Extracellular vesicles for the treatment of amyotrophic lateral sclerosis

Abstract

This specification discloses a method for treating ALS in a subject by administering a therapeutically effective amount of a composition to the subject, for example, nerve cells, such as EVs derived from neural progenitor cells. The EVs may be administered distally or peripherally to the CNS so that they cross the blood-brain barrier and exert their therapeutic function in the CNS. This method can reduce inflammation (e.g., NLRP3 inflammatory pathway signaling), reduce disease activity or progression, and / or improve motor or neurological function, signs or symptoms associated with ALS, or survival in the ALS subject compared to a control ALS subject. A method for inhibiting necroptosis in cells by contacting cells with a therapeutically effective amount of a composition containing EVs is also provided.

Description

[Technical Field]

[0001] (Related applications) This application claims priority to U.S. Provisional Application No. 63 / 512,483, filed on 7 July 2023. The entire contents of the aforementioned priority application are incorporated herein by reference. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disease characterized by selective degeneration of both upper and lower motor neurons, progressive atrophy of muscle fibers, and degeneration of both upper and lower motor neurons. ALS spreads throughout the motor system, causing neuronal loss at all levels of the motor system, from the cortex to the anterior horn of the spinal cord. ALS is a fatal disease with a median survival time of 3 years from the onset of debilitation. Medical complications such as aspiration pneumonia, respiratory muscle atrophy, and immobility contribute to the morbidity of patients with this disease.

[0003] The cause of ALS is unknown, but a family history of the disease is found in about 5% of patients, and twin studies have shown a genetic contribution with a heritability of about 61%. In some cases, ALS overlaps clinically, pathologically, and biologically with frontotemporal dementia and may share common biological mechanisms with Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases.

[0004] Currently, there is no cure for ALS, and treatment options are focused on slowing the progression of symptoms, preventing complications, and maintaining patients' daily living activities. For example, FDA-approved ALS drugs such as riluzole, edaravone, and sodium phenylbutyrate-taurursodiol only slow the rate of functional decline in ALS patients and delay disease progression by 2-3 months. Therefore, there is a great need for more treatment options that can effectively treat ALS. [Overview of the project]

[0005] This specification provides extracellular vesicles (EVs) and compositions and methods relating to the use of the same for treating ALS in a subject and reducing necroptosis and / or inflammation.

[0006] In a first aspect, a method for treating amyotrophic lateral sclerosis (ALS) in a subject is provided herein. The method comprises administering a therapeutically effective amount of a composition comprising nerve cell-derived extracellular vesicles (EVs) to a subject, thereby treating ALS.

[0007] In some embodiments, the composition is administered distally to the central nervous system (CNS) of the target. In some embodiments, the composition is administered intravenously, intranasally, intraperitoneally, orally, or in any combination thereof.

[0008] In some embodiments, compared to a control group, the subjects show improvement in neurological function, motor function, and / or one or more signs or symptoms associated with ALS, and / or extended survival. In some embodiments, one or more signs or symptoms include muscle weakness, difficulty walking, stumbling, falling, decreased grip strength, clumsiness, slurred speech, difficulty chewing, difficulty swallowing, and / or difficulty breathing.

[0009] In some embodiments, the levels or activity of neurofilament light chains, chitinase-3-like protein 1 (CHI3L1), nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3 (NLRP3), NLRP3 signaling pathway molecules, and / or pro-inflammatory cytokines are reduced in the subject. In some embodiments, the NLRP3 signaling pathway molecules include caspase-1, IL1-β, and / or IL-18. In some embodiments, the pro-inflammatory cytokines include TNFα and / or IL1-β.

[0010] In a second aspect, a method for inhibiting necroptosis in cells is provided herein. This method involves contacting cells with a composition comprising a therapeutically effective amount of extracellular vesicles (EVs) derived from nerve cells, thereby inhibiting necroptosis.

[0011] In some embodiments, levels of receptor interacting protein kinase 1 (p-RIPK1) are reduced in cells compared to control cells.

[0012] In some embodiments, cells are present within the subject. In some embodiments, the subject has amyotrophic lateral sclerosis (ALS), and compared to a control subject, the subject exhibits improved neurological function, motor function, and / or one or more signs or symptoms associated with ALS, and / or extended survival.

[0013] In some embodiments of the methods provided herein, the nerve cells are untransformed neural progenitor cells. In some embodiments, the nerve cells are derived from human pluripotent stem cells, human induced pluripotent stem cells, or human embryonic stem cells.

[0014] In some embodiments, EV comprises ecto-5'-nucleotidase (CD73). In some embodiments, EV further comprises one or more of the following: melanoma-associated chondroitin sulfate proteoglycan (MCSP), pentraxin-3 (PTX3), angiopoietin-1, insulin-like growth factor-binding protein 2 (IGFBP2), and macrophage colony-stimulating factor.

[0015] In some embodiments, the composition contains about 1 mg to about 750 mg of EVs. In some embodiments, the EVs comprise exosomes. In some embodiments, the EVs are about 10 nm to about 10 μm in size, for example, about 20 nm to about 250 nm.

[0016] In some embodiments, the EV further comprises cargo. In some embodiments, the cargo comprises one or more nucleotides, peptides, proteins, antibodies, and small molecules. In some embodiments, the nucleotide is one or more siRNAs, antisense oligonucleotides, mRNAs, plasmids, and cDNAs. [Brief explanation of the drawing]

[0017] [Figure 1] This graph shows the phosphorylated receptor-interacting protein kinase 1 (p-RIPK1):GAPDH ratio in HMC3 cells treated with TNFα in the presence of EVs derived from a vehicle or neural progenitor cells provided herein. [Figure 2] This graph shows the neurological deficit scores in SOD1-G93A mice (ALS mouse model) administered weekly doses of EV or vehicle at the onset of symptoms. * indicates p<0.05. [Figure 3] This graph shows the fall latency in SOD1-G93A mice (ALS mouse model) administered a weekly dose of EV or vehicle at the onset of symptoms. * indicates statistical significance at p<0.05. [Figure 4] This is the overall survival curve for SOD1-G93A mice (ALS mouse model) that were administered a weekly dose of EV or vehicle at the time of symptom onset. * indicates statistical significance at p<0.05. [Figure 5] This graph shows the serum neurofilament (NFL) levels in SOD1-G93A mice (ALS mouse model) that were administered EV or vehicle once a week at the onset of symptoms. * indicates p<0.05. * indicates statistical significance at p<0.05. [Figure 6A] Graphs show the levels of NLRP3 inflammasome pathway markers, NLRP3 (Figure 6A), caspase-1 (Figure 6B), IL1-β (Figure 6C), IL-18 (Figure 6D), and the inflammatory marker TNFα (Figure 6E) in the serum of SOD1-G93A mice (ALS mouse model) administered weekly doses of EV or vehicle at the time of onset. * indicates p<0.05. * indicates statistical significance at p<0.05. [Figure 6B] Graphs show the levels of NLRP3 inflammasome pathway markers, NLRP3 (Figure 6A), caspase-1 (Figure 6B), IL1-β (Figure 6C), IL-18 (Figure 6D), and the inflammatory marker TNFα (Figure 6E) in the serum of SOD1-G93A mice (ALS mouse model) administered weekly doses of EV or vehicle at the time of onset. * indicates p<0.05. * indicates statistical significance at p<0.05. [Figure 6C] Graphs show the levels of NLRP3 inflammasome pathway markers, NLRP3 (Figure 6A), caspase-1 (Figure 6B), IL1-β (Figure 6C), IL-18 (Figure 6D), and the inflammatory marker TNFα (Figure 6E) in the serum of SOD1-G93A mice (ALS mouse model) administered weekly doses of EV or vehicle at the time of onset. * indicates p<0.05. * indicates statistical significance at p<0.05. [Figure 6D]Graph showing the levels of NLRP3 inflammasome pathway markers, NLRP3 (Figure 6A), caspase-1 (Figure 6B), IL1-β (Figure 6C), IL-18 (Figure 6D), and the inflammatory marker TNFα (Figure 6E) in the serum of SOD1-G93A mice (ALS mouse model) administered weekly doses of EV or vehicle at onset. * indicates p < 0.05. * indicates significance at p < 0.05. [Figure 6E] Graph showing the levels of NLRP3 inflammasome pathway markers, NLRP3 (Figure 6A), caspase-1 (Figure 6B), IL1-β (Figure 6C), IL-18 (Figure 6D), and the inflammatory marker TNFα (Figure 6E) in the serum of SOD1-G93A mice (ALS mouse model) administered weekly doses of EV or vehicle at onset. * indicates p < 0.05. * indicates significance at p < 0.05. [Figure 7] Graph of the level of chitinase-3-like protein 1 (CHI3L1) in the serum of 80-day-old SOD1-G93A mice (ALS mouse model). Mice were administered EV or vehicle 5 times a week from 69 days of age. *** indicates p = 0.0002. [Figure 8] Schematic showing the potential mechanism by which the EV provided herein restores ALS-induced inflammation.

Mode for Carrying Out the Invention

[0018] I. Definitions To make the present disclosure more readily understandable, certain terms are first defined. Additionally, it should be noted that whenever a value or range of values of a parameter is recited, intermediate values and ranges within the recited values are also intended to be part of the present disclosure.

[0019] As used herein, the term “blood-brain barrier” refers to a highly selective, semipermeable boundary of endothelial cells that prevents molecules in the circulating blood from non-selectively entering the extracellular fluid of the CNS. The blood-brain barrier includes endothelial cells of the capillary wall, astrocytic end feet that cover capillaries with sheaths, and pericytes embedded in the capillary basement membrane. The blood-brain barrier prevents various peripherally administered exogenous molecules, including non-neuronal extracellular molecules, from reaching the CNS and achieving physiologically significant concentrations within it.

[0020] As used herein, the term “cargo” refers to one or more molecules that can be delivered to a target site in the body (e.g., the nervous system). Cargo may include, but are not limited to, small molecules (e.g., small molecule drugs), nucleic acids (e.g., mRNA, siRNA, shRNA, antisense RNA, antisense oligonucleotides, miRNA, plasmids, cDNA, DNA), or proteins or peptides (e.g., hormones, growth factors, enzymes, anticoagulants, interferons, interleukins, antibodies, antibody fragments, antibody-drug conjugates, etc.). In some embodiments, the cargo is a therapeutic molecule. In some embodiments, the cargo can be attached directly or indirectly to an EV. In other embodiments, the cargo is delivered using an EV as provided herein.

[0021] As used herein, the term “central nervous system” or “CNS” refers to all structures within the dura mater. Such structures include, but are not limited to, the cells and tissues of the brain and spinal cord. The CNS also includes the cerebrospinal fluid that fills the ventricles of the brain and the central canal of the spinal cord.

[0022] As used herein, the terms “distal” or “peripheral” in the context of administration of the compositions described herein refer to a site or route of administration that is different from a specific target site. For example, administration to a site distal to the brain refers to administration to a site other than the brain, such as intrathecal, intravenous, or intranasal administration, and can be contrasted with local administration to a site within the brain, for example, by direct injection into the brain and surrounding parenchyma. In some embodiments, the EV-containing compositions described herein may be administered to a site distal to the brain. In other embodiments, the EV-containing compositions described herein may be administered to a site distal to the CNS. For example, a distal route of administration to the CNS may include intravenous and intranasal administration. Other suitable routes of administration, including those distal to the brain and / or distal to the CNS, are described herein.

[0023] In the context of "intact blood-brain barrier," the term "intact" refers to a properly functioning blood-brain barrier that maintains its normal physiological function, non-selectively preventing molecules in the circulating blood from entering the extracellular fluid of the CNS and being delivered to the brain. Subjects containing an intact blood-brain barrier do not mean healthy or disease-free subjects, such as those without a neurological condition or disorder (e.g., ALS). Subjects may simultaneously have an intact blood-brain barrier and a disease or condition, such as a neurological disorder, e.g., ALS.

[0024] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount of a composition containing an agent, such as an EV, that is sufficient to reduce or reverse the severity and / or duration of a disorder or one or more of its symptoms, prevent the progression of a disorder, cause regression of a disorder, prevent the recurrence, onset, or progression of one or more symptoms associated with a disorder, or enhance or improve the preventive or therapeutic effect of another therapy. For example, an effective dose of an EV composition for inhibiting necroptosis or reducing inflammation may be an amount sufficient to inhibit necroptosis or reduce inflammation in the target cells exposed to the EV composition, as measured, for example, by the levels of markers of necroptosis or inflammation (e.g., p-RIPK1, NLRP3, caspase-1, IL-1β, IL-18, TNFα). In another example, an effective dose of an EV composition for treating ALS may be sufficient to slow the progression of ALS, maintain neurological or motor function, improve one or more signs or symptoms associated with ALS (e.g., muscle weakness, difficulty walking, stumbling, falling, decreased grip strength, clumsiness, slurred speech, difficulty chewing, difficulty swallowing, and / or difficulty breathing), and / or improve the patient's survival. For example, if a given clinical treatment is considered effective when there is a reduction of at least a certain percentage of a measurable parameter associated with ALS, then the effective therapeutic dose of a composition for treating ALS (e.g., an EV composition) is the amount necessary to obtain a reduction of at least a certain percentage of that parameter. Those skilled in the art can, in accordance with the clinical and scientific circumstances, select a specific percentage or range of percentages of reduction in a measurable parameter for the treatment to be considered effective.

[0025] The term "embryonic stem cell" or "ESC (embryonic stem cell)" refers to pluripotent cells isolated from blastocyst-stage embryos, preferably from primates including humans.

[0026] The terms “extracellular vesicles” and “EVs” are used herein to refer to vesicles approximately 10 nm to 10 μm in size surrounded by a lipid bilayer, e.g., a portion of the cell membrane. EVs may contain fluids, macromolecules, solutes, and metabolites from cells. The term “EVs” also includes lipid vesicles engineered to contain bioactive molecules found in cell-derived EVs, such as neural EVs. These terms encompass, but are not limited to, microvesicles (e.g., any vesicles detached from the cell's plasma membrane), exosomes (e.g., any vesicles derived from the endolysosomal pathway), apoptotic bodies (e.g., which can be obtained from apoptotic cells), microparticles (e.g., which may be derived from platelets), ectosomes (e.g., which may be derived from neutrophils and monocytes in serum), prostagosomes (e.g., which can be obtained from prostate cancer cells), or cardiosomes (e.g., which may be derived from cardiac cells). Furthermore, EVs may refer to extracellular vesicle mimes or cell membrane vesicles obtained by membrane extrusion or other techniques. The term "EV" encompasses vesicles isolated from recombinant cell sources that have been engineered or modified to produce / express exogenous activators (e.g., exogenous polypeptides), which are incorporated into the EVs. The term "EV" also encompasses vesicles that have been engineered (e.g., loaded) to contain exogenous activators (e.g., exogenous polypeptides) after isolation. The term "EV" also encompasses artificial lipid vesicles that have been engineered to contain bioactive molecules found in cell-derived EVs, such as neural EVs. The term "EV" encompasses both exosomes and ectosomes.

[0027] Exosomes (EVs) can be obtained from a suitable biological source using isolation techniques, such as a combination of methodologies including centrifugation, filtration, and ultracentrifugation. Exosomes are released by exocytosis of the multivesicular body (MVB). Ectosomes are vesicles assembled in a plasma membrane and released therefrom. In some examples, EVs are approximately 20 nm–10 μm, 20 nm–1 μm, 20 nm–500 nm, 30 nm–100 nm, 30 nm–160 nm, or 80 nm–160 nm in size. In some embodiments, EVs are exosomes approximately 20 nm–150 nm in size. EVs can be isolated from any suitable biological sample from mammals, including but not limited to whole blood, serum, plasma, breast milk, cerebrospinal fluid, amniotic fluid, ascites, or bone marrow. EVs can be isolated from cultured mammalian cells (e.g., nerve cells, dendritic cells (wild-type or immortalized), induced and uninduced pluripotent mesenchymal stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes, etc.). In certain embodiments, EVs can be isolated from nerve cells (e.g., neural progenitor cells, neural stem cells, glial cells, astrocytes, neurons, etc.).

[0028] The term "pluripotent stem cell" (PSC), with "embryonic stem cells" (ESCs) and induced pluripotent stem cells (iPSCs) as subsets, is characterized by its ability to originate from preembryonic, embryonic, fetal tissue, or adult stem cells (in the case of induced pluripotent stem cells) at any point after fertilization and, under appropriate conditions, produce offspring of several different cell types, particularly neural stem cells and precursors, neural crest cells, mesenchymal stem cells (MSCs), and associated proliferative and non-proliferative neurons. This term includes established strains of various types of stem cells and cells obtained from primary tissues that are pluripotent in the manner described. Human pluripotent stem cells include, for example, human embryonic stem cells (hESCs). Human pluripotent stem cells can also include pluripotent stem cells derived from adult cells, such as induced pluripotent stem cells (hiPSCs).

[0029] As used herein, “pharmaceutically acceptable” means a material, such as a carrier or diluent, that does not inhibit the biological activity or properties of a compound and is relatively non-toxic; that is, the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained. In some cases, “pharmaceutically acceptable” means a compound that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, immunogenicity, or complications commensurate with a reasonable benefit / risk ratio.

[0030] As used herein, the term “neuron” refers to cells associated with the nervous system. The term “neuron” encompasses neural stem cells, e.g., neural progenitor (NP) cells, and differentiated nerve cells. In some embodiments, differentiated nerve cells can be induced in vitro from neural stem cells, NP cells, or pluripotent stem cells. Exemplary nerve cells include neurons, glial cells, astrocytes, oligodendrocytes, microglia, Schwann cells, and glioma cells. In other embodiments, nerve cells can be neural progenitor (NP) cells or neural stem cells. The terms “neuron" and “neural stem cells” refer to pluripotent cells that have the ability to differentiate into a limited repertoire of neuronal and glial cell types. In some embodiments, NP cells can be induced in vitro from pluripotent stem cells, e.g., induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). In some embodiments, the nerve cells referred to herein are human nerve cells. In exemplary embodiments, the NP cells referred to herein are human NP cells. NP cells do not have to be transformed. As used herein, “transformed” refers to cells that have genetic changes resulting from the incorporation of exogenous genetic material. As used herein, “untransformed” refers to cells that do not have genetic changes or the incorporation of exogenous genetic material. Cell transformation may result in material changes in the composition or characteristics of EVs derived from the cell. In some embodiments, NP cells are proliferative. In some embodiments, NP cells maintain their phenotype without differentiation.

[0031] The terms “neuronal EV,” “neuronal cell EV,” or “neuronal cell-derived EV” are used to refer to EVs derived from nerve cells, such as neural stem cells, such as neural progenitor cells. “Neural EV” can be a “neuronal progenitor EV,” or an EV derived from other nerve cell types such as neurons, glial cells, astrocytes, oligodendrocytes, microglia, Schwann cells, or glioma cells, or their progenitor cells. The term also refers to vesicles that contain a sufficient number of bioactive molecules found in cell-derived neural EVs and have been manipulated to have substantially the same bioactivity.

[0032] The terms “neural progenitor EV,” “neural progenitor cell EV,” or “neural progenitor cell-derived EV” are used interchangeably to refer to EVs derived from neural progenitor cells. This term also refers to vesicles that contain a sufficient number of bioactive molecules found in cell-derived neural progenitor EVs and have been manipulated to have substantially the same bioactivity.

[0033] As used herein, “pharmaceutically acceptable” means a material, such as a carrier or diluent, that does not inhibit the biological activity or properties of a compound and is relatively non-toxic; that is, the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained. In some cases, “pharmaceutically acceptable” means a compound that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or complications commensurate with a reasonable benefit / risk ratio.

[0034] As used herein, the term “sample” refers to a specimen (e.g., cells (e.g., nerve cells), tissue (e.g., spinal cord, brain), blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, pancreatic juice, chorionic villi specimens, and bone marrow). In some embodiments, the sample is derived from or taken from a subject.

[0035] As used herein, the term “Subject” refers to any organism that is the target of administration or treatment. “Subject” can be an organism, for example, a mammal (e.g., human, non-human mammal, non-human primate, primate, laboratory animal, mouse, rat, hamster, cat, or dog). In one embodiment, the subject is a human subject. The term “Patient” refers to a human subject diagnosed with a disease or disorder including ALS, a human subject exhibiting one or more symptoms of any disorder or disease described herein (e.g., ALS), and / or a human subject under the treatment of a clinician, for example, a physician. The subject may be male or female.

[0036] In some embodiments of this disclosure, the subject requires delivery of an EV to the CNS. In some embodiments, the subject requires delivery of an EV-loaded exogenous cargo to the CNS. In some embodiments, the subject requires treatment of ALS or reduction of inflammation in the brain. In some embodiments, the subject has a neurological condition or symptoms associated with ALS.

[0037] The terms “to treat” and “treatment” refer to medical management of an object intended to improve, restore, stabilize (e.g., prevent exacerbation), prevent or cure a disease, pathological condition, or disorder. This term includes active treatment (treatment aimed at improving a disease, pathological condition, or disorder), causal treatment (treatment aimed at the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed to relieve symptoms), preventive treatment (treatment aimed at minimizing or partially or completely inhibiting the occurrence of the associated disease, pathological condition, or disorder), and supportive treatment (treatment used to complement another treatment). Treatment also includes reducing the degree of the disease or condition, whether detectable or undetectable, preventing the spread of the disease or condition, delaying or slowing the progression of the disease or condition, restoring or alleviating the disease or condition, and causing remission (whether partial or complete). To “recover” or “alleviate” a disease or condition means that the degree and / or undesirable clinical symptoms of the disease, disability, or condition are reduced and / or the progression is slowed or prolonged compared to the degree or course of the condition if no treatment is given. Treatment does not require complete recovery of symptoms or disease, but includes embodiments that reduce symptoms and / or underlying risk factors. “Treatment” can also mean extending survival compared to the survival expected if no treatment is given. Those requiring treatment include those who already have a condition or disability, those who are prone to developing a condition or disability, or those for whom a condition or disability can be prevented. The term “prevent” does not require 100% elimination of the possibility of the event. Rather, it indicates that the likelihood of the event occurring is reduced in the presence of the compound or method.

[0038] According to the present invention, conventional cell culture methods, chemical or synthetic methods, and other biological and pharmaceutical techniques within the scope of the art may be used. Such techniques are well known and otherwise described in detail in the literature. Standard techniques for cloning, DNA isolation, amplification, and purification, as well as various isolation techniques, for growing cells, separating cells, and, where applicable, for enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, etc., may be known and commonly used by those skilled in the art.Many standard techniques are used in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York, Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York, Wu (Ed.) 1993 Meth. Enzymol. 218, Part I, Wu (Ed.) 1979 Meth.Enzymol.68, Wu et al., (Eds.) 1983 Meth.Enzymol.100 and 101, Grossman and Moldave (Eds.) 1980 Meth.Enzymol.65, Miller (Ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, Old and Primrose, 1981 Principles of Gene Manipulation,University of California This information is found in Press, Berkeley, Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where used, are considered standard in the field and are commonly used in professional journals such as those cited herein.

[0039] The term "antibody" is used herein in its broadest sense and is not limited to, but encompasses a variety of structures that bind to a target antigen, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, monobodies, antibody mimes, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0040] Where a range of values ​​is provided, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of that range, and any other stated or intermediate values ​​within that stated range, are understood to be included within the invention as if they were explicitly stated. These upper and lower limits of a range may independently be included within that range and are also included within the invention, subject to any specifically excluded limits within the stated range. If a stated range includes one or both limits, a range excluding either of those included limits is also included within the invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. Any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this disclosure, but exemplary methods and materials are described herein.

[0042] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. For example, "an object" means one or more objects.

[0043] The terms "about" or "approximately" usually mean within 5%, or more preferably within 1%, of a given value or range.

[0044] II. Extracellular vesicles (EVs) Extracellular vesicles (EVs), such as exosomes, can be used to enhance the efficiency of nucleic acid delivery. Exosomes are small, membrane-bound vesicles of endocytosis origin that are released into the extracellular environment after fusion of the polyendoplasmic reticulum with the plasma membrane. Exosome production has been described from many types of cells, including immune cells such as B cells, T cells, and dendritic cells (DCs), tumor cells, mesenchymal stem cells (MSCs), and more recently, nerve cells.

[0045] EVs (extosomes) comprise a heterogeneous group of small structures surrounded by a lipid bilayer, such as a portion of the cell plasma membrane. EVs can range in size from approximately 10 nm to 10 μm in diameter, most commonly falling within the range of approximately 25 to 500 nm. EVs can be broadly classified into two classes: exosomes and ectosomes. Exosomes can be formed by cells through inward budding of the endosomal membrane during the maturation of the multivesicle endoplasmic reticulum (MVB). Exosomes can then be released into extracellular space by fusion of the MVB with the cell surface. Exosomes are typically 25 to 2500 nm in diameter, and in some embodiments can range from approximately 25 to 200 nm, approximately 30 to 150 nm, or approximately 50 to 200 nm. Ectosomes, also known as microvesicles, can be formed by cells via budding of the plasma membrane. Ectosomes can vary in size from approximately 10 nm to 10 μm, and in some embodiments, they can be in the range of approximately 10 to 1000 nm, approximately 10 to 500 nm, approximately 10 to 250 nm, or approximately 10 to 200 nm.

[0046] Extracellular vesicles (EVs) are involved in intercellular communication that enables the movement of substances from EVs to cells through fusion with the cell membrane or through cellular uptake including, but not limited to, caveolin-independent and dependent mechanisms, clathrin-independent and dependent mechanisms, macropinocytosis, and / or phagocytosis. EVs have been reported to be involved in numerous physiological processes, including immunomodulation, angiogenesis, endothelial cell migration associated with tumor growth, or damage reduction in ischemia-reperfusion injury. Many of these functions are mediated by nucleic acids, proteins, or lipids contained within or on vesicles.

[0047] EVs can carry cargo embedded in and / or attached to a lipid bilayer within their lumen. Cargo carried by EVs may include proteins, lipids, and / or nucleic acids such as mRNA or miRNA. When EVs are produced by cells, the composition of the cargo is highly cell-type dependent. For example, EVs derived from astrocytes, neural progenitor cells, and mesenchymal stem cells have been shown to contain distinct complements of protein cargo (see U.S. Patent Application Publication 2018 / 0327714(A1), the contents of which are incorporated herein by reference in their entirety). EVs derived from these different cell types also contain different profiles of nucleic acid molecules, including mRNA and / or miRNA. In embodiments where EVs are obtained from cells, the EV contains endogenous cargo that reflects the contents of the EV produced by the cell from which the EV originates. In some embodiments, EVs obtained from cells may also contain exogenous cargo. The exogenous cargo includes proteins, nucleic acids, small molecules, or lipids that are introduced into the EV by vesicle manipulation after release from the producing cell. In other embodiments, EVs obtained from cells may contain exogenous cargo that is packaged in the EV as a result of recombinant nucleic acids present in the cell from which the EV originates. For example, an EV containing a recombinant protein may originate from a cell containing recombinant nucleic acid encoding the protein. In embodiments in which EVs are produced synthetically, suitable cargo for inclusion in the vesicle can be selected. For example, in one embodiment, a synthetic EV may contain one or more proteins, lipids, or nucleic acids present in an EV derived from a nerve cell, such as a neural progenitor cell, neuron, glial cell, or astrocyte.

[0048] EVs suitable for use in the compositions and methods described herein can be derived from any suitable source. For example, EVs can be derived from nerve cells such as neural stem cells and neural progenitor cells, or differentiated nerve cells such as neurons, glial cells, or astrocytes. EVs suitable for use in the methods described herein can also be produced synthetically. In some embodiments, the EVs used in the compositions and methods described herein are derived from neural progenitor cells or neural stem cells.

[0049] Neural EVs, including neural progenitor EVs, contain complement of membrane proteins that are thought to facilitate the passage of neural EVs across the capillaries of the blood-brain barrier, although we do not wish to be constrained by theory. Conventional methods for preparing EVs for nucleic acid delivery, such as electroporation, may impair membrane integrity, resulting in a reduced ability to cross the blood-brain barrier. This specification provides neural EVs containing exogenous nucleic acids, their use in the treatment of neurological diseases, and methods for loading exogenous nucleic acids into EVs in a manner that preserves membrane integrity.

[0050] Cell-derived extracellular molecules (EVs), such as exosomes, contain a variety of biomolecules that reflect their cellular origin. In one embodiment, a population of EVs derived from nerve cells, e.g., neural progenitor cells, is provided herein. Such neural EVs contain a diverse protein environment derived from nerve cells, including cytokines and growth factors, as well as coding and non-coding RNA molecules. The cargo contained in neural EVs can influence neuronal and vascular function by providing neuroprotection, reducing inflammation, immunomodulating through action on T cell macrophages and microglia, reducing oxidative stress, improving vascular integrity, influencing metabolic activity, and inducing neurogenesis, cell migration, myelin regeneration, and differentiation. While we do not wish to be bound by theory, it is thought that the combination of native proteins expressed on the surface of EVs derived from nerve cells (e.g., neural progenitor cells, neural stem cells) allows the vesicles to localize to the CNS and cross the blood-brain barrier to a greater extent than EVs from other sources. Furthermore, the ability of neural EVs to target the CNS and / or cross the blood-brain barrier is not hindered by loading nucleic acids into the EVs. In some embodiments of this disclosure, EVs loaded with cargo containing EVs derived from nerve cells (e.g., neural progenitor cells, neural stem cells) can transport the functionally intact cargo across the blood-brain barrier for delivery to targets in the brain and central nervous system.

[0051] In some embodiments, nerve cell-derived EVs, such as exosomes, may contain membrane proteins including, but not limited to, CD63, CD81, and CD133. Therefore, in one embodiment, an EV suitable for use in the compositions and methods described herein, such as an exosome, comprises one or more of the following cell surface proteins: CD63, CD81, and CD133 (i.e., one or more, two or more, or all three). Furthermore, an EV, for example, an exosome, may contain one or more of the following miRNAs: hsa-miR-135a-2, hsa-miR-124-1, hsa-miR-124-2, hsa-miR-124-3, hsa-miR-489, hsa-miR-9-3, hsa-miR-9-2, hsa-miR-9-1, hsa-miR-219b, hsa-miR-219a-2, hsa-miR-363 and / or hsa-miR-20b (i.e., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all 12). In one embodiment, a population of isolated EVs is enriched for one or more of the aforementioned miRNAs. Concentration can be measured in absolute or relative amounts, such as when compared to unmodified exosomes derived from non-neuronal cells, for example, mesenchymal stem cells.

[0052] As described herein, neural progenitor cell-derived EVs (NP EVs) possess several miRNAs concentrated in their vesicles compared to EVs derived from parental NP cells or other cell sources. Therefore, in some embodiments, neural progenitor cell-derived EVs may contain one or more of the following miRNAs: miR-92a, miR-219a-2, miR-363, miR-21, miR-124, miR-135a-2, miR-130b, miR-148a, miR-25, miR-30a-5p, miR-30a-3p, miR-340, miR-122, and / or miR-31-5p. In some embodiments, NP EV may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen of the aforementioned miRNAs. In some embodiments, NP EV may contain one, two, three, or four of miR-219a-2, miR-363, miR-124, or miR-135a-2, or a combination thereof. In some embodiments, NP EV may contain miR30a-3p and / or miR-340. In some embodiments, NP EV may contain miR-122 and / or miR-31-5p. In some embodiments, the NP EV includes one, two, three, four, five, six, seven, or eight of the following: miR-219a-2, miR-363, miR-124, miR-135a-2, miR30a-3p, miR-340, miR-122, and miR-31-5p, or a combination thereof.

[0053] (i) Therapeutic effects of EV The extracellular entities (EVs) provided herein (e.g., neural EVs, neural progenitor EVs) can exhibit therapeutic activity, such as anti-inflammatory effects, without additional payload (e.g., exogenous cargo). For example, in some embodiments, the neural EVs (e.g., neural progenitor EVs) provided herein contain ecto-5'-nucleotidase (NT5E). NT5E, also known as AMPase and CD73, converts AMP to adenosine. While we do not wish to be bound by theory, during neurodegeneration, post-stroke, and other neurological injuries, dead and dying cells release large amounts of ATP into the extracellular space. ATP is a potent inflammatory signal that suppresses and / or kills anti-inflammatory regulatory T cells, while causing secondary injury after the acute phase. NT5E activity confers the ability of EVs to convert pro-inflammatory ATP to adenosine, which is anti-inflammatory and supports an immunosuppressive environment. NT5E (CD73) can be a potency marker of EVs.

[0054] In further embodiments, the neuronal extracellular proteins provided herein include melanoma-associated chondroitin sulfate proteoglycans (MCSPs), pentraxin-3 (PTX3), angiopoietin-1, insulin-like growth factor-binding protein 2 (IGFBP2), and other bioactive polypeptides or proteins such as macrophage colony-stimulating factor.

[0055] The extracellular genes (EVs) provided herein (e.g., neural EVs, neural progenitor EVs) can cross the blood-brain barrier (including intact blood-brain barriers) and exert therapeutic activity without additional payloads. EVs can reduce neuroinflammation, including by reducing pro-inflammatory T cells and increasing anti-inflammatory T cells and macrophages (Webb, RL, et.al. 2018 Transl Stroke Res. 9(5):530-539). In addition, or alternatively, EVs can promote neuroprotection, including by preventing cell death and enhancing neuronal survival by reducing oxidative stress (Webb, RL 2018 Stroke 49(5):1248-1256, Webb, RL, et.al. 2018 Transl Stroke Res 9(5):530-539). Furthermore, EVs can stimulate nerve regeneration, including increased neural stem cell proliferation that leads to nerve growth and myelin sheath reformation (Sun, M., et.al. 2020 Journal of Neurotrauma, doi.org / 10.1089 / neu.2019.6443).

[0056] (ii) EV production EVs suitable for use in the compositions and methods disclosed herein can be produced by various cell types (e.g., human neural progenitor cells, human neural stem cells, mouse neural progenitor cells, mouse neural stem cells, etc.) or can be produced synthetically. As described above, EVs can be purified from any cell source (e.g., cultured mammalian cells, including but not limited to primary cells, stem cells / progenitor cells, transformed cells, and established cell lines) using known methods. For example, EVs can be purified by procedures selected from a group of techniques including liquid chromatography (LC), high-performance liquid chromatography (HPLC), spin filtration, tangential flow filtration, hollow fiber filtration, centrifugation, immunoprecipitation, flow-field separation, dialysis, microfluidic-based separation, or any combination thereof. In one embodiment, the purification of EVs is carried out using a sequential combination of filtration (preferably ultrafiltration (UF), tangential flow filtration, or hollow fiber filtration) and size exclusion liquid chromatography (LC). This combination of purification processes results in optimized purification, which in turn leads to superior therapeutic activity. Furthermore, compared to ultracentrifugation (UC), which is routinely used to purify extracellular viables such as exosomes, continuous filtration-chromatography can scale up considerably faster to higher production volumes, a significant drawback of the more commonly used current UC methodology. Another purification methodology is tangential flow filtration (TFF), which offers scalability and purity and may be combined with other types of purification techniques such as filtration.

[0057] EVs suitable for use according to some embodiments of this disclosure may be derived from nerve cells, dendritic cells (wild-type or immortalized), artificial and non-induced pluripotent stem cells, fibroblasts, platelets, neutrophils, monocytes, immune cells, reticulocytes, cardiac cells, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes, and apoptotic cells. In some embodiments, EVs are derived from cultured cell lines, e.g., CHO cells, HEK cells. In some embodiments, EVs are derived from primary cells. In some embodiments, EVs are derived from cells isolated from a subject and cultured to produce exosomes. In some embodiments, EVs may be derived from nerve cells such as neural progenitor cells, neurons, glial cells, astrocytes, oligodendrocytes, microglia, Schwann cells, or glioma cells. In some embodiments, nerve cells are untransformed cells. In some embodiments, nerve cells are primary nerve cell cultures. In some embodiments, EVs can be produced by transformed cell lines, e.g., transformed neuronal cell lines. In other cases, EVs can be produced by engineered cell lines expressing exogenous polypeptides and / or nucleic acids, such as recombinant cell lines. In specific embodiments, the EVs are neural EVs derived from neural progenitor cells, or nerve cells such as neural stem cells, neurons, oligodendrocytes, microglia, Schwann cells, or astrocytes.

[0058] In some embodiments, EVs include exosomes. Exosomes may be derived from any of the aforementioned cell types. For example, exosomes suitable for use in the compositions and methods disclosed herein may be neural exosomes. Neural exosomes may be derived from nerve cells including, but not limited to, neural progenitor cells, neurons, astrocytes, oligodendrocytes, microglia, Schwann cells, or glioma cells. In other embodiments, exosomes may be produced synthetically.

[0059] Alternatively, EVs can be ectosomes, also known as microvesicles. Microvesicles can be derived from any of the aforementioned cell types. For example, a suitable microvein for use in the compositions and methods disclosed herein may be a neuron microvein. Neuron microvesicles can be derived from nerve cells, including but not limited to neural progenitor cells, neurons, astrocytes, oligodendrocytes, microglia, Schwann cells, or glioma cells. In other embodiments, microvesicles can be produced synthetically.

[0060] In some embodiments, the compositions and methods described herein may utilize a population of isolated and / or purified EVs containing exosomes and / or ectosomes.

[0061] In some cases, the disclosed EVs can be obtained by culturing cells, such as nerve cells, for a sufficient time for the cells to produce EVs. In some embodiments, the cells used to produce EVs may be derived from pluripotent stem cells, such as human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). EVs can be isolated from cell culture media or tissue culture supernatant. EVs produced from cells can be collected from the culture medium by any preferred method. Typically, isolated populations of EVs can be prepared from cell culture or tissue supernatant by centrifugation, size exclusion columns, microfluidic devices, polymer precipitation, filtration, or a combination of these methods, for example, as described in U.S. Patent Application Publication 2014 / 0356382, which is entirely incorporated herein by reference. For example, extraepithelial ions (EVs) can be prepared by fractional centrifugation, i.e., slow (<20,000 g) centrifugation to pelletize larger particles followed by high (>100,000 g) centrifugation to pelletize the EVs, size filtration using a suitable filter (e.g., a 0.22 μm filter), gradient ultracentrifugation (e.g., using a sucrose gradient), or a combination of these methods.

[0062] In some embodiments, the EV-producing cells disclosed herein are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or about 1, 2, 3, 4, 5, or 6 months, depending on the cells and their EV-producing capacity. The EV-producing cells may be cultured in a suitable medium and grown under conditions readily determined by those skilled in the art. Cell culture conditions may vary depending on the cell type, and the examples shown below illustrate suitable media and conditions. For example, CMRL 1066 medium (from INVITROGEN) containing exosome-depleted fetal bovine serum (e.g., 10%) and optionally supplemented with glutamine or a glutamine-containing mixture and antibiotics may be used. In some embodiments, cells can be grown attached to a surface; for example, they can be grown on a surface as a monolayer or multilayer (without feeder cells) and may be grown to 30, 40, 50, 60, 70, 80, 90, 95, or 100% confluent. In other embodiments, cells can be grown as cell aggregates in suspension culture or on microbeads.

[0063] Cell growth media are well known in the art and generally include at least minimal essential media plus one or more optional components (e.g., growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (if indicated and not excluded), activin A, transferrin, beta-mercaptoethanol, and other agents well known in the art and other agents as otherwise described herein). Preferred media in some embodiments are low-protein, serum-free growth media that support nerve cells. The growth factors used may be fibroblast growth factor 2 (FGF2) alone or preferably in combination with leukemia inhibitor factor (LIF).

[0064] In embodiments in which NPs or nerve cells are grown in growth medium, the inclusion of LIF is preferred but not required. Further preferred types of media include basal cell media that may contain serum, for example, about 0.1% to 20% (preferably about 2 to 10%) of fetal bovine serum, or, in the case of standardized media, do not contain fetal bovine serum and KSR, but optionally contain bovine serum albumin (about 1 to 5%, preferably about 2%). In some cases, the medium is standardized, serum-free, and low in protein content. In other cases, the medium is ARUNA BIOMEDICAL, INC. medium and supplement (e.g., ARUNA BIOMEDICAL, INC.'s AB2® Neural Cell Culture Media Kit, which contains AB2® Basal Neural Medium and ANS® Neural Medium Supplement), which allow nerve cultures to maintain a stable karyotype over multiple passages without the need for feeder cells, making them an excellent choice for a wide variety of research applications, including early-stage drug discovery. The components of the growth medium depend on the type of nerve cells being grown, all of which are well known in the field. In one case, the AB2® Neural Cell Culture Media Kit is used, which contains AB2® Basal Neural Medium and ANS® Neural Medium Supplement. In specific cases, the media and supplements described above are specifically manipulated for diversity to meet all nerve cell culture needs. AB2® Basal Neural Medium and ANS® Neural Medium Supplement can be used as the basis for specialized media to direct the differentiation of hNP cells (e.g., hNP1® strains) to various neural phenotypes. Each lot of media and supplements is pre-qualified for use by testing for cell growth, sterility, pH, osmotic pressure, and endotoxin.

[0065] Other agents that may be optionally added to the culture medium include, depending on the cell type being grown in the medium, several other components, such as nicotinamide, members of the TGF-β family (including TGF-β1, 2, and 3), activin A, nodal, bone morphogen proteins (BMP2-7), serum albumin, members of the fibroblast growth factor (FGF) family, platelet-derived growth factors-AA and -BB, platelet-rich plasma, insulin growth factor (IGF-I, II, LR-IGF), growth differentiation factors (GDF-5, -6, -8, -10, 11), glucagon-like peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetodes, exendin-4, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, and epidermal growth factor. Examples include factor (EGF), gastrin I and II, copper chelating agents (e.g., triethylenepentamine), forskolin, sodium butyrate, betacerlin, ITS, noggin, neurite growth factor, nodal, valproic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloids (e.g., cyclopamine (EMD, CA)), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, Notch pathway inhibitors, sonic hedgehog inhibitors, heregulin, or any one or more of these combinations. Each of these components, if included, is included in an effective amount.

[0066] In some cases, a suitable culture medium can be prepared from the following components, for example: Dulbecco's modified Eagle's medium (DMEM), GIBCO#11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), GIBCO#10829-018; Ham F12 / 50% DMEM basic medium, 200 mM L-glutamine, GIBCO#15039-027; non-essential amino acid solution, GIBCO 11140-050; β-mercaptoethanol, SIGMA#M7522; human recombinant basic fibroblast growth factor (bFGF), GIBCO#13256-029; etc. Other suitable reagents may include Neurobasal (GIBCO), BrainPhys (STEM CELL TECHNOLOGIES), and / or NeuroDiff (STEM CELL TECHNOLOGIES).

[0067] Cell media are commercially available and can be supplemented with commercially available components, including specified heterogeneous components available from INVITROGEN CORP. (GIBCO), CELL APPLICATIONS, INC., BIOLOGICAL INDUSTRIES, BETH HAEMEK, Israel, and CALBIOCHEM. Those skilled in the art can easily modify the cell media to produce any one or more target cell types used as a source of extracellular life (EVs).

[0068] The disclosed EV-producing cells can be cultured on a layer of feeder cells that support the cells in various ways. Approaches for culturing cells on a layer of feeder cells are well known in the art. Cells can grow on a cell support or matrix as an adhesive monolayer or as cell aggregates in suspension. In some cases, the use of a cell support may be preferred depending on the cells used to produce EVs. When used, the cell support preferably contains at least one substrate protein. Examples of substrate proteins include extracellular matrix proteins (which are proteins found in the extracellular matrix, e.g., laminin, tenascin, thrombospongin, and mixtures thereof), which exhibit growth-promoting activity and contain domains homologous to epidermal growth factor (EGF). Other substrate proteins that may be used include, for example, collagen, fibronectin, vibronectin, polylysine, polyornithine, and mixtures thereof. Furthermore, gels and other substances (e.g., methylcellulose of other gels containing one or more of these embryonic stem cell differentiation proteins at effective concentrations) may also be used. Examples of differentiated proteins or materials containing these differentiated proteins include, among others, recombinant laminin, BD Cell-Tak® Cell and Tissue Adhesive, BD® FIBROGEN Human Recombinant Collagen I, BD® FIBROGEN Human Recombinant Collagen III, BD Matrigel® Basement Membrane Matrix, BD Matrigel® Basement Membrane Matrix High Concentration (HC), BD® PuraMatrix® Peptide Hydrogel, Collagen I, Collagen I High Concentration (HC), Collagen II (bovine), Collagen III, Collagen IV, Collagen V, and Collagen VI.

[0069] Alternatively, these cells may be cultured in a culture system that does not contain feeder cells, or is essentially feeder cell-free, but nevertheless supports cell proliferation for EV production. Cell growth in feeder-free culture can be supported using a medium conditioned by pre-culturing with another cell type. Alternatively, growth of EV-producing cells in non-differentiated feeder-free culture can be supported using a chemically defined medium. These approaches are well known in the art. In certain embodiments of the present invention, cells are grown in a medium that does not contain feeder cells.

[0070] In some embodiments of this disclosure, EVs can be isolated from neurons derived from (e.g., differentiated from) pluripotent stem cells such as hESCs or iPSCs, e.g., neural progenitor cells. Pluripotent stem cells may express one or more markers detectable using stage-specific embryonic antigens (SSEAs) 3 and 4, and antibodies referred to as Tra-I-60 and Tra-1-81, as described by Thomson et al. (Science 282:1145, 1998). Differentiation of pluripotent stem cells in vitro results in loss of SSEA-4, Tra-I-60, and Tra-1-81 expression (if present), and increased SSEA-1 expression. Undifferentiated pluripotent stem cells typically possess alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde and then chromogenically developing them using Vector Red as a substrate, as described by the manufacturer (VECTOR LABORATORIES, Burlingame Calif.). Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as can be detected by antibody or RT-PCR. In some embodiments, EVs for use in the compositions and methods described herein can be produced from neural progenitor cells derived from hESCs. In other embodiments, EVs for use in the compositions and methods described herein can be produced from neural progenitor cells derived from iPSCs.

[0071] Types of pluripotent stem cells that can be used for EV generation include established pluripotent cell lines derived from post-fertilization tissue, including proembryonic tissue (e.g., blastocysts), embryonic tissue, or fetal tissue, collected at any point during pregnancy, typically but not necessarily, around 10–12 weeks prior to conception. Non-limiting examples include established hESC or human embryonic germ cell lines, such as hESC lines WA01, WA07, and WA09 (WICELL). Furthermore, this specification intends to use such cells during the initial establishment or stabilization of pluripotent stem cells, in which case the source cells are primary pluripotent cells collected directly from the source tissue. Cells collected from a pluripotent stem cell population already cultured in the absence of feeder cells are also suitable. For example, mutant hESC strains such as BG01v (VIACYTE, San Diego, CA), as well as normal hESC strains such as WA01, WA07, WA09 (WICELL, Madison, WI) and BG01, BG02 (VIACYTE, San Diego, CA), are also suitable.

[0072] hESC can be prepared by methods described in the art, for example, by Thomson et al. (U.S. Patent No. 5,843,780, Science 282:1145-1147, 1998, Curr Top Dev Biol 38:133-165, 1998, Proc Natl Acad Sci 92:7844-7848, 1995). Alternatively, they may be commercially available.

[0073] iPSCs are created by dedifferentiating adult somatic cells and returning them to a pluripotent state. iPSCs can be generated by any suitable methodology, including, but not limited to, the artificial expression of four genes: c-myc, Klf4, SOX2, and OCT4.

[0074] A method for producing human neural progenitor cells (hNPs) from hESCs is described, for example, in U.S. Patent No. 7,531,354, which is incorporated herein by reference in its entirety. hNPs can express markers associated with the earliest pluripotent neural stem cells, including, for example, nestin, Musashi-1, SOX1, SOX2, and SOX3. In one case, hNPs express SOX1 (SOX1 + In other cases, the hNP expresses SOX2(SOX2+). In some other cases, the hNP expresses SOX3(SOX3 + In some specific cases, hNPs express at least one of nestin, Musashi-1, SOX1, SOX2, and SOX3. In other cases, hNPs express two or more of nestin, Musashi-1, SOX1, SOX2, and SOX3. In yet another case, hNPs express three or more of nestin, Musashi-1, SOX1, SOX2, and SOX3. In some cases, hNPs express at least one of nestin, Musashi-1, SOX1, SOX2, and SOX3, but do not express OCT4. In some other cases, hNPs express at least two of nestin, Musashi-1, SOX1, SOX2, and SOX3, but do not express OCT4. In yet another case, the hNP expresses at least three of nestin, Musashi-1, SOX1, SOX2, and SOX3, but does not express OCT4. In a specific case, the hNP expresses SOX1, SOX2, and SOX3, but does not express OCT4. Neural progenitor cells can be cultured with or without feeder cells. In some cases, neural progenitor cells produced according to the method presented in U.S. Patent No. 7,531,354 do not contain feeder cells and do not contain embryoid bodies.

[0075] In some cases, the disclosed extracellular viable cells (EVs) can be obtained by culturing differentiated nerve cells, such as glial cells, directly or indirectly induced from pluripotent stem cells, in cell culture medium under conditions that produce EVs and for a sufficient period of time, and then isolating the EVs from the culture medium. Types of glial cells include oligodendrocytes, astrocytes, ependymal cells, Schwann cells, microglia, and satellite cells. In one case, the differentiated nerve cells (e.g., glial cells) include astrocytes. Differentiated nerve cells that can be used include hN2® nerve cells (ARUNA BIOMEDICAL INC), NeuroNet® neurons, and AstroPro® astrocytes (ARUNA BIOMEDICAL INC).

[0076] Extracellular vesicles (EVs) can be isolated from cell culture media or tissue culture supernatant. EVs produced from cells can be collected from the culture medium by any preferred method. Typically, isolated populations of EVs can be prepared from cell culture or tissue supernatant by centrifugation, size exclusion columns, microfluidic devices, polymer precipitation, filtration, or a combination of these methods. For example, EVs can be prepared as described in U.S. Patent Application No. 20140356382 (which is incorporated herein by reference in its entirety). For example, EVs can be prepared by fractional centrifugation, i.e., slow (<2,000 g) centrifugation to pellet larger particles followed by high (>100,000 g) centrifugation to pellet the EVs, size filtration using a suitable filter (e.g., a 0.22 μm filter), gradient ultracentrifugation (e.g., using a sucrose gradient), or a combination of these methods.

[0077] In some embodiments, the EV-producing NP cells and / or nerve cells disclosed herein are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or about 1, 2, 3, 4, 5, or 6 months, depending on the cells and their EV-producing capacity. The EV-producing cells may be cultured in a suitable medium and grown under conditions readily determined by those skilled in the art. Cell culture conditions may vary depending on the cell type, and the examples shown below illustrate suitable media and conditions. For example, CMRL 1066 medium (Invitrogen) containing fetal bovine serum (e.g., 10%) and optionally supplemented with glutamine or a glutamine-containing mixture and antibiotics may be used. In some embodiments, cells can be grown attached to a surface; for example, they can be grown as a monolayer to a multilayer on the surface (without feeder cells) and may be grown to 30, 40, 50, 60, 70, 80, 90, 95, or 100% confluence. In other embodiments, cells can be grown as cell aggregates in suspension culture.

[0078] Cell growth media are well known in the art and include at least minimal essential media plus one or more optional components (e.g., growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin (if indicated and not excluded), activin A, transferrin, beta-mercaptoethanol, and other agents well known in the art and other agents as otherwise described herein). Preferred media are low-protein, serum-free growth media that support nerve cells. The growth factors used may be fibroblast growth factor 2 (FGF2) alone or preferably in combination with leukemia inhibitor (LIF). Depending on the NPs or nerve cells being grown in the growth medium, the inclusion of LIF is preferred but not required. Examples of culture media include basal cell media that may contain serum, for example, about 0.1% to 20% (preferably about 2% to 10%) of fetal bovine serum, or, in the case of standardized media, do not contain fetal bovine serum and KSR, but optionally contain bovine serum albumin (about 1% to 5%, preferably about 2%). In some cases, the media are standardized, serum-free, and have a low protein content. In other cases, the media are Aruna media and supplements, which allow nerve cultures to maintain a stable karyotype over multiple passages without the need for feeder cells, making them an excellent choice for a wide variety of research applications, including early-stage drug discovery. The components of the growth medium depend on the type of nerve cells being grown, all of which are well known in the art. In one case, AB2® Neural A Cell Culture Media Kit is used, which contains AB2® Basal Neural Medium and ANS® Neural Medium Supplement. In specific cases, the media and supplements described above are specifically manipulated for diversity to meet all neuronal cell culture needs. AB2® Basal Neural Medium and ANS® Neural Medium Supplement can be used as the basis for specialized media to direct the differentiation of hNP1® strains towards various neuronal phenotypes.Each lot of culture medium and supplement is pre-qualified for use by testing for cell growth, sterility, pH, osmotic pressure, and endotoxins.

[0079] Other agents that may be optionally added to the culture medium, depending on the cell type being grown in the medium, include, for example, nicotinamide, members of the TGF-β family (including TGF-β1, 2, and 3), activin A, nodal, bone morphogen proteins (BMP2-7), serum albumin, members of the fibroblast growth factor (FGF) family, platelet-derived growth factors-AA and -BB, platelet-rich plasma, insulin growth factor (IGF-I, II, LR-IGF), growth and differentiation factors (GDF-5, -6, -8, -10, 11), glucagon-like peptides-I and II (GLP-I and II), GLP-1 and GLP-2 mimetodes, exendin-4, parathyroid hormone, insulin, progesterone, aprotinin, hydrocortisone, ethanolamine, and epidermal growth factor (EGF). Examples include gastrin I and II, copper chelating agents (e.g., triethylenepentamine), forskolin, sodium butyrate, beta-cerulin, ITS, noggin, neurite growth factor, nodal, valproic acid, trichostatin A, sodium butyrate, hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco, CA), steroid alkaloids (e.g., cyclopamine (EMD, CA)), keratinocyte growth factor (KGF), Dickkopf protein family, bovine pituitary extract, islet neogenesis-associated protein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors, Notch pathway inhibitors, sonic hedgehog inhibitors, heregulin, or any one or more of these combinations. Each of these components, if present, is present in an effective amount.

[0080] In some cases, a suitable culture medium can be prepared from the following components, for example, Dulbecco's Modified Eagle Medium (DMEM), Gibco #11965-092, Knockout Dulbecco's Modified Eagle Medium (KO DMEM), Gibco #10829-018, Ham F12 / 50% DMEM basic medium, 200 mM L-glutamine, Gibco #15039-027, non-essential amino acid solution, Gibco 11140-050, β-mercaptoethanol, Sigma #M7522, human recombinant basic fibroblast growth factor (bFGF), Gibco #13256-029, etc.

[0081] Cell media are commercially available and can be supplemented with commercially available components, including specified heterogeneous component-free components, such as those available from Invitrogen Corp. (GIBCO), Cell Applications, Inc., Biological Industries, Beth HaEmek, Israel, and Calbiochem. Those skilled in the art can readily modify the cell media to produce any one or more target cells in accordance with this disclosure.

[0082] The disclosed EV-producing cells can be cultured on a layer of feeder cells that support the cells in various ways. Approaches for culturing cells on a layer of feeder cells are well known in the art. Cells can grow on a cell support or matrix as an adhesive monolayer or as cell aggregates in suspension. In some cases, the use of a cell support may be preferred depending on the cells used to produce EVs. When used, the cell support preferably contains at least one substrate protein. Examples of substrate proteins include extracellular matrix proteins (which are proteins found in the extracellular matrix, e.g., laminin, tenascin, thrombospongin, and mixtures thereof), which exhibit growth-promoting activity and contain domains homologous to epidermal growth factor (EGF). Other substrate proteins that may be used include, for example, collagen, fibronectin, vibronectin, polylysine, polyornithine, and mixtures thereof. Furthermore, gels and other substances (e.g., methylcellulose of other gels containing one or more of these embryonic stem cell differentiation proteins at effective concentrations) may also be used. Examples of differentiated proteins or materials containing these differentiated proteins include, for example, recombinant laminin, BD Cell-Tak® Cell and Tissue Adhesive, BD® FIBROGEN Human Recombinant Collagen I, BD® FIBROGEN Human Recombinant Collagen III, BD Matrigel® Basement Membrane Matrix, BD Matrigel® Basement Membrane Matrix High Concentration (HC), BD® PuraMatrix® Peptide Hydrogel, Collagen I, Collagen I High Concentration (HC), Collagen II (bovine), Collagen III, Collagen IV, Collagen V, and Collagen VI.

[0083] Alternatively, these cells may be cultured in a culture system that does not contain feeder cells, or is essentially feeder cell-free, but nevertheless supports cell proliferation for EV production. Cell growth in feeder-free culture can be supported using a medium conditioned by pre-culturing with another cell type. Alternatively, growth of EV-producing cells in non-differentiated feeder-free culture can be supported using a chemically defined medium. These approaches are well known in the art. In certain embodiments, cells are grown in feeder cell-free medium.

[0084] EVs can be collected at various time intervals (for example, at intervals of approximately 1, 2, 4, 6, 8, or 3, 6, 9, 12 days or longer, depending on the rate of EV production). Exemplary EV yields, as otherwise described herein, range from at least about 1 ng EV / 1 million cells to at least about 10.0 μg EV / 1 million cells (e.g., about 10 ng EV / 1 million cells, about 50 ng EV / 1 million cells, about 100 ng EV / 1 million cells, about 500 ng EV / 1 million cells, about 750 ng EV / 1 million cells, about 800 ng EV / 1 million cells, about 900 ng EV / 1 million cells, about 1.0 μg EV / 1 million cells, about 1.5 μg EV / 1 million cells, about 2.0 μg EV / 1 million cells, about 2.5 μg EV / 1 million cells, e.g., about 3.0 μg EV / 1 million cells, about 5.0 μg) during a period of about 24 hours to 7 days of culture of proliferating and non-proliferating nerve cells. It can be in the range of EV / 1 million cells.

[0085] In many cases, extracellular viable cells (EVs) are collected and harvested by ultracentrifugation, fractional centrifugation, or any combination thereof, and pelletized EVs are collected. Optionally, the collected pelletized EVs are washed with a suitable medium. For example, EV preparations can be prepared from cell cultures or tissue supernatants by centrifugation, filtration, or a combination thereof. In some embodiments, EVs can be prepared by fractional centrifugation, i.e., slow (<20,000 g) centrifugation to pellet larger particles, followed by high (>100,000 g) centrifugation to pellet the EVs, size filtration using a suitable filter (e.g., a 0.22 μm filter), gradient ultracentrifugation (e.g., using a sucrose gradient), or a combination thereof. EVs can be purified by fractional centrifugation, microfiltration and ultrafiltration, polymer precipitation, microfluidic separation, immunocapture, and size exclusion chromatography. These and / or related methods for isolating and purifying extracellular vehicles (EVs) are described by Thery et al. (Current Protocols in Cell Biology 3.22.1-3.22.29 (2006), copyright 2006 by John Wiley & Sons, Inc.), Sokolova et al. (Colloids and Surfaces B Biointerfaces 87:146-150 (2011)), Wiklander et al. (J Extracellular Vesicles 4:26316 (2015)), and Boing et al. (J Extracellular Vesicles 3:23430 (2014)). Other separation methods, such as electric field, radiofrequency, and acoustic methods, may be developed.

[0086] Methods for producing synthetic vesicles, such as synthetic exosomes, are known in the art. Such methods may be used to produce synthetic vesicles suitable for use in the compositions and methods provided herein. It should be noted that the contents of EVs, i.e., EVs from which the lipid bilayer has been removed or eliminated, and the resulting contents may also be used to manipulate artificial EVs.

[0087] (iii) Cargo delivered to the CNS In some embodiments, EVs comprising a cargo, e.g., one or more therapeutic agents, for delivery to the CNS across an intact blood-brain barrier are provided herein. In some embodiments, the EVs are derived from nerve cells (e.g., neural progenitor cells or neural stem cells). In some embodiments, the EVs are derived from untransformed human neural progenitor cells. In some embodiments, the cargo is an exogenous cargo that can be introduced into the EV by recombinant expression of the cargo in the cells from which the EVs are derived, or that can be introduced into the EV after the EVs have been isolated from the cells. In some embodiments, the exogenous cargo may include one or more therapeutic agents. Substantially any molecular agent that can be used for the therapeutic management or treatment of a disease and / or disorder can be loaded into the EV. Suitable therapeutic agents for delivery via EV include a wide variety of activators, including (i) small molecule therapeutic agents synthesized by chemical synthesis, (ii) naturally occurring compounds that can be obtained, for example, by purification from natural sources, (iii) various types of nucleic acid-based compounds, such as oligonucleotides (siRNA, splice-switching RNA, CRISPR guide chains, short hairpin RNA (shRNA), antisense oligonucleotides, antisense RNA, microRNA (microRNA, miRNA), etc.), polynucleotides (mRNA, plasmids, cDNA, DNA, etc.), and nucleic acids including chemically synthesized and / or chemically modified nucleotides (2'-O-Me, 2'-O-allyl, 2'-O-M0E, 2'-F, 2'-CE, 2'-EA, 2'-FANA, LNA, CLNA, ENA, PNA, phosphorothioates, tricyclo-DNA, etc.), and (iv) any type of peptides and polypeptides (i.e., proteins or antibodies), including those obtained by peptide synthesis or recombinant protein production.

[0088] Therapeutic agents are essentially drugs in all areas that are pharmaceutically and / or pharmacologically and / or diagnostically relevant, such as anti-inflammatory agents, anti-fibrotic agents, anticancer agents, cell proliferation inhibitors, tyrosine kinase inhibitors, statins, NSAIDs, antibiotics, antifungal agents, antibacterial agents, anti-inflammatory agents, anti-fibrotic agents, antihypertensive agents, aromatase or esterase inhibitors, anticholinergic agents, SSRIs, BKT inhibitors, PPAR agonists, HER inhibitors, AKT inhibitors, BCR-ABL inhibitors, signaling inhibitors, angiogenesis inhibitors, synthase inhibitors, ALK inhibitors, BR These can be obtained from AF inhibitors, MEK inhibitors, PI3K inhibitors, neprilysin inhibitors, beta-2 agonists, CRTH2 antagonists, FXR agonists, BACE inhibitors, sphingosine-1-phosphate receptor modulators, MAPK inhibitors, Hedgehog signaling inhibitors, MDM2 antagonists, LSD1 inhibitors, lactamase inhibitors, TLR agonists, TLR antagonists, IDO inhibitors, ERK inhibitors, Chkl inhibitors, splicing modulators, DNA or RNA interfering substances, etc. Other non-limiting pharmacological agents according to the present invention include, in particular, everolimus, trabectedin, Abraxane, pazopanib, enzastaurin, vandetanib, FLT-3 inhibitors, VEGFR inhibitors, EGFR TK inhibitors, Aurora kinase inhibitors, PIK-1 modulators, Bel-2 inhibitors, HD AC inhibitors, c-MET inhibitors, PARP inhibitors, Cdk inhibitors, EGFR TK inhibitors, IGFR-TK inhibitors, anti-HGF antibodies, PI3 kinase inhibitors, AKT inhibitors, JAK / STAT inhibitors, checkpoint-1 or 2 inhibitors, adhesion plaque kinase inhibitors, MAP kinase (MEK) inhibitors, pemetrexed, erlotinib, dasatinib, nilotinib, decatanib, panitumumab, amrubicin, olegovomab, noratrexide, batablin, ofatumumab, zanorimab Bu, edotecarin, tetrandrin, lubitecan, tesmilifen, oblimersen, tisilimmumab, ipilimumab, gossypol, silenditide, gimatecan, lucanton, neuradziab, vitespan, talampanel, atrasentan, romidepsin, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, 5'-deoxy-5-fluorouridine,Vincristine, temozolonide, sericiclib, capecitabine, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, batalanib, goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, erlotinib, lapatanib, canertinib, ronafanib, tipifarnib, amifostine, suberoylanalide hydroxamic acid, valproic acid, trichostatin Sorafenib, Amsacrin, Anagrelide, Bleomycin, Buserelin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clodronate, Cyproterone, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Diethylstilbestrol, Epirubicin, Fludarabine, Fludrocortisone, Fluoxymesterone, Flutamide, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Leuprolide, Levamizole, Lomustine, Mechloretamine, Melphalan, 6-Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Niltamide, Octreotide, Oxaliplatin, Pamidronate, Pentostatin, Plicamycin, Porfimer, Procarbazine, Larcitrexed, Rituximab, Streptozocin, Teniposide, Testosterone, Thalidomide, Thioguanine, Thiotepa, Tretinoin, Vindesine, 13-Cis-Retinoic Acid, Phenylalanine Mustard, Uracil Mustard, Estramustine, Altretamine, Phloxuridine, 5-Deoxyuridine, Cytosine Arabinoside, 6-Mecaptopurine, Deoxycoformycin, Calcitriol, Barrubicin, Mithramycin, Vinblastine, Vinorelbine, Topotecan, Lazoxin, Marimast, COL-3, Neovastat, Squalamine, Endostatin, Vitaxin, Doroxifen, Idoxifen, Spironolactone, Finasteride, Cymutidine, Trastuzumab, Denileukin Difutitox, Gefitinib, BortezimibPaclitaxel, Cremohol-free paclitaxel, Docetaxel, Epitiron B, Droloxifen, 4-Hydroxytamoxifen, Pipendoxifen, Alzoxifen, Fulvestrant, Acorbifen, Lasofoxifen, Idoxifen, Topotecan, Rapamycin, Temsirolimus, Zolendronate, Prednisone, Lenalidomide, Gemtuzumab, Hydrocortisone, Dexrazoxane, Alemtuzumab, All-trans Retinoic Acid, Ketoconazole, Megestrol, Immunoglobulin, Nitrogen Mustard, Methylprednisolone, Ibritumomab tiuxetan Examples include tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium-89, casopitant, netsupitant, NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, drasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa, and darbepoetin alfa, as well as efavirinz. Furthermore, therapeutic agents also include nucleic acid-based drugs comprising naturally derived compounds that can be obtained, for example, through purification from natural sources, any kind of nucleic acid-based compounds, such as oligonucleotides including siRNA, splice-switching RNA, CRISPR guide chains, short hairpin RNA, antisense oligonucleotides, mRNA, in particular chemically synthesized and / or chemically modified nucleotides such as 2'-O-Me, 2'-O-allyl, 2'-O-M0E, 2'-F, 2'-CE, 2'-EA, 2'-FANA, LNA, CLNA, ENA, PNA, phosphorothioates, tricyclo-DNA, etc. Furthermore, peptides and polypeptides, as well as peptides and / or proteins that can be obtained through peptide synthesis,Peptides and proteins that can be obtained through recombinant protein production are also intended as therapeutic agents suitable for use in the compositions and methods of this disclosure.

[0089] In some embodiments, the EV comprises one or more inhibitory nucleic acids. For example, in some embodiments, the EV may include one or more inhibitory nucleic acids selected from short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide (ASO), and double-strand RNA (dsRNA).

[0090] In some embodiments, the EV comprises one or more therapeutic antibodies or their antigen-binding moieties, each containing an antibody fragment selected from the group consisting of Fab, F(ab')2, scFv, tandem scFv, diabody, minibody, and single-domain antibodies. In some embodiments, the antibody or its antigen-binding moiety is a humanized antibody or its antigen-binding moiety. In some embodiments, the antibody or its antigen-binding moiety is a fully human antibody or its antigen-binding moiety.

[0091] In some embodiments, EV comprises one or more neurotrophic agents. EV may include leukemia inhibitor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor-binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factor (NT3), and a tissue inhibitor of metalloproteinases. The drug may contain one or more agents selected from metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL-1-α, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factors beta (TGFβ-1), and TGFβ-3.

[0092] The aforementioned therapeutic agents and other cargoes can be loaded into the EV using methods recognized in the art. Such methods include, but are not limited to, lipofection, transfection, electroporation, click chemistry, conjugation to a hydrophobic moiety, or recombinant expression of the therapeutic agent as a fusion protein with an exosome sorting domain, such as the exosome sorting domain of CD9, CD63, CD81, or Lamp2b, or any further exosome sorting domains described herein (in the case of peptide or protein-based therapeutic agents). Peptide or protein therapeutic agents can also be recombinantly expressed as fusion proteins with exosome proteins (or their exosome sorting domains) typically found in the EV lumen, such as ALIX or syntenin. In other embodiments, therapeutic agents can be loaded into the EV by coupling the drug to a cell-permeable peptide, such as transportan, penetratin, CADY-1, or VP22. Further cell-permeable peptides are described, for example, in U.S. Patent Publication No. 20109 / 0388347(A1), the entirety of which is incorporated herein by reference.

[0093] Any of the aforementioned drugs, or combinations thereof, can be loaded into an EV as described herein for delivery, for example, across the blood-brain barrier to the brain and / or CNS.

[0094] III. Inhibition of necroptosis and / or reduction of inflammation A method is provided for inhibiting necroptosis in cells and / or reducing inflammation. The method involves contacting cells with a therapeutically effective amount of a composition comprising nerve vesicles (EVs), such as neural EVs and neural progenitor EVs, thereby inhibiting necroptosis and / or reducing inflammation.

[0095] As used herein, “necroptosis” refers to a programmed necrotizing cell death pathway. Receptor-interacting protein kinase 1 (RIPK1) modulates necroptosis by promoting the sequential activation of two downstream targets, receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL). Application of Nec-1, a highly specific inhibitor of RIPK1 kinase activity, blocks necroptosis and inflammation in vitro and in vivo (Ito et al. 2016 Science 353(6299):603-608). While we do not wish to be bound by theory, necroptosis may play a role in the pathogenesis of neurodegenerative diseases, including ALS, but further preclinical and clinical research is needed for confirmation. Several studies have detected strong activation of RIPK1, RIPK3, and MLKL proteins in preclinical and clinical studies of ALS, but other studies have not shown involvement of the necroptotic pathway in ALS. Studies demonstrating activation of the necroptosis pathway in ALS are based on consistent results evaluating all molecular stages of this cascade, namely RIPK1, p-RIPK1, RIPK3, MLKL, p-MLKL, or p-RIPK3, and tumor necrosis factor (TNF)-α, including in situ experiments selectively focusing on the cells of greatest interest, namely motor neurons in the anterior horn of the spinal cord.

[0096] In some embodiments, p-RIPK1 levels are reduced in cells contacted with a therapeutically effective amount of EV-containing composition compared to control cells.

[0097] In some embodiments, the cells are present in the subject. In some embodiments, the subject has amyotrophic lateral sclerosis (ALS), and compared to a control subject, the subject shows improvement in neurological function, motor function, and / or one or more signs or symptoms associated with ALS, and / or extended survival. The control subject may be a subject that does not contain cells that have been contacted with a therapeutically effective amount of the composition containing EV.

[0098] In some embodiments, inflammation is reduced in cells exposed to a therapeutically effective amount of the EV-containing composition compared to control cells. Control cells may be cells not exposed to a therapeutically effective amount of the EV-containing composition. For example, compared to control cells, cells exposed to the EV composition show an increased number of regulatory T cells, a decreased number of T helper cells, an increased number of M2 macrophages, suppressed inflammatory response in M1 macrophages, increased anti-inflammatory response in M2 macrophages, decreased levels of pro-inflammatory cytokines (e.g., IL-17), and / or increased levels of anti-inflammatory cytokines (e.g., IL-10).

[0099] IV. Formulation, Delivery, and Administration The EVs provided herein can be formulated in a pharmaceutical composition for delivery to a subject. In some embodiments of this disclosure, the subject requires delivery of EVs to the CNS. In some embodiments, the subject requires delivery of cargo loaded with EVs to the CNS. In some embodiments, the subject requires reduction of brain swelling, reduction of brain inflammation, or reduction of the volume of a brain lesion. In some embodiments, the subject has a neurological condition or symptoms associated with a neurological condition.

[0100] The pharmaceutical composition may contain a therapeutically effective amount of this EV (e.g., EV derived from nerve cells) and a pharmaceutically acceptable carrier. The formulations containing the disclosed EV may preferably be in the form of liquid, solid, semi-solid, or lyophilized powder, suitable for simple administration of precise doses, such as solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, and patches.

[0101] The pharmaceutical composition typically includes conventional pharmaceutical carriers and / or excipients, and may also include other drugs, carriers, adjuvants, additives, etc. The weight percentage ratio of EV to one or more excipients may be approximately 20:1 to 1:60, or approximately 15:1 to 1:45, or approximately 10:1 to 1:40, or approximately 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1 to approximately 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, preferably approximately 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, or 5:1. In some embodiments, the disclosed composition comprises a total EV of about 1 μg to about 1 g or more, about 500 μg to about 500 mg, about 1 mg to about 500 mg of total EV, about 5 mg to about 500 mg, about 10 mg to about 500 mg, about 25 mg to about 500 mg, about 50 mg to about 350 mg, about 75 mg to about 450 mg, about 50 mg to about 450 mg, or about 75 mg to about 325 mg, or about 100 mg to about 650 mg of total EV, and may optionally contain one or more suitable pharmaceutical carriers, additives, and / or excipients.

[0102] Injectable compositions for parenteral administration (e.g., intravenous, intramuscular, intranasal) typically contain EV and optionally additional components in a suitable IV solution, such as sterile physiological saline. The composition may also be formulated as a suspension in an aqueous emulsion.

[0103] Liquid compositions can be prepared by dissolving or dispersing a pharmaceutical composition containing EV and an optional pharmaceutical adjuvant in a carrier such as physiological saline, dextrose aqueous solution, glycerol, or ethanol to form a solution or suspension. For use in oral liquid preparations, compositions may be prepared as solutions, suspensions, emulsions, or syrups and supplied in either liquid form or dry form suitable for hydration in water or physiological saline. For intranasal or intrapulmonary administration, compositions may be provided as liquid compositions that can be sprayed into the nose, trachea, and / or lungs.

[0104] For oral administration, such excipients include pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc. If desired, the composition may also contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifiers, or buffers.

[0105] In some embodiments, the disclosed EV is contained in or on a biocompatible scaffold such as a hydrogel. In some embodiments, the disclosed EV is contained in or on a biocompatible scaffold containing collagen, fibrin, silk, agarose, alginate, hyaluronan, chitosan, biodegradable polyesters such as polylactic acid-coglycolic acid, polylactic acid, or polyglycolic acid, polyethylene glycol, polyvinylpyrrolidone, polyethersulfone, peptide-based biomaterials, glycosaminoglycans, fibronectin, laminin, or any combination thereof.

[0106] When a composition is used in the form of a solid preparation for oral administration, the preparation may be a tablet, granule, powder, capsule, etc. In tablet formulations, the composition is typically formulated with excipients such as sugars or cellulose preparations, binders such as starch paste or methylcellulose, fillers, disintegrants, and other additives commonly used in the manufacture of pharmaceutical preparations.

[0107] Methods for preparing such dosage forms are known or obvious to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co. 1985). The composition to be administered contains a certain amount of a selected compound in a therapeutically effective dose for therapeutic use in a biological system including a patient or subject, in accordance with the present invention.

[0108] Intravenous formulations may contain the EVs described herein, an isotonic medium, and one or more substances that prevent aggregation of the EVs. An exemplary intravenous fluid formulation may contain physiological saline (e.g., normal saline, NS; about 0.9% w / v NaCl, about 300 mOsm / L) and / or 4% dextrose in 0.18% saline, and optionally 1%, 2%, or 3% human serum albumin. In addition, the EVs may be broken down to obtain contents and contents for use in compositions according to the present invention.

[0109] In exemplary embodiments, the formulation of the present invention may contain an intravenous medium with an EV / mL of about 50 ng, which includes an intravenous medium with an EV / mL of about 100 ng to 1.0 μg, which includes an EV / mL of 1.0 μg to 100 μg or more.

[0110] In some embodiments, the intravenous formulation may contain an intravenous medium with an EV of approximately 0.1 to 20.0 μg or more, or an intravenous medium with an EV of approximately 20.0 μg or more.

[0111] In some embodiments, the pharmaceutical composition is a dosage form comprising at least 25 mg of extravalent protein (EV), at least 300 mg of EV, about 350 mg to 1 g of EV, or about 1 g (1,000 mg) or more of EV, alone or in combination with at least one additional bioactive agent in a therapeutically effective amount that may be useful for treating neurological disorders and injuries, including ALS. In some embodiments, the pharmaceutical composition comprises about 10 mg to about 750 mg of EV, about 25 mg to about 650 mg, or about 30 mg to about 500 mg, or about 35 mg to about 450 mg, most often about 50 mg to about 500 mg of EV.

[0112] Compositions containing EV provided herein include the auricle (ear), buccal, conjunctiva, skin, teeth, electroosmosis, intracervix, sinus, trachea, intestine, extraamniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intracystic, intracardiac, intracartilage, intracavitary, intracavitary, intracorneal, intracoronal (tooth), intracoronal, intracavernosum, intradermal, intraductal, intraduodenal, intraepidermal, intraesophageal, intragastric, intrastomal, intrastomal, intralesional, intrapulmonary, intracavitary, intralymphatic, intraocular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, nasal cavity, intracystic bursa, intratendinous, intratesticular, intrathoracic, intratubular, intratumoral, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous infusion, and ventricle. Administration can be performed distal to the CNS by any preferred route, including but not limited to internal, intravesical, intravitreal, iontophoresis, irrigation, larynx, nasal, nasogastric, occlusive dressing, ocular, oral, oropharyngeal, parenteral, percutaneous, periarticular, perineurial (peripheral), periodontal, rectal, respiratory (inhalation), retroocular, soft tissue, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureter, urethra, and / or vaginal administration, and / or any combination of the above routes of administration, which typically depends on the disease, sign, or symptom being treated. In some embodiments, the EV composition is administered directly to the CNS, for example, by intrathecal administration or direct injection into the brain.

[0113] The administration of EVs described herein is suitable for delivering EVs to the brain or CNS, and in some embodiments, across the blood-brain barrier. In some embodiments, the administration is performed at a site of affected and / or dysfunctional tissue (e.g., the brain). In some embodiments, the administration site is distal to the site of affected and / or dysfunctional tissue (e.g., intravenous or intranasal delivery). In one embodiment, the EV composition described herein is administered parenterally to the subject. In one embodiment, the EV composition described herein is administered intravenously to the subject. In another embodiment, the EV composition described herein is administered intranasally to the subject. In some embodiments, the subject has an intact or largely intact blood-brain barrier.

[0114] Injectable compositions for parenteral administration (e.g., intravenous, intramuscular, or intranasal) may contain the EV of this disclosure and optionally additional components in a suitable iv solution such as sterile physiological saline. In other embodiments, the composition is formulated as a suspension in an aqueous emulsion.

[0115] The pharmaceutical compositions containing EVs described herein may be administered to a subject as monotherapy (single agent) or as combination therapy, in which the pharmaceutical composition containing EVs is administered to the subject in combination with one or more additional agents. The pharmaceutical compositions containing EVs and one or more additional agents of this disclosure may be administered to the subject simultaneously, sequentially, or over time.

[0116] The therapeutically effective dose of a pharmaceutical composition containing the EV of this disclosure is sufficient to treat or restore one or more symptoms of the condition being treated (e.g., neurological disorders, e.g., Alzheimer's disease, Huntington's disease, Parkinson's disease, or neurological cancers, e.g., glioblastoma), but not exceeding a dose that could cause significant adverse effects. The therapeutically effective dose may depend on many factors, including the nature of the condition being treated and the specific individual being treated.

[0117] In certain embodiments, EVs of the Disclosure, e.g., neural EVs, neural progenitor EVs, EVs derived from non-transformed neural progenitor cells, or pharmaceutical compositions comprising EVs of the Disclosure, have superior ability to cross the blood-brain barrier and, when administered distal to the CNS, are delivered more efficiently to the target CNS compared to a reference molecule, e.g., blood-derived exosomes, achieving concentrations in the CNS, brain, brain-blood ratio, or plasma-blood ratio at least 10%, 15% to 1000%, or more than 1000% higher than the concentrations in the CNS, brain, brain-blood ratio, or plasma-blood ratio of the comparison molecule, e.g., blood-derived exosomes, non-neuronal EVs.

[0118] In some embodiments, delivery of the EV of the present invention, or a pharmaceutical composition containing the EV of the present invention, to the CNS results in a reduction of necroptosis or inflammation in the CNS. In some embodiments, delivery of the EV of the present invention, or a pharmaceutical composition containing the EV of the present invention, to the CNS results in an improvement of neurological conditions or symptoms associated with ALS in the subject (e.g., muscle weakness, difficulty walking, stumbling, falls, decreased grip strength, clumsiness of the hands, slurred speech, difficulty chewing, difficulty swallowing, and / or difficulty breathing). In some embodiments, delivery of the EV of the present invention, or a pharmaceutical composition containing the EV of the present invention, to the CNS results in an improvement in the subject's survival (e.g., prolonged survival). In some embodiments, any one of these effects in a subject brought about by the EV of the present invention, or a pharmaceutical composition containing the EV of the present invention (e.g., reduction of necroptosis or inflammation in the CNS, improvement of neurological conditions or symptoms associated with ALS, extension of survival) is at least 10% greater, 15% to 1000% greater, or more than 1000% greater than the effect brought about by a comparative molecule, e.g., a vehicle, blood-derived exosomes, non-neuronal EVs, or cargo administered without EVs.

[0119] V. Treatment of Amyotrophic Lateral Sclerosis (ALS) In some embodiments, this disclosure provides methods for treating subjects having amyotrophic lateral sclerosis (ALS) using EVs or compositions containing EVs as described herein. Any EV-containing composition or pharmaceutical composition described herein is suitable for use in any of the methods provided herein. In some embodiments, the EVs are derived from nerve cells, e.g., neural progenitor cells, neurons, or astrocytes. In other exemplary embodiments, the EVs are synthetically produced and contain one or more markers characteristic of neural EVs, e.g., one or more proteins or nucleic acids present in neural EVs but not present in EVs derived from mesenchymal stem cells. In other embodiments, the EVs contain one or more additional therapeutic agents, e.g., one or more of riluzole, edaravone, or sodium phenylbutyrate-taurulsodiol, or one or more drugs for reducing or controlling one or more signs or symptoms, including muscle spasms, constipation, fatigue, excessive oropharyngeal secretions, pain, depression, sleep disorders, mood disorders, urinary incontinence, or leg swelling.

[0120] In some embodiments, the present invention provides a method for treating amyotrophic lateral sclerosis (ALS) in a subject by administering to the subject a composition containing the EV described herein in an amount sufficient to treat the subject (e.g., curing, suppressing, restoring, delaying or preventing its progression, delaying or preventing its onset, or preventing its relapse or recurrence). An amount sufficient to treat the disease or disorder (e.g., ALS) is preferably an effective amount, such as a therapeutic effective amount provided herein.

[0121] As shown in Figure 8, EVs provided herein (e.g., neural EVs, neural progenitor cell-derived EVs) can treat ALS through several underlying mechanisms of action. While we do not wish to be bound by theory, EVs provided herein can inhibit pro-inflammatory cytokines (e.g., IL-1β, IL-6, TNFα), the NLRP3 pathway (e.g., NLRP3, caspase-1, IL-1β, IL-18), and / or necroptosis in cells or subjects.

[0122] EV compositions can be administered by any preferred formulation, route, and dosage to achieve the desired therapeutic effect. For example, EV compositions can be administered distally to the target central nervous system (CNS). In some embodiments, the composition is administered intravenously, intranasally, intraperitoneally, orally, or in any combination thereof. Alternatively or in addition, EV compositions can be administered directly to the CNS, such as by injection into the brain or intrathecal administration.

[0123] In some embodiments, the methods provided herein improve neurological function, motor function, and / or one or more signs or symptoms associated with ALS, and / or extend survival in subjects compared to a control subject. In some embodiments, one or more signs or symptoms include muscle weakness, difficulty walking, stumbling, falling, decreased grip strength, clumsiness of the hands, slurred speech, difficulty chewing, difficulty swallowing, and / or difficulty breathing.

[0124] In some embodiments, the levels or activity of neurofilament light chains (NFLs), chitinase-3-like protein 1 (CHI3L1), nucleotide-binding domain leucine-rich repeat-containing family pyrine domain-containing 3 (NLRP3), NLRP3 signaling pathway molecules, and / or pro-inflammatory cytokines are reduced in subjects by the methods provided herein. While we do not wish to be bound by theory, NFLs are a major byproduct of nerve axonal disruption, and NFL levels may indicate neurodegenerative activity in ALS. CHI3L1 is a marker of glial activation, and CHI3L1 levels may indicate neuroinflammatory status, disease severity, and ALS progression.

[0125] In some embodiments, the NLRP3 signaling pathway molecules include caspase-1, IL1-β, and / or IL-18. In some embodiments, the pro-inflammatory cytokines include TNFα and / or IL1-β.

[0126] The activity of the EVs described herein can be evaluated by methods known in the art. The amount of EV required for therapeutic use may vary depending not only on the specific cells from which the EVs are prepared, but also on the route of administration, the nature of the condition being treated, and the patient's age and condition, and is ultimately at the discretion of the attending physician or clinician. However, generally, the dose can range from about 0.01 mg / kg body weight to about 10 mg / kg body weight per day.

[0127] The change in symptoms as a result of treatment can be measured against any preferred control. For example, the change in symptoms can be measured against the frequency, severity, duration, or number of symptoms experienced by the same subject before the start of treatment. In other embodiments, the change in symptoms can be measured against the frequency, severity, duration, or number of symptoms experienced by a different subject or group of subjects with similar symptoms who have not received treatment, for example, a composition containing EV. In some embodiments, the degree of improvement is at least 5%, when determined against a preferred control.

[0128] In some embodiments, the composition containing EV is administered to the subject as a single dose. In some embodiments, the composition containing EV is administered in multiple doses. For example, in some embodiments, the composition may be administered once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every eight weeks, or once every twelve weeks.

[0129] In some embodiments, the EV compositions of this disclosure are administered in combination with a second therapy known to be effective in treating ALS or in preventing, alleviating, or treating one or more signs, symptoms, conditions, or complications associated with ALS. The EV compositions may be administered before, after, concurrently with, or in conjunction with the second therapy.

[0130] The second therapy may be an additional therapeutic agent for ALS, such as riluzole, edaravone, or sodium phenylbutyrate-taurulsodiol, or a drug to alleviate or control one or more signs or symptoms, including muscle spasms, constipation, fatigue, excessive oropharyngeal secretions, pain, depression, sleep disorders, mood disorders, urinary incontinence, and leg swelling. The EV composition and the additional therapeutic agent may be administered in combination within the same composition or as separate compositions. The second therapy may also be a non-pharmacological therapy, such as respiratory therapy, physical therapy, occupational therapy, speech therapy, nutritional therapy, and psychosocial therapy.

[0131] Other suitable modifications and adaptations of the methods of the present invention as described herein are obvious and will be readily apparent to those skilled in the art as being made using suitable equivalents without departing from the scope of the present invention or the embodiments disclosed herein. Although the present invention has been described in detail, it will be better understood by referring to the following examples, which are included solely for illustrative purposes and are not intended to limit the present invention. [Examples]

[0132] Example 1. Isolation of extracellular vesicles (EVs) The following examples describe the isolation of extracellular vesicles (EVs) for use in the compositions and methods described herein.

[0133] A purified population of extracellular viable cells (EVs) was obtained from neural progenitor cells according to the method provided in U.S. Patent Application Publication No. 2018 / 0327714(A1) (which is incorporated herein by reference in its entirety). Briefly, human neural progenitor cells were cultured, and the medium was collected from the confluent culture 24 hours after a medium change. The medium was filtered through a 0.22 μm filter unit and frozen at -20°C. Before purifying the EVs, the frozen medium was thawed overnight at 4°C. The EVs were purified from the filtered cell culture medium using tangential flow filtration (TFF) and chromatography.

[0134] Example 2. Neural EVs downregulate necroptosis markers in vitro. This example illustrates the effect of nerve extravasation (EV) on inhibiting necroptosis and inflammation in vitro.

[0135] Human microglial cell lines (HMC3) were pretreated for 2 hours with a vehicle or neural progenitor EV (neuronal EV) (medium and high doses) prepared in Example 1, then stimulated with TNF-α (10 ng / mL) for 4 hours, and p-RIPK1 was measured by Western blotting. 24 hours prior to treatment, 300,000 HMC3 cells (ATCC, CRL-3304) per well were seeded in a 6-well dish. Cells were then treated with a vehicle or neural EV (medium dose: 5 × 10⁶). 4 EV / cell, high dose: 1×10 5 Cells were treated with TNF-alpha (10 ng / mL, R&D Systems) for 4 hours in the presence of either EV / cell. After 4 hours, cells were lysed and proteins were quantified using a BCA assay. Cell lysates were run on a gel and stained for phospho-RIPK1 (ProteinTech, #28252-1-AP) and GAPDH (GeneTex, #GTX100118). The gel was quantified using ImageJ. Phospho-RIPK1 can be an indicator of the degree of necroptosis in cells (Chevin & Sebire 2021 Cell Death Discovery 7:79).

[0136] As shown in Figure 1, normalized immunoblot quantification (phospho-RIPK1 / GAPDH ratio) for GAPDH decreased in a dose-dependent manner in cells pretreated with EV, demonstrating the effect of EV in upregulating TNF-α-induced p-RIPK1 and downregulating necroptosis.

[0137] Example 3. Neural EVs delay disease progression and extend survival in an ALS mouse model. This example describes the effects of neuronal extravasation (EV) on slowing disease progression, restoring signs and symptoms, and improving survival in ALS mice.

[0138] B6SIL-Tg(SOD1 * The effect of neuronal extravasation (EV) on ALS progression was tested using G93A)1Gur / J mice. This transgenic model, which expresses large amounts of mutant superoxide dismutase 1 (SOD1), induces neurodegeneration of spinal motor neurons, resulting in progressive paralysis similar to that of ALS.

[0139] From 62 days of age, 40 B6SIL-Tg(SOD1) * G93A)1Gur / J male mice were subjected to the preparation of neural progenitor EVs (neuronal EVs) (2.7 × 10⁻¹⁰) in Example 1 during weeks 1-3. 11 Weekly intravenous (IV) administration of either particles / kg or vehicle (n=20 per group), and nerve EV (2×10) at weeks 4-9. 9 Mice received weekly intranasal (IN) administration of either total particles or vehicle (up to 9 doses in total). Disease progression was quantified using neurological deficit score (NDS) scoring and rotarod testing. From day 59, mouse body weight was measured and NDS was measured twice weekly. From day 90, mouse body weight was measured and NDS was checked daily until the humane endpoint. To quantify the decline in motor function, mice underwent rotarod testing weekly, starting in the same week as the NDS. This schedule was continued until the mice reached the humane endpoint or day 125-126, whichever came first. The mice were then euthanized and samples were collected for further analysis.

[0140] As shown in FIGS. 2-4, treatment with neural EVs demonstrated the effects of neural EVs in improving the NDS score (FIG. 2) and rotarod performance (FIG. 3), prolonging survival (FIG. 4), and delaying disease progression. Furthermore, treatment with neural EVs showed a significant decrease in serum neurofilament light chain levels on day 97 (FIG. 5), indicating a decrease in neurodegenerative activity. At the transcript level in the lumbar spinal cord, treatment with neural EVs significantly reduced the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα, FIG. 6E) and interleukin-1 beta (IL1-β, FIG. 6C). Neural EV treatment also resulted in a trend towards decreased levels of several targets associated with the NLRP3 inflammatory pathway, including NLRP3 (FIG. 6A), caspase 1 (FIG. 6B), IL-1β (FIG. 6C), and IL-18 (FIG. 6D).

[0141] In another experiment, starting at 69 days of age, 30 B6SJL-Tg(SOD1 * G93A)1Gur / J male mice were administered intranasally (IN) either neural progenitor EVs (neural EVs) prepared in Example 1 (4.2×10 11 particles / kg, 1.1×10 10 total particles; n = 20) or vehicle (EV formulation buffer, n = 10) five times a week (i.e., Monday-Friday) until they reached the humane endpoint. Serum samples were collected on day 80, and the protein level of chitinase-3-like protein 1 (CHI3L1), a marker of glial activation and ALS disease progression, was quantified by ELISA. As shown in FIG. 7, mice treated with neural EVs had significantly (p = 0.0002) lower levels of CHI3L1 compared to vehicle-treated mice, indicating that intranasally administered neural EVs delay glial cell activation and disease progression in the ALS mouse model.

[0142] In summary, these results indicate that neural EVs reduce inflammation (e.g., NLRP3 inflammasome signaling), delay disease progression, delay motor dysfunction, and increase in vivo survival time in a mouse model of ALS.

[0143] Built-in by reference All references, patents, pending patent applications, and publications cited throughout this application are expressly incorporated herein by reference in their entirety.

[0144] Equivalents Those skilled in the art can recognize or confirm many equivalents to the specific embodiments of the invention described herein by means of routine experiments. Such equivalents are intended to be covered by the following claims.

Claims

1. A method for treating amyotrophic lateral sclerosis (ALS) in a subject, comprising administering to the subject a therapeutically effective amount of a composition containing extracellular vesicles (EVs) derived from nerve cells, thereby treating ALS.

2. The method according to claim 1, wherein the composition is administered distally to the central nervous system (CNS) of the subject.

3. The method according to claim 2, wherein the composition is administered intravenously, intranasally, intraperitoneally, orally, or in any combination thereof.

4. The method according to any one of claims 1 to 3, wherein, compared to a control subject, one or more signs or symptoms related to neurological ability, motor ability, and / or ALS are improved, and / or survival is extended.

5. The method according to claim 4, wherein one or more of the signs or symptoms include muscle weakness, difficulty walking, stumbling, falling, decreased grip strength, clumsiness of the hands, slurred speech, difficulty chewing, difficulty swallowing, and / or difficulty breathing.

6. The method according to any one of claims 1 to 5, wherein the levels or activity of neurofilament light chains, chitinase-3-like protein 1 (CHI3L1), nucleotide-binding domain leucine-rich repeat-containing family pyrine domain-containing 3 (NLRP3), NLRP3 signaling pathway molecules, and / or pro-inflammatory cytokines are reduced in the subject.

7. The method according to claim 6, wherein the NLRP3 signaling pathway molecule comprises caspase-1, IL1-β, and / or IL-18, and / or the pro-inflammatory cytokine comprises TNLα and / or IL1-β.

8. A method for inhibiting necroptosis in cells, comprising contacting the cells with a composition comprising a therapeutically effective amount of extracellular vesicles (EVs) derived from nerve cells, thereby inhibiting necroptosis.

9. The method according to claim 8, wherein the level of phosphorylation receptor-interacting protein kinase 1 (p-RIPK1) is reduced in cells compared to control cells.

10. The method according to claim 8 or 9, wherein the cells are located within the target.

11. The method according to claim 10, wherein the subject has amyotrophic lateral sclerosis (ALS), and compared to a control subject, the subject shows improvement in neurological function, motor function, and / or one or more signs or symptoms associated with ALS, and / or survival is extended.

12. The method according to any one of claims 1 to 11, wherein the nerve cells are untransformed neural progenitor cells.

13. The method according to any one of claims 1 to 12, wherein the nerve cells are derived from human pluripotent stem cells, human induced pluripotent stem cells, or human embryonic stem cells.

14. The method according to any one of claims 1 to 13, wherein the EV comprises ecto-5'-nucleotidase (CD73).

15. The method according to claim 14, wherein the EV further comprises one or more of the following: melanoma-associated chondroitin sulfate proteoglycan (MCSP), pentraxin-3 (PTX3), angiopoietin-1, insulin-like growth factor-binding protein 2 (IGFB2), and macrophage colony-stimulating factor.

16. The method according to any one of claims 1 to 15, wherein the composition comprises about 1 mg to about 750 mg of EV.

17. The method according to any one of claims 1 to 16, wherein the EV comprises an exosome.

18. The method according to any one of claims 1 to 17, wherein the EV has a size of about 10 nm to about 10 μm.

19. The method according to claim 18, wherein the EV has a size of about 20 nm to about 250 nm.

20. The method according to any one of claims 1 to 19, wherein the EV further includes a cargo.

21. The method according to claim 20, wherein the cargo comprises one or more of nucleotides, peptides, proteins, antibodies, and small molecules.

22. The method according to claim 21, wherein the nucleotide is one or more of siRNA, antisense oligonucleotide, mRNA, plasmid, and cDNA.

Images