Antioxidant and antiviral compositions and methods

Abstract

To provide a composition for treating tissue damage caused by oxidative stress in a subject at risk of having tissue damage caused by oxidative stress.SOLUTION: Compositions comprising exosomes and / or PEP can be used to treat a subject having, or at risk of having, a condition or tissue damage caused at least in part by oxidative stress. In addition, compositions comprising exosomes and / or PEP can be used to treat a subject having, or at risk of having, a viral infection.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 879,033, filed July 26, 2019, which is incorporated herein by reference in its entirety. Summary of the Invention

[0002] In one aspect, the present disclosure describes a method for treating tissue damage caused by oxidative stress in a subject at risk of having such tissue damage. Generally, the method includes administering to the subject a composition comprising exosomes and / or purified exosome products (PEPs) having at least one antioxidant protein in an amount effective to reduce the likelihood or severity of the tissue damage compared to a subject not administered the composition.

[0003] In another aspect, the present disclosure describes a method for treating a condition caused by oxidative stress in a subject at risk of having the condition. Generally, the method includes administering to the subject an effective amount of a composition comprising exosomes and / or PEP containing at least one antioxidant protein. In some cases, an effective amount of the composition is an amount effective to reduce the likelihood that the subject will experience symptoms or clinical signs of the condition caused by oxidative stress, compared to a subject not administered the composition. In other cases, an effective amount of the composition is an amount effective to reduce the severity of symptoms or clinical signs of the condition caused by oxidative stress, compared to a subject not administered the composition.

[0004] In another aspect, the present disclosure describes a method for treating tissue damage caused by oxidative stress in a subject. Generally, the method includes administering to the subject a composition comprising exosomes and / or PEPs containing at least one antioxidant protein in an amount effective to reduce the severity of the tissue damage compared to a subject not administered the composition.

[0005] In another aspect, the present disclosure describes a method for treating a condition caused by oxidative stress in a subject. Generally, the method includes administering to the subject a composition comprising exosomes and / or PEP containing at least one antioxidant protein in an amount effective to reduce the severity of symptoms or clinical signs of the condition caused by oxidative stress compared to a subject not administered the composition.

[0006] In some embodiments of any of the aspects summarized above, the exosomes and / or PEP are provided in an amount effective to reduce apoptosis in cells of the oxidatively stressed tissue.

[0007] In another aspect, the present disclosure describes a method for treating a subject at risk of having a viral infection. Generally, the method includes administering to the subject a composition comprising an effective amount of exosomes and / or PEP with at least one antiviral protein. In some cases, the effective amount is an amount effective to reduce the likelihood that the subject will experience symptoms or clinical signs of a viral infection compared to a subject not administered the composition. In other cases, the effective amount is an amount effective to reduce the severity of symptoms or clinical signs of a condition caused by a viral infection compared to a subject not administered the composition. In some embodiments, the antiviral protein may include interferon-inducible transmembrane protein-1 (IFITM-1), IFITM-3, MX1, or viperin.

[0008] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples that can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0010] [Figure 1] Schematic of the mechanism of exosome formation and free radical generation. (A) Schematic of exosome production and secretion by cells. (B) Schematic of free radical generation and antioxidants that inhibit this free radical chain reaction. The compound LY83583 is a superoxide generator when administered to cells in culture and was used in several studies to demonstrate that PEP can suppress the toxic effects of oxidative stress. [Figure 2] Antioxidant expression in three different PEP preparations. (A) Western blot analysis of antioxidant proteins in PEP samples: heme oxygenase-1 (HO-1), Cu / Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), and extracellular superoxide dismutase (SOD3). (B) Quantification of catalase activity in several PEP samples. [Figure 3]PEP reduces oxidative stress in primary neural cells. Mouse neurons were treated in vitro with or without PEP before treatment with the superoxide generator LY83583 (1 μM) for 24 hours. Cell death was detected using a caspase-3 / 7-specific dye, which stains apoptotic cells red. (A) Phase-contrast microscopy of mouse neurons. (B) Fluorescence microscopy image of (A). Cells treated with LY83583 alone display the red caspase-3 / 7 dye. (C) Neurons were also labeled with NucLight green to detect cell nuclei. (D) Merged images of B–D. (E) Quantification of caspase-3 / 7-positive cells using an INCUCYTE S3 cell imager (Essen Bioscience, Inc., Ann Arbor, MI). In the absence of PEP, LY83583 induces neuronal apoptosis after 24 hours of LY83583 treatment. In contrast, a 1-hour PEP pretreatment suppresses oxidative stress. [Figure 4] Dose-dependent effects of PEP on oxidative stress in human embryonic kidney 239T (HEK239T or 293T) cells. (A) Pretreatment of 293T cells with PEP dose-dependently inhibits LY83583-induced oxidative stress. Mouse neurons were treated in vitro with or without PEP before treatment with the superoxide generator LY83583 (1 μM) for 24 hours. (B) Confocal microscopy images showing apoptotic cells after treatment with LY83583 alone. (C) Confocal microscopy images showing apoptotic cells after treatment with LY83583 and 10% PEP. (D) Confocal microscopy images showing apoptotic cells after treatment with LY83583 and 20% PEP. Cell death was detected using a caspase 3 / 7-specific dye, which stains dying cells red. Analysis was quantified using an INCUCYTE S3 Cell Imager (Essen Bioscience, Inc., Ann Arbor, MI). [Figure 5]PEP suppresses oxidative stress in primary human umbilical endothelial cells (HUVECs). HUVECs were treated in vitro with or without PEP before treatment with the superoxide generator LY83583 (20 μM) for 5 hours. Live-cell imaging was performed to observe tube formation over time using an INCUCYTE S3 Cell Imager (Essen Bioscience, Inc., Ann Arbor, MI). Representative fluorescent images of endothelial tube formation. [Figure 6] PEP suppresses oxidative stress in primary human umbilical vein endothelial cells (HUVECs). In vitro, HUVECs were treated with or without PEP before treatment with the superoxide generator LY83583 (20 μM) for 5 hours. (A) Quantification of vessel percentage area. (B) Quantification of vessel length. Tube formation was measured using ImageJ software. PEP pretreatment significantly enhanced tube formation in the setting of oxidative stress, as determined by total vessel percentage and vessel length. [Figure 7] Characterization of exosomes in PEP. (A) NANOSIGHT (Malvern Panalytical Ltd., Salisbury, UK) image of exosomes in PEP. (B) NANOSIGHT quantification of exosome size and number in 20% PEP (18001-B2). (C) Western blot analysis of known markers of exosomes in three separate formulations of PEP. [Figure 8] Schematic diagram showing exosome-packaged interferon-inducible transmembrane proteins 1 and 3 (IFITM), as well as antiviral proteins including MX1 and viperin, which are contained within exosomes and are also included in PEP formulations. [Figure 9] Schematic showing the mechanism by which PEP inhibits virus production. Pre- and post-treatment with PEP inhibits virus entry into cells. The antiviral protein in PEP is responsible for this inhibition. [Figure 10] Characterization of antiviral proteins in glioblastoma (GBM) and adipose-derived mesenchymal stem cell (aMSC) cell lines. (A) IFITM3 was detected in both whole cell lysates (WCL) and PEP prepared from aMSC, but not in PEP prepared from GBM. (B) IFITM1 was found to be expressed in three different preparations of PEP. [Figure 11] Characterization of antiviral proteins in six different cell lines. Western blot of cell lysates for IFITM-1, IFITM-3, MX1, and viperin. Lane 1: human embryonic kidney cells (HEK 293T); Lane 2: human umbilical vein endothelial cells (HUVEC); Lane 3: normal human lung fibroblasts (NHLF); Lane 4: normal human dermal fibroblasts (NHDF); Lane 5: adipose-derived mesenchymal stem cells (aMSC); Lane 6: umbilical cord-derived mesenchymal stem cells (uMSC). Estimated infectivity is derived from the amount of antiviral protein shown on the Western blot. Lane 1 (HEK 293T) has the lowest amount of IFITM proteins and is therefore predicted to be the most susceptible to infection. [Figure 12] Characterization of protein expression in PEP. (A) Western blot comparing the protein expression levels of the exosome marker CD63 and the antiviral proteins MX1 and viperin in 293T cells, HeLa cells, and PEP. (B) Comparison of IFITM1 expression in three different production batches of PEP. [Figure 13] Schematic diagram summarizing the in vitro experimental design. (A) Pretreatment of 293T cells with PEP. (B) Treatment of 293T cells with PEP after viral infection. [Figure 14]Pretreatment of cells with PEP inhibits viral infection. 293T cells were seeded at 300,000 cells / well in 6-well plates. Cells were pretreated with PEP for 96, 72, 48, or 24 hours before infection with VSV-GFP at a multiplicity of infection (MOI) of 10. VSV-GFP (2.37 × 105 PFU / ml) was diluted in serum-free medium to an MOI of 10. 24 hours after infection, cells were fixed with 2% PFA and subjected to flow cytometry to determine the number of infected cells that turned green after infection. (A) Negative control: unstained cells. (B) Positive control: 293T cells infected with VSV-GFP at an MOI of 10 without PEP pretreatment. (C) 293T cells treated with PEP for 96 hours before viral infection. (D) 293T cells treated with PEP for 72 hours before viral infection. (E) 293T cells treated with PEP for 48 hours before virus infection. (F) 293T cells treated with PEP for 24 hours before virus infection. PEP pretreatment significantly inhibited virus infection, especially within 48 hours of exposure. [Figure 15] Treatment of cells with PEP after viral infection inhibits viral spread. 293T cells were transduced with VSV-GFP (MOI 10) and treated with PEP simultaneously, 1 h after transduction, 2 h after transduction, or 3 h after transduction. PEP posttreatment significantly protected cells from viral infection. 24 h after transduction, cells were fixed with 2% PFA and subjected to flow cytometry to determine the number of infected cells that turned green after infection. (A) Negative control: unstained cells. (B) Positive control: 293T cells infected with VSV-GFP at an MOI of 10 without PEP pretreatment. (C) 293T cells treated with PEP simultaneously with virus exposure. (D) 293T cells treated with PEP 1 h after virus exposure. (E) 293T cells treated with PEP 2 h after virus exposure. (F) 293T cells treated with PEP 3 h after exposure to virus. [Figure 16]In vivo uptake of nebulized PEP into mouse lungs. Nebulized PEP is taken up by the epithelium and penetrates the densely packed lung tissue (alveolar bed). DiR-labeled PEP in saline was administered at various doses over a 5-minute period using a ventilator (FLEXIVENT; SCIREQ Scientific Respiratory Equipment, Inc., Montreal, Quebec, Canada). Images were acquired using a XENOGEN imager (IVIS 200, Caliper Life Sciences, Inc., Hopkinton, MA). [Figure 17] A time-course study was conducted to examine DiR-labeled PEP accumulation in the lung via the nebulized route. Doses of 5% or 10% PEP were administered for 5, 10, 15, 20, or 25 days. PEP accumulation in the lung plateaued at 20 days. PEP was administered 5 minutes per day, 5 days per week. Each dose was administered as a PEP solution (approximately 0.6-0.8 ml) delivered via nebulization over 5 minutes. [Figure 18] PEP is detected in both type I and type II pneumocytes. (A) Schematic of alveolar anatomy. (B) Immunohistochemistry: Minimal endogenous CD63 staining (green) in a control mouse lung. (C) Immunohistochemistry: CD63 staining (PEP - green), as well as surfactant protein C (SPC, red cytosolic staining) and T1 alpha protein (red membrane staining). (D) Immunohistochemistry: Strong CD63 staining (green) after 3 days of PEP nebulization (5 min / day at 20% PEP). (E) Immunohistochemistry: Close-up image of (C) (63x magnification). PEP was detected using an antibody against CD63; T1 alpha protein is a specific marker for type I pneumocytes; SPC is a marker for type II pneumocytes. DETAILED DESCRIPTION OF THE INVENTION

[0011] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Exosomes are microvesicles (40-100 nm in diameter) secreted by all different cell types and provide intercellular communication signals. A variety of different cargo molecules, including miRNAs and proteins, can be transported between cells via exosomes. However, current knowledge regarding exosome function in wound healing remains limited.

[0012] Figure 1A is a schematic diagram showing the production of exosomes by cells. PEP (Purified Exosome Product) is a modified exosome product with a unique physical structure compared to standard exosomes. For example, the preparation of PEP from human blood cells is described in detail in International Patent Application No. PCT / US2018 / 065627 (International Publication No. WO 2019 / 118817 A1).

[0013] PEP can be formulated into pharmaceutical compositions for many uses. For example, PEP can increase the proliferation of mesenchymal stem cells (MSCs) and / or dermal fibroblasts to a greater extent than conventional therapies (e.g., platelet lysate) or fetal bovine serum. Similarly, PEP may induce osteogenic, chondrogenic, and / or adipogenic differentiation to a greater extent than conventional therapies (e.g., platelet lysate) or fetal bovine serum. PEP can also maintain myoblast proliferation to a greater extent than conventional therapies (e.g., platelet lysate) or fetal bovine serum. PEP can be used to enhance the proliferation profile of cells used in immunotherapy, including but not limited to CAR-T, TRuC-T, NK-CAR, and hematopoietic stem cells. (International Patent Application No. PCT / US2018 / 065627; International Publication No. WO2019 / 118817 A1).

[0014] PEP compositions and formulations can induce a wide range of cellular responses, primarily focused on proliferation, anti-apoptosis, immunomodulation, and new blood vessel formation. In the presence of PEP, damaged tissues tend to regenerate. This response is exemplified by observations demonstrating enhanced expression of transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (EFG), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF). While the response is not limited to these factors, the observation that these factors are induced in different tissues is an example of the regenerative influence of PEP.

[0015] The present disclosure describes the antioxidant and antiviral properties of exosomes, both natural exosomes and PEPs, leading to additional therapeutic and / or prophylactic uses. While described herein in the context of exemplary embodiments of PEPs and / or exosomes, the compositions and methods described herein may include any extracellular vesicles containing one or more proteins involved in imparting antioxidant or antiviral properties to PEPs and / or exosomes. For example, the compositions and methods described herein may include extracellular vesicles regardless of their origin and mechanism of release from cells. While exosomes are generally 50 nm to 150 nm in size and have a specific biological mechanism of origin, extracellular vesicles have multiple biological mechanisms of origin and range in size from 50 nm to 1000 nm.

[0016] Antioxidant activity For example, cardiovascular disease is a leading cause of mortality and morbidity worldwide. A harmful environment is present in pathologies where elevated levels of free radicals cause oxidative stress and increased immune cell activity. This increase in immune cell activity may contribute to the development of cardiovascular disease. Antioxidants counter the effects of free radicals by catalyzing their conversion to stable nonradical compounds (Figure 1B). Exosomes and PEP contain antioxidants and evade the immune response, respectively, helping to mitigate the harmful disease environment.

[0017] Figure 7A shows a representative NANOSIGHT (Malvern Panalytical Ltd., Salisbury, UK) image of PEP particles. Figure 7B shows NANOSIGHT quantification of the size and number of PEP particles in a 20% reconstituted PEP formulation (PEP diluted to 20% of the stock concentration in serum-free medium). Figure 7C shows Western blot analysis of known marker exosomes in three separate formulations of PEP.

[0018] Figure 2 shows data demonstrating the presence of antioxidant expression in different PEP formulations. Each formulation was prepared in the same manner, but the starting material from each formulation was derived from a different batch of PEP production. Figure 2A shows Western blot analysis of five antioxidant proteins in PEP samples: catalase, heme oxygenase-1 (HO-1), Cu / Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), and extracellular superoxide dismutase (SOD3). Figure 2B shows quantification of catalase and superoxide dismutase (SOD) expression in each of the eight different PEP formulations.

[0019] Figure 3 shows the dose-dependent effect of PEP on oxidative stress in 293T cells. Cells pretreated with PEP exhibited less apoptosis after LY83583-induced oxidative stress (Figure 3A-D). Furthermore, the effect of PEP pretreatment is dose-dependent. Confocal microscopy images confirm that apoptosis decreases dose-dependently as neurons are pretreated with increasing concentrations of PEP. Figures 4-6 provide data demonstrating the antioxidant effect of PEP in other 293T cells (Figure 4) and human umbilical vein endothelial cells (HUVECs; Figures 5 and 6).

[0020] In some embodiments, the antioxidant may be an inducible antioxidant within the exosomes from which the exosomes or PEP are generated. While exosomes and / or PEP may contain endogenous antioxidants, the antioxidants in exosomes and / or PEP can be upregulated by preconditioning the exosome source to stress. These stress inducers may include, but are not limited to, hypoxia, hyperthermia, chemical-induced stress, or radiation.

[0021] Therefore, exosomes and / or PEP can be used to suppress oxidative stress in diseased tissues. Pre-treating cells with antioxidant-containing exosomes and / or PEP reduces the effects of oxidative stress associated with many diseases.

[0022] Thus, the present disclosure describes methods for treating a subject having or at risk of having a disease caused at least in part by oxidative stress. As used herein, the term "at risk" refers to a subject who may or may not actually have the described risk. Thus, for example, a subject "at risk" of a condition caused at least in part by oxidative stress is a subject who has one or more risk factors associated with the condition, such as, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history.

[0023] Thus, compositions comprising exosomes and / or PEP can be administered before, during, or after a subject first exhibits symptoms or clinical signs of a condition caused, at least in part, by oxidative stress. Treatment initiated before a subject first exhibits symptoms or clinical signs associated with the condition is considered prophylactic treatment, reducing the likelihood that the subject will experience clinical evidence of the condition, reducing the severity of the symptoms and / or clinical signs of the condition, and / or resulting in complete recovery from the condition, compared to subjects not administered the composition. Treatment initiated after a subject first exhibits symptoms or clinical signs associated with the condition is considered therapeutic, reducing the severity of the symptoms and / or clinical signs of the condition, and / or resulting in complete recovery from the condition, compared to subjects not administered the composition.

[0024] Thus, the method involves administering to a subject having or at risk of having a particular condition an effective amount of a composition comprising exosomes and / or PEP. In this embodiment, an "effective amount" is an amount effective to alleviate, inhibit the progression of, ameliorate, or reverse to some extent the symptoms or clinical signs associated with the condition.

[0025] Antiviral activity The interferon (IFN) system is the first line of defense against animal viruses in humans. Binding of type I or type III IFNs to their receptors (IFNAR1 / 2 and IL-28Rα / IL-10Rβ, respectively) induces an antiviral state in cells by inducing the transcription of IFN-stimulated genes, including interferon-inducible transmembrane proteins (IFITM-1, IFITM-3, and IFITM-5), viperin, RNA-activated protein kinase (PKR), ribonuclease L (RNase L), myxoma resistance protein 1 (MX1), and oligoadenylate synthetase (OAS).

[0026] Figure 8 is a schematic diagram showing antiviral proteins, such as interferon-inducible transmembrane proteins 1 and 3 (IFITM), MX1, and viperin, packaged in exosomes. These antiviral proteins are also present in PEP. Figure 9 is a schematic diagram showing the mechanism by which PEP and / or exosomes can inhibit viral production. Pre- and post-treatment with PEP inhibits viral entry into cells. Antiviral proteins in PEP and / or exosomes are responsible for this inhibition.

[0027] The interferon-inducible transmembrane protein IFITM is a member of the IFITM family (interferon-inducible transmembrane proteins) and is encoded by the IFITM gene. The human IFITM gene is located on chromosome 11 and has four members: IFITM1, IFITM2, IFITM3, and IFITM5. IFITM proteins have been identified as antiviral restriction factors for influenza virus A replication. IFITM3 knockout increases influenza virus A replication, and IFITM3 overexpression inhibits influenza virus A replication. IFITM proteins can also inhibit infection by several other enveloped viruses belonging to various viral families. These viruses include flaviviruses (dengue virus and West Nile virus), filoviruses (Marburg virus and Ebola virus), coronaviruses (SARS-CoV), and lentiviruses (human immunodeficiency virus). IFITM3 knockout increases swine influenza virus replication, while overexpression reduces viral levels.

[0028] Interferon-inducible GTP-binding protein Mx1 is a protein encoded by the MX1 gene in humans. In mice, the interferon-inducible Mx protein is involved in a specific antiviral state against influenza virus infection. The human protein is similar to the mouse protein as determined by its antigenic relatedness, induction conditions, physicochemical properties, and amino acid analysis. This cytoplasmic protein is a member of both the dynamin and large GTPase families.

[0029] Viperin (viral inhibitory protein, endoplasmic reticulum-associated, interferon-inducible), also known as RSAD2 (radical SAM domain-containing 2), is a multifunctional protein in viral processes. Viperin is a cellular protein that can inhibit many DNA and RNA viruses, including CHIKV, HCMV, HCV, DENV, WNV, SINV, influenza, and the HIV-1 LAI strain.

[0030] In some cases, exosomes or PEPs can be converted into antiviral particles capable of inhibiting viral entry and replication. Exosomes may contain endogenous antiviral proteins that remain present during PEP preparation. Exosomes and / or PEPs can be further modified to contain polynucleotides encoding miRNAs that disrupt viral replication. Suitable miRNAs include, but are not limited to, miR-127-3p, miR-486-5, miR-593-5p, miR-196, miR-199a-3p, miR-296, miR-351, miR-431, and miR-448.

[0031] Figure 11 shows the characterization of antiviral proteins in six different cell lines. The cell lines tested were human embryonic kidney cells (HEK 293T), human umbilical vein endothelial cells (HUVEC), normal human lung fibroblasts (NHLF), normal human dermal fibroblasts (NHDF), adipose-derived mesenchymal stem cells (aMSC), and umbilical cord-derived mesenchymal stem cells (uMSC). Inhibition of viral replication in humans may be particularly useful as an antiviral preventative (pre-infection) or therapeutic (post-infection) treatment. Different cell types contain different antiviral proteins found in exosomes and / or PEP. Thus, the antiviral cargo of PEP preparations can be designed, at least in part, by the cell type used as the starting material for preparing the PEP.

[0032] Figure 13 shows the experimental design for a study on the in vitro antiviral activity of PEP. Figure 13A shows the experimental design for testing whether prophylactic pretreatment of cells with PEP can inhibit viral infection. Viral infection was monitored by infecting cells with VSV-GFP, which causes infected cells to express green fluorescent protein (GFP). Figure 14 shows that cells pretreated with antiviral PEP before VSV-GFP transduction showed a significant decrease in the number of GFP-positive cells compared to the positive control. Cells treated 24 hours before transduction showed few or no GFP-positive cells and were more similar to negative control cells.

[0033] Figure 13B shows the experimental design for testing whether PEP administered after cells were exposed to VSV-GFP can inhibit viral growth. Figure 15 shows that PEP administered with or up to 3 hours after exposure to VSV-GFP caused a significant decrease in the amount of GFP-positive cells compared to the positive control, with a profile nearly identical to the negative control in all cases.

[0034] Therefore, exosomes and / or PEP can be used to treat viral infections. Viral infections treatable with PEP and / or exosomes include Orthomyxoviridae, including but not limited to influenza A, influenza B, and influenza C; Flaviviridae, including but not limited to West Nile virus, dengue virus, Zika virus, and hepatitis C virus; Rhabdoviridae, including but not limited to vesicular stomatitis virus, rabies virus, and Lagos bat virus; Filoviridae, including but not limited to Marburg virus and Ebola virus; and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and SARS-CoV-2. These include, but are not limited to, infections caused by viruses of the Coronaviridae family, including but not limited to: Human Immunodeficiency Virus (HIV)-1, Moloney Leukemia Virus, and Jaagsiekte Ovine Retrovirus; Arenaviridae family, including but not limited to Lassa Virus, Machupo Virus, Lymphocytic Choriomeningitis Virus, and Lujo Virus; Togaviridae family, including but not limited to Semliki Forest Virus; Bunyaviridae family, including but not limited to La Crosse Virus, Hantaan Virus, Andes Virus, Rift Valley Fever Virus, and Crimean-Congo Hemorrhagic Fever Virus; and Reoviridae family, including but not limited to Reovirus.

[0035] Treatment of viral infections can be preventative or can be initiated after a subject shows one or more symptoms or clinical signs of a condition caused by viral infection. Preventative treatment (e.g., before a subject shows symptoms or clinical signs of a condition, such as while the infection remains asymptomatic) is referred to herein as treatment of a subject "at risk" of the condition. As used herein, the term "at risk" refers to a subject who may or may not actually have the described risk. Thus, for example, a subject "at risk" of an infectious condition is a subject who is present in an area where other individuals have been identified as having an infectious condition, and / or a subject who is likely to be exposed to an infectious virus, even if the subject has not yet shown detectable signs of infection, regardless of whether the subject may have a subclinical level of infection.

[0036] Thus, compositions containing exosomes and / or PEP can be administered before, during, or after a subject's initial contact with an infectious virus. Treatment initiated before a subject's initial contact with an infectious virus may reduce the likelihood that the subject will experience clinical evidence of viral infection, reduce the severity of symptoms and / or clinical signs of a condition caused by viral infection, and / or completely reverse the viral infection, compared to subjects to whom the composition is not administered. Treatment initiated after a subject's initial contact with an infectious virus may reduce the severity of symptoms and / or clinical signs of a condition caused by viral infection, and / or completely reverse the viral infection, compared to subjects to whom the composition is not administered.

[0037] Thus, the method involves administering to a subject having or at risk of having a viral infection an effective amount of the composition, In this aspect, an "effective amount" is an amount effective to alleviate, inhibit the progression of, ameliorate, or reverse to some extent the symptoms or clinical signs associated with the condition caused by the viral infection.

[0038] PEP and / or exosomes can be formulated with a pharmaceutically acceptable carrier to form a pharmaceutical composition. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspending agent, colloid, etc. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as a conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., capable of being administered to an individual, together with PEP and / or exosomes, without causing undesirable biological effects or interacting in a deleterious manner with any of the other ingredients in the pharmaceutical composition in which it is contained.

[0039] Whether intended to treat conditions caused by or associated with oxidative stress or conditions associated with or caused by viral infection, pharmaceutical compositions containing PEP and / or exosomes can be formulated in various forms compatible with the preferred route of administration. Thus, pharmaceutical compositions can be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). Pharmaceutical compositions can be administered to mucosal surfaces, for example, by administration to the nasal or respiratory mucosa (e.g., via spray or aerosol). Pharmaceutical compositions can also be administered via sustained or delayed release.

[0040] Therefore, the pharmaceutical composition can be provided in any suitable form, including, but not limited to, a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The pharmaceutical composition can be delivered in a formulation containing any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation can be delivered in a conventional topical dosage form, such as a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, etc. The formulation may further include one or more additives, including, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, etc.

[0041] The formulations can be conveniently presented in unit dosage form and can be prepared by methods well known in the art of pharmacy.The method for preparing compositions containing pharmaceutically acceptable carriers includes combining PEP and / or exosomes with one or more auxiliary ingredients.In general, the formulations can be prepared by uniformly and / or intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0042] The amount of PEP and / or exosomes administered can vary depending on various factors, including, but not limited to, the content and / or source of the PEP and / or exosomes administered, the subject's weight, physical condition, and / or age, and / or route of administration. Thus, the absolute weight of PEP and / or exosomes contained in a given unit dosage form can vary widely and depends on factors such as the subject's species, age, weight, and physical condition, and / or method of administration. Therefore, it is not practical to generally describe the amount that constitutes an effective amount of PEP and / or exosomes for all possible uses. However, those skilled in the art can easily determine the appropriate amount, taking such factors into due consideration.

[0043] In some embodiments, the method can include administering sufficient PEP and / or exosomes to provide a subject with a dose ranging from, for example, about a 0.01% solution to a 100% solution, although in some embodiments, the method can be practiced by administering a dose of PEP and / or exosomes outside this range. As used herein, a 100% solution of PEP refers to PEP solubilized in 1 ml of a liquid carrier (e.g., water, phosphate-buffered saline, serum-free medium, etc.). For comparison, a dose of 0.01% PEP is approximately equivalent to a standard dose of exosomes prepared using conventional methods, such as isolating exosomes from cells in vitro using standard cell-conditioned medium.

[0044] Thus, in some embodiments, the method may comprise administering sufficient PEP and / or exosomes to provide a minimum dose of at least 0.01%, at least 0.05%, at least 0.1%, at least 0.25%, at least 0.5%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, or at least 70%.

[0045] In some embodiments, the method may include administering sufficient PEP and / or exosomes to provide a maximum dose of 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0046] In some embodiments, the method may include administering sufficient PEP and / or exosomes to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in some embodiments, the method may include administering sufficient PEP and / or exosomes to provide 1% to 50% of the dose, e.g., 5% to 20% of the dose. In certain embodiments, the method may include administering sufficient PEP and / or exosomes to provide a dose equal to any minimum dose or any maximum dose described above. Thus, for example, the method may include administering a dose of 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100%.

[0047] In some embodiments, PEP and / or exosomes can be administered, for example, in a single dose or multiple doses per week, although in some embodiments, the method can be practiced by administering PEP and / or exosomes at a frequency outside this range. When administered multiple times within a given period, the doses can be the same or different. For example, a 1 mg daily dose can be administered as a single dose of 1 mg, two doses of 0.5 mg, or a first dose of 0.75 mg followed by a second dose of 0.25 mg. Furthermore, when administered multiple times within a given period, the intervals between doses can be the same or different.

[0048] In certain embodiments, PEP and / or exosomes can be administered as a one-time or monthly dose, or once daily to multiple times daily, depending on the application. For example, PEP and / or exosomes can be administered as a one-time treatment for acute myocardial infarction. In other embodiments, PEP-exosomes can be administered multiple times daily for wound healing or cosmetic uses.

[0049] In some embodiments, the methods may involve administering a mixture of exosomes and / or PEP prepared from various cell types, each cell type having a unique antiviral protein profile. In this manner, the exosome and / or PEP composition may provide a broader spectrum of antiviral activity than if the exosome and / or PEP composition were prepared from a single cell type. [Example]

[0050] Preparation of PEP Preparations of PEP were prepared as previously described (International Publication No. WO 2019 / 118817 A1; U.S. Patent No. 10,596,123 B2; U.S. Patent Application Publication No. US 2016 / 0324794 A1).

[0051] Western blot analysis PEP and other cell line pellets were reconstituted in lysis buffer containing 50 mmol / L NaCl, 50 mmol / L NaF, 50 mmol / L sodium pyrophosphate, 5 mmol / L EDTA, 5 mmol / L EGTA, 2 mmol / L Na3VO4, 1% Triton X-100, 0.5 mmol / L PMSF, 10 mmol / L HEPES, and 10 μg / ml leupeptin (pH 7.4). Soluble protein extracts (20 μg per sample) were loaded onto 12.5% polyacrylamide gels (Bio-Rad Laboratories, Inc., Hercules, CA). The gels were then transferred to polyvinylidene fluoride (PVDF) membranes. Primary antibodies against various antigens were incubated overnight, followed by 1 hour of probing with the appropriate secondary antibodies and visualization using enhanced chemiluminescence.

[0052] NANOSIGHT analysis of PEP The size and number of PEP exosomes were analyzed using a NANOSIGHT 300 particle analyzer (Malvern Panalytical Ltd., Salisbury, UK). PEP was reconstituted with 5 ml of water to obtain a 20% PEP solution, which was further diluted 1:1000 before analysis. Each sample was analyzed in triplicate and averaged.

[0053] Live cell imaging and apoptosis detection Real-time imaging of cells was performed using an INCUCYTE S3 imaging system (Essen Bioscience, Inc., Ann Arbor, MI) according to the manufacturer's instructions. Caspase 3 / 7 dye reagent was used according to the manufacturer's guidelines (Essen Bioscience, Inc., Ann Arbor, MI).

[0054] DiR labeling of PEP for xenogen studies For XENOGEN (Caliper Life Sciences, Inc., Hopkinton, MA) image analysis, PEPs were labeled with the far-red dye DiR (Thermo Fisher Scientific, Inc., Waltham, MA) and the near-infrared fluorescent lipophilic carbocyanine DiOC according to the manufacturer's guidelines. 18 (7) ("DiR") is weakly fluorescent in water but highly fluorescent and photostable when incorporated into membranes. PEP was reconstituted in dH2O and filtered through a 0.20 μm filter. After incubation with DiR dye for 30 minutes at room temperature on a rotator, the PEP-DiR solution was spun down at maximum speed (14,800 rpm) in a temperature-controlled countertop minicentrifuge for 30 minutes and washed once with dH2O.

[0055] Flow cytometry Cells were harvested and washed with PBS and FACS buffer (PBS containing 1.8% BSA, 1 m m EDTA). Prior to flow cytometry analysis, cells were resuspended in 100 μl of buffer and fixed with 2% paraformaldehyde.

[0056] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements, or a combination of any two or more of the listed elements. The terms "comprises," "comprising," and variations thereof, should be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably to mean one or more. And, the recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0057] In the foregoing description, for clarity, certain embodiments may be described in isolation. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless expressly specified that a feature of a particular embodiment is incompatible with a feature of another embodiment.

[0058] For any method disclosed herein that includes separate steps, the steps can be performed in any practicable order, and, if desired, any combination of two or more steps can be performed simultaneously.

[0059] The complete disclosures of all patents, patent applications, publications, and electronically available materials cited herein (including, for example, nucleotide sequence submissions in GenBank and RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, PDB, etc., and translations from the annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of a conflict between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described. Variations obvious to those skilled in the art will be encompassed within the invention as defined by the claims.

[0060] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the scope of the claims to equivalents, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0061] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible, however, all numerical values inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0062] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

Claims

1. 1. A method of treating tissue damage caused by oxidative stress in a subject at risk of having tissue damage caused by oxidative stress, comprising: administering to the subject a composition comprising exosomes and / or purified exosome products (PEP) comprising at least one antioxidant protein in an amount effective to reduce the likelihood or severity of tissue damage compared to a subject not administered the composition. A method comprising:

2. 1. A method of treating an oxidative stress-induced condition in a subject at risk of having the oxidative stress-induced condition, comprising: compared to the subject not administered the composition, reducing the likelihood that the subject will experience symptoms or clinical signs of the condition caused by oxidative stress; or To reduce the severity of the symptoms or clinical signs of the condition caused by oxidative stress. administering to the subject an effective amount of a composition comprising exosomes and / or PEPs containing at least one antioxidant protein. A method comprising:

3. 1. A method of treating tissue damage caused by oxidative stress in a subject having tissue damage caused by oxidative stress, comprising: administering to the subject a composition comprising exosomes and / or PEPs comprising at least one antioxidant protein in an amount effective to reduce the severity of tissue damage compared to a subject not administered the composition. A method comprising:

4. 1. A method of treating an oxidative stress-induced condition in a subject having the oxidative stress-induced condition, comprising: administering to the subject a composition comprising exosomes and / or PEPs comprising at least one antioxidant protein in an amount effective to reduce the severity of symptoms or clinical signs of the condition caused by oxidative stress compared to a subject not administered the composition. A method comprising:

5. 5. The method of claim 1, wherein the antioxidant protein comprises catalase, heme oxygenase-1 (HO-1), Cu / Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), or extracellular superoxide dismutase (SOD3).

6. 6. The method of any one of claims 1 to 5, wherein the exosomes and / or PEPs comprise catalase, heme oxygenase-1 (HO-1), Cu / Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), and extracellular superoxide dismutase (SOD3).

7. The method of any one of claims 1 to 6, wherein the exosomes and / or PEP are provided in an amount effective to reduce apoptosis in cells of a tissue undergoing oxidative stress.

8. 1. A method of treating a subject at risk of having a viral infection, comprising: administering to the subject a composition comprising exosomes and / or PEPs comprising at least one antiviral protein in an amount effective to reduce the likelihood that the subject will experience symptoms or clinical signs of a viral infection compared to a subject not administered the composition. A method comprising:

9. 1. A method of treating a subject having a viral infection, comprising: administering to the subject a composition comprising exosomes and / or PEPs comprising at least one antiviral protein in an amount effective to reduce the severity of symptoms or clinical signs of a condition caused by a viral infection compared to a subject not administered the composition. A method comprising:

10. The method of claim 8 or 9, wherein the exosome and / or PEP comprises interferon-inducible transmembrane protein-1 (IFITM-1), IFITM-3, MX1, or viperin.

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