Examples of helminth parasites include, but are not limited to, Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus, Oesophagostomum, Onchocerca, Opisthorchis, Ostertagia, Parafilaria, Paragonimus, Parascaris, Physaloptera, and/or Protostrongylus.

The exosome compositions of the disclosure (e.g., any of the MSC Compositions, as described herein) may be administered by any suitable route or method of administration. Administration to a human or animal subject may be selected from rectal, buccal, vaginal, parenteral, intramuscular, intracerebral, intravascular (including intravenous), intracutaneous, subcutaneous, intranasal, intracardiac, intracerebroventricular, intraperitoneal intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial routes, or transdermal administration, and/or via an implanted reservoir.

The exosomes (e.g., MSC-Exos) may be delivered as a composition (e.g., any of the MSC Compositions, as described herein). The composition may be formulated for any suitable route or method of administration, including rectal, buccal, vaginal, parenteral, intramuscular, intracerebral, intravascular (including intravenous), intracutaneous, subcutaneous, intranasal, intracardiac, intracerebroventricular, intraperitoneal intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial routes, or transdermal administration, and/or via an implanted reservoir. Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. The exosomes of the disclosure (e.g., MSC-Exos) may be formulated in a pharmaceutical composition (e.g., any of the MSC Compositions, as described herein), which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and other pharmaceutically acceptable carriers or excipients and the like in addition to the exosomes.

A “pharmaceutically acceptable carrier” (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to a subject. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methyl cellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc ); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents.

The compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions provided herein.

A therapeutically effective amount of composition can be administered. The therapeutically effective amount of the produced exosomes (e.g., MSC-Exos) is that amount that achieves a desired effect in a subject being treated. For instance, this can be the amount of exosomes necessary to treat a disease, including, for instance, any eye disease such as DED. The amount of exosomes necessary to treat a disease may also include the amount necessary to inhibit advancement, or to cause regression of, cancer, or which is capable of relieving symptoms caused by cancer. This can be the amount of exosomes necessary to inhibit advancement, or to cause regression, of an autoimmune or alloimmune disease, or which is capable of relieving symptoms caused by an autoimmune disease, such as pain and inflammation. It can also be of the amount of exosomes necessary to inhibit advancement, or to cause regression, of a microbial infection, or which is capable of relieving symptoms caused by a microbial infection.

The produced exosomes (e.g., MSC-Exos) can be administered in treatment regimens consistent with the disease, for example a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to inhibit disease progression and prevent disease recurrence. The dose may be determined according to various parameters, especially according to the severity of the condition, age, and weight of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. Optimum dosages may vary depending on the relative potency of individual constructs and can generally be estimated based on ECsos found to be effective in in vitro and in in vivo animal models. In general, dosage is from 0.01 mg/kg to 100 mg per kg of body weight. A typical daily dose is from about 0. 1 to 50 mg per kg, preferably from about 0.1 mg/kg to 10 mg/kg of body weight, according to the potency of the specific construct, the age, weight and condition of the subject to be treated, the severity of the disease and the frequency and route of administration. Different dosages of the construct may be administered depending on whether administration is by topical administration, intramuscular injection, systemic (intravenous or subcutaneous) injection, and/or any other route or method of administration or combination thereof. In some cases, the dose of single or multiple systemic injections is in the range of 10 to 100 mg/kg of body weight.

In some cases, the individual may have to be treated repeatedly, for example once or more daily, weekly, monthly or yearly. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the construct in bodily fluids or tissues. Following successful treatment, it may be desirable to have the individual undergo maintenance therapy, wherein the exosomes (e.g., MSC-Exos) and/or any composition (e.g., any of the MSC Compositions, as described herein) containing such exosomes are administered in maintenance doses, ranging from 0.01 mg/kg to 100 mg per kg of body weight, once or more daily, to once every 20 years.

MSC Compositions for treating DED

As described above herein, one or more compositions containing MSCs, MSC-Exos, and/or MSC-Exos-derived biological compounds (referred to herein as “MSC Compositions”) can be used for the treatment of one or more diseases and/or disorders, including eye disorders such as DED.

In at least one embodiment, the MSC Compositions are composed in a solution that can be delivered topically to a patient’s eye. Such solutions need not be biologies, but can be over-the- counter (OTC) drug products manufactured under current Good Manufacturing Practices (cGMP) and regulated by the Food and Drug Administration (FDA). Accordingly, the MSC Compositions can provide safe and effective eye-drops that are immediately ready for clinical use.

In at least one embodiment, the MSC Compositions may include any one or more types of MSCs described herein, one or more types of MSC-Exos described herein, and/or one or more MSC-Exos-derived biological compounds (e.g., active agents, bioactive agents, growth factors, immunoregulatory proteins, drugs, etc.) described herein.

Growth factors, cytokines, and other molecules

Non-limiting examples of MSC-Exos-derived biological compounds include growth factors, cytokines, and/or other similar molecules derived or sourced from MSC-Exos.

Growth factors and their receptors control a wide range of biological functions, regulating cellular proliferation, survival, migration and differentiation. Growth factors found in exosomes (e.g., MSC-Exos) can play a critical role in fetal growth and development.

A non-limiting list of growth factors includes epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), vascular endothelial growth factor A (VEGF-A), tumor necrosis factor A (TNF-a), hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7), matrix metallopeptidase (MMP-9), granulocyte-colony stimulating factor (GCSF), matrix metalloproteinase-7 (MMP-7), matrix metalloproteinase- 13 (MMP-13), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P), fibroblast growth factor 4 (FGF- 4), endocrine gland-derived vascular endothelial growth factor (EG-VEGF), interleukin 8 (IL-8), fibroblast growth factor 21 (FGF-21), angiopoietin-2 (ANG-2), Glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor 19 (FGF-19), TIMP metallopeptidase inhibitor 2 (TIMP- 2), angiopoietin-1 (ANG-1), transforming growth factor beta 1 (TGFpi), macrophage colonystimulating factor (M-CSF), angiotensinogen, platelet derived growth factor-AA (PDGF-AA), and stem cell factor (SCF).

Epidermal growth factor (EGF) is a small polypeptide hormone with mitogenic properties in vivo and in vitro. EGF elicits biologic responses by binding to a cell surface receptor which is a transmembrane glycoprotein containing a cytoplasmic protein tyrosine kinase. EGF responses are mediated by ligand binding and activation of this intrinsic protein kinase. The receptor can be phosphorylated by other protein kinases, and this may regulate receptor function. Stimulation of the receptor tyrosine kinase activity by ligand binding must regulate the activity of an as yet undefined molecule(s) responsible for transmitting a mitogenic signal to the nucleus (Todderud G., et al., Biofactors. 1989, 2(1): 11-5).

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), was originally described as an endothelial cell-specific mitogen. VEGF is produced by many cell types including tumor cells, macrophages, platelets, keratinocytes, and renal mesangial cells. The activities of VEGF are not limited to the vascular system; VEGF plays a role in normal physiological functions such as bone formation, hematopoiesis, wound healing, and development (Duffy A.M., et al., In: Madame Curie Bioscience Database [Internet], Austin (TX): Landes Bioscience (2000)).

TGF-a has a structure similar to EGF and binds to the same receptor. The amnion cells of the umbilical cord express EGF, TGF-a, and the functional EGF/TGF-a receptor, suggesting the possibility of a regulating role of the amnion in fetal growth and development. EGF and TGF-a have also been shown to stimulate the production of surfactant components.

TGFpi is believed to induce terminal differentiation of intestinal epithelial cells and to accelerate the rate of healing of intestinal wounds by stimulating cell migration. TGFpi may also stimulate IgA production. VEGF-A is a signal protein that stimulates vasculogenesis and angiogenesis (Hoeben Am, et al., Pharmacol Rev. 2004, 56:549-580).

Transforming growth factor-beta (TGF-P) is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. Many cells synthesize TGF- P and essentially all of them have specific receptors for this peptide. TGF-P regulates the actions of many other peptide growth factors and determines a positive or negative direction of their effects (Sporn M.B., et al., Science 1986, 233(4763) 532-534).

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. A recent study showed its immunomodulation potential of amniotic fluid stem cells (Maraldi T., et al., Stem Cells Transl. Med., 4(6):539-47 (2015)).

Fibroblast growth factors (FGFs) that signal through FGF receptors (FGFRs) regulate a broad spectrum of biological functions, including cellular proliferation, survival, migration, and differentiation. The FGF signal pathways are the RAS/MAP kinase pathway, PI3 kinase/ AKT pathway, and PLCy pathway, among which the RAS/MAP kinase pathway is known to be predominant. Several studies have recently implicated the in vitro biological functions of FGFs for tissue regeneration. Many current applications of FGF are in regeneration of tissues, including skin, blood vessel, muscle, adipose, tendon/ligament, cartilage, bone, tooth, and nerve tissues (Yun Y.R., et al., J. Tissue Eng. 2010: 1(1)).

Matrix metalloproteinases (MMPs), also called matrixins, function in the extracellular environment of cells and degrade both matrix and non-matrix proteins. They play central roles in morphogenesis, wound healing, tissue repair and remodeling in response to injury, e.g., after myocardial infarction, and in progression of diseases such as atheroma, arthritis, cancer and chronic tissue ulcers. They are multi-domain proteins and their activities are regulated by tissue inhibitors of metalloproteinases (TIMPs) (Nagase H., et al., Cardiovascular Research, European Society of Cardiology, 562-573 (2006)).

Exosomes (e.g., MSC-Exos) may also contain many pro- and anti-inflammatory cytokines. Pro- and anti-inflammatory cytokines play important immunoregulatory roles. Inflammation is characterized by interplay between pro- and anti-inflammatory cytokines. Cytokines are commonly classified in one or the other category: interleukin- 1 (IL-1), tumor necrosis factor (TNF), gamma-interferon (IFN-y), IL-12, IL-18, and granulocyte-macrophage colony stimulating factor are well characterized as pro-inflammatory cytokines, whereas IL4, IL- 10, IL- 13, IFN-a and TGF-0 are recognized as anti-inflammatory cytokines.

Exemplary pro-inflammatory cytokines include Eotaxin-2 (CCL24), interleukin 6 (IL-6), pulmonary and activation-regulated chemokine PARC or chemokine (C-C motif) ligand 18 (CCL18), total GRO which consisted of three subunits GROa/CXCLl, GRO0/CXCL2, and GROy/CXCL3, expression of the neutrophil-activating CXC chemokine (ENA-78/CXCL-5), chemokine (C-C motif) ligand 21 (CCL21or 6Ckine), macrophage inflammatory protein 3 alpha (MIP-3 or CCL20), monokine induced by gamma (MIG or CXCL-9), MIP-l , chemokine (C-C motif) ligand 5 (CCL-5), also known asRANTES (regulated on activation, normal T cell expressed and secreted), Interleukin- 1 alpha (IL- la), macrophage inflammatory protein- 10 (MIP-10 or CCL4), tumor necrosis factor (TNF-a), and monocyte chemotactic protein 2 (MCP-2 or CCL8).

Exemplary anti-inflammatory cytokines include interleukin 8 (IL-8), interleukin 13 (IL- 13), interleukin 27 (IL-27), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), vascular endothelial growth factor D (VEGF-D), interleukin- 1 receptor antagonist (IL-IRa), transforming growth factor beta 1 (TGF01), interleukin 5 (IL-5), and interleukin 21 (IL -21).

Formulations In at least one embodiment, the MSC Compositions can be administered in concentrated form, diluted with sterile water or buffer, formulated as a solution, gel, ointment, or suspension. It can include additional therapeutic, prophylactic or diagnostic agents, either in the solution, gel, ointment or suspension, or as particles (nanoparticles, liposomes, microparticles) or implants. Representative excipients include solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.

Solutions, gels, ointments, and suspensions

Numerous ophthalmological formulations are known and available. The MSC Compositions can be concentrated or diluted with water, buffered saline, or an equivalent, formed into a gel with a polysaccharide such as alginate or hyaluronic acid, polyvinyl pyrrole, or ointment such as petrolatum or mineral oil, or emulsified with lipid or oil. Ophthalmic emulsions are generally dispersions of oily droplets in an aqueous phase. There should be no evidence of breaking or coalescence.

In at least one example, the MSC Compositions are solutions manufactured under current Good Manufacturing Practices (cGMP), regulated and reviewed by the FDA. The MSC Compositions are then sterilized to ensure a safe, sterile product. The MSC Compositions may, in at least one example, contain one or more osmoprotectants. The one or more osmoprotectants may include a variety of compounds, such as natural water (e.g., undiluted and/or pure water), one or more sugars (e.g., sucrose, trehalose, gentiobiose, melibiose, maltose, turanose, raffinose, stachyose, verbascose, altrose, palatinose, cellobiose) and/or their derivatives, one or more amino acids (e.g., glutamine, proline, alanine, carnitine) and/or their derivatives, one or more polyols (e.g., glycerol, arabitol, inositol, mannitol, sorbitol, maltitol) and/or their derivatives, one or more heterosides (e.g., glucosylglycerol, mannosucrose) and/or their derivatives, glycine betaine, and/or trimethylglycine. The one or more osmoprotectants may also be, for instance, one or more naturally occurring solutes that are compatible with, and can be internalized by, cells (e.g., one or more cells of the eye and/or glands associated with the eye. One or more tonicity adjusting agents may be added to provide the desired ionic strength and/or to ensure the MSC Compositions are hypotonic. Tonicity-adjusting agents for use include those which display no or only negligible pharmacological activity after administration. Both inorganic and organic tonicity adjusting agents may be used. The MSC Compositions can also include excipients and/or additives. Examples of excipients are surfactants, stabilizers, complexing agents, antioxidants, or preservatives which prolong the duration of use of the MSC Compositions, flavorings, vitamins, or other additives known in the art. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof. Preservatives include, but are not limited to, those that protect the solution from contamination with pathogenic particles, including benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate. Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E or salts or esters thereof.

In at least one example, the MSC Compositions include one or more pharmaceutically acceptable salts, such as, for instance, one or more chloride, acetate, and/or citrate salts. Further non-limiting examples of pharmaceutically acceptable salts include, for example, sodium chloride, potassium chloride, calcium chloride (e.g., calcium chloride dihydrate), magnesium chloride (e.g., magnesium chloride hexahydrate), sodium acetate (e.g., sodium acetate trihydrate), and/or sodium citrate (e.g., sodium citrate dihydrate).

In at least one example, the MSC Compositions include one or more physiological buffers, such as a phosphate (e.g., monobasic sodium phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate).

In at least one example, the MSC Compositions include hyaluronic acid (e.g., crosslinked hyaluronic acid), sodium hyaluronate, chondroitin sulfate, dermatan sulfate, heparin sulfate, keratin sulfate, hydroxylpropylmethylcellulose, recombinant human collagen, and combinations thereof.

In at least one example, the MSC Compositions include one or more stabilizers, which can maintain and/or improve the physical and/or chemical stability of the solutions. The one or more stabilizers may be hydrated in an aqueous solvent. Non-limiting examples of stabilizers include carboxymethylcellulose, hydroxypropylmethyl cellulose, cellulose-based compounds (e.g., hydroxyethyl cellulose), polyvinyl-based compounds (e.g, polyvinyl alcohol, polyvinylpyrrolidone), acrylic compounds (e.g., one or more carbomers), gum compounds (e.g., gellan gum, xanthangum), and polysaccharides (e.g, hyaluronic acid, sodium hyaluronate, sodium alginate, dextran).

In at least one example, the MSC Compositions include ophthalmically acceptable demulcents, ophthalmically acceptable excipients/emollients, ophthalmically acceptable astringents, and/or ophthalmically acceptable vasoconstrictors. Non-limiting examples of ophthalmically acceptable demulcents include, for instance, carboxymethylcellulose sodium, hydroxyethyl cellulose, hypromellose, methyl cellulose, dextran 70, gelatin, glycerin, polyethylene glycol 300, polyethylene glycol 400, polysorbate 80, propylene glycol, polyvinyl alcohol, and povidone. Non-limiting examples of ophthalmically acceptable excipients/emollients include, for instance, anhydrous lanolin, lanolin, light mineral oil, mineral oil, paraffin, petrolatum, white ointment, white petrolatum, white wax, and yellow wax. Non-limiting examples of ophthalmically acceptable astringents include, for instance, zinc sulfate. Non-limiting examples of ophthalmically acceptable vasoconstrictors include, for instance, ephedrine hydrochloride, naphazoline hydrochloride, phenylephrine hydrochloride, and tetrahydrozoline hydrochloride.

In at least one example, the MSC Compositions may take the form of a solution (e.g., a liquid solution, an aqueous solution), a gel (e.g., a viscoelastic gel), and/or a film (e.g., a viscoelastic film).

In at least one example, the MSC Compositions may be balanced and/or buffered. The pH may be, for instance, a physiological pH such as, for instance, about 6.0 to about 8.0, or at any pH and/or pH range therebetween (e.g., pH 7.0). Thus, the pH of the MSC Compositions may be, for example, about 6.5 to about 7.8, about 6.5 to about 7.5, about 6.8 to about 7.6, about 7.0 to about 7.4, about 7.0 to about 7.2, or about 6.8 to about 7.2. The MSC Compositions may further include one or more balanced salt solutions. Non-limiting examples of salts that can be incorporated into the one or more balanced salt solutions include, for instance, sodium chloride, potassium chloride, calcium chloride dehydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, and/or sodium citrate dihydrate. The pH of the balanced salt solution may have a pH of about 6.0 to about 8.0, and the balanced salt solution may include sodium hydroxide and/or hydrochloric acid to adjust pH. In at least one example, the MSC Compositions may have an osmolality ranging from about 200 mOsm/kg to about 400 mOsm/kg, such as, for instance, 300 mOsm/kg. The MSC Compositions may include one or more buffer solutions (e.g., an infusion buffer solutions). Such buffer solutions may be, for instance, a phosphate buffer solution, a buffer solution containing sodium chloride, and/or a buffer solution containing potassium chloride. Further suitable physiological buffers may be used. The buffer solutions may have a concentration ranging from, for instance, about 0.005 M to about 1.0 M, about 0.01 M to about 1.0 M, about 0.01 M to about 0.5 M, about 0.05 M to about 1.0 M, about 0.1 M to about 0.5 M, or about 0.5 M to about 1.0 M.

In at least one example, the MSC Compositions have a viscosity ranging from about 50,000 milliPascal-seconds (mPa sec) to about 160,000 mPa sec, about 50,000 mPa sec to about 75,000 mPa sec, about 50,000 mPa sec to about 55,000 mPa sec, about 90,000 mPa sec to about 110,000 mPa sec, about 100,000 mPa sec to about 150,000 mPa sec, about 125,000 mPa sec to about 150,000 mPa sec, about 130,000 mPa sec to about 140,000 mPa sec, or about 120,000 mPa sec to about 140,000 mPa sec.

In at least one example, the concentration of one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos-derived biological compounds (e.g., growth factors, cytokines) ranges from about 0.001 mg/mL to about 10 mg/mL, about 0.001 mg/mL to about 1.0 mg/mL, about 0.005 mg/mL to about 0.1 mg/mL, about 0.01 mg/mL to about 1.0 mg/mL, about 0.05 mg/mL to about 2.0 mg/mL, about 0.07 mg/mL to about 2.5 mg/mL, about 0.1 mg/mL to about 3 mg/mL, about 0.5 mg/mL to about 1 mg/mL, about 1 mg/mL to about 6 mg/mL, about 1 mg/mL to about 5 mg/mL, about 1.5 mg/mL to about 4.5 mg/mL, about 2 mg/mL to about 4 mg/mL, about 3 mg/mL to about 5 mg/mL, about 4 mg/mL to about 5 mg/mL, about 2 mg/mL to about 4 mg/mL, about 0.5 mg/mL to about 2.5 mg/mL, about 1 mg/mL to about 2 mg/mL, about 3.5 mg/mL to about 5 mg/mL, or about 5 mg/mL to about 10 mg/mL.

In at least one example, the concentration of one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos-derived biological compounds (e.g., growth factors, cytokines) ranges from about 10 pg/mL to about 500 pg/mL, about 50 pg/mL to about 250 pg/mL, about 100 pg/mL to about 500 pg/mL, about 150 pg/mL to about 300 pg/mL, or about 250 pg/mL to about 500 pg/mL. In at least one example, the total volume of one or more aliquots, samples, and/or doses of the MSC Compositions ranges from about 0.050 mb to about 2.0 mL, such as, for example, about 1.0 mL. Thus, the amount of the MSC Compositions administered to a patient’s eye at any one given time may range from about 0.050 mL to about 2.0 mL, such as, for example, about 1.0 mL.

Thus, in at least one example, the dose of one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos-derived biological compounds (e.g., growth factors, cytokines) administered to a patient’s eye ranges from about 0.2 mg/100 pL (2 mg/mL) to about 0.6 mg/100 pL (2 mg/mL) per treatment, about 0.25 mg/100 pL to about 0.5 mg/100 pL, about 0.25 mg/100 pL to about 0.55 mg/100 pL, about 0.28 mg/100 pL to about 0.475 mg/100 pL, about 0.3 mg/100 pL to about 0.5 mg/100 pL, about 0.35 mg/100 pL to about 0.45 mg/100 pL, about 0.25 mg/100 pL to about 0.5 mg/100 pL, about 0.45 mg/100 pL to about 0.5 mg/100 pL, or about 0.3 mg/100 pL to about 0.4 mg/100 pL. Further non-limiting examples of dosages include, for instance, from about 0.005 mg/mL to about 2.0 mg/mL, about 0.025 mg/mL to about 1.0 mg/mL, or about 0.05 mg/mL to about 0.2 mg/mL.

In at least one example, the dose of one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos-derived biological compounds e.g., growth factors, cytokines) administered to a patient’s eye ranges from about 0.005 mg (5 pg) to about 2.0 mg per total delivery volume of the MSC Compositions, about 0.01 mg to about 2 mg, about 0.05 mg to about 0.5 mg, about 0.1 mg to about 1.0 mg, about 0.5 mg to about 1.8 mg, about 0.75 mg to about 1.5 mg, about 0.5 mg to about 1 .25 mg, about 0.75 mg to about 1 mg, about 0.5 mg to about 0.75 mg, about 0.25 mg to about 0.75 mg, about 0.6 mg to about 1.2 mg, about 0.9 mg to about 1.3 mg, or about 1.5 mg to about 1.8 mg per total delivery volume of the MSC Compositions. As non-limiting examples, the total delivery volume of the MSC Compositions may range from about 0.1 mL to about 2.0 mL, about 0.1 mL to about 0.5 mL, about 0.2 mL to about 0.35 mL, 0.25 mL to about 0.75 mL, about 0.25 mL to about 0.45 mL, about 0.5 mL to about 1.0 mL, about 0.5 mL to about 2.0 mL, about 1.0 mL to about 2.0 mL, or about 1.0 mL to about 1.5 mL per application e.g., one or more applications) or per eye (e.g., administered in one or more applications). For instance, the volume of the MSC Compositions administered may be less than 1 mL, such as, for instance, in a range of about 0.1 mL to about 0.9 mL. Ophthalmic suspensions generally contain solid particles dispersed in a liquid vehicle; they must be homogeneous when shaken gently and remain sufficiently dispersed to enable the correct dose to be removed from the container. A sediment may occur, but this should disperse readily when the container is shaken, and the size of the dispersed particles should be controlled. The active ingredient and any other suspended material must be reduced to a particle size small enough to prevent irritation and damage to the cornea.

Ophthalmic ointments are generally sterile, homogeneous, semi-solid preparations intended for application to the conjunctiva or the eyelids. They are usually prepared from nonaqueous bases, e.g., soft paraffin (Vaseline), liquid paraffin, and wool fat. They may contain suitable additives, such as antimicrobial agents, antioxidants, and stabilizing agents.

When the solution is dispensed in a multidose container that is to be used over a period of time longer than 24 hours, a preservative must be added to ensure microbiologic safety over the period of use.

Ideally, the pH of ophthalmic drops should be equivalent to that of tear fluid, which is 7.4. However, the decision to add a buffering agent should be based on stability considerations. The pH selected should be the optimum for both stability of the active pharmaceutical ingredient and physiological tolerance. If a buffer system is used, it must not cause precipitation or deterioration of the active ingredient. The influence on the lachrymal flow should also be taken into account.

Although solutions with the same pH as lacrimal fluid (7.4) are ideal, the outer surfaces of the eye tolerate a larger range, 3.5 to 8.5. The normal useful range to prevent corneal damage is 6.5 to 8.5. The final pH of the solution (e.g., of one or more MSC Compositions) is often a compromise, because many ophthalmic drugs have limited solubility and stability at the desired pH of 7.4. Buffers or pH adjusting agents or vehicles can be added to adjust and stabilize the pH at a desired level. Ophthalmic solutions are ordinarily buffered at the pH of maximum stability of the drug(s) they contain. The buffers are included to minimize any change in pH during the storage life of the drug; this can result from absorbed carbon dioxide from the air or from hydroxyl ions from a glass container. Changes in pH can affect the solubility and stability of drugs; consequently, it is important to minimize fluctuations in pH. The buffer system should be designed sufficient to maintain the pH throughout the expected shelf-life of the product, but with a low buffer capacity so that when the ophthalmic solution is instilled into the eye, the buffer system of the tears will rapidly bring the pH of the solution back to that of the tears. Low concentrations of buffer salts are used to prepare buffers of low buffer capacity.

The preparation of aqueous ophthalmic drops (which may be one form of the MSC Compositions disclosed herein) requires careful consideration of the need for isotonicity, a certain buffering capacity, the desired pH, the addition of antimicrobial agents and/or antioxidants, the use of viscosity-increasing agents, and the choice of appropriate packaging. Ophthalmic drops are considered isotonic when the tonicity is equal to that of a 0.9% solution of sodium chloride. The eye can usually tolerate solutions equivalent to 0.5-1.8% of sodium chloride (NaCl).

Solutions that are isotonic with tears are preferred. An amount equivalent to 0.9% NaCl is ideal for comfort and should be used when possible. The eye can tolerate tonicities within the equivalent range of 0.6- 2% NaCl without discomfort. There are times when hypertonic ophthalmic solutions are necessary therapeutically, or when the addition of an auxiliary agent required for reasons of stability supersedes the need for isotonicity. A hypotonic ophthalmic solution will require the addition of a substance (tonicity adjusting agent) to attain the proper tonicity range.

The most widely used ophthalmic buffer solutions are boric acid vehicle and Sorensen’s modified phosphate buffer. The boric acid vehicle is a 1.9% solution of boric acid in purified water or preferably sterile water. It is isotonic with tears. It has a pH of approximately 5 and is useful when extemporaneously compounding ophthalmic solutions of drugs that are most stable at acid pH. This vehicle does not possess large buffer capacity, but it is sufficient to stabilize pH for the short expiratory periods used for compounded solutions, without overwhelming the natural buffers in lacrimal fluid. The second most commonly used buffer solution is the Sorensen’s modified phosphate buffer and is used for drugs needing pH values between the range of 6.5-8.0. This buffer uses two stock solutions, one acidic containing NaH2PO4, and one basic containing Na2HPO4. The formulas for the stock solutions and their respective proportions used to obtain specific pH values are generally known.

In some instances, the MSC Compositions are distributed or packaged in a liquid form. Alternatively, formulations of the MSC Compositions for ocular administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration. The MSC Compositions (for instance, in solution, suspension, and/or emulsion form) for ocular administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.

The MSC Compositions (for instance, in solution, suspension, and/or emulsion form) for ocular administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. Solutions, suspensions, or emulsions for ocular administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives are known in the art, and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.

The MSC Compositions (for instance, in solution, suspension, and/or emulsion form) for ocular administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.

The ophthalmic drug may be present in its neutral form, or in the form of a pharmaceutically acceptable salt. In some cases, it may be desirable to prepare a formulation containing a salt of an active agent due to one or more of the salt’s advantageous physical properties, such as enhanced stability or a desirable solubility or dissolution profile.

Generally, pharmaceutically acceptable salts can be prepared by reaction of the free acid or base forms of an active agent with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of an active agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704. Examples of ophthalmic drugs sometimes administered in the form of a pharmaceutically acceptable salt include timolol maleate, brimonidine tartrate, and sodium diclofenac.

Particles and implants containing one or more therapeutic, prophylactic or diagnostic agents dispersed in a polymer matrix

Particles can also be formed containing one or more therapeutic, prophylactic or diagnostic agents dispersed or encapsulated in a polymeric matrix. The matrix can be formed of non- biodegradable or biodegradable matrices, although biodegradable matrices are preferred. The polymer is selected based on the time required for in vivo stability, e.g., that time required for distribution to the site where delivery is desired, and the time desired for delivery.

Representative synthetic polymers include: poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol), polyalkylene oxides such as poly(ethylene oxide), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes and co-polymers thereof, derivatized celluloses such as alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt (jointly referred to herein as “synthetic celluloses”), polymers of acrylic acid, methacrylic acid or copolymers or derivatives thereof including esters, poly(m ethyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as “polyacrylic acids”), poly (butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), copolymers and blends thereof. As used herein, “derivatives” include polymers having substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxyl ati on s, oxidations, and other modifications routinely made by those skilled in the art.

Non-limiting examples of preferred biodegradable polymers include polymers of hydroxy acids such as lactic acid and glycolic acid, and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(lactide-co- caprolactone), blends and copolymers thereof.

Non-limiting examples of preferred natural polymers include proteins such as albumin and prolamines, for example, zein, and polysaccharides such as alginate, cellulose and polyhydroxyalkanoates, for example, polyhydroxybutyrate.

The in vivo stability of the matrix can be adjusted during the production by using polymers such as polylactide co glycolide copolymerized with polyethylene glycol (PEG). PEG if exposed on the external surface may elongate the time these materials circulate since it is hydrophilic.

Non-limiting examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Particles having an average particle size of between 10 nm and 1000 microns are useful in the compositions described herein. In preferred embodiments, the particles have an average particle size of between 10 nm and 100 microns, more preferably between about 100 nm and about 50 microns, more preferably between about 200 nm and about 50 microns. In certain embodiments, the particles are nanoparticles having a diameter of between 500 and 700 nm. The particles can have any shape but are generally spherical in shape.

Microparticle and nanoparticles can be formed using any suitable method for the formation of polymer micro- or nanoparticles known in the art. The method employed for particle formation will depend on a variety of factors, including the characteristics of the polymers present in the polymer-drug conjugate or polymer matrix, as well as the desired particle size and size distribution. The type of therapeutic, prophylactic or diagnostic agent(s) being incorporated in the particles may also be a factor as some therapeutic, prophylactic or diagnostic agents are unstable in the presence of certain solvents, in certain temperature ranges, and/or in certain pH ranges.

In circumstances where a monodisperse population of particles is desired, the particles may be formed using a method which produces a monodisperse population of nanoparticles. Alternatively, methods producing poly disperse nanoparticle distributions can be used, and the particles can be separated using methods known in the art, such as sieving, following particle formation to provide a population of particles having the desired average particle size and particle size distribution.

Common techniques for preparing microparticles and nanoparticles include, but are not limited to, solvent evaporation, hot melt particle formation, solvent removal, spray drying, phase inversion, coacervation, and low temperature casting. Suitable methods of particle formulation are briefly described below. Pharmaceutically acceptable excipients, including pH modifying agents, disintegrants, preservatives, and antioxidants, can optionally be incorporated into the particles during particle formation.

Implants can be formed from one or more polymers. In preferred embodiments, the implants are intraocular implants. Suitable implants include, but are not limited to, rods, discs, wafers, and the like.

Implants can also be formed from a polymeric matrix having one or more therapeutic, prophylactic or diagnostic agents dispersed or encapsulated therein. The matrix can be formed of any of the nonbiodegradable or biodegradable polymers described above, although biodegradable polymers are preferred. The composition of the polymer matrix is selected based on the time required for in vivo stability, e.g., that time required for distribution to the site where delivery is desired, and the time desired for delivery. Implants can also be formed from blends of polymer- drug conjugates with one or more of the polymers described above herein.

The implants may be of any geometry such as fibers, sheets, films, microspheres, spheres, circular discs, rods, or plaques. Implant size is determined by factors such as toleration for the implant, location of the implant, size limitations in view of the proposed method of implant insertion, ease of handling, etc.

Where sheets or films are employed, the sheets or films will be in the range of at least about 0.5 mm x 0.5 mm, usually about 3 to 10 mm x 5 to 10 mm with a thickness of about 0. 1 to 1.0 mm for ease of handling. Where fibers are employed, the fiber diameter will generally be in the range of about 0.05 to 3 mm and the fiber length will generally be in the range of about 0.5 to 10 mm.

The size and shape of the implant can also be used to control the rate of release, period of treatment, and drug concentration at the site of implantation. Larger implants will deliver a proportionately larger dose, but depending on the surface to mass ratio, may have a slower release rate. The particular size and geometry of the implant are chosen to suit the site of implantation.

Intraocular implants may be spherical or non-spherical in shape. For spherical-shaped implants, the implant may have a largest dimension (e.g., diameter) between about 5 pm and about 2 mm, or between about 10 pm and about 1 mm for administration with a needle, greater than 1 mm, or greater than 2 mm, such as 3 mm or up to 10 mm, for administration by surgical implantation. If the implant is non-spherical, the implant may have the largest dimension or smallest dimension be from about 5 pm and about 2 mm, or between about 10 pm and about 1 mm for administration with a needle, greater than 1 mm, or greater than 2 mm, such as 3 mm or up to 10 mm, for administration by surgical implantation.

The vitreous chamber in humans is able to accommodate relatively large implants of varying geometries, having lengths of, for example, 1 to 10 mm. The implant may be a cylindrical pellet (e.g., rod) with dimensions of about 2 mm x 0.75 mm diameter. The implant may be a cylindrical pellet with a length of about 7 mm to about 10 mm, and a diameter of about 0.75 mm to about 1.5 mm. In certain embodiments, the implant is in the form of an extruded fdament with a diameter of about 0.5 mm, a length of about 6 mm, and a weight of approximately 1 mg. In some embodiments, the dimension are, or are similar to, implants already approved for intraocular injection via needle: diameter of 460 microns and a length of 6 mm and diameter of 370 microns and length of 3.5 mm.

Intraocular implants may also be designed to be least somewhat flexible so as to facilitate both insertion of the implant in the eye, such as in the vitreous, and subsequent accommodation of the implant. The total weight of the implant is usually about 250 to 5000 pg, more preferably about 500-1000 pg. In certain embodiments, the intraocular implant has a mass of about 500 pg, 750 pg, or 1000 pg.

Implants can be manufactured using any suitable technique known in the art. Examples of suitable techniques for the preparation of implants include solvent evaporation methods, phase separation methods, interfacial methods, molding methods, injection molding methods, extrusion methods, coextrusion methods, carver press method, die cutting methods, heat compression, and combinations thereof. Suitable methods for the manufacture of implants can be selected in view of many factors including the properties of the polymer/polymer segments present in the implant, the properties of the one or more therapeutic, prophylactic or diagnostic agents present in the implant, and the desired shape and size of the implant. Suitable methods for the preparation of implants are described, for example, in U.S. Patent No. 4,997,652 and U.S. Patent Application Publication No. US 2010/0124565.

In certain cases, extrusion methods may be used to avoid the need for solvents during implant manufacture. When using extrusion methods, the polymer/polymer segments and therapeutic, prophylactic or diagnostic agent are chosen so as to be stable at the temperatures required for manufacturing, usually at least about 85 degrees Celsius. However, depending on the nature of the polymeric components and the one or more therapeutic, prophylactic or diagnostic agents, extrusion methods can employ temperatures of about 25°C to about 150°C, more preferably about 65°C to about 130°C.

Implants may be coextruded in order to provide a coating covering all or part of the surface of the implant. Such coatings may be erodible or non-erodible, and may be impermeable, semi- permeable, or permeable to the therapeutic, prophylactic or diagnostic agent, water, or combinations thereof. Such coatings can be used to further control release of the therapeutic, prophylactic or diagnostic agent from the implant.

Compression methods may be used to make the implants. Compression methods frequently yield implants with faster release rates than extrusion methods. Compression methods may employ pressures of about 50-150 pounds per square inch (psi), more preferably about 70-80 psi, even more preferably about 76 psi, and use temperatures of about 0°C to about 1 15°C, more preferably about 25°C.

Storage

In at least one embodiment, the MSC Compositions can be stored in frozen conditions at about -20° C. to about -80° C. In addition, the MSC Compositions may be distributed in vials equipped with special rubber stoppers for sterile lyophilization. Lyophilization is generally carried out in a sterile environment. The rubber stoppers on the vials are then automatically pushed down in the freeze dryer to definitively close them. Then, an aluminum cap is sealed on each vial to protect its sterile content. In such a lyophilized state, the MSC Compositions may be stored at +4° C. or room temperature for at least one year without decrease of one or more components thereof, such as, for example, one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos- derived biological compounds (e.g., growth factors, cytokines). Before use, lyophilized versions of the MSC Compositions may be reconstituted by adding the initial volume of sterile water to the powder in order to restore a transparent and homogeneous physiological liquid.

In at least one example, the MSC Compositions contain growth factors and other biological components that are stabilized against degradation (e.g., chemical and/or enzymatic degradation). Molecules contained within the fluid are stabilized against degradation, avoiding the need for chemical or physical modification to maintain the biological activity of the molecules over extended periods of time. Therefore, the MSC Compositions can be stored and/or distributed for long periods of time, allowing for a broad range of application and/or treatment methods.

In at least one example, the MSC Compositions can be stored in refrigerated conditions at about 1° C. to about 10° C. For instance, the MSC Compositions can be refrigerated at 4° C. for up to 12 months and more.

In at least one example, the MSC Compositions can be stored at room temperature for over a week, 2 weeks, 3 weeks, a month, 2 months, 3 months, 6 months, or up to 12 months or more, while still retaining most biologically active components such as, for example, one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos-derived biological compounds (e.g., growth factors, cytokines). The biological activity of such room temperature-stored MSC Compositions is preferably comparable to that of MSC Compositions refrigerated at about 1° C. to about 10° C. and/or MSC Compositions stored at about -20° C. to about -80° C. For example, fluids purified according to the described methods retain the biological properties of the component molecules over extended periods of storage, ideally without the need for freeze/thawing.

In at least one example, storage of the MSC Compositions, at any temperature and/or temperature range described herein, does not reduce the quantity and/or biological activity of one or more MSCs, one or more MSC-Exos, and/or one or more MSC-Exos-derived biological compounds (e.g., growth factors, cytokines). Therefore, in at least one example, little or no statistically significant changes in biological activity are observed when storing the MSC Compositions at 4° C. or at room temperature for up to a day, 2 days, 3 days, 4 days, 5 days, 6 days, up to one week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to one month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, or more than 6 months. In at least one example, the MSC Compositions are stored, without degradation, in any of the storage conditions described herein for at least about 1 day, at least about 2 days, at least about 3 days, at least about 5 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 3 years, at least about 4 years, or at least about 5 years. During such storage times, degradation of one or more components of the MSC Compositions (e.g., one or more MSCs, one or more MSC-Exos, and/or one or more MSC- Exos-derived biological compounds) is less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, less than about 2%, or less than about 1%.

Methods of administration

The compositions and methods disclosed herein, such as the MSC Compositions, are suitable for treating any eye disease (e.g., DED) and/or any discomfort, pain, dryness, excessive tearing, injuries, infections, and/or bums associated with the eye. In some embodiments, the MSC Compositions are used to alleviate pain, facilitate healing, and/or reduce or inhibit scarring.

In at least one example, the MSC Compositions may be applied to any portion of the eye and/or any bodily structure associated with the eye, including, for instance, the eye itself, the cornea, endothelial tissue, anterior chamber segment tissue, the posterior chamber of the eye, the retina, the epithelium, the native comeal epithelium, the epithelial cells, the lacrimal glands, the meibomian glands, and/or the mucin-producing goblet cells.

In at least one example, the MSC Compositions are formulated in a dosage between about 0.1 ml and about 100 ml, inclusive; or between about 0.1 ml and 1 ml, inclusive; or between about 1 ml and about 10 ml, inclusive; or between about 10 ml and about 50 ml, inclusive. In at least a further example, the formulation is combined with any amount of between about between about 0.1 ml and about 100 ml, inclusive; or between about 0.1 ml and 1 ml, inclusive; or between about 1 ml and about 10 ml, inclusive; or between about 10 ml and about 50 ml, inclusive, of sterile water, or saline solution.

In at least one example, the MSC Compositions are packaged into sterile dosage units which can be stored and distributed for use by attending physicians and/or other healthcare professionals. Lyophilized or fluid formulations can be in the form of sterile packaged ampule ready for use. A filled ampoule can contains a formulation of the MSC Compositions. Generally, such solutions are in one or more pharmaceutically acceptable carriers and buffered for human use to a pH of about 3.5-10.0, preferably about pH 6.0-8.0. In at least one example, the formulations of the MSC Compositions are free of preservatives where such preservatives may exert opposite effects to that required by the formulations. Water or saline solution can be used to provide the carrier.

Generally, volumes used herein refer to MSC Compositions at 1 x strength without any dilution or concentration. In at least one example, where lyophilized formulations of MSC Compositions are used, these volumes refer to the volume of fluid when the lyophilized powder is reconstituted with the initial volume of sterile water, i.e., 1 x strength. The MSC Compositions can be administered in concentrated form, diluted with sterile water, saline or buffer. The formulation may also include additional therapeutic, prophylactic, or diagnostic agents. Said agent(s) may be in-mixed with the formulations or mixed in separate containers to be used in conjunction with the MSC Compositions. The efficacy of administration is determined by physician evaluations, patient self-evaluations, imaging studies, and/or quality of life evaluations.

In at least one example, the MSC Compositions may be administered to one or more eyes of a patient for various periods of time per treatment. As non-limiting examples, the periods of time per treatment may be at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, or at least 10 minutes or more. Any given patient and/or their eyes may be treated multiple times per day, such as, for instance, once per day, twice per day, three times per day, five times per day, or more than five times per day. Further, any given patient and/or their eyes may be treated with the MSC Compositions over a total period of time lasting at least one day, at least 24 hours, at least 2 days, at least 3 days, at least one week, at least 3 weeks, at least 6 weeks, or at least 12 weeks or more.

Eye diseases and disorders

The compositions and methods disclosed herein, such as the MSC Compositions, are also suitable for prophylactic uses. In some embodiments, the MSC Compositions are used to relieve discomfort associated with extended computer use in human subjects. Non-limiting examples of eye disorders that may be treated according to the compositions and methods disclosed herein, such as the MSC Compositions, include DED (including severe DED), amoebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, onchorcercal keratitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, corneal dystrophic diseases, Fuchs’ endothelial dystrophy, meibomian gland dysfunction, anterior and posterior blepharitis, conjunctival hyperemia, conjunctival necrosis, cicatrical scaring and fibrosis, punctate epithelial keratopathy, filamentary keratitis, corneal erosions, thinning, ulcerations and perforations, Sjogren’s syndrome, Stevens-Johnson syndrome, autoimmune dry eye diseases, environmental dry eye diseases, corneal neovascularization diseases, post- corneal transplant rejection prophylaxis and treatment, autoimmune uveitis, infectious uveitis, anterior uveitis, posterior uveitis (including toxoplasmosis), pan-uveitis, an inflammatory disease of the vitreous or retina, endophthalmitis prophylaxis and treatment, macular edema, macular degeneration, age-related macular degeneration, proliferative and nonproliferative diabetic retinopathy, hypertensive retinopathy, an autoimmune disease of the retina, primary and metastatic intraocular melanoma, other intraocular metastatic tumors, open angle glaucoma, closed angle glaucoma, pigmentary glaucoma, and combinations thereof. Other disorders including injury, burn, or abrasion of the cornea, cataracts and age related degeneration of the eye or vision associated therewith.

In some embodiments, the MSC Compositions can be applied to the eye dissolve cataracts, reducing cataracts about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90%, in size. In other embodiments, the MSC Compositions dissolve cataracts, eliminating the need for an operation to remove cataracts. In some embodiments, the MSC Compositions are used to assist recovery from a cataract removal procedure.

The MSC Compositions may be administered to animals, especially mammalian animals for treating or alleviating pain, disease, disorder, infection, or injury of the eye. Mammalian subjects, include, but are not limited to, humans, primates such as monkeys and apes, canines such as dogs, felines such as cats, bovines such as cows, equines such as horses, swine such as pigs, and rodents such as mice and rats. In some embodiments, the MSC Compositions are used to relieve/treat dry eye, treat eye infection, improve vision, or assist recovery from a surgical procedure on the eye in mammals such as dogs, cats, rabbits, and horses. Administration of the MSC Compositions, as described herein, helps treat DED and/or alleviate the symptoms thereof. After administration with MSC Compositions, one or more objective measurements (e.g., tear film breakup time (TBUT), tear film thickness) can be taken to determine the effects of such administration. TBUT is preferably less than about 10 seconds or less than about 5 seconds after one or more courses of treatment with one or more d-MAPPS solutions. Generally, tear film thickness can increase as TBUT increases, as noted by Creech J.L., et al., “In vivo tear-film thickness determination and implications for tear film stability,” Curr. Eye Res. 17:1058-66 (1998). Tear film thickness can be used to measure the liquid layer, the lipid layer, and/or a combination of the liquid layer and the lipid layer. Tear film thickness can be measured by a slit lamp and a video camera. This can be done by, for instance, instilling fluorescein dye in the form of an eyedrop and videotaping the tear meniscus in profile. Tear-film breakup can then be videotaped through the ocular port of the slit lamp and evaluated based on a severity scale. Tear film thickness can also be measured by using an optical interferometer (e.g., a wavelengthdependent optical interferometer). A non-limiting example of measuring tear film thickness using such interferometers is provided in King-Smith, P.E., et al., “The Thickness of the Human Precorneal Tear Film: Evidence from Reflection Spectra,” Invest. Ophthalmol. Vis. Sci. 41(1 l):3348-59 (2000). Specifically, multiple measurements are taken of an area of a subject’s eye, where the area has a predetermined length and width. Such lengths and/or widths may range from about 10 pm, about 20 pm, about 30 pm, about 100 pm, or more than about 100 pm. The area may be located in any suitable area of the eye such as, for example, the apex of the cornea. The measurements may be taken over a window of time such as, for instance, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, or more than about 1 minute. The number of measurements may be, for instance, about 10, about 20, about 30, about 50, or more than about 50. Accordingly, a measurement may be taken every about 10 ms, about 20 ms, about 50 ms, about 100 ms, about 200 ms, about 300 ms, about 400 ms, about 500 ms, or more than about 500 ms. Such tear film thickness measurements may be taken both before and after MSC Composition-based treatment. Before treatment, tear film thickness may be, for example, less than about 2 microns, or less than about 1 micron. After treatment, tear film thickness may be, for example, about 5 microns, about 6 microns, about 8 microns, about 10 microns, about 12 microns, or more than about 12 microns. Further, case studies have shown an immediate positive disease modification for patients with mild to moderate and severe DED, glaucoma, Sjogren’s syndrome, possible Ankylosing spondylitis and age-related declining vision. Due to the viscosity of at least some of the MSC Compositions, drops applied directly onto the eye adhere to the ocular surface longer than common over the counter (“OTC”) artificial tear formulas. The capacity to adhere to the ocular surface is paramount when treating injuries and diseases such as Sjogren’s syndrome and chemical burns. Some unexpected results reported in the study were perceptible improvement to clarity of vision which had been diminished in several patients. Relief from varying levels of ocular discomfort or pain was observed. Nine (9) patients were administered Snell Eye Chart exams at the start and completion of the initial 30 day study of therapy. Five (5) of the nine demonstrated enriched visual acuity and consistently conveyed improvements in visual clarity, distance and reading ability.

Improvements of one to several lines on the test charts were recorded. Only two patients tested at undetectable improvement levels. Visual acuity appeared to be correlated to the level of corneal integrity of the recipient. This can be an unexpected benefit from MSC Composition therapy and treatments. Other unexpected benefits were being able to read at night for the first time in years and regaining the visibility required to drive a car. Most participants were able to discontinue or drastically reduce the amount and frequency of using additional applications of artificial tears (“AT”) drops and or alternate curatives. One participant diagnosed with mild dry eye exhibited no signs of the disease at the end of the initial 30 day trial.

Ocular bums

In some embodiments, the compositions and methods described herein, such as the MSC Compositions, are used for assisting recovery from ocular burns, or from procedures managing ocular burns such as autolimbal or allolimbal transplantation.

Ocular bums such as thermal and chemical burns represent potentially blinding ocular injuries. Thermal burns result from accidents associated with firework explosions, steam, boiling water, or molten metal (commonly aluminum). Chemical burns may be caused by either alkaline or acidic agents. Common alkaline agents include ammonium hydroxide used in fertilizer production, sodium hydroxide (caustic soda) used for cleaning drains and pipes, and calcium hydroxide found in lime plaster and cement. Alkaline agents are particularly damaging as they have both hydrophilic and lipophilic properties, which allow them to rapidly penetrate cell membranes and enter the anterior chamber. Alkali damage results from interaction of the hydroxyl ions causing saponification of cell membranes and cell death along with disruption of the extracellular matrix. Common acidic agents causing injury include sulphuric acid found in car batteries, sulphurous acid found in some bleaches, and hydrochloric acid used in swimming pools. Acids tend to cause less damage than alkalis as many corneal proteins bind acid and act as a chemical buffer. In addition, coagulated tissue acts as a barrier to further penetration of acid. Acid binds to collagen and causes fibril shrinkage.

Recovery of ocular surface bums depends upon the causative agent and the extent of damage to corneal, limbal, and conjunctival tissues at the time of injury. Damage to intraocular structures influences the final visual outcome. Thus, in some embodiments, the MSC Compositions are used to speed the recovery from an ocular burn.

Ocular blast injuries

Ocular blast injuries can be primary, from the blast wave itself; secondary, from fragments carried by the blast wind; tertiary, due to structural collapse or being thrown against a fixed object; or quaternary, from burns and indirect injuries. In some embodiments, the MSC Compositions are used in the management of injuries inflicted by blasts and explosions for preventative and/or therapeutic purposes.

Eye surgery

The MSC Compositions are suitable for use in the management of eye surgeries. Eye surgery, ocular surgery, or ophthalmologic surgery, refers to any surgery that is performed on the eye or its adnexa. Exemplary ocular surgeries include laser eye surgery, cataract removal, glaucoma surgery such as canaloplasty, refractive surgery such as LASIK®, corneal surgery, vitreo-retinal surgery, eye muscle surgery, oculoplastic surgery such as eye lid surgery and orbital surgery, surgery involving the lacrimal apparatus, and eye removal.

In some embodiments, the MSC Compositions are used prior, during or after one or more ocular surgeries. Thus, in some embodiments, the MSC Compositions are used along with one or more systemic drugs. For example, at least some of the MSC Compositions are applied as eye drops while the patient is on non-steroidal anti-inflammatory drugs such as ibuprofen. In some embodiments, the MSC Compositions are used to assist recovery from an ocular surgery. In some embodiments, the MSC Compositions are used to prevent, reduce, or alleviate one or more symptoms from an ocular surgery. For example, the MSC Compositions can be used during recovery after a surgical procedure of amniotic membrane graft onto the ocular surface. In some embodiments, the MSC Compositions are used to prevent one or more potential complications from an ocular surgery such as an infection. In some embodiments, the MSC Compositions are used to assist local tissue repair, and/or minimize scarring of the surgical site.

Eye infections

The compositions and/or methods described herein (e.g., the MSC Compositions) are suitable for use in the management of eye infections. Eye infections include infections from bacteria, fungi, and viruses. Eye infections can occur in different parts of the eye and can affect just one eye or both. Exemplary eye infections include conjunctivitis, stye, caratitis, and ocular herpes.

In some embodiments, the MSC Compositions are for prophylactic purposes to prevent an outset of a suspected eye infection. For example, if one person with an eye infection, e.g., conjunctivitis, is identified, anyone who has been recently in contact with that person can use the disclosed formulation for prophylactic purposes. In some embodiments, the MSC Compositions are used to prevent, reduce, or alleviate one or more symptoms from an eye infection.

Drug-induced eye conditions

The MSC Compositions are also suitable for use in the management of eye problems that arise as a side effect of using one or more systemic drugs.

Thus, in some embodiments, the MSC Compositions are used prior, during or after taking one or more systemic drugs. Exemplary drugs that can cause ocular side effects include corticosteroids, antihistamines, antipsychotic medications, antimalarials, blood pressure medications, herbal medicines, erectile dysfunction drugs, anticholinergics, immunosuppressants, antibiotics, anti arrhythmic agents, and anti-cancer drugs/treatment. Some specific examples are bisphosphonate, amiodarone, tamsulosin, topiramate, ethambutol, minocycline, cyclosporine and tacrolimus.

Corticosteroids used for many conditions such as asthma, allergies, arthritis and skin conditions can cause swelling in the back of the eye or retina and potentially lead to cataracts. Antihistamines, used for conditions such as allergies, can raise certain patients’ risk for glaucoma. Antipsychotic medications, such as THORAZINE® and MELLARIL® can be toxic to the retina. Antimalarials, such as PLAQUENIL® (hydroxychloroquine), used to treat malaria, lupus and rheumatoid arthritis, is a known retinal toxin, and the effects are irreversible. FOSAMAX®, a bisphosphonate that is prescribed for post-menopausal women to prevent calcium bone loss, can cause orbital inflammation, uveitis and scleritis.

Cyclosporine and Tacrolimus, commonly used in patients who have undergone organ or bone marrow transplants, can cause posterior reversible encephalopathy syndrome. These patients will present with bilateral vision loss. Minocycline is a tetracycline derivative and is commonly used to treat acne. Minocycline can cause increased intracranial pressure and papilledema, which can cause permanent vision loss if not reversed. Ethambutol is widely used to treat mycobacterial disease, including tuberculosis. If it is not taken at safe doses, it is an optic nerve toxin. Topiramate (Topamax) is used to treat epilepsy and migraine headaches, and it is used off-label for weight loss. It can cause angle-closure glaucoma soon after starting treatment.

Tamsulosin (Flomax), which is used to treat prostate enlargement and improve urinary flow in men. The well-known syndrome, intraoperative floppy iris syndrome, used to occur only in men who were on medicine to relax their prostate. Women with these drugs can at the time of cataract surgery, make surgical risk much higher. Amiodarone (Cordarone) effectively treats cardiac arrhythmias. It causes the appearance of a whorl in the cornea, which does not usually cause symptoms, although some people can have a little bit of blurred vision.

Anticholinergics, e.g., dicyclomine (BENTYL®), and other drugs with anticholinergic effects, are administered to patients who have stomach conditions that require stomach relaxers and to patients with Parkinson’s disease. Young patients taking these drugs will develop difficulty with accommodation. Erectile dysfunction drugs, e.g., sildenafil citrate (VIAGRA®) and tadalafil (CIALIS®) are often prescribed for men with erectile dysfunction. Some of the ocular side effects include blue vision, and ischemic optic neuropathy. Further, blood pressure medications can cause glaucoma.

In some embodiments, the compositions and methods disclosed herein, such as the MSC Compositions, are used for treating, alleviating, and/or preventing one or more ocular symptoms that arise as a side effect from taking a systemic drug. In some embodiments, the compositions and methods disclosed herein, such as the MSC Compositions, are used for treating, alleviating, and/or preventing one or more ocular symptoms in patients with DED, including, for instance, severe DED. Such symptoms include, for instance, dry eye discomfort, pain, swelling, and ocular surface damage. Other exemplary ocular manifestations that can be treated include moderate to severe keratoconjunctivitis sicca, bilateral marginal keratitis, anterior uveitis, corneal ulceration or neovascularization. Thus, in some embodiments, the MSC Compositions are suitable for treating, alleviating, and/or preventing keratoconjunctivitis sicca, bilateral marginal keratitis, anterior uveitis, corneal ulceration or neovascul ari zati on .

Dosing and dosing regimens

In at least one embodiment, the MSC Compositions have been developed for topical application to the eye, for the treatment of ocular diseases and injuries including DED, Sjogren’s syndrome, cataracts, burns and injuries to the eye tissues. The method can involve the application of one or more formulations of the MSC Compositions directly to the eye(s), preferably as a liquid ocular solution, much like a common liquid eye drops, lubricant or gel. The MSC Compositions delivered to the surface of the eye can alleviate or prevent at least one symptom of a number of ocular injuries and diseases, including in addition to DED, chronic dry eye, Sjogren’s syndrome, and burns or injuries, corneal neovascular disorders, corneal opacities (including corneal haze), and/or prolonged redness and inflammation of the eye(s).

In at least one embodiment, the MSC Compositions have been tested and shown to contain over 300 human growth factors, which can stimulate the proliferation of stem cells, thereby accelerating healing and contributing to modifying the advancement of disease. Due to the viscosity of at least one of the MSC Compositions, drops applied directly onto the eye adhere to the ocular surface longer than common OTC artificial tear formulas. The capacity to adhere to the ocular surface is paramount when treating injuries and diseases such as Sjogren’s syndrome and chemical burns.

Unlike Human Amniotic Membrane (HAM) treatments, in a preferred embodiment, one or more MSC Compositions are provided as a single daily application provided by a licensed ophthalmic profession for in-home use by patients. Therefore, nonsurgical ophthalmologists and optometrists can dispense and oversee the therapy, giving patients greater choices and access to treatment. In addition, unlike the surgical application of HAM, daily applications of the MSC Compositions deliver a sustainable level of beneficial growth factors. Further, the MSC Compositions require much less manipulation during processing and is sterilized without the harsh terminal irradiation or e-beam required for HAM.

As demonstrated by the applications, the concentration and dosage (number of times per day of amount of formulation for period of time) will vary depending on the condition to be treated, the severity of the condition, and the inclusion of other therapeutic, prophylactic or diagnostic agents. The appropriate amounts are determined on an individual basis, measuring response to treatment over time, as demonstrated in the examples. In most cases, two to three drops of solution will be administered once or twice daily as needed.

The dilution ratio of at least some of the MSC Compositions will be dependent on the severity of the disorder or injury; for example, non-severe DED, early to moderate dry eye or chronic redness, surface inflammation and, intraocular inflammation may be best treated with a low concentration, whereas severe DED, Sjogren’s syndrome, a corneal neovascular disorder, or corneal opacity may dictate a higher concentration of these MSC Compositions.

In the case of sustained or controlled release formulations, ointments, implants or injections into the eye, the dosages will be modified to deliver a therapeutically equivalent amount.

Additional therapeutic, prophylactic, and/or diagnostic agents

In at least one embodiment, MSC Compositions may further comprise, and/or may be used in combination with, one or more additional therapeutic, diagnostic, and/or prophylactic agents to alleviate pain (e.g., pain associated with one or more diseases, including, for instance, eye diseases such as DED), facilitate healing, and/or to reduce or inhibit scarring. For instance, such therapeutic, diagnostic, and/or prophylactic agents can be delivered to one or more tissues in a patient via MSC- Exos. In at least an additional embodiment, the MSC Compositions comprise one or more additional compounds to prevent or treat one or more eye diseases e.g., DED), and/or to relieve symptoms such as inflammation. Non-limiting examples include antimicrobial agents, analgesics, local anesthetics, anti-inflammatory agents, antioxidants, immunosuppressants, anti-allergenic agents, enzyme cofactors, essential nutrients, and growth factors.

In some cases, one or more additional active agents may be dispersed in, or otherwise associated with particles in, the MSC Compositions. In certain embodiments, one or more additional active agents may also be dissolved or suspended in the pharmaceutically acceptable carrier.

In at least one embodiment, the active agents include, for instance, small molecules, biomolecule, peptides, sugar, glycoproteins, polysaccharides, lipids, nucleic acids, and/or combinations thereof. Suitable small molecule active agents include, but are not limited to, organic and organometallic compounds. In at least one instance, the aforementioned small molecule active agent has a molecular weight of less than about 2000 g/mol, more preferably less than about 1500 g/mol, and most preferably less than about 1200 g/mol. The small molecule active agent can be a hydrophilic, hydrophobic, or amphiphilic compound. In at least one example, one or more additional agents may be dispersed, dissolved, and/or suspended in one or more MSC Compositions, including, for instance, being delivered in MSC-Exos contained in the one or more MSC Compositions.

In some cases, the active agent is a diagnostic agent imaging or otherwise assessing the eye. Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast media.

When used for the treatment of ocular diseases (e.g., DED), the MSC Compositions may contain one or more ophthalmic drugs to treat, prevent or diagnose a disease or disorder of the eye. Non-limiting examples of ophthalmic drugs include anti-glaucoma agents, anti-angiogenesis agents, anti-infective agents, anti-inflammatory agents, an analgesic, a local anesthetic, growth factors, immunosuppressant agents, anti-allergic agents, an anti-oxidant, a cytokine, and combinations thereof.

The volume of administration of the MSC Compounds may be tissue-specific and dependent on the disease, disorder, and/or condition to be treated. Dosages can be readily determined by those of skill in the art. See, e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th ed.), Williams and Wilkins (1995). Additionally, one or more of the MSC Compositions may be administered in conjunction with other types of cells, e.g., other exogenous stem cells, pluripotent cells, somatic cells, and/or combinations thereof. In at least one embodiment, one or more therapeutic, prophylactic, and/or diagnostic agents is administered prior to, in conjunction with, and/or subsequent to treatment with one or more MSC Compositions. In other embodiments, one or more therapeutic active agents such as an anti-glaucoma agent, an anti-angiogenesis agent, an anti-infective agent, an anti-inflammatory agent, an analgesic agent, a local anesthetic, a growth factor, an immunosuppressant agent, an anti-allergic agent, an anti-oxidant, and a cytokine are administered prior to, in conjunction with, subsequent to, or alternation with treatment with one or more MSC Compositions.

In other embodiments, one or more therapeutic active agents such as an anti-glaucoma agent, an anti-angiogenesis agent, an anti-infective agent, an anti-inflammatory agent, an analgesic agent, a local anesthetic, a growth factor, an immunosuppressant agent, an anti-allergic agent, an anti-oxidant, and a cytokine are administered prior to, in conjunction with, subsequent to, or alternation with treatment with the MSC Compositions.

In at least one embodiment, the aforementioned therapeutic, prophylactic, and/or diagnostic agents may be administered in a neutral form, or in the form of a pharmaceutically acceptable salt. In at least one example, it may be desirable to prepare a formulation containing a salt of an agent due to one or more of the salt's advantageous physical properties, such as, for example, enhanced stability, a desirable solubility, and/or a desirable dissolution profile.

In at least one embodiment, pharmaceutically acceptable salts are prepared by reaction of the free acid or base forms of an active agent with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media such as, for example, ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of an active agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Adejare et al., Remington: The Science and Practice of Pharmacy (23rd ed.), Academic Press (2020).

In at least one embodiment, the MSC Compositions comprise one or more local anesthetics. Non-limiting examples of such local anesthetics include tetracaine, lidocaine, amethocaine, proparacaine, lignocaine, and bupivacaine. In at least one example, one or more additional agents, such as, e.g., a hyaluronidase enzyme, is also added to the MSC Compositions to accelerate and/or improve dispersal of the local anesthetic. In some cases, the active agent is an anti-allergic agent such as olopatadine and/or epinastine. Anti-glaucoma agents

In some embodiments, the one or more additional active agents is one or more antiglaucoma agents. Representative anti-glaucoma agents include prostaglandin analogs (such as travoprost, bimatoprost, and latanoprost), beta-andrenergic receptor antagonists (such as timolol, betaxolol, levobetaxolol, and carteolol), alpha-2 adrenergic receptor agonists (such as brimonidine and apraclonidine), carbonic anhydrase inhibitors (such as brinzolamide, acetazolamine, and dorzolamide), miotics (e.g., parasympathomimetics, such as pilocarpine and ecothiopate), seretonergics muscarinics, dopaminergic agonists, and adrenergic agonists (such as apraclonidine and brimonidine).

Anti-angiogenesis agents

In some embodiments, the one or more additional active agents is one or more antiangiogenesis agents. Representative anti-angiogenesis agents include, but are not limited to, antibodies to vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and other anti-VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanim sodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigment epithelium derived factor(s) (PEDF); COX-2 inhibitors such as celecoxib (CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin- 12 (IL- 12); thalidomide (THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozyme inhibitors such as ANGIOZYME® (Sirna Therapeutics); multifunctional anti angiogenic agents such as NEOVASTAT® (AE-941) (Aetema Laboratories, Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors such as sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib (Nexavar®) and erlotinib (Tarceva®); antibodies to the epidermal grown factor receptor such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®), as well as other anti-angiogenesis agents known in the art.

Anti-infective agents

In at least one embodiment, the MSC Compositions are used in combination with one or more antimicrobial agents. An antimicrobial agent, at least in the context of the present disclosure, is a substance that inhibits the growth of microbes including, for instance, bacteria, fungi, viruses, and/or parasites. Accordingly, antimicrobial agents include, for example, antiviral agents, antibacterial agents, antiparasitic agents, and anti-fungal agents. Non-limiting examples of antiviral agents include, e.g., ganciclovir and acyclovir. Non-limiting examples of antibiotic agents include, for example, aminoglycosides (e.g., streptomycin, amikacin, gentamicin, and tobramycin), ansamycins e.g., geldanamycin and herbimycin), carbacephems, carbapenems, cephalosporins, glycopeptides (e.g., vancomycin, teicoplanin, and telavancin), lincosamides, lipopeptides (e.g., daptomycin, macrolides such as azithromycin, clarithromycin, dirithromycin, and erythromycin), monobactams, nitrofurans, penicillins, polypeptides (e.g., bacitracin, colistin, and polymyxin B), quinolones, sulfonamides, and tetracyclines.

Other exemplary antimicrobial agents include, for instance, iodine, silver compounds, moxifloxacin, ciprofloxacin, levofloxacin, cefazolin, tigecycline, gentamycin, ceftazidime, ofloxacin, gatifloxacin, amphotericin, voriconazole, natamycin.

Anesthetics

In at least one embodiment, the MSC Compositions are administered in combination with one or more local anesthetics. A local anesthetic, at least in the context of the present disclosure, is a substance that causes reversible local anesthesia and has the effect of loss of sensation of pain. Non-limiting examples of local anesthetics include ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and combinations thereof. In at least another aspect of this embodiment, the MSC Compositions include an anesthetic agent in an amount of, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8% about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10% by weight of the total composition. The concentration of local anesthetics in the MSC Compositions can be therapeutically effective, meaning that the concentration is adequate to provide a therapeutic benefit without inflicting harm to the patient.

Ophthalmic anesthetics are agents that act locally to block pain signals at the nerve endings in the eyes. Some exemplary ophthalmic anesthetics are lidocaine, proparacaine, and tetracaine.

Anti-inflammatory agents

In at least one embodiment, the MSC Compositions are administered in combination with one or more anti-inflammatory agents. Anti-inflammatory agents reduce inflammation and include, for instance, steroidal and non-steroidal drugs. Suitable steroidal active agents include, for example, glucocorticoids, progestins, mineralocorticoids, and corticosteroids. Other nonlimiting examples of anti-inflammatory agents include triamcinolone acetonide, fluocinolone acetonide, prednisolone, dexamethasone, loteprednol, fluoromethoIone, ibuprofen, aspirin, and naproxen. Non-limiting examples of immune-modulating drugs include cyclosporine, tacrolimus, and rapamycin. Non-limiting examples of non-steroidal anti-inflammatory drugs (NSAIDs) include ketorolac, nepafenac, and diclofenac.

In at least one embodiment, anti-inflammatory agents are anti-inflammatory cytokines. Non-limiting examples of such cytokines include IL-10, IL-17, TNF-a, TGF-0, IL-35, and others described herein. Anti-inflammatory cytokines in the context of biomaterial implants and tissue grafts are cytokines that induce an anti-inflammatory immune environment or suppress an inflammatory immune environment. Activation of regulatory T cells, Tregs, is involved in the prevention of rejection, and the induction and maintenance of peripheral tolerance of the allograft. Thl7 cells are a subset of T helper cells which is characterized by the production of IL-17. Thl7 cells have been suggested to play a role in allograft rejection. In some embodiments, cytokines to be added to the MSC Compositions are those that induce Tregs activation (e.g., IL-25) and suppress Thl7 activation (e.g., IL-10) for minimizing rejection.

Growth factors

In at least one embodiment, the MSC Compositions are administered in combination with one or more growth factors. As mentioned above herein, growth factors are proteins and/or glycoproteins capable of stimulating cellular growth, proliferation, and/or cellular differentiation. Non-limiting examples of growth factors include transforming growth factor beta (TGF-0), transforming growth factor alpha (TGF-a), granulocyte-colony stimulating factor (GCSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF).

Cofactors and essential nutrients

In at least one embodiment, the MSC Compositions are administered in combination with one or more enzyme cofactors, and/or one or more essential nutrients. Non-limiting examples of such cofactors include vitamin C, biotin, vitamin E, and vitamin K. Non-limiting examples of such essential nutrients include amino acids, fatty acids, etc.

Cells and tissues

In at least one embodiment, the MSC Compositions comprise at least one eukaryotic cell type, including, for instance, at least one cell type other than one or more types of MSCs. Nonlimiting examples of such eukaryotic cell types include non-mesenchymal stem cells, immune cells (e.g., T lymphocytes, B lymphocytes, natural killer cells, macrophages, dendritic cells), and combinations thereof. In at least an additional embodiment, the cells used are cells that dampen one or more inflammation responses (e.g., regulatory T cells). In at least a further embodiment, exosomes are generated ex vivo from one or more types of MSCs (e.g., MSC-Exos). Such exosomes may include, for instance, one or more biological compounds (e.g., MSC-Exos-derived biological compounds, active agents, bioactive agents and/or substances, etc.).

Kits

Any of the compositions described herein (e.g., MSC Compositions) may be comprised in a kit. In a nonlimiting example, cells, reagents to produce cells, exosomes, and reagents to produce exosomes, and/or components thereof may be comprised in a kit. In certain embodiments, exosomes (e.g., MSC-Exos) may be comprised in a kit, and they may or may not yet express one or more bioactive substances. Such a kit may or may not have one or more bioactive substances to be loaded into the exosomes, including reagents to generate same and/or reagents to manipulate the exosomes for loading of the agents. Such agents include small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and/or a combination thereof, for example. In particular embodiments, the kit comprises the exosome-based therapy of the disclosure (e.g., administration with one or more MSC Compositions which may contain, for instance, MSC- Exos) and also another therapy. In some cases, the kit, in addition to the exosome-based therapy embodiments, also includes a second therapy, such as chemotherapy, hormone therapy, immunotherapy, and/or antimicrobial therapy, for example. The kit(s) may be tailored to a particular disease for an individual and comprise respective second therapies for the individual.

The article of manufacture or kit can further comprise a package insert comprising instructions for using the exosomes to treat or delay progression of disease, for example, one or more eye diseases (e.g., DED), cancer, an infection, or an immune disorder, in an individual or to enhance treatment of an individual having cancer, an infection, or an immune disorder. Any of the exosomes described herein (e.g., MSC-Exos) may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or HASTELLOY®). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., any biological compounds, treatments, drugs, and/or substances for treating one or more eye diseases such as DED, a chemotherapeutic substance, an anti -neoplastic agent, an anti-microbial agent, and the like). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.

Embodiments of the present disclosure will be further understood by reference to the following non-limiting examples.

Examples

It should be known 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 disclosure. 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 disclosure.

Example 1: Preparation of MSC-Exos

In some embodiments, MSC-Exos were obtained from MSCs, preferably placental tissue- derived mesenchymal stem cells (“PL-MSCs”), and amniotic fluid of healthy human donors. PL- MSC were grown in complete DMEM. Low passage (<5) PL-MSCs were grown to 60%-80% confluence in multiflasks before isolation. Fresh PL-MSC media were layered and collected after 48 to 72 h (conditioned medium). Exosomes (“Exos”) were isolated by ultracentrifugation (100,000g at 4°C for 70 min). The isolation of exosomes was performed by positive selection using the //MACS™ Separator (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat. No. 130-042-602) and the Exosome Isolation Kit Pan, human (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat. No. 130-110-912) which contained a cocktail of MicroBeads conjugated to the tetraspanin proteins CD9, CD63, and CD81.

Umbilical cord tissue can be extremely attractive and low-cost alternative source of MSCs (UC-MSCs). Generally, UC-MSCs show a higher expansion capacity and a greater exosome yield per cell than BM-MSCs. Given the advantages of UC-MSCs over BM-MSCs as a source of exosomes, UC-Exos can be used, in some aspects, as delivery vehicles in tumor therapy, while in other aspects related to selected diseases and situations, BM exosomes can be used.

Example 2: Exosome electroporation to load miRNAs

One strategy for producing Exos-miRNAs relies on transducing MSCs with lentivirus (LV) containing the cDNA of the mi-RNA, followed by isolation of the mi-RNA from the supernatant. In other cases, mature miRNAs are directly loaded into exosomes by electroporation. Standard operating procedures (SOPs) were utilized for the electroporation of miRNAs into UC-Exos. Specifically, UC-MSCs were cultured, supernatant collected, and UC-Exos isolated by centrifugation. Human miRNA double stranded mature miR-mimic (Sigma Aldrich) was electroporated into UC-Exos. Each electroporation reaction contained approximately 1-2 pg of total exosomal protein. To assess the amount of miRNA loaded into the UC-Exos, the miRNA is treated with RNase (or without RNase as control) to eliminate any free miRNA, total RNA was isolated using TRIZOL™ or the like, and RT-qPCR was performed using primers specific for a particular miRNA. Samples with known quantities of the miRNA are simultaneously assayed to develop a standard curve. Based on the results, electroporation programs that consistently have the lowest Ct value across all replicates were identified. Isolation and use of miRNAs proved very successful. For example, treatment with miR-182 significantly increased tumor toxicity. Further, administration of miR-23b to a subject induced dormancy ofBM2 breast cancer cells and promoted resistance to docetaxel. In addition to loading exosomes with miRs, synthetic interfering RNAs (siRNA) can also be loaded into MSC-derived exosomes.

Example 3: Providing umbilical cord mesenchymal stem cell-derived exosomes to treat toxicities

In addition to the capacity of UC-Exos to deliver bioactive substances, the potential of UC- MSCs to mitigate treatment induced CNS toxicities is also reported. Based on recent evidence indicating that exosomes are capable of reversing traumatic brain injury and inflammation, in some aspects, treatment with MSC-derived exosomes e.g., MSC-Exos) may be as effective as MSCs at reversing chemoradiation-induced brain injury. These data suggested that, in some embodiments, bioactive substances delivered by exosomes such as UC-Exos may be effective in the treatment of neurocognitive toxicities secondary to radiation and chemotherapy.

Example 4: Preparation of MSC Compositions

Materials and methods

A non-limiting example of a MSC Composition is a solution that is an engineered biological product obtained from MSCs (e.g., placental tissue-derived MSCs (“PL-MSC” or “PL- MSCs”)) and/or the amniotic fluid from healthy human donors. Human placental tissue and amniotic fluid was collected from healthy human donor Caesarean sections, as described above. Amniotic fluid and placental tissue were stored in refrigerated condition at 2.5° C. to 6.5° C. prior to the clarification and filtration process. Regarding amniotic fluid preparation, amniotic fluid was centrifuged at 5,000 to 10,000 rpm for 20 minutes to 1 hour in 50 mL to 250 mL receptacles. The supernatant was collected. When collecting the supernatant, it was important to avoid detaching or aspirating insoluble components. If the supernatant contained residual insoluble components, they were pre-filtered with 5 to 10 p cellulose ester capsule pre-filters without TRITON® surfactant to avoid contamination in the filtration process. The liquid phase was collected and filtered with poly ether sulfone 1.0 p capsule filters and the liquid was collected. The liquid was then filtered with poly ether sulfone 0.25 p capsule filter. The filtrate was transferred to vials and sealed with stoppers aseptically. Four samples from the final filtrate were taken to test whether the sterile filtered human amniotic fluid retained exogenous immune cells of interest. Generally, the concentration of the exogenous immune cells in the sterile filtered amniotic fluid was from about 20 pg/mL to about 2400 pg/mL. The concentrations of all the exogenous immune cells in the four samples were in the range of 20-150 pg/mL.

The MSC Compositions may also include MSC-Exos and/or MSC-Exos-derived biological compounds (e.g., bioactive factors, bioactive substances, growth factors, proteins, nucleic acids, cytokines, etc.). PL-MSC were grown in complete DMEM. Low passage (<5) PL-MSCs were grown to 60%-80% confluence in multiflasks before isolation. Fresh PL-MSC media were layered and collected after 48 to 72 h (conditioned medium). Exosomes (“Exos”) were isolated by the ultracentrifugation protocol (100,000g at 4°C for 70 min). The isolation of exosomes was performed by positive selection using the //MACS™ Separator (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat. No. 130-042-602) and the Exosome Isolation Kit Pan, human (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat. No. 130-1 10-912), which contained a cocktail of MicroBeads conjugated to the tetraspanin proteins CD9, CD63, and CD81.

The MSC Compositions can be further prepared by collecting MSCs and/or amniotic fluid. De-cellularization was then performed to remove only cells and particulate matter by a series of centrifugation and filtration steps (e.g., centrifuged at 5,000 to 15,000 rpm for 15 minutes to 1 hour; filtration of supernatant with 5 to 10 p cellulose ester capsule filters). Next, the de- cellularized liquid was incubated and/or stored from 1°C to 20°C, from 2°C to 8°C, at 4°C, or at room temperature, for one or more days, weeks, months, or up to a year.

Before isolation of MSCs, blood samples of healthy donors were tested by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) and found negative using United States (U.S.) Food and Drug Administration (FDA) licensed tests for detection of: Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus Types 1/2, and Treponema Pallidum. All of the samples contain one or more types of MSCs, one or more MSC-Exos (e.g., AF-MSC Exosomes), and one or more MSC-derived and/or MSC-Exos-derived biological compounds e.g., AF-MSC-derived growth factors and/or cytokines), manufactured under current Good Manufacturing Practices (cGMP), regulated and reviewed by the FDA.

As stated above herein, the growth factors derived from MSCs/MSC-Exos may include, for instance, IL-IRa, sTNFRI, sTNFRII, GRO-y, fatty acid-binding protein 1 (FABP1), and/or platelet factor 4 (PF4).

As stated above herein, the cytokines derived from MSCs/MSC-Exos may include, for instance, one or more members of the IL- 12 cytokine family (e.g., IL- 12, IL -23, IL-27, IL-35), one or more CSC chemokines (e.g., CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17), and/or other immunostimulatory molecules known in the art.

Results

MSC Compositions were provided to DED patients for treatment (e.g., attenuating pain, dryness, grittiness, scratchiness, soreness, irritation, burning, watering, and/or eye fatigue. MSC Compositions contain growth factors that (1) suppress IL- 10 and TNF-a-driven inflammation, (2) prevent the generation of inflammatory Thl and Thl7 cells, (3) support tear stability, and (4) reduce ocular surface epithelial damage. The MSC Compositions may further attenuate, prevent, and/or remedy one or more symptoms of DED (e.g., discomfort, pain, ocular damage, etc.).

Patients can be treated with the MSC Compositions for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, or at least about 3 months. The MSC Compositions can be administered as eye drops 3-4 times per day (e.g., at about 10 pg/drop), every day. Changes in eye condition can be observed by measuring, for instance, ocular surface disease index, conjunctiva redness scores, ocular surface staining, best corrected visual acuity, tear secretion, tear meniscus height, tear film breakup time (TBUT), and/or tear film thickness.

TBUT is preferably less than about 10 seconds or less than about 5 seconds after one or more courses of treatment with one or more MSC Compositions. Improvements in TBUT may be seen in at least some patients after, for instance, 21 days after topical administration of one or more MSC Compositions. Tear film thickness, which can be used to measure the liquid layer, the lipid layer, and/or a combination of the liquid layer and the lipid layer, can increase as TBUT increases. Tear film thickness can be measured by, for instance, ( 1) a slit lamp and a video camera, and/or (2) an optical interferometer (e.g., a wavelength-dependent optical interferometer), as described above herein. Specifically, multiple measurements can be taken of an area of a subject’s eye using an optical interferometer. The area has a predetermined length and width (e.g., about 20 pm, about 30 pm, about 50 pm, about 100 pm) and may be located in a suitable area of the eye (e.g., the apex of the cornea). Multiple measurements (e.g., at least about 10, at least about 20, at least about 30, at least about 50) can be taken over a window of time (e.g., 20 seconds to 1 minute). Such tear film thickness measurements may be taken both before and after MSC Compositions-based treatment. Before treatment, tear film thickness may be, for example, less than about 2 microns, or less than about 1 micron. After treatment, tear film thickness may be, for example, about 5 microns, about 6 microns, about 8 microns, about 10 microns, about 12 microns, or more than about 12 microns.

Overall, significantly improved visual acuity, relieved ocular pain, and complete healing of corneal epithelial defects may be noticed in at least some patients. Further, the MSC Compositions can improve the viability of injured corneal epithelial cells and alleviate the symptoms elicited by corneal injury. In addition, the MSC Compositions may (e.g., after four weeks of treatment) result in improved visual acuity and in decreased ocular pain.

Example 5: Treatment of dry eye disease (DED) patients with MSC Compositions

Materials and methods

The MSC Compositions used in this Example are bio-engineered biologic products obtained from amniotic fluid derived MSCs (AF-MSCs), previously collected from healthy human donors. Blood samples were given by the donor prior to, or at the time of, collection. These samples were tested by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) and found negative using United States (U.S.) Food and Drug Administration (FDA) licensed tests for detection of at minimum: Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus Types 1/2, Treponema Pallidum. AF samples were obtained with patient consent and stored at 4°C until processed. The MSC Compositions are then bio-engineered as an AF-MSC-derived sterile product containing, among other components, AF-MSC-Exos and AF-MSC-derived biological compounds e.g., cytokines and growth factors). The MSC Compositions may contain additional components (e.g., osmoprotectants) as described above herein. The MSC Compositions may also have specific properties (e.g., pH) as described above herein. The MSC Compositions may also include one or more solutions as described specifically above herein in previous Examples. The MSC Compositions are manufactured under current Good Manufacturing Practices (cGMP), regulated and reviewed by the FDA. The MSC Compositions are then sterilized to provide for a safe, sterile product that can be introduced as eye drops into the eyes of patients.

Specifically, a total of 131 DED patients were recruited (27 male and 104 female), with a median age of 62 years (ranging from 19 to 85 years of age). Patients received MSC Compositions and were followed up for 12 months. The principles of Good Clinical Practice and the Declaration of Helsinki were adhered to at all times during the study. All patients were under continuous medical supervision by either their ophthalmologist or optometrist.

Results

Patients’ subjective symptoms were graded numerically using VAS (Visual Analogue pain Score). The scale ranges from 0 (defined as the absence of pain) to 10 (defined as maximum pain). Patients were asked to describe their discomfort or pain using VAS.

The Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED) questionnaire was also used to evaluate patients’ dry eye-related symptoms. The SPEED questionnaire asks about symptoms including dryness or grittiness or scratchiness, soreness or irritation, burning or watering, and eye fatigue. The frequency that patients experience these symptoms is scored from 1 through 3. A score of 1 equates to a frequency of “sometimes,” a score of 2 equates to a frequency of “often,” and a score of 3 equates to a frequency of “constant.” The severity of the symptoms is scored from 0 through 4. A score of 0 represents “no problems or symptoms,” a score of 1 represents a “tolerable” severity, a score of 2 represents an “uncomfortable” severity, a score of 3 represents a “bothersome” severity, and a score of 4 represents an “intolerable” severity.

All patients treated with the MSC Compositions experienced significantly reduced VAS and SPEED scores after 3 months (p < 0.001). Specifically, VAS scores declined from a baseline of between 8-9 to between 3-4 after 3 months. SPEED scores declined from a baseline score of between 15-20 to between 5-10 after 3 months. These results indicate that the MSC Compositions managed to improve patient symptoms including pain, dryness, grittiness, scratchiness, soreness, irritation, burning, watering and eye fatigue.

Importantly, the MSC Compositions continued to have beneficial effects after 3 months and, indeed, during the entire 12-month observational period. Specifically, these beneficial effects appear to be significantly increased during the last 6 months of this 12-month period, with the highest reduction in VAS and SPEED scores observed after 12 months of MSC Compositions- based therapy. Indeed, patients’ VAS and SPEED scores were lower after 12 months (p < 0.001) than after 3 months or 6 months. These results indicate that MSC Compositions-based treatment provides long-lasting beneficial effects in alleviating ocular symptoms in DED patients.

Example 6: Treatment of moderate dry eye

A 71 year-old female patient with moderate dry eye resulted from sustained work at a computer for the past 20 years. She had not attained a very comfortable level with the traditional dry eye treatment and had been seeking better therapy. She had a history of allergies. She completed a four-week therapy of MSC Compositions drops (twice a day) along with artificial tears. She used artificial tears more than eight times a day initially with a gradual declining frequency over time.

After the therapy, she observed great improvement in her eye condition. She reached homeostasis and her eyes were comfortable throughout the day. She was almost free of dry eye towards the end of her therapy period although she felt further improvement if the drops were used. Example 7: Treatment of dry eyes

A 64 year-old female patient with dry eyes as a result of her hysterectomy at the age of 38 was treated. She had been diabetic for the past 25 years and had been using metformin. She also had rheumatoid arthritis.

Prior to the study, she was less than comfortable in appearance and semi-squinting constantly. She also had complaints of scratchy, sore and burning eyes.

She completed a four-week therapy of MSC Compositions drops (twice a day) along with artificial tears.

The use of artificial tears declined over time. She had a much improved vision, sunlight sensitivity, comfort levels and appearance after therapy.

Example 8: Treatment of dry eyes and mouth

A 40 year-old female patient diagnosed with Sjogren’s syndrome in 2003 was treated. She noted dry mouth and subsequently dry eye problems. She was overall in good health with no joint pain or swelling, although her appearance was uncomfortable with constant squinting and blinking. She had severe light sensitivity and burning sensation in her eyes. She preferred to keep her eyes closed if possible.

She completed a four-week therapy of MSC Compositions drops (twice a day) along with artificial tears, which were applied eight times a day for four weeks.

After the therapy, the patient reported improvement in redness and light sensitivity, comfort level and abilities. Clinical examination identified a significant staining present, suggesting analgesic benefits to the MSC Compositions that suppressed the clinical evidence of corneal staining.

Example 9: Treatment of dry eye and light sensitivity

A 59 year-old female patient with questionable health conditions was treated. She had a recent weight loss with unexplained reasons, chronic back pain from previous injury as well as rheumatoid arthritis.

Prior to the study, she had dry eye for more than 10 years along with a severe light sensitivity. She also had mild redness in her eyes, swollen superior lid appearance and clumping of eyelashes due to anterior blepharitis. She complained of severe discomfort in her eyes and had no relief from traditional artificial tears. The chief source of her problem was the meibomian gland dysfunction of the “obstructive” type that rendered her inadequate protection of tear evaporation.

She completed a four- week therapy of MSC Compositions drops (twice a day) combined with artificial tears. Artificial tears was used 10 times a day but was later reduced to three times a day during the therapy period.

An improvement in appearance and comfort levels was observed upon the completion of the therapy.

Example 10: Treatment of Sjogren’s syndrome A 74 year old female patient with Sjogren’s syndrome and a severe dry eye condition was treated. She had been forced to compromise some areas in her life such as driving, reading, etc.

After a five-week therapy of MSC Compositions drops (twice a day) combined with artificial tears (six to eight times a day), she commented that she was able to drive and that her light sensitivity improved after four and a half weeks after therapy. Further, after five weeks of therapy, she reported that she had started reading again after years of inability to do so.

Example 11: Treatment of glaucoma

An 80 year-old female patient with glaucoma for 10 years, experiencing loss of vision and dry eye, was treated. After a five and a half-week therapy of MSC Compositions drops (twice a day) combined with artificial tears (six times a day), her reading ability, eye staining, dry eye symptoms and standard examination scores have improved.

She had been unable to read prior to therapy, and was back to reading after therapy. She had significant central and inferior corneal staining in punctate and patches prior to therapy, and the patches were all cleared with only less serious punctate fine staining after therapy. She had superficial cornea edema appearing three weeks after therapy, which vanished with a mild hypertonic solution. For alleviating edema, the topical glaucoma medication could be removed and changed to oral acetazolamide in the future.

Overall summary of the studies

The MSC Compositions, including those described above herein administered in drop form, provide definite and real improvement for DED, dry eye discomfort, tear hyperosmolarity, and/or tear hyperosmolarity-induced changes in patients suffering from dry eye discomfort. Artificial tears have been the mainstay of dry eye therapy and patients would report that these artificial tears are of no help to their condition, while most clinicians feel they offer no therapeutic benefit. The MSC Compositions feature immediate benefits, e.g., within four days of use, and cumulative improvement as therapy progresses. Patients quickly begin to make lifestyle changes by venturing out more, are not as hindered, note improvements in performance and sustainability during tasks such as using a computer or the ability to stay up later in the evening. Patients’ attitudes improve and expectations rise as they sense greater comfort and greater freedom in life, and people are pleased now and at a point of homeostasis. Cosmetic enhancements are noted with all patients due to less injection of bulbar and palpebral conjunctiva. Improvements are noted among a difficult subset of people knows as severe dry eye patients.

Severe DED patients often present with compromised appearances due extreme discomfort. Indications of this are habitual squinting, gaze in downward position versus straight ahead, listening to conversation with eyes closed instead of eyes open with good eye contact, high blinking frequency, etc.

A noticeable change in the appearance was apparent in patients in these studies by the end of two weeks of therapy. Other people would comment to these patients that their eyes were looking better. Most patients expressed improvements and increased comfort with therapy. Most patients expressed satisfaction and interest in continuing on the therapy. The majority of the 9 patients studied showed improvements in light sensitivity. One patient reported after two weeks of therapy being able to return to driving after years of avoiding it due to eye discomfort from dryness, sunlight, etc.

The dry eye ocular surface disease index (OSDI) showed a general trend of improvement in OSDI scores as therapy continued.

Frequency of artificial tear use among patients showed a general trend that patients will use less artificial tears after initiating this therapy. This was a surprise early in therapy, often volunteered without prompting. Despite the patients “feeling” like they do not need their previous artificial tears as much as prior to amniotic eye drop therapy, there is objective evidence the patient may benefit from the use more than they are aware. The advantages some artificial tears are meant to provide seem to still benefit the patient, even when the patients are experiencing a new level of soothing and comfort from the use of the MSC Compositions. Supplemental therapy with artificial tears for the moderate dry eye patient, who had no objective clinical evidence of dry eye remaining after three weeks of therapy, showed further improvement in comfort when artificial tears were applied. This observation verifies the hypothesis of what may or may not be accomplished in dry eye therapy. For instance, the forces of evaporation still present challenges to the ocular surface, which are aided by this type of therapy control and management.

Improved reading performance was noted in the majority of the patients, while the other patients had early cataracts developed prior to therapy. Improvements in visual acuity (VA) were noted in the majority of the patients with at least one line on the Snellen chart and in others, two or more. Visual acuity improvements seem closely correlated to corneal integrity levels. When central corneal integrity is compromised as evidenced by corneal staining, visual acuity levels are also compromised. As corneal integrity improves with good therapy, visual acuity also improves as indicated. The MSC Compositions used help heal the corneal surface integrity issues, but are not expected to rehydrate these tissues, and traditional methods of dry eye care may still be advantageous to treat this aspect of dry eye disease. All patients demonstrated improvements in palpebral and bulbar injection levels in essentially all patients within the study.

These and other objectives and features of the invention are apparent in the disclosure, which includes the above and ongoing written specification. The foregoing description details certain embodiments of the invention. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to including any specific characteristics of the features or aspects of the invention with which that terminology is associated.

The invention is not limited to the particular embodiments illustrated in the drawings and described above in detail. Those skilled in the art will recognize that other arrangements could be devised. The invention encompasses every possible combination of the various features of each embodiment disclosed. One or more of the elements described herein with respect to various embodiments can be implemented in a more separated or integrated manner than explicitly described, or even removed or rendered as inoperable in certain cases, as is useful in accordance with a particular application. While the invention has been described with reference to specific illustrative embodiments, modifications and variations of the invention may be constructed without departing from the spirit and scope of the invention as set forth in the following claims.

Claims

What is claimed is:

1. A method of treating, alleviating, and/or preventing dry eye disease (DED), the method comprising: administering to a subject in need thereof an effective amount of a composition, the composition comprising: one or more types of mesenchymal stem cell-derived exosomes (MSC-Exos), and/or one or more biological compounds derived from the one or more types of MSC- Exos.

2. The method of claim 1, wherein the one or more types of MSC-Exos are derived from one or more human tissues, wherein the one or more human tissues comprise lacrimal glands.

3. The method of claim 1, wherein the one or more biological compounds comprise one or more bioactive compounds, wherein the one or more bioactive compounds are selected from the group consisting of: proteins, nucleic acids, growth factors, immunoregulatory molecules, cytokines, chemokines, and combinations thereof.

4. The method of claim 1, wherein the administration results in at least one of: attenuating nitric oxide (NO), TNF-a, IL-ip, and/or reactive oxygen species (ROS) production in eye-infiltrated immune cells, inhibiting expression of co-stimulatory molecules in one or more types of immune cells, suppressing synthesis of pro-Thl and Thl7 cytokines, and impeding expansion of Thl and Thl7 cells.

5. The method of claim 4, wherein the pro-Thl and Th 17 cytokines are selected from the group consisting of: IL- 12, IFN-y, IL-ip, IL-6, IL- 12, and combinations thereof.

6. The method of claim 1, wherein the one or more types of MSC-Exos result in at least one of: triggering ocular surface epithelial repair, improving tear film stability, and preventing inflammation-induced apoptosis of corneal epithelial cells (CECs).

7. The method of claim 1, wherein the one or more types of MSC-Exos result in decreased levels of inflammatory cytokines, wherein the inflammatory cytokines are selected from the group consisting of: IL-ip, IL-6, IL- la, TNF-a, and combinations thereof.

8. The method of claim 1, wherein the one or more types of MSC-Exos result in at least one of: enhancing the production of immunosuppressive IL- 10 to suppress generation of an inflammatory phenotype in eye-infdtrated neutrophils and monocytes, reducing expression of NLRP3, IL-ip, and/or IL-18 in one or more eye tissues, and attenuating NLRP3-driven inflammation.

9. The method of claim 1, wherein the one or more types of MSC-Exos result in suppression of caspase-3, thereby inhibiting caspase-3 -driven apoptosis.

10. The method of claim 9, wherein the inhibition of caspase-3 -driven apoptosis results in at least one of: preventing loss of corneal epithelial cells (CECs), and enabling enhanced regeneration of the ocular surface epithelial barrier.

11. The method of claim 1, wherein the one or more types of MSC-Exos suppress T cell-driven inflammation by at least one of: promoting expansion of immunosuppressive Tregs in one or more inflamed eyes of the subject, decreasing serum levels of Thl 7-related inflammatory cytokines, increasing serum levels of immunosuppressive TGF-P, downregulating presence of Th 17 cells, suppressing activation of the Jak-Stat signaling pathway in IL-17-producing Thl7 cells to cause G0/G1 cell cycle arrest, and increasing Treg:Thl7 ratio in the one or more inflamed eyes of the subject.

12. The method of claim 1, wherein the one or more types of MSC-Exos suppress T cell-driven inflammation by delivering IL- 10, thereby inducing generation of a tolerogenic phenotype in dendritic cells (DCs).

13. The method of claim 12, wherein the tolerogenic DCs create an immunosuppressive environment in one or more eye tissues of the subject.

14. The method of claim 1, wherein the one or more types of MSC-Exos alter a secretory profile of T cells by at least one of: downregulating production of one or more inflammatory cytokines, and increasing production and/or secretion of IL-10 and/or TGF-p.

15. A method of treating, alleviating, and/or preventing dry eye disease (DED), the method comprising: administering to a subject in need thereof one or more types of mesenchymal stem cell- derived exosomes (MSC-Exos), wherein the MSC-Exos contain one or more biological compounds that are delivered to one or more eye tissues of the subject.

16. The method of claim 15, wherein the one or more biological compounds comprise growth factors selected from the group consisting of: VEGF, TGF-P, PDGF, and combinations thereof.

17. The method of claim 15, wherein the MSC-Exos provide bioactive lipids that integrate into tear film of the subject, wherein the bioactive lipids are selected from the group consisting of: phospholipids, cholesterol, glycolipids, and combinations thereof.

18. The method of claim 15, wherein the one or more biological compounds comprise microRNAs, wherein the microRNAs comprise miRNA-125b.

19. The method of claim 18, wherein the miRNA-125b results in at least one of: reducing the number of auto-antibody-producing plasma cells, reducing the amount of auto-antibody production, and attenuating one or more B cell-driven autoimmune responses.

20. The method of claim 18, wherein the miRNA-125b results in at least one of: preventing and/or reducing interactions between PR domain zinc finger protein 1

(PRDM1) mRNA and ribosomes, and inhibiting synthesis ofPRDMl.

21. The method of claim 20, wherein the miRNA-125b regulates development, recruitment, and/or activation of B cells and impairs B cell differentiation in auto-antibody- secreting plasma cells.

22. The method of claim 15, wherein the one or more biological compounds comprise at least one bioactive substance, wherein the MSC-Exos are produced by: transfecting or transducing one or more types of mesenchymal stem cells (MSCs) to load at least one bioactive substance into the one or more types of MSCs, to generate transfected or transduced MSCs; culturing the transfected or transduced MSCs, to generate cultured MSCs; and collecting exosomes from the cultured MSCs, wherein the exosomes (i) are generated from the transfected or transduced MSCs, and (ii) comprise the at least one bioactive substance.

23. The method of claim 15, wherein the one or more biological compounds comprise at least one bioactive substance, wherein the MSC-Exos are produced by: culturing one or more types of mesenchymal stem cells (MSCs), to generate cultured MSCs; collecting exosomes from the cultured MSCs; and electroporating the collected exosomes to load at least one bioactive substance into the collected exosomes.

24. A pharmaceutical composition comprising: one or more types of MSC-derived exosomes (MSC-Exos) and/or one or more biological compounds derived from the one or more types of MSC-Exos; one or more pharmaceutically acceptable excipients; and one or more pharmaceutically acceptable carriers.

25. The pharmaceutical composition of claim 24, wherein the one or more types of MSC-Exos and/or one or more biological compounds derived from the one or more types of MSC-Exos are formulated in a formulation for topical ocular administration.

26. The pharmaceutical composition of claim 25, wherein the formulation is a topical solution, and wherein the topical solution comprises one or more osmoprotectants.

27. The pharmaceutical composition of claim 24, wherein the one or more types of MSC-Exos are encapsulated in a lipid bilayer.

28. The pharmaceutical composition of claim 24, further comprising one or more additional agents, wherein the one or more additional agents are selected from the group consisting of: an adjuvant, an antigen, an excipient, a vaccine, an allergen, an antibiotic, a gene therapy vector, a kinase inhibitor, a co-stimulatory molecule, a Toll-like receptor (TLR) agonist, a TLR antagonist, a therapeutic agent, a prophylactic agent, a diagnostic agent, an antimicrobial agent, an analgesic, a local anesthetic, an anti-inflammatory agent, an anti-oxidant agent, an immunosuppressant agent, an anti-allergenic agent, an enzyme cofactor, an essential nutrient, a growth factor, and combinations thereof.

29. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition is administered prior to, in conjunction with, subsequent to, or alternating with, one or more therapeutic, prophylactic, and/or diagnostic agents.

30. The pharmaceutical composition of claim 29, wherein the one or more therapeutic, prophylactic, and/or diagnostic agents is selected from the group consisting of: an anti-glaucoma agent, an anti-angiogenesis agent, an anti-infective agent, an anti-inflammatory agent, an analgesic agent, a local anesthetic, a growth factor, an immunosuppressant agent, an anti-allergic agent, an anti-oxidant, a cytokine, and combinations thereof.

31. The pharmaceutical composition of claim 24, wherein the one or more biological compounds comprises anti-apoptotic factors that inhibit caspase-3 activation.

32. The pharmaceutical composition of claim 24, wherein the one or more biological compounds comprises a therapeutic agent for treating dry eye disease (DED).

33. A kit comprising : a container containing a composition comprising one or more types of mesenchymal stem cell-derived exosomes (MSC-Exos) and/or one or more biological compounds derived from the one or more types of MSC-Exos; and an applicator for administering the composition to an eye of a subject.

34. The kit of claim 33, wherein the applicator minimizes contamination and delivers an accurate dosage to the eye.

35. The kit of claim 33, wherein the container contains one or more single, sterile unit doses, wherein the one or more single, sterile unit doses is in the form of eye drops.

36. The kit of claim 33, wherein the subject has dry eye disease (DED), and wherein the composition induces an anti-inflammatory phenotype in one or more eye-infdtrated immune cells of the subject.

37. The kit of claim 36, wherein the anti-inflammatory phenotype is characterized by an increase in one or more anti-inflammatory cytokines and/or a decrease in one or more pro- inflammatory cytokines.

38. The kit of claim 33, wherein the one or more types of MSC-Exos modulate endothelial cell expression of selectins in the subject, and wherein the selectins comprise E and P selectins,

39. The kit of claim 33, wherein the one or more biological compounds comprise microRNAs, and wherein the microRNAs comprise miRNA-125b.

40. The method of claim 1, wherein the one or more types of MSC-Exos are derived from one or more types of mesenchymal stem cells (MSCs) isolated from a tissue selected from the group consisting of: adipose tissue, bone marrow, umbilical cord, and combinations thereof.