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WO2025151822A1 - Administration de médicament exosome dérivé de cellules souches mésenchymateuses pour la sécheresse oculaire et d'autres troubles - Google Patents

Administration de médicament exosome dérivé de cellules souches mésenchymateuses pour la sécheresse oculaire et d'autres troubles

Info

Publication number
WO2025151822A1
WO2025151822A1 PCT/US2025/011244 US2025011244W WO2025151822A1 WO 2025151822 A1 WO2025151822 A1 WO 2025151822A1 US 2025011244 W US2025011244 W US 2025011244W WO 2025151822 A1 WO2025151822 A1 WO 2025151822A1
Authority
WO
WIPO (PCT)
Prior art keywords
msc
exos
cells
exosomes
eye
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/011244
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English (en)
Inventor
Carl Randall HARRELL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MAM Holdings of West Florida LLC
Original Assignee
MAM Holdings of West Florida LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MAM Holdings of West Florida LLC filed Critical MAM Holdings of West Florida LLC
Publication of WO2025151822A1 publication Critical patent/WO2025151822A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/04—Artificial tears; Irrigation solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources

Definitions

  • the present invention relates generally to methods and compositions for immunotherapy and drug delivery.
  • 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).
  • DED dry eye disease
  • 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.
  • a primary function of the mammalian cornea and its surrounding structures is to moisten the eye.
  • DED dry eye disease
  • 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.
  • HAM Human amniotic membrane
  • HAM Human amniotic membrane
  • these procedures usually impose severe vision impairment during treatment as the amniotic membrane is non-transparent.
  • 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.
  • MSC Mesenchymal stem cells
  • 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).
  • sources e.g., bone marrow, amniotic fluid, placental tissue, spinal cord, umbilical cord blood, umbilical cord tissue, adipose tissue, etc.
  • biological compounds e.g., carbohydrates, proteins and peptides, lipids, lactate, pyruvate, electrolytes, enzymes, hormones, and various growth factors.
  • 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).
  • 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).
  • bioactive molecules e.g., microRNAs, enzymes, cytokines, chemokines, immunomodulatory, trophic, growth factors, and the like
  • MSC-Exos 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.
  • exosomes including, for instance, MSC-sourced exosomes and/or MSC-derived exosomes.
  • 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.
  • 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.
  • 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.
  • a treatment e.g., for one or more eye disorders such as dry eye disease (DED)
  • 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).
  • various ophthalmic and ocular conditions and/or diseases e.g., dry eye disease (DED), including severe DED.
  • 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.
  • 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.
  • the exosome e.g., MSC-Exos
  • the exosome is isolated from autologous cells of a subject, from a cell line, from a primary cell culture, and/or from a mesenchymal stem cell.
  • the at least one plasmid is an RNA plasmid, a DNA plasmid, or any combination thereof.
  • any medical disorder e.g., DED
  • the exosomes e.g., MSC-Exos
  • 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)
  • exosomes e.g., MSC-Exos
  • 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.
  • 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.
  • the MSCs are from umbilical cord tissue or amniotic fluid.
  • the one or more biological compounds and/or bioactive substances is miRNA, siRNA, shRNA, protein, peptides, drug, lipids, DNA, RNA, or a combination thereof.
  • 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.
  • the protein comprises an antibody or antibody fragment.
  • the one or more biological compounds and/or bioactive substances is miRNA, a nucleic acid therapeutic, and/or a protein therapeutic.
  • 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.
  • 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.
  • 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).
  • a method for prevention and treatment of a disease 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.
  • endogenous immune cells e.g., dendritic cells, macrophages, natural killer cells, T cells, and the like
  • administration of an effective amount of MSC Compositions improves one or more symptoms of one or more eye diseases in the subject.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions are administered with a pharmaceutically acceptable carrier.
  • such compositions are administered as a solution, suspension, ointment, or gel, with or without an implant.
  • 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.
  • 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
  • one or more MSC Compositions in combination with one or more therapeutic, prophylactic or diagnostic agents are also described.
  • 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.
  • 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.
  • the one or more diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, contrast media.
  • Figure 1 shows various molecular and cellular effects that occur in dry eye disease (DED), according to at least one embodiment of the disclosure.
  • FIG. 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.
  • MSCs mesenchymal stem cells
  • FIG 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.
  • FIG 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.
  • MDSCs myeloid-derived suppressor cells
  • FIG. 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.
  • T-cell driven inflammation e.g., eye inflammation in DED patients
  • administering 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.
  • 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.
  • 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.
  • biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient or subject.
  • biodegradable polymer 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.
  • 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).
  • metastasis e.g., spread to other locations in the subject’s body via lymph or blood.
  • 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.
  • cancer 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.
  • 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.
  • abate 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.).
  • 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”).
  • APC antigen-presenting cell
  • tDC tumor-associated dendritic cells
  • an agent e.g., including one or more MSC Compositions described herein
  • 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.
  • 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).
  • 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.).
  • 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.
  • 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)).
  • 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)).
  • immunological refers 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.
  • a beneficial humoral i.e., antibody-mediated
  • a cellular response e.g., mediated by immune cells, such as antigen-specific T cells, or their secretion products
  • 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.
  • MHC major histocompatibility complex
  • 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.
  • proliferation assays e.g, CD4+ T cells
  • CTL cytotoxic T lymphocyte
  • 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.
  • implant 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.
  • 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.
  • nanoparticle 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.”
  • parenteral administration refers to a type of administration by any method other than through the digestive tract or non-invasive topical or regional routes.
  • parenteral administration may include administration to a subject via intravenous, intradermal, intraperitoneal, intrapleural, intratracheal, intraarticular, intrathecal, intramuscular, subcutaneous, subjunctival, injection, and/or infusion.
  • peptide refers to a polymer of amino acid residues.
  • the amino acid residues may be naturally occurring and/or non-naturally occurring.
  • polypeptide peptide
  • protein protein
  • 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.
  • pharmaceutically acceptable 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.
  • subject refers 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.
  • 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.
  • 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.
  • 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.
  • the subject is a laboratory animal/organism, such as, for example, a mouse, rabbit, guinea pig, or rat.
  • a subject includes, for instance, farm animals, domestic animals and/or pets (e.g., cats, dogs).
  • 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.
  • 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.
  • treating refers, 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.
  • treatment with the disclosed one or more agents, formulations, and/or methods results in a clinical improvement in one or more eye diseases in a subject.
  • Dry eye disease also known as dry eye syndrome or keratoconjunctivitis sicca
  • DED Dry eye disease
  • 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.
  • DED patients 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.
  • Injured epithelial cells e.g., injured corneal and conjunctival epithelial cells
  • DAMPs damaged associated molecular patterns
  • alarmins e.g., alarmins
  • 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.
  • TNF-a tumor necrosis factor alpha
  • IL-1 interleukin 1 beta
  • Thl cell-sourced IFN-y 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.
  • inflammatory mediators e.g., nitric oxide (NO), reactive oxygens species (ROS), TNF-a, IL-10, IL-6, IL-12, IL-23
  • Such increased synthesis may favor the generation of an inflammatory phenotype in eye-infiltrated monocytes/macrophages, shown at block 116.
  • 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.
  • CD8+ cytotoxic lymphocytes 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.
  • chronic T cell-driven inflammation shown at block 134
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MGD meibomian gland dysfunction
  • 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.
  • 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 “h
  • MSC Mesenchymal stem cells
  • DED dry eye disease
  • MSC-Exos MSC-sourced exosomes
  • 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.
  • MSCs, MSC-Exos, and/or MSC-Exos-derived biological compounds e.g., the bioactive factors referred to above herein and described further below herein
  • MSC-Exos-derived biological compounds are new remedies in regenerative ophthalmology.
  • compositions containing MSCs, MSC-Exos, and/or MSC-Exos-derived biological compounds 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.
  • 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.
  • MSCs can be most frequently derived from bone marrow (BM), umbilical cord (UC), amniotic fluid (AF), and adipose tissue (AT).
  • BM- MSC bone marrow
  • UC umbilical cord
  • AF amniotic fluid
  • AT adipose tissue
  • 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.
  • UC-MSC UC-derived MSC
  • AT- MSC AT-derived MSC
  • AT-MSC AT-derived MSC
  • BM-MSC and AT-MSC may differentiate into corneal epithelial cells.
  • SHEM hormonal epidermal medium
  • DMEM Dulbecco's Modified Eagle Medium
  • ATRA all- trans-retinoic acid
  • epithelial markers e.g., cytokeratin (CK)12, CK3, CK19, E- cadherin
  • mesenchymal markers e.g., Vim, snail and alpha smooth muscle actin (a-SMA)
  • MSC SHEM SHEM
  • MSC ATRA ATRA-supplemented medium
  • MSC DMEM standard culture conditions
  • human corneal epithelial cells 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 .
  • 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.
  • TGF-P receptor type-1 TGF-P receptor type-2
  • Ras-related C3 botulinum toxin substrate 1 Ras-related C3 botulinum toxin substrate 2 derived from UC-MSC
  • Ras-related C3 botulinum toxin substrate 2 derived from UC-MSC
  • JNK Jun-N-terminal kinase
  • 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.
  • immunomodulatory factors e.g., IL-10, TGF-0, growth related oncogene (GRO), indoleamine 2,3 dioxygena
  • 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.
  • FOXP3 also referred to as “scurfin” is a protein that assists in regulation of regulatory pathways, including, for example, development of Tregs.
  • 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.
  • 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.
  • co-stimulatory molecules e.g., CD80 and CD86
  • pro-Thl and pro-Thl7 cytokines e.g., IL-12, IL-ip, IL-6, IL-23
  • MSC are also able to efficiently inhibit proliferation and cytotoxicity
  • 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.
  • pro-apoptotic and toxic molecules e.g., perforins and granzymes
  • cytotoxic cytokines e.g., TNF-a and IFN-y
  • Juxtacrine communication 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.
  • PDL programmed death-ligand
  • FasL Fas ligand
  • 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.
  • 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.
  • 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.
  • MSCs have enormous therapeutic potential, several side effects caused by engrafted MSCs can limit their present clinical use in DED treatment.
  • 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.
  • MHC major histocompatibility class
  • 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.
  • spontaneous differentiation of MSCs in chondrocytes and osteocytes can compromise tissue structure, integrity and function.
  • 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.
  • pro-angiogenic factors e.g., angiopoietin, vascular endothelial growth factor (VEGF), IL-6
  • VEGF vascular endothelial growth factor
  • IL-6 vascular endothelial growth factor
  • MSC-Exos MSC-derived exosomes
  • EVs extracellular vesicles
  • 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).
  • proteins e.g., growth factors, immunoregulatory molecules, cytokines, chemokines
  • lipids e.g., 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 D
  • 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.
  • 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 can attenuate cell-driven eye inflammation in DED processes.
  • BAC benzalkonium chloride
  • 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)).
  • PBS phosphate-buffered saline
  • 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.
  • CECs corneal epithelial cells
  • 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.
  • inflammatory cytokines e.g., IL-10, IL-6, IL-1 a, TNF-a, IFN-y
  • 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.
  • 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.
  • MSC-Exos 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.
  • anti-inflammatory cells e.g., tolerogenic DCs and forkhead box P3 (FoxP3)-expressing CD4+CD25+T regulatory cells (Tregs)
  • 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.
  • pro-Thl and pro- Thl7 cytokines e.g., IL-12, IL-23, IL-10, IL-6
  • IDO indoleamine 2,3 -dioxygenase
  • IDO insulin receptor RI
  • TRP tryptophan
  • KYN kynurenine
  • 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).
  • 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).
  • various inflammatory cytokines e.g., IL-ip, IL-6, IL-la, and TNF-a
  • 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.
  • 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.
  • 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.
  • MDSCs myeloid-derived suppressor cells
  • cytokines e.g., TGF-P and IL-10
  • 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.
  • immunoregulatory factors e.g., arginase-1 and NO
  • Such immunoregulatory factors can then inhibit the proliferation of activated Thl and Thl7 cells, as shown at block 408.
  • 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.
  • MDSCs can, as shown at block 422, inhibit the expansion of effector Thl and Thl 7 cells.
  • 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.
  • OE-MSC-Exos 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.
  • MHC class II and co-stimulatory molecules e.g., CD40, CD80, CD86
  • 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.
  • TLR-4 toll like receptor
  • 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.
  • MSC-Exos-dependent polarization of T cells can also contribute to the beneficial effects of MSC-Exos in the treatment of DED.
  • tolerogenic DCs in an IDO-dependent manner, can induce the generation of FoxP3 -expressing Tregs and prevent their transdifferentiation in inflammatory Thl 7 cells.
  • MSC-Exos can prevent maturation and induce the generation of a tolerogenic phenotype in DCs.
  • 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.
  • MSC-Exos can attenuate DED-related symptoms via, for instance, MSC-Exos-dependent enhancement of Treg-driven immunosuppression of inflammatory Thl and Thl7 cells.
  • 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).
  • MNCs can previously be isolated from the blood of patients suffering with various diseases (e.g., primary Sjogren's syndrome (pSS)).
  • pSS primary Sjogren's syndrome
  • 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.
  • LG-MSC-Exos may significantly increase saliva flow rate in experimental animals.
  • 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.
  • Thl7-related inflammatory cytokines e.g., IL-6 and IL- 17
  • 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.
  • inflammatory cytokines e.g., IL- 17, IL-6, TNF-a, IL-6
  • 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.
  • 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.
  • 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.
  • various inflammatory cytokines e.g., IFN-y, TNF-a, IL-6, IL-17A, and IL-17F
  • UC-MSC-Exos UC-MSC-Exos
  • oGVHD ocular graft versus disease
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a gas regulator allows for, if desired, continual infusion of one or more gases into the cell culture reactor.
  • 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).
  • exosomes are enriched or concentrated from the medium of cultured MSCs using differential ultracentrifugation followed by filtration through a sucrose gradient.
  • 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.
  • 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.
  • D2O Tris/sucrose/heavy water
  • 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.
  • 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).
  • the exosomes may be further modified.
  • the exosomes are further modified to harbor and/or carry one or more bioactive substances, including any of the biological compounds described herein.
  • the MSCs are modified (e.g., transfected, transduced, electroporated, etc.), and modified exosomes are generated by the modified MSCs.
  • 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.
  • 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.
  • bioactive substances may also be referred to as “biological compounds,” “agents,” and/or “therapeutic agents” interchangeably throughout this specification.
  • the exosomes are of a specific size such that their size determines the type of bioactive substances that they can carry.
  • 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.
  • 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.
  • 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.
  • exosomes may also be loaded by directly transforming or transfecting the exosomes with a nucleic acid construct that expresses the bioactive substance(s).
  • the nucleic acid construct encoding the bioactive substance(s) is comprised in a vector.
  • 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.
  • 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.
  • 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
  • a suitable vector is capable of crossing the blood-brain barrier.
  • 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).
  • adenoviruses provide a convenient platform for gene delivery systems.
  • 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.
  • 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.
  • 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.
  • the vector 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.
  • 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.
  • the MSCs and/or exosomes are loaded by electroporation.
  • 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.
  • an electroporation system may be controlled to create electric current and send it through a cell- or exosome-containing solution.
  • a static electroporation apparatus is used.
  • a flow electroporation apparatus is used.
  • 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.
  • 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.
  • Pulse duration 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.
  • electrical pulses can also be characterized by pulse number, pulse width, pulse shape, pulse pattern, and pulse polarity.
  • 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 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the pulse generator generates an exponential decay wave pulse, and the pulse width is a function of a rate of exponential decay.
  • 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,
  • 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,
  • 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).
  • 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.
  • RT-qPCR realtime quantitative PCR
  • 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.
  • the exosomes are able to be loaded with any type of bioactive substance(s).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the exosomes 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.
  • 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
  • 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.
  • 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.
  • a bioactive substance may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mit
  • 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
  • non-limiting examples of antimicrobial agents include, for instance, an antibiotic, an antifungal, an antiviral, 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.
  • 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.
  • anionic peptides e.g., dermicidin
  • linear cationic a-helical peptides e.g, LL37
  • cationic peptides enriched for proline arginine, phenylalanine, glycine, or tryptophan
  • 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 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.
  • polyene antifungals e.g, amphotericin B, nystatin, natamycin, and the like
  • flucytosine e.g., imidazoles (e.g.
  • 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),
  • 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).
  • a non-coding RNA molecule (guide) RNA which sequence-specifically binds to DNA
  • a Cas protein e.g., Cas9
  • nuclease functionality e.g., two nuclease domains
  • 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.
  • 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
  • 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.
  • the exosomes are useful for one or more cancers.
  • exosomes derived from umbilical cord tissue-derived MSCs 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.
  • 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.
  • the cancer may be primary, metastatic, resistant to therapy, and so forth.
  • 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.
  • 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.
  • 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
  • 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
  • cancers for which the present exosomes are useful is glioblastoma multiforme (GBM).
  • GBM glioblastoma multiforme
  • 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 daunting 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.
  • BBB/BTB blood brain/blood tumor barriers
  • exosomes used to treat GBM are loaded with the anti-GMB miRNA miR-124.
  • 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.
  • 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.
  • the exosomes e.g., MSC-Exos
  • the exosomes derived from umbilical cord tissue-derived MSCs 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.
  • 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, celiacdynamis-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,
  • 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.
  • 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.
  • 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, P
  • 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.
  • protozoa examples include, but are not limited to, Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium.
  • 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.
  • exosome compositions of the disclosure 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 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” 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.);
  • compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • the dose of single or multiple systemic injections is in the range of 10 to 100 mg/kg of body weight.
  • 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.
  • 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.
  • compositions containing MSCs, MSC-Exos, and/or MSC-Exos-derived biological compounds can be used for the treatment of one or more diseases and/or disorders, including eye disorders such as DED.
  • the MSC Compositions are composed in a solution that can be delivered topically to a patient’s eye.
  • 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).
  • OTC over-the- counter
  • cGMP current Good Manufacturing Practices
  • FDA Food and Drug Administration
  • 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.
  • MSC-Exos-derived biological compounds e.g., active agents, bioactive agents, growth factors, immunoregulatory proteins, drugs, etc.
  • 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
  • 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 metall
  • EGF Epidermal growth factor
  • 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).
  • VEGF Vascular endothelial growth factor
  • VPF vascular permeability factor
  • 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): Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX): Austin ( Austin (TX):
  • 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).
  • TGF-P Transforming growth factor-beta
  • TGF-P Transforming growth factor-beta
  • 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.
  • HGF Hepatocyte growth factor
  • Fibroblast growth factors 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.
  • Matrix metalloproteinases 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)).
  • TIMPs tissue inhibitors of metalloproteinases
  • Exosomes 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 (M
  • 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).
  • IL-8 interleukin 8
  • IL- 13 interleukin 13
  • IL-27 interleukin 27
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • VEGF-D vascular endothelial growth factor D
  • IL-IRa interleukin- 1 receptor antagonist
  • TGF01 transforming growth factor beta 1
  • IL-5 interleukin 5
  • IL -21 interleukin 21
  • 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.
  • 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 tri
  • the MSC Compositions include one or more pharmaceutically acceptable salts, such as, for instance, one or more chloride, acetate, and/or citrate salts.
  • 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).
  • the MSC Compositions include one or more physiological buffers, such as a phosphate (e.g., monobasic sodium phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate).
  • physiological buffers such as a phosphate (e.g., monobasic sodium phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate).
  • 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.
  • hyaluronic acid e.g., crosslinked hyaluronic acid
  • sodium hyaluronate e.g., crosslinked hyaluronate
  • chondroitin sulfate e.g., dermatan sulfate
  • heparin sulfate e.g., keratin sulfate
  • keratin sulfate hydroxylpropylmethylcellulose
  • recombinant human collagen recombinant human collagen
  • 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.
  • 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).
  • cellulose-based compounds e.g., hydroxyethyl cellulose
  • polyvinyl-based compounds e.g, polyvinyl alcohol, polyvinylpyrrolidone
  • acrylic compounds e.g., one or more
  • 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.
  • 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.
  • mPa sec milliPascal-seconds
  • 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 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.
  • the MSC Compositions are distributed or packaged in a liquid form.
  • 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.
  • Suitable preservatives 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.
  • PHMB polyhexamethylenebiguanidine
  • BAK benzalkonium chloride
  • Purite® stabilized oxychloro complexes
  • phenylmercuric acetate chlorobutanol
  • sorbic acid chlorhexidine
  • chlorhexidine benzyl alcohol
  • parabens parabens
  • thimerosal and mixtures thereof.
  • 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
  • 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.
  • 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.
  • the particles may be formed using a method which produces a monodisperse population of nanoparticles.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • extrusion methods may be used to avoid the need for solvents during implant manufacture.
  • 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.
  • 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.
  • psi pounds per square inch
  • 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.
  • 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.
  • degradation e.g., chemical and/or enzymatic degradation.
  • the MSC Compositions can be stored in refrigerated conditions at about 1° C. to about 10° C.
  • the MSC Compositions can be refrigerated at 4° C. for up to 12 months and more.
  • 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.
  • 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.
  • storage of the MSC Compositions 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.
  • 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.
  • 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.
  • degradation of one or more components of the MSC Compositions 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%.
  • compositions and methods disclosed herein 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.
  • the MSC Compositions are used to alleviate pain, facilitate healing, and/or reduce or inhibit scarring.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • volumes used herein refer to MSC Compositions at 1 x strength without any dilution or concentration.
  • 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.
  • the MSC Compositions may be administered to one or more eyes of a patient for various periods of time per treatment.
  • 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.
  • 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.
  • 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.
  • TBUT tear film breakup time
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions and methods described herein 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.
  • Acids tend to cause less damage than alkalis as many corneal proteins bind acid and act as a chemical buffer.
  • coagulated tissue acts as a barrier to further penetration of acid. Acid binds to collagen and causes fibril shrinkage.
  • the MSC Compositions are used to speed the recovery from an ocular burn.
  • 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.
  • the MSC Compositions are used prior, during or after one or more ocular surgeries.
  • the MSC Compositions are used along with one or more systemic drugs.
  • 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.
  • the MSC Compositions are used to assist recovery from an ocular surgery.
  • the MSC Compositions are used to prevent, reduce, or alleviate one or more symptoms from an ocular surgery.
  • the MSC Compositions can be used during recovery after a surgical procedure of amniotic membrane graft onto the ocular surface.
  • the MSC Compositions are used to prevent one or more potential complications from an ocular surgery such as an infection.
  • the MSC Compositions are used to assist local tissue repair, and/or minimize scarring of the surgical site.
  • compositions and/or methods described herein 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.
  • 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.
  • 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.
  • the MSC Compositions are used prior, during or after taking one or more systemic drugs.
  • 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.
  • 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.
  • VIPAGRA® sildenafil citrate
  • CIALIS® tadalafil
  • 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 .
  • 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).
  • 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.
  • 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.
  • the dosages will be modified to deliver a therapeutically equivalent amount.
  • 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.
  • pain e.g., pain associated with one or more diseases, including, for instance, eye diseases such as DED
  • therapeutic, diagnostic, and/or prophylactic agents can be delivered to one or more tissues in a patient via MSC- Exos.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the MSC Compositions may contain one or more ophthalmic drugs to treat, prevent or diagnose a disease or disorder of the eye.
  • 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.
  • 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.
  • 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.
  • the aforementioned therapeutic, prophylactic, and/or diagnostic agents may be administered in a neutral form, or in the form of a pharmaceutically acceptable salt.
  • 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.
  • 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).
  • the MSC Compositions comprise one or more local anesthetics.
  • local anesthetics include tetracaine, lidocaine, amethocaine, proparacaine, lignocaine, and bupivacaine.
  • 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.
  • the active agent is an anti-allergic agent such as olopatadine and/or epinastine.
  • Anti-glaucoma agents are examples of local anesthetics.
  • 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).
  • prostaglandin analogs such as travoprost,
  • 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; ribozy
  • the MSC Compositions are used in combination with one or more antimicrobial agents.
  • An antimicrobial agent is a substance that inhibits the growth of microbes including, for instance, bacteria, fungi, viruses, and/or parasites.
  • 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.
  • 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.
  • aminoglycosides e.g., streptomycin, amikacin, gentamicin, and to
  • antimicrobial agents include, for instance, iodine, silver compounds, moxifloxacin, ciprofloxacin, levofloxacin, cefazolin, tigecycline, gentamycin, ceftazidime, ofloxacin, gatifloxacin, amphotericin, voriconazole, natamycin.
  • 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,
  • 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.
  • 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.
  • 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.
  • the MSC Compositions are administered in combination with one or more growth factors.
  • growth factors are proteins and/or glycoproteins capable of stimulating cellular growth, proliferation, and/or cellular differentiation.
  • 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).
  • TGF-0 transforming growth factor beta
  • TGF-a
  • compositions described herein may be comprised in a kit.
  • cells, reagents to produce cells, exosomes, and reagents to produce exosomes, and/or components thereof may be comprised in a kit.
  • exosomes e.g., MSC-Exos
  • exosomes 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.
  • 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.
  • eye diseases e.g., DED
  • Any of the exosomes described herein 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®).
  • 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.
  • 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.
  • 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.
  • LV lentivirus
  • mature miRNAs are directly loaded into exosomes by electroporation.
  • Standard operating procedures SOPs
  • 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.
  • the miRNA is treated with RNase (or without RNase as control) to eliminate any free miRNA, total RNA was isolated using TRIZOLTM 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.
  • miR-23b administration of miR-23b to a subject induced dormancy ofBM2 breast cancer cells and promoted resistance to docetaxel.
  • siRNA synthetic interfering RNAs
  • UC-Exos 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.
  • MSC-derived exosomes e.g., MSC-Exos
  • Example 5 Treatment of dry eye disease (DED) patients with MSC Compositions
  • 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.
  • Example 11 Treatment of glaucoma

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Abstract

La présente invention concerne de manière générale des compositions, des formulations et des méthodes d'immunothérapie et d'administration de médicament, comprenant le traitement de maladies oculaires telles que la sécheresse oculaire (DED). En particulier, la présente invention concerne des procédés de production d'exosomes à partir de cellules souches mésenchymateuses (MSC-Exos) et, facultativement, de chargement desdits exosomes avec une ou plusieurs substances bioactives. Les exosomes peuvent être chargés à l'aide d'une électroporation avec une ou plusieurs substances bioactives telles que des protéines, des miARN et/ou un ARNsi. Dans des modes de réalisation spécifiques, les exosomes, et/ou un ou plusieurs composés biologiques dérivés des exosomes, peuvent être fournis à un individu en ayant besoin, y compris dans le cadre d'une ou de plusieurs compositions contenant des cellules souches mésenchymateuses (MSC). L'individu en ayant besoin peut être un individu ayant un trouble médical tel que le DED, des troubles immunitaires, un cancer et d'autres troubles. Par conséquent, les compositions contenant des MSC peuvent être utilisées pour une application topique sur l'œil. Les compositions peuvent contenir des MSC, des MSC-Exos, un ou plusieurs composés biologiques dérivés des MSC (par exemple des facteurs de croissance, des protéines, etc.).
PCT/US2025/011244 2024-01-12 2025-01-10 Administration de médicament exosome dérivé de cellules souches mésenchymateuses pour la sécheresse oculaire et d'autres troubles Pending WO2025151822A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210186877A1 (en) * 2017-11-16 2021-06-24 Board Of Regents, The University Of Texas System Methods for production of msc-derived exosomes
US20230137723A1 (en) * 2021-11-01 2023-05-04 MAM Holdings of West Florida, L.L.C. Mesenchymal stem cells for the prevention and targeted treatment of cancer and other disorders
WO2024145054A1 (fr) * 2022-12-29 2024-07-04 MAM Holdings of West Florida, L.L.C. Méthodes et compositions pour traiter une maladie du greffon contre hôte et d'autres pathologies oculaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210186877A1 (en) * 2017-11-16 2021-06-24 Board Of Regents, The University Of Texas System Methods for production of msc-derived exosomes
US20230137723A1 (en) * 2021-11-01 2023-05-04 MAM Holdings of West Florida, L.L.C. Mesenchymal stem cells for the prevention and targeted treatment of cancer and other disorders
WO2024145054A1 (fr) * 2022-12-29 2024-07-04 MAM Holdings of West Florida, L.L.C. Méthodes et compositions pour traiter une maladie du greffon contre hôte et d'autres pathologies oculaires

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HARRELL CARL RANDALL, DJONOV VALENTIN, VOLAREVIC VLADISLAV: "Therapeutic Potential of Mesenchymal Stem Cells in the Treatment of Ocular Graft-Versus-Host Disease", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL (MDPI), BASEL, CH, vol. 23, no. 21, Basel, CH , pages 13254, XP093337928, ISSN: 1422-0067, DOI: 10.3390/ijms232113254 *
JAFFET JILU, MOHANTY APARNA, VEERNALA INDUVAHI, SINGH SWATI, ALI MOHAMMAD JAVED, BASU SAYAN, VEMUGANTI GEETA K., SINGH VIVEK: "Human Lacrimal Gland Derived Mesenchymal Stem Cells – Isolation, Propagation, and Characterization", INVESTIGATIVE OPTHALMOLOGY & VISUAL SCIENCE, ASSOCIATION FOR RESEARCH IN VISION AND OPHTHALMOLOGY, US, vol. 64, no. 10, 13 July 2023 (2023-07-13), US , pages 12, XP093337942, ISSN: 1552-5783, DOI: 10.1167/iovs.64.10.12 *
XING YIXIAO, LI BOYA, HE JING, HUA HONG: "Labial Gland Mesenchymal Stem Cell Derived Exosomes-Mediated miRNA-125b Attenuates Experimental Sjogren’s Syndrome by Targeting PRDM1 and Suppressing Plasma Cells", FRONTIERS IN IMMUNOLOGY, FRONTIERS MEDIA, LAUSANNE, CH, vol. 13, Lausanne, CH , XP093337934, ISSN: 1664-3224, DOI: 10.3389/fimmu.2022.871096 *

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