Cell-free allosome exosome formulations and methods of use thereof

Abstract

There is provided a method of treating or ameliorating a symptom of neuronal damage in a subject, the method comprising administering to the subject a therapeutically effective amount of an acellular allogeneic formulation (UCF or UCF-E) comprising exosome wherein the acellular allogeneic formulation is derived from mixed human umbilical cord blood.

Description

Technical Field

[0001] The present technology relates to a cell-free allogeneic exosome preparation for treating neuronal damage, in particular ischemic brain damage such as stroke or cerebrovascular accident (CVA). The present technology also relates to the use of a cell-free allogeneic exosome (extracellular vesicle (EV)) preparation for the manufacture of a drug for treating neuronal damage (such as stroke or CVA).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to Australian Provisional Patent Application No. 2022902095 filed on July 27, 2022, which is incorporated herein by reference in its entirety. Background Art

[0004] Globally, stroke is the second leading cause of death and the third leading cause of death and disability combined, expressed as disability-adjusted life years lost (DALYs). The global cost of stroke is estimated to exceed US$ 721 billion, or 0.66% of global GDP. From 1990 to 2019, the absolute number of stroke cases increased substantially (stroke incidence increased by 70%, stroke mortality increased by 43%, stroke prevalence increased by 102.0%, and DALYs increased by 143.0%).

[0005] A stroke is caused by a blockage or rupture of a cerebral artery that blocks blood flow to the brain, resulting in a lack of oxygen, which causes the sudden death of some brain (nerve) cells. The most common types of stroke are ischemic stroke and hemorrhagic stroke. In an ischemic stroke, the lack of oxygen flow to the brain causes apoptosis and necrosis of brain tissue cells, resulting in infarction. Similar to cardiovascular ischemia, cerebral ischemia can be caused by a variety of factors, such as blood clots, thrombosis, embolism, atherosclerotic plaques, or other blockages in the blood vessels. Hypercholesterolemia, hypertension, diabetes, and obesity are risk factors for ischemic stroke. A hemorrhagic (cerebral hemorrhage) stroke is caused by a weakened blood vessel (artery) that ruptures and bleeds into the surrounding brain. The blood then accumulates and compresses the surrounding brain tissue, causing the part of the body controlled by the damaged area to not function or work properly. There are two main types of hemorrhagic stroke. Intracerebral hemorrhage refers to bleeding inside the brain due to weakened blood vessels associated with an aneurysm, while arteriovenous malformations (AVMs) and subarachnoid hemorrhages refer to bleeding between the brain and the membrane covering the brain.

[0006] Regardless of the cause, a stroke attack results in the death of nerve cells, especially at the site of blockage or bleeding. In addition, the biochemical reactions that occur in the blood vessels after a stroke attack may lead to edema, hemorrhagic transformation, and further damage to the nerve tissue. The nerve damage and neuronal cell death caused by a stroke can cause physical and mental damage to the subject. Among other things, a stroke can cause problems with emotional control, consciousness, sensory perception, speech, hearing, vision, cognition, movement and mobility, and can lead to paralysis.

[0007] The current treatment for ischemic stroke, tissue plasminogen activator (tPA or PLAT), while effective, is only effective if administered within three hours of the ischemic event. Therefore, treatment with tPA remains insufficient for patients who cannot be diagnosed and treated within that time frame. tPA is also not indicated for patients with hemorrhagic stroke who require alternative interventional radiology or neurosurgery procedures (to stop bleeding and reduce brain pressure) as well as medications to reduce swelling, prevent seizures, and relieve pain. Given the limitations of tPA and the high mortality rate among stroke patients, it is clear that there is a clear, unmet need to reduce mortality, restore neurological function, and extend the current window of opportunity for treatment.

[0008] The present invention is based on the inventors' discovery that cell-free allogeneic exosome preparations derived from human umbilical cord plasma, such as human umbilical cord plasma containing exosomes (UCF) or exosomes only (UCF-E), can be used to treat stroke. SUMMARY OF THE INVENTION

[0010] In a first aspect, a method for treating or ameliorating symptoms of neuronal injury in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an exosome-containing cell-free allogeneic preparation (UCF or UCF-E) consisting of cell-free human umbilical cord plasma (UCF) or only exosomes from human umbilical cord plasma (UCF-E), wherein the exosome-containing cell-free allogeneic preparation (UCF or UCF-E) is derived from pooled human umbilical cord blood, wherein the neuronal injury is ischemic brain injury, hemorrhagic brain injury or traumatic brain injury, and wherein the UCF or UCF-E is first administered 3 to 90 days after the neuronal injury.

[0011] Ischemic brain injury or hemorrhagic brain injury can be a stroke.

[0012] In some embodiments, no immunosuppressants are administered.

[0013] In some embodiments, UCF or UCF-E is administered intrathecally.

[0014] The cell-free human umbilical cord plasma preparation (UCF) and the umbilical cord plasma exosome-only preparation (UCF-E) are derived from plasma characterized by levels of one or more of GM-CSF, HGF, MIP-1b (CCL4), and PDGF-bb that are at least 20% higher than in normal adult human serum.

[0015] The cell-free human umbilical cord plasma preparation (UCF) and the umbilical cord plasma exosome-only preparation (UCF-E) are derived from plasma characterized by IP-10 (CxCL10) levels that are at least 60% of the IP-10 levels in normal adult human serum.

[0016] The cell-free allogeneic umbilical cord preparations (UCF and UCF-E) containing exosomes may comprise exosomes having a mean, median or mode particle size of about 30-150 nm or about 75-95 nm.

[0017] In one embodiment, the cell-free allogeneic umbilical cord plasma (UCF) or umbilical cord exosome-only (UCF-E) preparation containing exosomes is first administered 3 to 30 days after neuronal injury, preferably 3 to 10 days after neuronal injury, and even more preferably 7 or 8 days after neuronal injury.

[0018] In some embodiments, the first administration of the exosome-containing cell-free allogeneic umbilical cord plasma (UCF) or umbilical cord exosomes-only (UCF-E) preparation is 7 to 10 days after neuronal injury.

[0019] In some embodiments, the acellular allogeneic UCF or UCF-E formulation is administered to the subject on a regular basis. For example, the acellular allogeneic UCF or UCF-E formulation can be administered weekly, every two weeks, every three weeks, monthly, or a combination thereof. Alternatively, the administration cycle can be daily, every 5 days, every 10 days, every 15 days, every 20 days, every 25 days, about every 30 days, or a combination thereof.

[0020] The method may comprise administering the acellular allogeneic UCF or UCF-E preparation a total of two, three, four, five, six, seven, eight, nine, or ten times.

[0021] The symptom may be selected from dystonia, aphasia, infarct size, inflammation, loss of white matter, loss of grey matter, apoptotic cells in the peri-infarct area, and CD16 / CD32 positive cells in the peri-infarct area.

[0022] Administration of the acellular allogeneic UCF or UCF-E preparation can reduce any one or more of dystonia, aphasia, infarct volume, systemic inflammation, inflammation in the infarct or peri-infarct area, gray matter and / or white matter loss in a subject.

[0023] Administration of acellular allogeneic UCF or UCF-E preparations can reduce the number of apoptotic cells in the peri-infarct area of ​​a subject and / or the number of CD16 / CD32 positive cells in the peri-infarct area of ​​a subject.

[0024] The method can also include administering the acellular allogeneic UCF or UCF-E preparation with an additional therapeutic agent (e.g., an anti-inflammatory agent, a steroid, dipyridamole, astaxanthin, dabigatran, losartan, nimodipine, policosanol, rivaroxaban, or ticlopidine).

[0025] In a second aspect, a method for improving an mRS score or a NIHSS score in a subject with ischemic brain injury is provided, the method comprising administering to the subject a therapeutically effective amount of a cell-free allogeneic UCF or UCF-E preparation, wherein the cell-free allogeneic UCF and UCF-E preparations are derived from pooled human umbilical cord blood, and wherein the UCF or UCF-E is first administered within 3 to 90 days after the ischemic brain injury.

[0026] In one embodiment, no immunosuppressants are administered.

[0027] The cell-free allogeneic human umbilical cord plasma used to isolate UCF or UCF-E is characterized by an IP-10 (CxCL10) level that is at least 60% of the IP-10 level in normal adult human serum.

[0028] The cell-free allogeneic exosome preparation may include exosomes having a mean, median, or mode particle size of about 30-150 nm.

[0029] In one embodiment, the first administration of exosome-containing cell-free allogeneic plasma (UCF) or exosome-only preparation (UCF-E) is 3 to 30 days after neuronal injury, preferably 3 to 10 days after neuronal injury, and even more preferably 7 or 8 days after neuronal injury.

[0030] In some embodiments, the first administration of acellular allogeneic UCF or UCF-E is 7 to 10 days after the ischemic brain injury.

[0031] In some embodiments, a cell-free allogeneic plasma (UCF) or exosome-only (UCF-E) preparation containing exosomes is administered to a subject on a regular basis. For example, a cell-free allogeneic exosome preparation can be administered weekly, every two weeks, every three weeks, monthly, or a combination thereof. Alternatively, the administration cycle can be daily, every 5 days, every 10 days, every 15 days, every 20 days, every 25 days, about every 30 days, or a combination thereof.

[0032] The method may comprise administering a cell-free allogeneic plasma (UCF) or exosome-only (UCF-E) preparation containing exosomes two, three, four, five, six, seven, eight, nine, or ten times.

[0033] In some embodiments, UCF or UCF-E is derived from blood from a single umbilical cord, or from a mixture of blood from multiple umbilical cords. The mixture can be from umbilical cords of the same blood type or from umbilical cords of different blood types.

[0034] In a third aspect, provided is a use of a cell-free allogeneic plasma preparation containing exosomes (UCF) or exosomes only (UCF-E) derived from pooled human umbilical cord blood in the manufacture of a medicament for treating or ameliorating symptoms of neuronal injury, wherein the neuronal injury is ischemic brain injury, hemorrhagic brain injury or traumatic brain injury, and wherein the medicament is first administered 3 to 90 days after the neuronal injury.

[0035] In a fourth aspect, provided is a use of a cell-free allogeneic plasma preparation (UCF or UCF-E) containing exosomes derived from pooled human umbilical cord blood in the manufacture of a medicament for improving the mRS score or NIHSS score of a subject with ischemic brain injury, wherein the medicament is first administered 3 to 90 days after the ischemic brain injury.

[0036] definition

[0037] Throughout the specification, unless the context clearly requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps; but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0038] Throughout the specification, the term "consisting of" means consisting of only.

[0039] The term "consisting essentially of" means including the recited elements, integers or steps, but may also include other elements, integers or steps that do not substantially change or contribute to the practice of the invention.

[0040] Any discussion of documents, acts, materials, devices, articles and the like included in this specification is intended solely to provide background to the present technology. This should not be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present technology as it existed before the priority date of each claim of this specification.

[0041] Unless the context requires otherwise or clearly stated to the contrary, integers, steps or elements of the present technology recited herein as a singular integer, step or element expressly encompass both the singular and the plural form of the recited integer, step or element.

[0042] In the context of this specification, the term "a" is used to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, reference to "an element" means one element or more than one element.

[0043] In the context of this specification, the term "about" means that the referenced number or value should not be regarded as an absolute number or value, but includes a range of variation above and below the number or value, which is consistent with the understanding of those skilled in the art in accordance with the art, including within the typical error range or instrument limit. In other words, the use of the term "about" should be understood to refer to a range or approximate value that a person skilled in the art considers to be equivalent to the stated value in the context of achieving the same function or result.

[0044] The terms "treating", "treatment" and "therapy" as used herein refer to curative therapy, prophylactic therapy, palliative therapy and preventive therapy. Therefore, in the context of the present disclosure, the term "treatment" encompasses curing, improving or alleviating the severity of ischemic brain injury or one or more of its associated symptoms.

[0045] The term "therapeutically effective amount" or "pharmacologically effective amount" or "effective amount" refers to the amount of an agent sufficient to produce the desired therapeutic or pharmacological effect in the subject being treated. These terms are synonymous and are intended to define the amount of each agent to achieve the goal of improving disease severity and / or frequency of onset relative to treatment with each agent alone, while preferably avoiding or minimizing adverse side effects, including those typically associated with other therapies.

[0046] "Subject" includes any human being and is used interchangeably with the term "patient."

[0047] The term "neuronal injury" refers to damage to a component of the central or peripheral nervous system. Neuronal injury can be caused by physical (including mechanical, electrical or thermal), ischemic, hemorrhagic, chemical, biological or biochemical insults. Examples of neuronal injury include ischemic and hemorrhagic stroke, spinal cord, brain, cranial nerve and peripheral nerve injury.

[0048] In the context of this specification, the term "administering" and its variants, including "administer" and "administration", include contacting, applying, delivering or providing the compound or composition of the present invention to a subject by any appropriate means.

[0049] Those skilled in the art will appreciate that the technology described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the technology includes all such variations and modifications. For the avoidance of doubt, the technology also includes all steps, features and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations of any two or more of said steps, features and compounds.

[0050] In order to more clearly understand the present technology, the following description is given in conjunction with preferred embodiments.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Optical microscopy images of mixed umbilical cord blood middle layer (A), top layer (B), cell-free human umbilical cord plasma (UCF) containing exosomes (C), and water (D).

[0053] Figure 2 : Cytokine profiles of three cell-free plasma samples compared to normal human (adult) serum (Serum-hAb (Biocore)) as control.

[0054] Figure 3 : Starting material (cell-free plasma), concentrated product (exosomes), and final product (freeze-dried exosomes) samples characterized by particle size distribution.

[0055] Figure 4 .Modified Neurological Severity Score (mNSS) evaluation.

[0056] Figure 5 Functional recovery assessed by the cylinder test (A) and limb placement score (B).

[0057] Figure 6. Evaluation of infarct volume by TTC (2,3,5-triphenyltetrazolium chloride) staining (6A). Samples were compared with the control group. The intracranial injection (ic) group was compared with the intraperitoneal injection (ip) group (6B).

[0058] Figure 7. Magnetic resonance imaging (MRI) was used to evaluate the lesion size 1 day and 21 days after the experimental procedure (7A). No statistically significant differences were found (7B).

[0059] Figure 8. Representative micrographs of microtubule-associated protein 2 (MAP2) and myelin basic protein (MBP) staining 21 days after treatment (8A). Quantification of MAP2-positive and MBP-positive area loss is expressed as the ratio of ipsilateral / contralateral area (8B). Data represent mean ± SEM. Sham n=3, control saline-ip n=3, control saline-ic n=3, UCF-ip n=3, UCF-ic n=3, UCF-E-ip n=3, and UCF-E-ic n=3. *P<0.05 compared with the control saline group, **P<0.01 compared with the control saline group, ***P<0.001 compared with the control saline group; φP<0.05 compared with the drug treatment group.

[0060] Fig. 9 After 14 days of treatment, apoptotic cells in the peri-infarct area were quantified by TUNEL staining.

[0061] Fig.10 GFAP-positive cells were quantified in the para-infarct area 14 days after treatment.

[0062] Fig.11 Arg-1 (11A) and CD16 / 32 (11B) positive cells were quantified in the peri-infarct area 14 days after treatment. DETAILED DESCRIPTION

[0063] The methods disclosed herein involve administering cell-free human umbilical cord plasma (UCF) or exosomes derived from cell-free human umbilical cord plasma (UCF-E). The inventors surprisingly discovered that administering UCF or UCF-E can treat or improve one or more symptoms of neuronal injury, such as ischemic brain injury (e.g., stroke).

[0064] It will be appreciated by those skilled in the art that various changes and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the embodiments of the present invention are to be considered illustrative and not restrictive in all respects.

[0065] Exosome preparation

[0066] Cell-free plasma for the methods described herein can be prepared from mixed human umbilical cord blood. In particular, plasma is obtained from umbilical cord blood collected during normal healthy pregnancy delivery. Plasma is not obtained from umbilical cord blood of pregnancies that are considered high risk or encounter difficulties during delivery. Specifically, umbilical cord blood is collected under sterile conditions, then mixed, centrifuged to separate plasma, red blood cells and buffy coat. The plasma and buffy coat are mixed and frozen at, for example, -80°C, then thawed and centrifuged at, for example, 3000g for 20 minutes to remove cells, membranes and cell / cell debris. The supernatant is filtered through a 0.1 to 0.22 micron filter (with or without pre-filtration, for example, using a 5 micron filter). The resulting hUC-P (umbilical cord factor plasma) preparation containing exosomes can be used immediately or lyophilized.

[0067] In some embodiments, the allogeneic cell-free plasma preparations (particularly UCF) are characterized by the presence of certain proteins, including at least one of IL-1α, IL-1-β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, eotaxin (CCL11), G-CSF, GM-CSF, basic FGF (FGF-2), HGF, IFN-γ, IP-10 (CxCL10), MCP-1 (CCL2), MIP-1b (CCL4), PDGF-bb, RANTES (CCL5), THF-α and VEGF, for example, detected by ELISA.

[0068] In some embodiments, the allogeneic cell-free plasma preparation (UCF plasma) is characterized by a level of one or more of GM-CSF, HGF, MIP-1b (CCL4), and PDGF-bb that is at least 20% higher than the level found in normal adult serum, for example, the level may be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher than the level found in normal adult serum. Alternatively, the exosome preparation (UCF-E) is characterized by a level of IP-10 (CxCL10) that is at least 60% of the level found in normal adult serum. For example, the level may be 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the level found in normal adult serum.

[0069] Exosomes (or extracellular vesicles (EVs)) are released by cells into the blood under normal and pathological conditions and carry RNA, lipids, and proteins from their host cells that can represent the molecular composition of the parent cell.

[0070] Cell-free allogeneic exosomes (UCF and UCF-E) are derived from a variety of different cells and share common structural and functional proteins, such as Rab GTPase, SNARE, annexin, Alix, Tsg101, and tetraspanins (CD9 and CD63), as well as glycosylphosphatidylinositol anchors, raft proteins, cholesterol, sphingomyelin, and hexosylceramide. Lipoprotein markers APOB (chylomicrons, VLDL, IDL, and LDL) and APOE (chylomicrons, VLDL, and HDL) are used to distinguish exosomes from lipoproteins.

[0071] Exosomes can be isolated by any means known in the art, including but not limited to ultracentrifugation, ultrafiltration, size exclusion chromatography, polymer-based precipitation, and affinity capture on antibody-coupled magnetic beads. For example, conventional methods for EV isolation include differential and buoyant density centrifugation, ultrafiltration, size exclusion, precipitation, and immunoaffinity separation. Size exclusion chromatography uses a biological fluid as a mobile phase relative to a porous stationary phase to elute molecules at a reverse speed differential associated with their size. That is, larger particles will elute first, and then smaller vesicles will enter and flow through the pores, resulting in a longer path and thus increasing the elution time. Differential ultracentrifugation relies on separating EV subpopulations via gradually increasing acceleration. Polyethylene glycol (PEG)-based precipitation uses a solution to promote a large number of polymer-entrapped vesicle aggregates. Immunoaffinity capture uses antibodies targeting exosome surface proteins to separate specific vesicle populations. Microfluidics uses chips with specific antibody-mediated binding to effectively capture exosomes. Ultrafiltration relies on filters of specific pore sizes, which produce vesicle-rich filtrates specific to the desired size.

[0072] The differential ultracentrifugation procedure begins with several clean-up spin steps designed to remove cells, cell debris, apoptotic bodies, and microvesicles. This is achieved by gradually separating the pellet and supernatant at increasing speeds: 300–400 × g for 10 minutes, then 2000 × g, and finally 10,000 × g to isolate the supernatant, which contains a relatively high concentration of exosomes, although it is still contaminated with microvesicles, lipoprotein fractions, and other protein aggregates. After this step, final exosome sedimentation is achieved by spinning the sample at 100,000–200,000 × g for 70 minutes to two hours. The pellet obtained here can be resuspended in phosphate-buffered saline (PBS) and ultracentrifuged again, which will increase the purity but reduce the yield of isolated exosomes. The exosomes found at this level range in size from 20-250nm and contain RNA and microRNA species, as well as some common protein markers associated with exosomes: Flotillin-1, Alix, TSG101, CD81, CD63 and CD9, etc. However, some exosome populations may not express these proteins, such as CD81 - exosomes, and / or these markers can be found on other types of EVs as well as exosomes.

[0073] Ultrafiltration technology relies on the use of membranes with specified pore diameters to separate particles of a predetermined size range. Larger particles are first removed by using filters with pore diameters of 0.8 and 0.45 μm, leaving a filtrate that is relatively rich in exosomes. The exosomes obtained are defined by the largest and smallest size ranges that pass through the first and last pores of the membrane filter. This protocol can be used as a complement to ultracentrifugation to separate large microvesicles and exosomes, although it can be used as a stand-alone technique.

[0074] Another approach is to use nano-ultrafiltration, which relies on sequential filtration to isolate exosomes, known as cross-flow filtration or tangential flow filtration. The technique begins with dead-end filtration of cells and their debris, as well as large vesicles with a diameter of, for example, 1000 nm. Next, tangential flow-based filtration is performed to remove contaminants (mainly proteins) with diameters smaller than the membrane cutoff size and place them into a waste chamber. The filtrate containing the exosomes is then repeatedly passed through an exclusion filter, thereby concentrating the input solution. Finally, using a specified and consistent pore size track-etched membrane (e.g., 50-250 nm in diameter), the exosomes can be further fractionated. The recovery of exosomes depends on the type of filter, as there are different membrane types and pore sizes. For example, cellulose membranes with a pore size of 10 kDa often have the highest recovery efficiency.

[0075] Polyethylene glycol (PEG)-based precipitation uses aqueous PEG solutions to promote the formation of exosome aggregates, which can then be pelleted by low-speed centrifugation (e.g., 1500×g). The size range of isolated exosomes is consistent with other methods (e.g., differential ultracentrifugation).

[0076] Exosomes (UCF-E) can be used immediately or lyophilized.

[0077] In some embodiments, the allogeneic cell-free exosome preparation (particularly UCF-E) is characterized by the presence of certain proteins, including at least one of CD63 and TSG101, and the absence of the lipoprotein markers APOB (chylomicrons, VLDL, IDL, and LDL) and APOE (chylomicrons, VLDL, and HDL).

[0078] In some embodiments, the particle size of the exosome is about 10-200nm, for example, the particle size can be about 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm or about 200nm. Preferably, the particle size of the exosome is about 30-150nm, for example, the particle size can be about 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm or about 150nm. The particle size can be the average particle size or the median particle size. Alternatively, the particle size can be the mode (i.e., the particle size that most often occurs in a group of particles).

[0079] In some embodiments, lyophilization does not significantly change the particle size distribution of the exosomes.

[0080] Exosomes and exosome-containing plasma preparations are cell-free, i.e., no cells are visible under a light microscope (or no living cells or cells / cell debris or membranes are present).

[0081] method

[0082] The methods described herein involve administering a therapeutically effective amount of human umbilical cord plasma and exosomes (hUC-P or UCF) or exosomes alone (hUC-E or UCF-E) to a subject in order to treat or ameliorate at least one symptom of neuronal damage.

[0083] The term "therapeutically effective" or "therapeutic effect" refers to an amount of UCF or UCF-E that will alleviate one or more symptoms of the neuronal injury being treated to some extent. In another embodiment, the term "therapeutically effective dose" refers to an amount of UCF or UCF-E that, when administered to a subject, is effective to at least partially treat the neuronal injury suffered by the individual, or to at least partially alleviate or ameliorate the symptoms of such injury. As is understood in the art, the therapeutically effective amount of UCF or UCF-E depends at least in part on the mode of administration, any carrier or vehicle employed, the specific injury, other medications taken by the subject, and the specific characteristics (age, weight, condition, sex, etc.) of the subject to whom the compound is to be administered.

[0084] UCF or UCF-E can be administered parenterally or intranasally. For parenteral administration, including intravenous, intraperitoneal, intracranial, intrathecal, intramuscular, subcutaneous or intraperitoneal administration, UCF, UCF-E or both are formulated as injectable solutions.

[0085] UCF, UCF-E, or both are suitable for injection after a final filtration step (e.g., after filtering through a 0.1 to 0.2 micron filter). Alternatively, lyophilized UCF or UCF-E can be reconstituted in any suitable injectable vehicle. Suitable vehicles include water for injection, saline, buffered saline (e.g., phosphate buffered saline or 85% phosphate buffered saline), Ringer's solution, or lactated Ringer's solution. In some embodiments, water or 85% PBS is a preferred vehicle for reconstitution of lyophilized UCF or UCF-E.

[0086] Preferably, administration is intrathecal or intravenous.

[0087] In some embodiments, the amount of allogeneic cell-free plasma and / or exosome preparation (UCF or UCF-E) or both administered and the dosage regimen for treating neuronal injury depend on a variety of factors, including the age, weight, sex, and medical condition of the subject; the severity of the injury; the route and frequency of administration; the specific compound employed; and the pharmacokinetic properties (e.g., absorption, distribution, metabolism, excretion) of the subject, and may therefore vary widely. Such treatment may be administered at a frequency and for a period of time deemed necessary by the treating physician. One skilled in the art will appreciate that for each individual, the dosage regimen or therapeutically effective amount of UCF, UCF-E, or both to be administered may need to be optimized for each individual. In some embodiments, UCF or UCF-E may be administered at a dose of about 0.01 mg / kg to 100 mg / kg body weight, typically about 0.1 mg / kg to about 50 mg / kg body weight may be appropriate, depending on the route and frequency of administration.

[0088] The dose to be administered is preferably in the form of a single bolus, or in the form of an infusion of UCF, UCF-E, or both over a period of time, in the form of periodic dosing, or a combination thereof. The concentration of any given dose depends on the frequency of administration.

[0089] In some embodiments, UCF or UCF-E is administered regularly, such as weekly, biweekly, three-weekly, monthly, or a combination thereof. Alternatively, UCF or UCF-E may be administered daily, every 5 days, every 10 days, every 15 days, every 20 days, every 25 days, about every 30 days, or a combination thereof.

[0090] In one embodiment, treating neuronal damage comprises administering UCF, UCF-E, or both multiple times, such as two, three, four, five, six, seven, eight, nine, or ten times, or more.

[0091] In some embodiments, treatment begins at least 1 day after neuronal injury when UCF, UCF-E, or both are first administered. For example, treatment can begin at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, about 30, about 60 days, or at least about 90 days after neuronal injury. In some embodiments, treatment begins at about 3, 4, 5, 6, 7, 8, 9, or 10 days after injury (e.g., 7 or 8 days after injury) when UCF, UCF-E, or both are first administered.

[0092] UCF and / or UCF-E may be administered with additional therapeutic agents, such as anti-inflammatory agents, steroids, dipyridamole, astaxanthin, dabigatran etexilate, losartan, nimodipine, prilocaol, rivaroxaban, or ticlopidine.

[0093] In some embodiments, UCF and / or UCF-E can be administered with one or more therapeutic agents that temporarily disrupt the blood-brain barrier, because blood-brain barrier disruption (BBBD) is effective and safe for the delivery of therapeutic agents. Suitable therapeutic agents for BBBD include one or more of mannitol, intracarotid arterial hyperosmolarmannitol (ICAHM), RMP-7 (a synthetic analog of bradykinin), and regadenoson.

[0094] In one embodiment, UCF, UCF-E, or both are administered without an immunosuppressant.

[0095] The UCF and UCF-E described herein can be used to treat a range of neuronal injuries, including ischemic brain injury, hemorrhagic brain injury, or traumatic brain injury.

[0096] In particular, the UCF and UCF-E described herein can be used to treat ischemic or hemorrhagic stroke.

[0097] These treatments can improve one or more symptoms of neuronal damage. For example, treatment can improve one or more of the following:

[0098] Dystonia - wherein the improvement is a decrease in the frequency or severity of involuntary muscle contractions compared to the same patient before treatment or to a control patient.

[0099] Aphasia - where the improvement is an increase in the ability to understand or produce language compared to the same patient before treatment or to a control patient.

[0100] • Infarct size - where the improvement is a decrease in infarct size.

[0101] • Inflammation - where the improvement is a decrease in inflammation systemically or in the area of ​​the infarct.

[0102] • White matter loss - where the improvement is a decrease in the amount of white matter lost due to injury.

[0103] Gray matter loss - where the improvement is a reduction in the amount of gray matter lost as a result of the injury.

[0104] - Apoptotic cells in the peri-infarct area - wherein the improvement is a decrease in the number of apoptotic cells in the peri-infarct area.

[0105] • CD16 / CD32 positive cells in the peri-infarct area - wherein the improvement is a decrease in the number of CD16 / CD32 positive cells in the peri-infarct area.

[0106] Dystonia is generally considered to be a movement disorder characterized by motor manifestations, mainly continuous or intermittent muscle contractions leading to abnormal, usually repetitive movements, postures or both. However, increasing evidence shows the importance of non-motor components to dystonia, including abnormal sensory and perceptual functions, as well as abnormalities in the fields of neuropsychiatry, cognition and sleep. Dystonia can be assessed by any means known in the art. This can include the use of a unified dystonia rating scale (UDRS), which includes a detailed assessment of the severity of dystonia in each body part. For example, the severity and duration of dystonia in each body part are rated. Proximal limbs and distal limbs are given separate ratings, respectively. The scores for each body part are added together to obtain an overall rating of dystonia severity. Therefore, the method described herein can lead to an improved UDRS.

[0107] Aphasia is the inability or impaired ability to understand or produce speech, particularly due to neuronal damage (e.g., stroke). Aphasia can be assessed informally (via interaction and observation) by the treating physician, or can be assessed using one or more known tests. These tests include the Psycholinguistic Assessment of Language Processing (PALPA), the Western Aphasia Battery, the Boston Naming Test, the Boston Diagnostic Aphasia Examination, the Aachen Aphasia Test, the Minnesota Aphasia Differential Diagnostic Test, the Acute Aphasia Screening Protocol, the Mississippi Aphasia Screening Test, the Mount Wilga Advanced Language Screening Test, Aphasia Language Performance Scale, NIH Stroke Scale, Bedside Assessment Screening Test, Boston Diagnostic Aphasia Examination, Porch Communication Ability Index, Stroke Burden Scale, Pyramid and Palm Trees, Caulfield Cognitive Language, Aphasia Rapid Assessment, Cognitive Language Rapid Test, Reitan-Indiana Aphasia Screening Test, Communication Activities of Daily Living, ScreeLing, Communication Effectiveness Index, Sheffield Language Disorders Screening Test, Comprehensive Aphasia Test, Sklar Aphasia Rating Scale, Frenchay Aphasia Screening Test, Receptive Grammar Test, Functional Assessment of Communication Skills in Adults, Aphasia Screening Test, Functional Communication Profile, Ullevaal Aphasia Screening Test, Wechsler Personal Achievement Test, Information Language Processing Screen (ILPS), Western Aphasia Battery, Inpatient Functional Communication Interview, Whurr Aphasia Screening Test, Cognitive Language Ability Measure. The methods described herein may result in improved performance on any one or any combination of these tests.

[0108] Infarct size (volume) is typically assessed using MRI or CT scan methods known in the art and available to treating physicians. For example, infarct volume can be assessed using the Alberta Stroke Program Early Computed Tomography Score (ASPECTS), which is applied to images obtained with noncontrast computed tomography (NCCT), postcontrast CT (PCCT), and diffusion-weighted imaging (DWI). DWI stroke volume is measured semiquantitatively using manually outlined hyperintense lesions. Infarct core volume is calculated using a threshold apparent diffusion coefficient value of 600 × 10 –6 mm 2 The intraclass correlation coefficient (ICC) can be estimated to assess the inter-reader reliability of ASPECTS score and DWI stroke volume.

[0109] Inflammatory markers, such as C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis factor (TNF)-α and fibrinogen, are upregulated after neuronal damage such as stroke. These biomarkers are known to be associated with increased mortality, recurrent vascular risk and poor functional outcomes. Any known method can be used to measure these biomarkers. The methods described herein can result in a reduction in these biomarkers compared to untreated subjects.

[0110] As is well known, neuronal damage such as ischemic stroke can lead to gray matter and white matter damage. White matter damage is a risk factor leading to adverse neurological consequences. Most of the damage caused by stroke is located in the subcortical region, and white matter can occupy nearly half of the average infarct volume. White matter is very sensitive to ischemia and is usually more seriously damaged than gray matter. The clinical symptoms associated with white matter damage include cognitive dysfunction, emotional disorders, sensory motor impairment, and urinary incontinence and pain, all of which are closely related to the destruction and remodeling of white matter connectivity. White matter (and gray matter) damage can be detected non-invasively by MRI, which provides a three-dimensional evaluation of its morphology, metabolism and function.

[0111] After acute stroke, a variety of immune cells can enter the brain parenchyma in an orderly manner. Peripheral immune cells, including myeloid dendritic cells, monocytes / macrophages, and neutrophils, appear within 1 day after stroke. Subsequently, a small increase in T and B lymphocytes can be detected. Once the brain environment is affected by pathophysiological changes such as stroke, infiltrating cells can differentiate into macrophages with special functions, including the production of inflammatory mediators and phagocytosis. Infiltrating immune cells are mainly differentiated into two phenotypes: classical proinflammatory phenotype (M1) and alternative anti-inflammatory phenotype (M2). Phenotypic markers of M1 include CD16, CD32, CD86, MHCII, and iNOS, while CD206, Arg1, and Ym1 are identified as M2 phenotype markers. As shown herein, the method of the present invention can reduce the number of CD16 / CD32 positive cells in the peri-infarct area.

[0112] The modified Rankin Scale (mRS) is a single-item, global outcome rating scale for subjects after stroke. It is used to classify the level of functional independence based on pre-stroke activities rather than on observed performance on specific tasks.

[0113] The routine administration of the mRS is a guided interview process. Assessment is performed by asking patients about their activities of daily living, including outdoor activities. Information should be obtained about the patient's neurological deficits on examination, including aphasia and intellectual deficits. All aspects of the patient's physical, mental presentation, and speech should be considered in selecting a single MRS grade.

[0114] Individual mRS grades should be assigned according to the following criteria (Dromerick, AW, Edwards, DF, Diringer, MN (2003). Sensitivity to changes in disability after stroke: A comparison of four scales useful in clinical trials. Journal of Rehabilitation Research and Development, 40(1), 1-8.)

[0115] Table 1: mRS grading

[0116]

[0117]

[0118] The methods disclosed herein can result in an improvement in mRS score.

[0119] The National Institutes of Health Stroke Scale or NIH Stroke Scale (NIHSS) is a tool used to objectively quantify the damage caused by a stroke. The NIHSS consists of 11 items, each of which scores a specific ability between 0 and 4. For each item, a score of 0 generally indicates normal function of that specific ability, while higher scores indicate impairment. The individual scores for each item are added together to calculate the patient's total NIHSS score. The highest possible score is 42 and the lowest score is 0. The methods described in this article can lead to improvements in NIHSS scores.

[0120] Table 2: NIHSS grading

[0121]

[0122] The items assessed in the NIHSS are as follows.

[0123] 1a. Level of consciousness. A score of 3 is given only if the patient does not make any movements (except reflexive postures) in response to noxious stimuli.

[0124] 1b.LOC questions. Ask the patient the month and his / her age. The answer must be correct. Aphasic and comatose patients who cannot understand questions are scored as 2. Patients who are unable to speak due to endotracheal intubation, orotracheal trauma, severe dysarthria of any cause, language impairment, or any other problem not secondary to aphasia are scored as 1.

[0125] 2. Optimal gaze. Only horizontal eye movements are tested. Voluntary or reflex (oculocephalic) eye movements will be scored, but caloric testing will not be performed. A score of 1 is given if the patient has a conjugate eye deviation that can be overcome by voluntary or reflex activity. A score of 1 is given if the patient has an isolated peripheral nerve palsy (CN III, IV, or VI). Gaze testing can be performed on all patients with aphasia. Patients with ocular trauma, bandages, preexisting blindness, or other visual or visual field impairments should be tested with reflex movements and at the investigator's option. Establishing eye contact and then moving the patient from side to side can sometimes clarify whether a partial gaze palsy is present.

[0126] 3. Visual fields. Visual fields (superior and inferior quadrants) are tested by standoff, using finger counting or visual threats as appropriate. The patient must be encouraged, but if they look appropriately at the side that moves the finger, they can be scored as normal. In case of unilateral blindness or enucleation, the visual field of the remaining eye is scored. A score of 1 is given only if significant asymmetry is found (including quadrantanopia). If the patient is blind for any reason, a score of 3 is given. Simultaneous bilateral stimulation is performed at this time. If neglect is present, the patient is scored as 1 and the result is also used to answer question 11.

[0127] 4. Facial paralysis. Ask or use hand gestures to encourage the patient to show teeth or raise eyebrows and close eyes. For patients who are unresponsive or unable to comprehend, score for symmetry of expression in response to noxious stimuli. If facial trauma / bandages, orotracheal tubes, tape, or other physical obstructions obscure the face, remove them whenever possible.

[0128] 5 & ​​6. Movement of arms and legs. Test each limb in turn, starting with the nonparetic arm (if known). Place the limb in the appropriate position: extended arm (palm down) 90 degrees (if sitting) or 45 degrees (if supine), and leg at 30 degrees (always test in the supine position). If the arm falls within 10 seconds or the leg falls within 5 seconds, score it as drift. Use urgent sounds and gestures but no noxious stimuli to encourage the aphasic patient. A score of "9" is given only in the case of shoulder or hip amputation or joint fusion, and the examiner must clearly write the reason for the score of "9".

[0129] 7. Limb Ataxia. This item is designed to look for evidence of a unilateral cerebellar lesion. Perform the test with both eyes open. In the case of visual impairment, ensure that the test is performed in the full visual field. Perform the finger-nose-finger test and the heel-shin test bilaterally, and score ataxia only if the ataxia is out of proportion to the weakness. Patients who are unable to comprehend or paralyzed will not have ataxia. Although the use of a non-testable item is discouraged, in the case of amputation, joint fusion, or certain fractures, this item may be scored as a "9" and the examiner must clearly write the explanation of the non-score. In the case of blindness, the test may be performed by touching the nose in an outstretched arm position.

[0130] 8. Sensation. Sensation to pinprick or expression of pain during testing, or flinching in response to noxious stimuli in patients with confusion or aphasia. Only sensory loss attributable to stroke is scored as abnormal, and the examiner should test as many body parts as necessary (arms (not hands), legs, trunk, face] to accurately test for hemiplegia. A score of 2, "severe or complete," should be given only when severe or complete loss of sensation can be clearly demonstrated. Thus, a comatose aphasic patient might receive a score of 1 or 0. A patient with a brainstem stroke and bilateral sensory loss receives a score of 2. If the patient is unresponsive and has quadriplegia, the score is 2. Comatose patients (item la=3) are arbitrarily given a score of 2 on this item.

[0131] 9. Optimal Language. A great deal of information regarding comprehension will be obtained during the first few parts of the examination. Ask the patient to describe what is happening in the picture, name items on a naming list, and read a string of sentences. Have the patient name all of the items on the naming list and read all of the phrases on two reading lists. Comprehension is judged based on responses here and on all of the commands given previously in the general neurologic examination. If visual loss interferes with the test, ask the patient to identify an object placed in the hand, repeat it, and speak it. Intubated patients should be asked to write. Comatose patients (question la=3) will be arbitrarily scored a 3 on this item. The examiner must select a score for a comatose patient or one with limited cooperation, but a score of 3 should only be used if the patient is mute and does not follow any one-step command.

[0132] 10. Dysarthria. If the patient is considered normal, adequate speech samples must be obtained by asking the patient to read or repeat the words from a list. If the patient has severe aphasia, the articulation clarity of spontaneous speech may be assessed. This item may be scored as "9" only if the patient is intubated or has other physical impairments that prevent speech, and the examiner must clearly write the reason for not scoring. Do not tell the patient why he or she is being tested.

[0133] 11. Neglect and inattention. Sufficient information was available during the previous test to identify neglect. If the patient has severe visual loss, is unable to perform visual bilateral stimulation, and skin stimulation is normal, score as normal. If the patient has aphasia but seems to be able to pay attention to both sides, score as normal. The presence of visuospatial neglect or visual agnosia can also be considered evidence of abnormality. Since abnormalities are scored only when present, this item is testable.

[0134] Example

[0135] Example 1: De-identified and pooled cord serum collected from a cord blood bank

[0136] Individual umbilical cord blood (UCB) was collected according to the standard protocol of the American Association of Blood Banks (AABB). See Badowski M.S., Harris DT (2012) Collection, Processing, and Banking of Umbilical Cord Blood Stem Cells for Transplantation and Regenerative Medicine, cited in Singh S. (eds) Somatic Stem Cells. Methods in Molecular Biology (Methods and Protocols), vol 879.

[0137] An aliquot of cord blood is removed from each individual for disease and blood typing testing.

[0138] After blood typing and disease-free status assessment, individual samples were pooled and stored frozen at -80°C.

[0139] Example 2: Plasma Preparation

[0140] Plasma was prepared from pooled cord blood by centrifugation.

[0141] Human UCB containing citrate phosphate glucose adenine as an anticoagulant was obtained from an umbilical cord blood bank. Only fresh UCB provided within 24 h postpartum were used in this study.

[0142] UCB was centrifuged at 1500 xg for 10 min at room temperature and separated into three layers: plasma (UCB-PL top layer), a mixture of leukocytes and platelets (middle layer), and erythrocytes (bottom layer).

[0143] The UCB-PL layer was carefully collected, aliquoted, and stored as UCF at -80 °C until use.

[0144] Cell-free plasma (UCF) was prepared by centrifugation of UCB-PL at 3000 g for 20 min.

[0145] use Cell counts were performed on each layer and cell-free plasma using a cell analyzer (Luminex). The results are listed in Table 3 and Figure 1 , which shows optical microscope images of the middle layer, top layer, cell-free plasma and water respectively.

[0146] Table 3: Cell count

[0147]

[0148]

[0149] Cytokine profiling of cell-free plasma was performed, and the results were as Figure 2 As shown, normal human (adult) serum was used as control.

[0150] Example 2: Exosome preparation

[0151] Cell-free frozen umbilical cord plasma samples (UCF) were thawed and pooled. Exosomes (UCF-E) were prepared using a combination of centrifugation, precipitation, and microfiltration steps. In some cases, cryoprotectants were added prior to freeze-drying (lyophilization) of the exosomes.

[0152] Lyophilized exosomes were stable for more than 36 months at 4°C. Samples stored at 4°C or room temperature showed no changes compared to the initial evaluation. Given this finding, exosomes remain stable under a variety of conditions.

[0153] The total number of exosome particles was subsequently quantified by NTA (Nanoparticle Tracking Analysis) using Nano Sight LM10. Lyophilization did not affect the stability of purified exosomes ( Figure 2 ). Exosome Standards Lyophilized exosome standards are of high purity, easy to reconstitute, easy to transport and store at 4°C.

[0154] Table 4: Lyophilized UCF

[0155]

[0156] Samples of the starting material (cell-free plasma), concentrated product (exosomes), and final product (freeze-dried exosomes) were characterized by nanoparticle tracking analysis (NTA) using the Nanosight NS300 (Malvern Instruments Ltd). NTA analysis provided estimates of EV concentration and size distribution, as provided in Table 5.

[0157] Table 5: NTA data summary

[0158]

[0159] These data are presented graphically in Figure 3 This is explained in .

[0160] Example 3: Freeze-drying

[0161] The cell-free plasma or exosome preparations were first frozen to -80°C and then quickly placed in a Labconco Freezone 12-liter vertical freeze dryer, where the condenser was maintained at -35°C, 100 mTorr (vacuum enough to freeze water rapidly) for 24 hours, and then the vials were resealed. No heat was applied, and no organic solvents or salts were added. The samples were lyophilized by maintaining a strong vacuum freeze during drying with the aid of a -45°C condenser.

[0162] Use the following protocol to lyophilize cell-free plasma or exosome preparations.

[0163] Table 6: Heat treatment steps

[0164]

[0165]

[0166] The freezing temperature was -40°C and the additional freezing time was 20 minutes. The condenser set point was -40°C and the vacuum set point was 150 mTorr.

[0167] Table 7: Primary Drying Steps

[0168] step Temperature(℃) Time(sec) Vac(mTorr) Temperature rise / fall / constant temperature 1 -35 100 100 H 2 -30 570 100 H 3 -35 390 100 H 4 -10 300 100 R 5 -10 240 100 H 6 0 100 100 R 7 0 630 100 H 8 -99 0 0 H 9 -99 0 0 H 10 -99 0 0 H 11 -99 0 0 H 12 -99 0 0 H 13 -99 0 0 H 14 -99 0 0 H 15 -99 0 0 H 16 -99 0 0 H Afterheat 20 1200 70

[0169] The secondary temperature is 30°C.

[0170] Example 4: Reconstruction Solution

[0171] 285 mg of lyophilized exosome-containing cell-free plasma (UCF) was reconstituted with 5 mL of water for injection. The powder completely dissolved and took on the appearance of serum (straw (yellow) color). The reconstituted cell-free plasma containing exosomes was filtered through a 0.2 μm filter. Sterile filtered cell-free plasma (UCF) containing exosomes can be used immediately or stored at 2-8°C for two weeks.

[0172] 86 mg of lyophilized exosomes (UCF-E) were reconstituted with modified 0.9% saline (85% saline) - 0.85x0.9% saline solution with water for injection (8.5 mL 0.9% saline: 1.5 mL water for injection). Prior to reconstitution, 85% saline was warmed to 37°C. 5 mL of warmed (37°C) saline was added to completely dissolve the contents. In some cases, incubation at 37°C may be required, with occasional or no vortexing. The powder was completely dissolved and had an off-white colloidal liquid appearance. The reconstituted exosome preparation was a homogeneous turbid solution filtered through a 0.2 μm filter. Sterile filtered exosomes (UCF-E) can be used immediately or stored at 2-8°C for two weeks.

[0173] Example 5: Animal study on the treatment of middle cerebral artery infarction in rats with cell-free plasma and exosomes

[0174] Adult male Sprague-Dawley rats (250-300 g, n = 84) were obtained from the Laboratory Animal Research Center of the Chinese Academy of Medical Sciences (Beijing, China). Housing conditions included a 12-hour light / dark cycle, controlled temperature and humidity, and free access to food and water. Rats were randomly assigned to two groups and subjected to transient focal cerebral ischemia and reperfusion, followed by treatment.

[0175] Cerebral ischemia was induced by transient intraluminal vascular occlusion using intravascular suture technology. Briefly, under halothane (Lunan Pharmaceutical, Jinan, China) anesthesia, the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were exposed. The ECA was cut with microscissors, and a monofilament nylon line with a diameter of 0.25 mm (Sunbio Biotech, Beijing, China) was inserted into the stump of the ECA. The nylon line was inserted into the ICA until resistance was encountered (1.8-2.1 cm from the bifurcation of the carotid artery). After a period of occlusion (120 minutes), the nylon line was pulled out. A silk suture was tied at the bifurcation of the external carotid artery to stop bleeding. The skin was sutured to close the neck incision. Two groups of rats undergoing middle cerebral artery occlusion (MCAO) surgery were administered recommended doses of buprenorphine and cefazolin to relieve pain and prevent infection. No immunosuppressants were administered.

[0176] Then the modified neurological severity score (mNSS) test was performed after surgery. Rats that underwent transient MCAO and had mNSS scores of around 10-12 were randomly assigned to four groups (AD groups, and for groups A, B, and C, the animals were further divided into intracranial and intraperitoneal subgroups), as shown in Table 8.

[0177] Reconstituted exosome-containing cell-free plasma (UCF) or exosomes alone (UCF-E as described above) were administered to rats with permanent MCAO (pMCAO) in the early subacute stage, which is equivalent to 3 days after pMCAO in rats (7 days after infarction in humans). The treatment regimen is shown in Table 8.

[0178] On the 3rd day after ischemia (PID), MCAO animals were randomly divided into 8 experimental groups. For the intraperitoneal treatment group, cell-free plasma (which contains exosomes, UCF) (0.5 mL), exosomes only (UCF-E) (0.5 mL) or saline (0.5 mL) was administered into the peritoneal cavity within 5 minutes. For the intracerebral treatment group, three holes were drilled on the right side of the rat skull (A was 30 μl, B was 30 μl, and C was 40 μl-see Table 9) (Table 9), and cell-free plasma (UCF) (0.1 mL), exosomes only (UCF-E) (0.1 mL) or saline (0.1 mL) were stereotaxically injected. All targets were selected around the infarct edge area.

[0179] Table 8: Treatment regimen for rats

[0180]

[0181] *Contains exosomes

[0182] Table 9: Hole coordinates

[0183] R(mm)AP (mm) DV(mm) V(μL) A3 +2 3 30 B3 +0.5 5 30 C3 -0.5 3 40

[0184] R: right of midline; AP: anterior to bregma; DV: inferior to dura mater; V: volume

[0185] 5.1 Functional evaluation

[0186] Modified Neurological Severity Score (mNSS) Test

[0187] The mNSS test is a standard and globally accepted method for assessing the severity of injury and recovery after stroke. The test combines motor, sensory, reflex and balance tests. The cumulative score of all tests determines the severity of the injury. An mNSS score of 13-18 indicates severe injury, 7-12 indicates moderate injury, and 1-6 indicates mild injury. The mNSS test was performed on both groups of rats before ischemia and at regular intervals (1 day, 7 days, 14 days and 21 days) until reperfusion on day 21.

[0188] Cylinder test

[0189] The cylinder test was used to measure forelimb asymmetry by observing the movements of the rats during 3-min intervals in a clear, 18-cm wide and 30-cm high Plexiglas cylinder, which was of sufficient diameter to allow movement and to facilitate upright and wall exploration. A mirror behind the cylinder allowed forelimb movements to be observed and recorded while the rats were facing away from the examiner. A touchdown was scored for the limb that first touched the ground after a period of standing and wall exploration, or for both limbs if both limbs touched the ground simultaneously. Percent use scores for the intact and the impaired limb were calculated relative to the total number of movements. The percentage use of the impaired limb was subtracted from the percentage use of the intact limb to give a total limb deviation score. Wall exploration and touchdown movements were analyzed separately. Rats were tested 1–4 weeks after transplantation with local ischemia.

[0190] Limb placement test

[0191] The limb placing test assesses sensorimotor integration of the forelimbs and hindlimbs by examining responses to tactile and proprioceptive stimulation, and is performed according to the method described by De Ryck et al. (Brain Research. 1992; 573(1):44-60) and modified by Puurunen K et al. (Neuropharmacology. 2001; 40(4):597-606, and Exp Neurol. 2001; 167(2):348-55). Both sides of the body are tested 24 hours after local ischemia. The test consists of seven limb placing tasks, which are scored by a tester who is blinded to the treatment group. The following scores are used to detect impairment of the hindlimbs and forelimbs: 0, no placement; 1, incomplete or delayed placement; 2, complete, immediate placement. The average placement score (forelimbs and hindlimbs combined) for each weekly test was calculated for each rat.

[0192] TTC staining

[0193] Rats treated with plasma (UCF) and exosomes (UCF-E) were deeply anesthetized with pentobarbital and decapitated. The brain was removed, placed in adult rat brain matrix (Kent Scientific Corporation, USA), frozen at about -70 ° C for about 8-10 minutes, and cut into 2 mm thick coronal slices. Coronal brain slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution in the dark for 30-45 minutes, and then images were captured using an Olympus SZX12 research stereo microscope. The area of ​​ischemic and non-ischemic areas of the ipsilateral hemisphere and the contralateral hemisphere of each slice was tracked and measured using Image J analysis software (NIH). The total volume of each area of ​​the rat brain was calculated. The percentage of infarct size for each rat was calculated using the following formula: Infarct size (%) = {(volume of the contralateral hemisphere)-(volume of the non-ischemic ipsilateral hemisphere)} x 100 / volume of the contralateral hemisphere. This formula accounts for the possible interference of brain edema on infarct volume. The ipsilateral hemisphere swelling of each rat was calculated using the following formula: Swelling (%) = {(ipsilateral hemisphere volume) - (contralateral hemisphere volume)} x 100 / contralateral hemisphere volume.

[0194] MRI scan

[0195] Seven days after surgery or treatment, rats were anesthetized with 1.5%-2% isoflurane for MRI. During the MRI procedure, rats were maintained at 37°C and an MRI-compatible breathing sensor was used to control the animals. MRI experiments included three-dimensional (3D) T2-weighted images (T2WI), a series of diffusion-weighted imaging (DWI) to calculate the apparent diffusion coefficient (ADC) map, and a series of perfusion-weighted imaging to calculate cerebral blood flow maps. Perfusion experiments were performed using arterial spin labeling without contrast injection. All MRI experiments were performed on a BIOSPEC BMT 47 / 40 (Bruker, Ettlingen, Germany) spectrometer operating at 4.7T and equipped with an 11.2 cm actively shielded gradient system capable of providing a gradient strength of 200 mT / m with a rise time of 80 seconds. A 7 cm birdcage radiofrequency coil was used for transmission and reception. ParaVision 3.0.1 (Bruker, Ettlingen, Germany) was used for T2WI, DWI, and perfusion-weighted imaging image analysis. ADC maps were calculated from DWI series using the image sequence analysis tool of the ParaVision package. Cerebral blood flow maps were calculated from arterial spin labeling images. The calculations were performed using Matlab 7.3 (MathWorks, Inc., Natick, MA), and the data are presented as percentages of affected brain volume.

[0196] Neuroprotection

[0197] To assess gray and white matter damage, coronal paraffin sections (6 μm) were incubated with MAP2 (Sigma Aldrich) or mouse anti-myelin basic protein (MBP, Sternberger Monoclonals, Lutherville, MD) and visualized with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Brain lesions were analyzed at −1.58 mm from bregma in adult mice using ImageJ software (Rasband WS, ImageJ, NIH, Bethesda, Maryland; http: / / rsb.info.nih.gov / ij / , 1997–2009). Ipsilateral MAP2 and MBP area losses were calculated as described.

[0198] Cell Death

[0199] Cell death was detected in the peri-infarct area by TUNEL staining (TdT-FragEL DNA fragmentation detection kit, Oncogene Research Products). Cell counts were performed on one section of each rat (n = 10 per group), which was taken 1.6 mm posterior to the anterior bregma. The number of positive cells was counted in at least 10 different microscopic fields based on nuclear morphology and dark color using a 40× objective and image analysis software (Image-Pro Plus 4.1, Media Cybernetics).

[0200] Neuroinflammation

[0201] Microglial activation was evaluated by measuring the shape of microglial transitions. At 1 and 7 days after treatment, 25 microglia were randomly selected from a 300 μm wide area of ​​the peri-infarct region in each of 6 coronal sections spaced 240 μm apart (150 microglia per animal) (n = 4 per group). The nuclei were placed at the center of 10 concentric grids spaced 5 μm apart, and the number of intersections between cytoplasmic processes and grid lines was recorded using a 100× oil immersion lens.

[0202] Glial scar

[0203] GFAP expression at the border of the infarct zone was quantified by measuring the integrated optical density and area fraction using ImageJ software (National Institute of Mental Health, Bethesda, MA).

[0204] Statistical analysis

[0205] All data are presented as mean ± SEM. Functional outcomes measured with the cylinder stand test were analyzed using two-way ANOVA analysis with Fisher's least significant difference post-test. P < 0.05 was considered statistically significant. Histological measurements were analyzed using one-way ANOVA with Bonferroni post-test. P < 0.05 was considered statistically significant.

[0206] 5.2 Results

[0207] Neurological findings

[0208] mNSS after MCAO Figure 4 All six groups gradually recovered neurological function over time (see Figure 4 ). At 14 days after MCAO (control-ip vs. UCF-ip, UCF-ic, UCF-E-ip and UCF-E-ip, P<0.05, or control-ic vs. UCF-ip, UCF-ic, UCF-E-ip and UCF-E-ic, P<0.05) and 21 days (control-ip vs. UCF-ip, UCF-ic and UCF-E-ip P<0.05, or control-ic vs. UCF-ip, UCF-ic and UCF-E-ip P<0.05; control-ic vs. UCF-E-ic, control-ip vs. UCF-E-ic, P<0.01), the mNSS of the animal groups treated with saline was significantly higher than that of the animals treated with UCF (cell-free plasma containing exosomes) and UCF-E (exosomes only) (P<0.05). The higher the mNSS, the greater the severity of neurological damage. The score of the UCF-E-ic group (3.5 ± 1.5) was lower than that of the other three treatment groups (4.5 ± 0.7 for the UCF-ic group, 4.2 ± 0.9 for the UCF-ip group, and 4.6 ± 0.3 for the UCF-E-ip group) 21 days after treatment. However, no significant differences were found between rats treated with intracranial or intraperitoneal UCF and UCF-E.

[0209] Using cylinder and limb placement scores, rats that received intracranial and intraperitoneal injections of UCF (cell-free plasma containing exosomes) and UCF-E (exosomes alone) on days 14 and 21 showed better functional outcomes than saline controls ( Figure 5 AB). Notably, rats that received UCF-E-ic also performed significantly better on behavioral tests than rats that received UCF-ic, UCF-ip, or UCF-E-ip on days 14 and 21 after stroke ( Figure 5 AB). However, the better test scores were not immediately apparent at the beginning of the behavioral testing.

[0210] Infarct volume assessment

[0211] At 14 and 21 days after treatment, the infarct volumes in the UCF-ic and UCF-E-ip groups were significantly lower than those in the saline-ic and saline-ip groups (UCF-ic vs. control-ic, UCF-ip vs. control-ic, UCF-ic vs. control-ip, UCF-ip vs. control-ip, **P<0.05; UCF-E-ic vs. control-ic, UCF-E-ip vs. control-ic, **P<0.01, Figure 6). However, no significant differences were found between rats treated with intracranial UCF and UCF-E injections or at 7 days after treatment. At 14 and 21 days after injection, rats that received intraperitoneal UCF or UCF-E infusions showed larger lesion sizes than those in the intracranial UCF or UCF-E injection groups.

[0212] The lesion size was evaluated by MRI 1 day and 14 days after the experimental procedure (Figure 7). The MRI scan data were similar to the TTC staining data.

[0213] At 14 and 21 days after injection, rats that received intraperitoneal UCF or UCF-E infusion showed greater infarction rates than those in the intracranial UCF or UCF-E injection groups.

[0214] Neuroprotection

[0215] The effects of UCF and UCF-E treatment on lesion size were determined 21 days after cerebral ischemia by analyzing the loss of ipsilateral gray matter (mouse anti-microtubule-associated protein 2 (MAP2)) and white matter (mouse anti-myelin basic protein (MBP)).

[0216] In mice treated with saline, the ipsilateral MAP2 area loss was 39.13 ± 3.5% and 37.86 ± 7.1%, respectively, with no difference in the values ​​between the two groups (ip vs ic). Treatment with UCF-E (ic and ip) or UCF (ip) significantly reduced the lesion volume compared with saline treatment (as shown in Figure 8, ***P < 0.001, **P < 0.01, *P < 0.05, respectively). There was no significant difference in the ipsilateral MAP2 area loss between the four treatment groups.

[0217] In rats treated with saline, the ipsilateral MBP area loss was 35.26 ± 4.2% and 36.91 ± 3.3%, respectively (Fig. 8), and the values ​​did not differ between the two groups (ip vs ic). Treatment with UCF-E (ic and ip) or UCF (ic and ip) significantly reduced the lesion volume compared with saline treatment (***P < 0.001, **P < 0.01, *P < 0.05, respectively, as shown in Fig. 8). There was no significant difference in ipsilateral MAP2 area loss between the UCF-ip group, the UCF-ic group, or the UCF-E-ip group. However, 21 days after treatment, the ipsilateral MAP2 area loss in rats treated with UCF-E-ic was significantly lower than that in rats treated with UCF-ip, UCF-ic, or UCF-E-ip.

[0218] TUNEL staining

[0219] TUNEL staining was used to determine the number of apoptotic cells in the peri-infarct area 14 days after treatment ( Fig. 9 ). TUNEL positive cells / mm 2 The average number of TUNEL-positive cells was as follows: 467 ± 43.2 (control ip), 8.25 ± 2.693 (sham), 20.45 ± 10.308 (ischemia), 20.15 ± 9.67 (vehicle), and 10.0 ± 3.974 (treatment). The four treatment groups had fewer apoptotic cells than the two control groups. There was a significant difference in the number of TUNEL-positive cells between the control and treatment groups (e.g. Fig. 9 14 days after treatment, the number of TUNEL-positive cells in the UCF-E-ic treatment group was significantly lower than that in the UCF-ip, UCF-ic or UCF-E-ip treatment groups.

[0220] Glial scar

[0221] 21 days after treatment, there was no significant difference in GFAP-positive cells between the different groups ( Fig.10 ).

[0222] Macrophage phenotype

[0223] To investigate whether the administration of UCF-E or UCF (ip or ic) affects macrophage phenotype, the expression profile of macrophages was evaluated by immunofluorescence staining with CD16 / 32 and arginase-1 (Arg-1). At 14 days after injury, higher numbers of Arginase-1-positive cells were found in the four treatment groups compared to the two control groups (all P < 0.01). At 14 days after treatment, the number of Arg-1-positive cells in the UCF-ip treatment group was significantly higher than that in the UCF-ic, UCF-E-ic, or UCF-E-ip treatment groups (all P < 0.01). In addition, the number of CD16 / 32-positive cells was significantly reduced in animals that received UCF-ic, UCF-ip, UCF-E-ic, or UCF-E-ip injections compared to animals in the two saline control groups. No significant differences were observed between the four treatment groups. See Fig.11 .

[0224] 5.3 Conclusion

[0225] Both UCF (cell-free plasma containing exosomes) and UCF-E (exosomes alone) were safe for intraperitoneal or intracranial injection, and no injection-related complications or deaths occurred.

[0226] Intraperitoneal or intracranial injection of UCF and UCF-E promoted functional recovery after stroke in rats. Although there was no statistically significant difference between the two (which may be related to the relatively small number of experimental rats and the short observation period), intracranial injection of UCF-E had a higher absolute value score.

[0227] The repair mechanism of UCF and UCF-E may be related to neuroprotection, as evidenced by the reduction in injury area, staining of neuron-related markers, and decreased apoptosis.

[0228] UCF and UCF-E have little effect on gliosis, so they have little effect on reducing glial scar formation in the chronic phase.

[0229] UCF and UCF-E have a strong regulatory effect on the local immune inflammatory response in the central nervous system.

[0230] 6. Human studies

[0231] A small-scale study was conducted in six volunteers to observe the safety and preliminary efficacy of UCF and UCF-E in humans. The study passed the ethical review of the PLA General Hospital Seventh Medical Center (No. 2021-1368-6) and was conducted in strict accordance with the ethical review opinions and experimental protocols of the PLA General Hospital Seventh Medical Center.

[0232] The inclusion criteria for this study were as follows:

[0233] 1. The stroke lasted for at least 3 months but no more than 24 months and was accompanied by motor neurological dysfunction.

[0234] 2. Men and women aged 30-65 years.

[0235] 3. Females must have a negative serum pregnancy test result and take appropriate contraceptive measures or be infertile (postmenopausal for at least two years or have undergone hysterectomy, oophorectomy or sterilization).

[0236] 4. A history of complete ischemic stroke with or without ischemic lesions in the subcortical area supplied by the middle cerebral artery (MCA) or lenticulostriate arteries, as detected by head MRI.

[0237] 5. Modified grading score: 2, 3 or 4.

[0238] 6.FMMS score is not higher than 55.

[0239] 7. NIHSS score fluctuates between plus 4 and minus 4 when evaluated twice at least 3 weeks before surgery.

[0240] 8. Able and willing to complete all follow-up visits.

[0241] 9. Able and willing to undergo post-operative physical therapy and rehabilitation.

[0242] The exclusion criteria for this study were as follows:

[0243] 1. Any psychological or psychiatric illness that may interfere with the research.

[0244] 2. Patients with a history of more than one symptomatic stroke other than transient ischemic attack (TIA).

[0245] 3. Patients with a history of other serious neurological diseases or injuries.

[0246] 4. The area of ​​cerebral infarction is greater than 8 cm at any measurement.

[0247] 5. History of myocardial infarction within 3 months.

[0248] 6. Patients with a history of epilepsy or taking anti-epileptic drugs.

[0249] 7. Patients who have participated in any other clinical research on drugs or medical devices within 30 days.

[0250] 8. In addition to blood transfusions, the patient also received other cell infusions.

[0251] 9. Any concomitant illness or condition, such as:

[0252] a. Coagulation dysfunction and preoperative INR>1.4

[0253] b. Panel reactive antibodies (PRA) > 20% during initial screening

[0254] c. Active infection before surgery

[0255] d. Hypotension requiring blood pressure-raising therapy

[0256] e. Skin damage in the surgical area

[0257] f. Malignant tumor or history of malignant tumor

[0258] g. Primary or secondary immunodeficiency

[0259] h. Continuous MRI artifacts occur before or after surgery, clear MRI images cannot be obtained, or MRI examination cannot be performed.

[0260] i. Serum creatinine>115μmol / L, liver function (aspartate aminotransferase / Alt, total bilirubin, alkaline phosphatase)>2 times the upper limit of normal, hematocrit (%) / (g / L) hemoglobin<30 / 10, total white blood cell count<4×10 9 / L, platelet count <100×10 9 / L), uncontrolled hypertension, systolic blood pressure>180MMHG or diastolic blood pressure>100MMHG or uncontrolled diabetes (hbalc>8%), gastrointestinal bleeding found in occult blood test, positive tuberculosis (TB skin test: PPD / tuberculin test), hepatitis B or C or human HIV positive.

[0261] 10. Any of the following:

[0262] a. Recent drug or alcohol abuse

[0263] b. Unstable health status

[0264] c. Unstable psychiatric illness, including psychosis and untreated major depression.

[0265] 11. Any situation that the researcher or attending physician believes may affect the subject's participation in the clinical study or cause harm to the subject.

[0266] 12. Any condition that the surgeon believes may cause postoperative complications.

[0267] 13. The patient is known to be allergic to tacrolimus or methylprednisolone.

[0268] 14. The patient is unable or unwilling to attend physical and / or occupational therapy as planned, and is unable or unwilling to return to the hospital for follow-up as planned.

[0269] 15. Failure to provide informed consent as required by the screening program.

[0270] 16. Take antiplatelet drugs within 2 weeks before surgery.

[0271] The evaluation criteria are as follows:

[0272] Safety Assessment

[0273] Safety was assessed by adverse events, clinical testing, vital signs, and physical examinations.

[0274] Follow-up endpoints related to intravenous administration were reported, including 30-day survival, wound infection, seizure, meningitis, cerebral hematoma, new ischemic stroke, disease progression, and secondary surgery.

[0275] Other safety endpoints included the incidence of complications related to:

[0276] a. Related to immunosuppressive therapy, including but not limited to nephrotoxicity (creatinine ≥177 mol / L [2 mg / dL], or ≥2 times baseline creatinine) or renal failure (requiring renal replacement therapy).

[0277] b. Hyperkalemia or hypomagnesemia caused by renal insufficiency.

[0278] c. Opportunistic infections (bacterial, fungal, or viral, including cytomegalovirus).

[0279] d. Tacrolimus-induced central nervous system neurotoxicity (including posterior leukoencephalopathy syndrome, epilepsy, focal neurological dysfunction, tremor and headache).

[0280] e. New onset of glucose intolerance, hyperglycemia, or diabetes mellitus.

[0281] f. Gastrointestinal disorders (including anorexia, nausea, vomiting, diarrhea and abdominal discomfort).

[0282] g. Malignant tumors (squamous cell carcinoma or malignant lymphoproliferative diseases) and malignant hypertension.

[0283] h. New complications related to stroke or neurological function, including worsening motor dysfunction (motor function score results), worsening sensory dysfunction (neurological examination), or worse cognitive test results (using a neuropsychological battery).

[0284] i. Other complications such as urinary tract infection, deep vein thrombosis (DVT), pulmonary embolism, gastric ulcer, bedsore, respiratory tract infection (pneumonia or tracheobronchitis), death within 30 days of surgery, and prolonged hospital stay.

[0285] Efficacy evaluation

[0286] The main efficacy indicators are:

[0287] a. Adverse events.

[0288] b. Serious adverse events (SAEs).

[0289] c. Regular brain MRI scans (conventional scans and brain perfusion imaging).

[0290] d. Serology and hematology.

[0291] e. Clinical assessment of brain injury, including the National Institutes of Health Stroke Scale (NIHSS), modified Rankin Score (MRS), Fugl-Meyer Stroke Scale, and cognitive battery.

[0292] f. Fluorodeoxyglucose uptake was detected by positron emission tomography (FDG-PET).

[0293] Secondary efficacy indicators include:

[0294] a. Graft survival of the transplanted site was determined by MRI (with or without gadolinium-based contrast) and dissection.

[0295] b. Evaluate the effectiveness of immunosuppressive therapy by detecting donor-specific HLA antibodies.

[0296] Patient Information

[0297] Three patients who met the inclusion criteria were selected and provided informed consent. The patient's demographic and clinical information is listed in Table 10, and the results after administration of UCF-E are listed in Table 11.

[0298] One patient received 200 μL of UCF-E intrathecally, and the other two patients received 400 μL of UCF-E intrathecally.

[0299] Table 10: Patient Information

[0300]

[0301]

[0302] mRS: Modified Rankin Scale. NIHSS: NIH (National Institutes of Health) Stroke Scale

[0303] Table 11: UCF-E post-injection assessment

[0304]

[0305] mRS: Modified Rankin Scale. NIHSS: NIH (National Institutes of Health) Stroke Scale

[0306] 7. Ongoing human studies

[0307] The patients described above are part of a larger study, the design of which is described below and is currently ongoing.

[0308] Table 12: UCF-E intrathecal injection

[0309] ID Grouping Dose per injection Total number of injections frequency A(n=2) 200μL 3 30 days / 1 injection B(n=2) 400μL 3 30 days / 1 injection C(n=20) 800μL 3 30 days / 1 injection

[0310] Table 13: UCF-E intravenous injection

[0311] ID Grouping Dose per injection Total number of injections frequency A(n=2) 10mL 3 30 days / 1 injection B(n=2) 20mL 3 30 days / 1 injection C(n=20) 30mL 3 30 days / 1 injection

[0312] A patient who received 3 intrathecal injections (total dose volume 1.5 ml) of exosomes (UCF-E) 4 months after a hemorrhagic stroke regained the ability to walk independently for short distances. Formal evaluation of the patient after exosome injection (UCF-E) was delayed due to Covid restrictions. Video footage was collected and filmed by family members.

[0313] Table 14: UCF intrathecal injection

[0314] ID Grouping Dose per injection Total number of injections frequency A(n=2) 1000μL 3 30 days / 1 injection B(n=2) 2000μL 3 30 days / 1 injection C(n=20) 3000μL 3 30 days / 1 injection

[0315] Table 15: UCF intravenous injection

[0316] ID Grouping Dose per injection Total number of injections frequency A(n=2) 50mL 3 30 days / 1 injection B(n=2) 100mL 3 30 days / 1 injection C(n=20 200mL 3 30 days / 1 injection

[0317] Six patients have been treated with UCF-E (Table 16), of which three of the six are patients described in Table 10. These patients showed good improvement after the injection of UCF-E, and it is expected that patients with higher doses will achieve greater improvement.

[0318] Table 16: Patients treated with intrathecal exosomes (UCF-E) every 14 days

[0319] Grouping Dose per injection Total number of injections frequency A(n=3) 400μL 3 14 days C(n=3) 1600μL 3 14 days

[0320] Table 17: Patients treated with intrathecal exosomes (UCF-E) every 7 days

[0321] Grouping Dose per injection Total number of injections frequency A(n=3) 400μL(Low) 3 7 days B(n=3) 1600μL(High) 3 7 days

[0322] After admission, hematoma puncture and drainage were performed under robot assistance, and rt-PA was injected to dissolve the hematoma. Three days later, most of the hematoma was drained without bleeding and other complications. Seven days after surgery, UCF-E was injected intrathecally. The injection volume was 400 μL or 800 μL, and the injection was repeated on the 14th and 21st days after surgery.

[0323] Table 18: Patient Information

[0324]

[0325] Table 19: Functional evaluation after UCF-E injection

[0326]

[0327]

[0328] All six patients received intrathecal injections of UCF-E, and none of them experienced complications. No significant hemodynamic or pulmonary complications were observed. At the end of follow-up, there were no signs of rebleeding or worsening of hemorrhagic infarction or cerebral hemorrhage. No seizures were observed in any patient. Five patients in the study group developed a low-grade fever (38.5°C) within the first 24 hours after injection. The fever in all five patients subsided within 2 days without the need for specific medical intervention.

[0329] In addition, the effects of an exosome preparation (UCF-E) are being investigated in a rat model of hemorrhagic stroke according to the table below.

[0330] Table 20: Overview of studies on hemorrhagic stroke in rats

[0331] Grouping Dose per injection Total number of injections frequency Control group (n=3) 20μL 3 3 days UCF-E group (n=3) 20μL 3 3 days UCF-E group (n=3) 80μL 3 3 days Sham operation (n=3) - - -

[0332] In another study, brain atrophy was evaluated 36 days after injury. The UCF-E group had significantly less brain atrophy than the saline control group (p = 0:0893; Fig.12 ), indicating that the UCF-E treatment group had a neuroprotective effect.

[0333] 8. Human Case Studies

[0334] Table 21: Patient Information

[0335]

[0336] Treatment Process

[0337] After admission, hematoma puncture and drainage were performed under robot guidance, and rt-PA was injected to dissolve the hematoma. After a follow-up examination three days later, the hematoma was basically drained, and there were no complications such as re-bleeding. UCF-E was administered to the L3-4 intervertebral space by intrathecal injection 7 days after surgery, and the patient was injected with a dose of 1600μL. After releasing an equal amount of cerebrospinal fluid, the exosomes were injected directly into the intrathecal area at a rate of 400μL per minute, and it took a total of 4 minutes to complete the injection. Regular rehabilitation training was then performed.

[0338]

[0339]

Claims

1. A method for treating or ameliorating a neuronal injury symptom in a subject, the method comprising administering to the subject a therapeutically effective amount of a cell-free allogeneic preparation (cell-free human umbilical cord plasma (UCF) containing exosomes or exosomes only from human umbilical cord plasma (UCF-E)), wherein the cell-free allogeneic exosome preparation (UCF or UCF-E) is derived from human umbilical cord blood, wherein the neuronal injury is ischemic brain injury, hemorrhagic brain injury or traumatic brain injury, and wherein UCF or UCF-E is first administered 3 to 90 days after the neuronal injury.

2. The method of claim 1, wherein UCF or UCF-E is first administered 7 to 10 days after neuronal injury.

3. The method of claim 1 or 2, wherein UCF or UCF-E is administered intrathecally.

4. The method of any one of claims 1 to 3, wherein UCF or UCF-E is administered to the subject on a regular basis.

5. The method of claim 4, wherein the administration cycle is weekly, biweekly, triweekly, monthly, or a combination thereof.

6. The method of claim 5, wherein the administration period is every day, every 5 days, every 10 days, every 15 days, every 20 days, every 25 days, about every 30 days, or a combination thereof.

7. The method of any one of claims 1 to 6, wherein the ischemic or hemorrhagic brain injury is a stroke.

8. The method of any one of claims 1 to 7, wherein no immunosuppressant is administered.

9. The method of any one of claims 1 to 8, wherein the cell-free human umbilical cord plasma (UCF) containing exosomes is characterized by levels of one or more of GM-CSF, HGF, MIP-1b (CCL4), and PDGF-bb that are at least 20% higher than the levels found in normal adult human serum.

10. The method of any one of claims 1 to 8, wherein the cell-free allogeneic preparations containing exosomes (UCF) and containing only exosomes (UCF-E) are characterized by IP-10 (CxCL10) levels of at least 60% of the levels found in normal adult human serum.

11. The method of any one of claims 1 to 10, wherein the exosomes in the allogeneic exosome-containing preparation (UCF or UCF-E) comprise at least one of CD63 and TSG101 and lack the lipoprotein markers APOB and APOE.

12. The method of any one of claims 1 to 11, wherein the cell-free allogeneic preparations containing exosomes (UCF and UCF-E) comprise exosomes having a mean, median or mode particle size of about 30-150 nm.

13. The method of any one of claims 1 to 12, wherein the method comprises administering the cell-free allogeneic preparations (UCF and UCF-E) containing exosomes a total of two, three, four, five, six, seven, eight, nine or ten times.

14. The method of any one of claims 1 to 13, wherein the symptom is selected from dystonia, aphasia, infarct size, inflammation, white matter loss, gray matter loss, apoptotic cells in the peri-infarct area, and CD16 / CD32 positive cells in the peri-infarct area.

15. The method of claim 14, wherein administration of the allogeneic formulation containing exosomes (UCF or UCF-E) reduces dystonia in the subject.

16. The method of claim 14, wherein administration of the allogeneic preparations (UCF and UCF-E) containing exosomes reduces aphasia in the subject.

17. The method of claim 14, wherein administration of the allogeneic formulations (UCF and UCF-E) containing exosomes reduces infarct volume in the subject.

18. The method of claim 14, wherein administration of the allogeneic formulations (UCF and UCF-E) containing exosomes reduces systemic inflammation in the subject or reduces inflammation in the infarct area or peri-infarct area of ​​the subject.

19. The method of claim 14, wherein administration of the allogeneic preparations (UCF and UCF-E) containing exosomes reduces the loss of gray matter and / or white matter in the subject.

20. The method of claim 14, wherein administration of the allogeneic formulations (UCF and UCF-E) containing exosomes reduces the number of apoptotic cells in the peri-infarct area of ​​the subject.

21. The method of claim 14, wherein administration of the allogeneic preparations (UCF and UCF-E) containing exosomes reduces the number of CD16 / CD32 positive cells in the peri-infarct area of ​​the subject.

22. The method of any one of claims 1 to 21, wherein the method further comprises administering an allogeneic preparation containing exosomes (UCF and UCF-E) and at least one additional therapeutic agent.

23. The method of claim 22, wherein the additional therapeutic agent is an anti-inflammatory agent, a steroid, dipyridamole, astaxanthin, dabigatran etexilate, losartan, nimodipine, prilocaol, rivaroxaban, or ticlopidine.

24. The method of claim 22 or 23, wherein the additional therapeutic agent is mannitol, intracarotid hyperosmotic mannitol (ICAHM), RMP-7, or regadenoson.

25. A method for improving the mRS score or NIHSS score of a subject with ischemic brain injury, the method comprising administering to the subject a therapeutically effective amount of a cell-free allogeneic preparation (UCF or UCF-E) containing exosomes, wherein the cell-free allogeneic preparation (UCF or UCF-E) is derived from pooled human umbilical cord blood, and wherein UCF or UCF-E is first administered 3 to 90 days after ischemic brain injury.

26. The method of claim 25, wherein UCF or UCF-E is first administered 7 to 10 days after the ischemic brain injury.

27. The method of claim 25 or 26, wherein no immunosuppressant is administered.

28. The method of any one of claims 25 to 27, wherein the subject is periodically administered an allogeneic preparation containing exosomes (UCF or UCF-E).

29. The method of claim 28, wherein the administration period is weekly, biweekly, triweekly, monthly, or a combination thereof.

30. The method of claim 29, wherein the administration period is every day, every 5 days, every 10 days, every 15 days, every 20 days, every 25 days, about every 30 days, or a combination thereof.

31. The method of any one of claims 25 to 30, wherein the method comprises administering the cell-free allogeneic preparation containing exosomes (UCF or UCF-E) a total of two, three, four, five, six, seven, eight, nine or ten times.

32. The method of any one of claims 1 to 31, wherein the UCF or UCF-E is derived from a single umbilical cord blood, or a pool of blood from multiple umbilical cords.

33. The method of claim 32, wherein the mixed blood is from umbilical cords having the same blood type or from umbilical cords having different blood types.

34. Use of a cell-free allogeneic preparation containing exosomes derived from pooled human umbilical cord blood (UCF or UCF-E) in the preparation of a medicament for treating or ameliorating symptoms of neuronal injury, wherein the neuronal injury is ischemic brain injury, hemorrhagic brain injury or traumatic brain injury, and wherein the medicament is first administered 3 to 90 days after the neuronal injury.

35. Use of a cell-free allogeneic preparation containing exosomes (UCF or UCF-E) derived from mixed human umbilical cord blood in the preparation of a drug for improving the mRS score or NIHSS score of a subject with ischemic brain injury, wherein: The drug is first administered 3 to 90 days after ischemic brain injury.

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