Composition for alleviating neurological diseases comprising neural crest cell-derived exosomes containing nerve growth factor

Abstract

The present invention relates to a technology for developing a next-generation extracellular vesicle-based therapeutic agent, and relates to a composition for alleviating neurological diseases, comprising exosomes derived from neural crest cells. When the extracellular vesicles of the present invention are used, neuropathy can be effectively prevented or treated.

Description

Composition for improving neurological diseases containing exosomes containing nerve growth factor derived from neural crest cells

[0001] The present invention was carried out under the support of the Ministry of Health and Welfare and the Ministry of Science and ICT of the Republic of Korea under project number 2710003628 and sub-project number 00331850, the specialized research management agency for the above project is the Korea Regenerative Medicine Technology Development Center, the research project name is "Korea Regenerative Medicine Technology Development Center (Ministry of Health and Welfare, Ministry of Science and ICT)," the research project title is "Advancement of Endogenous Stem Cell Activation Platform Based on Immunomodulation and Tissue Regeneration Enhancement for Treatment of Intractable Neuropathies and Development of Source Technology for Inducing Factor Delivery," the lead institution is the Industry-Academic Cooperation Foundation of Konkuk University, and the research period is April 1, 2024 - December 31, 2026.

[0002] The present invention was carried out under project number 2025-RISE-01-001-01 with the support of the Ministry of Education of the Republic of Korea, the research management agency for the said project is the Seoul RISE Center, the research project name is "Seoul Regional Innovation Center University Support System (RISE)", the research task name is "Composition for treating neuropathy containing exosomes derived from neural crest cells", the lead institution is Konkuk University (RISE Project Group), and the research period is 2025.06.01-2026.02.28.

[0003] This patent application claims priority to Korean Patent Application No. 10-2024-0133348 filed with the Korean Intellectual Property Office on September 30, 2024, the disclosures of said patent application are incorporated herein by reference.

[0004] The present invention relates to a technology for developing next-generation extracellular vesicle-based therapeutic agents, and to a composition for improving neurological diseases comprising exosomes rich in neurogrowth factors derived from neural crest cells.

[0005]

[0006] Exosomes are microvesicles with a lipid bilayer structure secreted from various eukaryotic cells, including humans, animals, insects, plants, and microorganisms. They have a nano-sized particle size (approximately 30–150 nm) and perform the function of intercellular information transmission while stably containing various physiologically active substances such as proteins, nucleic acids, lipids, and carbohydrates. In particular, stem cell-derived exosomes contain numerous key factors related to anti-inflammatory and tissue regeneration functions, and are attracting attention as promising bio-derived therapeutic agents for regenerative medicine and the treatment of intractable diseases.

[0007] Existing stem cell-based therapies involve directly transplanting living cells into the lesion site, which has several limitations, such as cell procurement, quality maintenance, and immune response induction. In contrast, stem cell-derived exosomes can partially replicate the therapeutic function of stem cells without the need for cells, making them a promising new therapeutic strategy that can overcome the shortcomings of cell therapies. However, since the number of exosomes typically secreted per nucleated cell is only around 500 to 1,000, securing a large quantity of high-purity exosomes required for treatment remains a technical challenge.

[0008]

[0009] The inventors have made diligent research efforts to develop a substance effective against neuropathy. As a result, they have completed the present invention by identifying that using extracellular vesicles derived from neural crest cells is effective in preventing or treating various neurological diseases, such as stroke and neuropathic pain, by regulating the inflammatory response of nerve cells and inducing neuroprotective and regenerative effects.

[0010] Therefore, the object of the present invention is to provide an extracellular vesicle with enhanced expression of nerve growth factor (NGF).

[0011] Another objective of the present invention is to provide a method for manufacturing extracellular vesicles.

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neuropathy comprising the extracellular vesicles.

[0013] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0014]

[0015] The present invention provides the inventions of 1 to 20 below.

[0016] 1. Extracellular vesicle with enhanced expression of nerve growth factor (NGF).

[0017] 2. The extracellular vesicle of 1, wherein the extracellular vesicle expresses a protein of SOX10, HNK1, PAX6, AP2α, BAF1, or a combination thereof.

[0018] 3. An extracellular vesicle in which, in 1 or 2, the average diameter of the extracellular vesicle is 50 to 300 nm.

[0019] 4. An extracellular vesicle having characteristics of promoting neuronal differentiation, regulating inflammatory response, inhibiting apoptosis, or a combination thereof, in any one of 1 to 3.

[0020] 5. An extracellular vesicle derived from a neural crest cell in any one of 1 to 4.

[0021] 6. In any one of 1 to 5, the extracellular vesicles wherein the neural crest cells have enhanced expression of nerve growth factor (NGF).

[0022] 7. An extracellular vesicle in any one of 1 to 6, wherein the neural crest cell is differentiated from a human adult stem cell (hASC), a human embryonic stem cell (hESCs), or a human induced pluripotent stem cell.

[0023] 8. An extracellular vesicle, wherein in any one of 1 to 7, the induced pluripotent stem cells are induced pluripotent stem cells derived from urine cells.

[0024] 9. An extracellular vesicle that is absorbed by microglia, neuroblastoma, or a combination thereof, in any one of 1 to 8.

[0025] 10. A method for preparing an extracellular vesicle comprising the following steps:

[0026] (a) a step of dedifferentiating human induced pluripotent stem cells from urine stem cells;

[0027] (b) a step of differentiating neural crest cells from human induced pluripotent stem cells; and

[0028] (c) A step of obtaining extracellular vesicles from the culture medium.

[0029] 11. A method for preparing an extracellular vesicle according to 10, further comprising the step of transfecting human induced pluripotent stem cells after step (a) to overexpress a neurotrophic factor selected from the group consisting of the following:

[0030] Nerve growth factor (NGF), Brain-Derived Neurotrophic Factor, Glial cell-the-line-Derived Neurotrophic Factor, Neurotrophin-3, Ciliary Neurotrophic Factor, and combinations thereof.

[0031] 12. A method for manufacturing extracellular vesicles, wherein, in 10 or 11, the manufacturing method has an improved amount of extracellular vesicle secretion.

[0032] 13. A method for manufacturing an extracellular vesicle, wherein, in any one of 10 to 12, the extracellular vesicle maintains the characteristics of a neural crest cell.

[0033] 14. A pharmaceutical composition for the prevention or treatment of neuropathy comprising any one of the extracellular vesicles of 10 to 13.

[0034] 15. A pharmaceutical composition for the prevention or treatment of neuropathy according to 14, wherein the neuropathy is peripheral neuropathy, diabetic neuropathy, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, sciatic neuropathy, neuropathy due to cervical or lumbar disc herniation, entrapment neuropathy, inflammatory neuropathy, secondary neuropathy due to central nervous system injury, cancer-related neuropathy, or a combination thereof.

[0035] 16. A pharmaceutical composition for the prevention or treatment of neuropathy, wherein, in 15, the neuropathy is neuropathy caused by physical injury.

[0036] 17. A method for the prevention or treatment of neuropathy comprising the step of administering any one of 1 to 9 extracellular vesicles, or any one of 14 to 16 pharmaceutical compositions, to a subject requiring treatment.

[0037] 18. A method for the prevention or treatment of neuropathy in which, in paragraph 17, the neuropathy is a neuropathy caused by physical injury.

[0038] 19. Use of any one of 1 to 9 extracellular vesicles, or any one of 14 to 16 pharmaceutical compositions for the prevention or treatment of neuropathy.

[0039] 20. Use in manufacturing a drug for the prevention or treatment of neuropathy, wherein the neuropathy in 19 is a neuropathy caused by physical injury.

[0040] In one embodiment of the present invention, an extracellular vesicle with enhanced expression of nerve growth factor (NGF) is provided.

[0041] The inventors have made diligent research efforts to develop a substance effective against neuropathy. As a result, it has been discovered that using extracellular vesicles derived from neural crest cells is effective in preventing or treating various neurological diseases, such as stroke and neuropathic pain, by regulating the inflammatory response of nerve cells and inducing neuroprotective and regenerative effects.

[0042] The term "extracellular vesicle" in the present invention refers to a microvesicle with a lipid bilayer structure secreted from a eukaryotic cell, which performs physiological functions such as mediating intercellular signal transmission and contains or possesses on its surface various biomolecules such as proteins, lipids, nucleic acids, and sugars. Extracellular vesicles generally have a diameter ranging from 30 nm to several hundred nm. The extracellular vesicles may be, for example, exosomes, microvesicles, or apoptotic bodies, but are not limited thereto, and in this specification, these are collectively referred to as "extracellular vesicles."

[0043] The term "nerve growth factor (NGF)" in this invention refers to a representative neurotrophic factor, a protein that plays a key role in the survival, growth, differentiation, and regeneration of nerve cells. NGF is primarily involved in the growth and maintenance of neurons in the central and peripheral nervous systems, and also influences the formation of neuronal axons and the regulation of synaptic function. Furthermore, NGF exhibits physiological activities that induce regeneration after nerve injury and regulate inflammatory responses, and is attracting attention as a potential therapeutic target for the treatment of various neurological diseases.

[0044] In the present invention, "extracellular vesicles with enhanced expression of nerve growth factor" refers to extracellular vesicles secreted from cells that express nerve growth factor at a high level, and are characterized by containing NGF protein or a bioactive factor derived therefrom at a relatively high concentration within said extracellular vesicles. At this time, said nerve growth factor may be encapsulated inside the extracellular vesicle, surface-displayed on the surface membrane of the extracellular vesicle, or may include both of these forms.

[0045] Such enhancement of expression can be induced, for example, through gene introduction technology that overexpresses the NGF gene in cells, and accordingly, extracellular vesicles derived from such cells can exhibit superior neurodifferentiation-promoting effects, neuroprotective effects, and anti-inflammatory effects compared to general extracellular vesicles.

[0046] Therefore, the extracellular vesicle of the present invention can effectively deliver various biological activities related to nerve regeneration mediated by NGF by enhancing NGF expression, and this can be utilized as a practical therapeutic means for the prevention or treatment of nerve damage and neurodegenerative diseases.

[0047] The extracellular vesicle of the present invention is p75 NTR It is derived from a cell expressing [the receptor], characterized in that the receptor exists as a surface or intrinsic protein of the EV. Such an EV, through the expressed p75 NTR, enables specific interaction with neurons, enables receptor-ligand-based targeted delivery, and can mediate biological effects such as promoting neuronal differentiation, repairing neuronal damage, and suppressing inflammation.

[0048] The term "p75 neurotrophin receptor (NTR)" in this specification refers to a common receptor for neurotrophin that can bind to ligands such as NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3, and NT-4 / 5, and is known to regulate a wide range of physiological responses, unlike Trk receptors. In particular, p75 NTR mediates various signaling pathways in neurogenesis, ranging from axon guidance to cell survival or apoptosis, and plays a key role in the growth, differentiation, and regeneration of neurons.

[0049] p75 NTR is widely expressed during embryonic development, but in adults, it is selectively expressed primarily in neural crest cell (NCC)-derived cells or around damaged neurons, and is involved in the regulation of neuroregeneration and inflammatory responses. Due to these biological characteristics, p75 NTR can be utilized as a marker for cells or their derived components that possess the ability to differentiate or regenerate neurons.

[0050] The presence and relative expression levels of surface markers expressed in the extracellular vesicles can generally be confirmed through Western blot, flow cytometry, ELISA, or nanoparticle-based immunocapture assays. Among these, Western blot analysis is widely used to qualitatively and quantitatively evaluate the presence of surface markers by detecting proteins present on the EV surface using antibodies. Additionally, by utilizing single-particle level immunoassay-based EV profiling technologies such as the ExoView® platform, the presence or absence of multiple surface markers can be quantitatively analyzed at the EV level.

[0051] In one embodiment of the present invention, the extracellular vesicle expresses a protein of SOX10, HNK1, PAX6, AP2α, BAF1, or a combination thereof.

[0052] The aforementioned proteins are primarily known as characteristic markers of neural crest cells (NCCs), which are a group of pluripotent cells that differentiate into various cell lineages during embryonic development; in particular, they are intermediate stem cells that differentiate into the peripheral nervous system, glial cells, melanocytes, cartilage, and some smooth muscle.

[0053] The term "SOX10 (SRY-box transcription factor 10)" in this specification is a transcription factor that is critical to the maintenance and differentiation of neural crest cells and is involved in the differentiation process into various cell lineages, such as melanocytes, as well as supporting cells of the peripheral nervous system, such as Schwann cells and satellite cells. The expression of SOX10 is considered a representative indicator of the identity of NCC-derived cells.

[0054] The term "HNK1 (Human Natural Killer-1)" in this specification refers to a cell surface antigen of a sugar chain structure, expressed particularly in early NCC and premyelinated Schwann cells. The presence of HNK1 serves as a marker indicating that the cell is in an undifferentiated NCC state.

[0055] The term "PAX6 (Paired box 6)" in this specification refers to a transcription factor that regulates the development of neuroectodermal-derived tissues and is closely related to the development of the eyes, spinal cord, and brain. It is expressed in some NCC-derived cells and can be interpreted as an indicator reflecting cell differentiation potential and neural differentiation pathways.

[0056] The term "AP2α (Activator protein 2 alpha)" in this specification refers to a transcriptional regulatory protein involved in the development of neural crest cells and epidermal cells, and is particularly important for regulating the development and motility of cranial NCCs. AP2α expression suggests that NCCs have the ability to differentiate into epidermal-derived tissues or peripheral nervous system structures.

[0057] The term "BAF1 (Brahma-related gene 1-associated factor 1)" in this specification is a component of the SWI / SNF complex, a chromatin reorganization complex, and plays an important role in determining cell fate and regulating transcription. During the NCC differentiation process, BAF1 influences the maintenance of cell pluripotency and the selection of differentiation pathways.

[0058] In this way, the extracellular vesicle of the present invention, by expressing one or more of the above proteins, complements the specificity of cell origin that is difficult to clearly identify with simple p75 NTR expression alone, and enables differentiated biological activity as an NCC-derived EV in physiological functions of EV, such as promoting nerve regeneration, regulating anti-inflammatory responses, and recovering peripheral nerve damage.

[0059] Furthermore, when these marker proteins are delivered by EVs, they induce genetic and epigenetic regulatory mechanisms in recipient cells, thereby further enhancing the effects of improving the cellular environment and inducing regeneration in target tissues for therapeutic applications.

[0060] Therefore, extracellular vesicles expressing the aforementioned marker protein are highly useful for implementing more precise and efficient extracellular vesicle-based therapeutics for various indications, such as neuropathic diseases, nerve damage, and inflammatory neurological diseases.

[0061] In one embodiment of the present invention, the average diameter of the extracellular vesicle is 50 to 300 nm.

[0062] According to one embodiment of the present invention, the extracellular vesicle has the characteristic of having an average diameter of 50 to 300 nm. Here, "average diameter" refers to the diameter corresponding to the central tendency value (arithmetic mean or mode value) among the size distributions of all particles as a result of quantitatively analyzing the particle size within the prepared extracellular vesicle sample. Generally, extracellular vesicles are nanometer-sized vesicles surrounded by a lipid bilayer, and their size can vary widely depending on cell origin, culture conditions, acquisition method, etc., and these size characteristics have a significant impact on the in vivo mobility, tissue penetration, target specificity, and physiological activity of the extracellular vesicles.

[0063] The diameter of the extracellular vesicles of the present invention was measured using the Nanoparticle Tracking Analysis (NTA) method. NTA analysis is a technique that irradiates a sample with a laser to track the Brownian motion of nanoparticles in real time and quantifies particle size and concentration based on the tracked trajectory information, providing high precision and reproducibility in the size analysis of extracellular vesicles.

[0064] The average diameter of the extracellular vesicles may be, for example, 50 to 300 nm, 50 to 270 nm, 50 to 240 nm, 50 to 210 nm, 50 to 180 nm, 50 to 150 nm, 50 to 120 nm, 50 to 90 nm, 50 to 60 nm, 80 to 300 nm, 110 to 300 nm, 140 to 300 nm, 170 to 300 nm, 200 to 300 nm, 230 to 300 nm, 260 to 300 nm, 290 to 300 nm, 100 to 200 nm, or 120 to 180 nm, but is not limited thereto.

[0065] In one embodiment of the present invention, the extracellular vesicle has characteristics of promoting neurogenesis, regulating inflammatory responses, inhibiting apoptosis, or a combination thereof. These characteristics suggest that the extracellular vesicle of the present invention can be utilized as a therapeutic factor with the potential to treat various diseases, going beyond being a simple intercellular signaling mediator.

[0066] The term "neurodifferentiation promotion" in this specification refers to the ability to have a positive effect on the process of differentiation of stem cells or neural progenitor cells into mature neurons or nerve cells. In the embodiments of the present invention, the induced extracellular vesicles were shown to increase the expression of neuronal-specific markers MAP2 and Tuj1, which demonstrates that the extracellular vesicles possess physiological activity that induces or promotes neuronal differentiation.

[0067] The term "regulation of inflammatory response" in this specification refers to a function of regulating the immune environment in a way that alleviates excessive or chronic inflammatory responses and maintains tissue homeostasis. The extracellular vesicles of the present invention have experimentally shown effects of suppressing the expression of inflammation-related cytokines and increasing the expression of anti-inflammatory cytokines (e.g., IL-10). These characteristics support the possibility of therapeutic applications in inflammatory diseases, autoimmune diseases, etc.

[0068] The term "inhibition of apoptosis" in this specification refers to the ability to inhibit programmed cell death induced by external stimuli or endogenous factors. It was confirmed that under conditions treated with the extracellular vesicles of the present invention, cell viability is increased to prevent tissue damage and maximize regenerative effects.

[0069] These complex biological functions suggest the potential of the extracellular vesicles of the present invention as important therapeutic agents capable of expecting broad therapeutic effects in various diseases, particularly neurological diseases, inflammatory diseases, and ischemic injuries.

[0070] In one embodiment of the present invention, the extracellular vesicle is derived from a neural crest cell.

[0071] The term "neural crest cell" in this specification is defined as a population of multipotent stem cells that arise at the dorsal edge of the neural tube during the formation of the neural tube in vertebrate development. These cells exist temporarily and possess very high motility and differentiation ability, moving to various tissues throughout the body to differentiate into various cell types.

[0072] NCCs can differentiate into various cells, such as neurons and Schwann cells of the peripheral nervous system, ganglion cells and melanocytes of the autonomic nervous system, chromaffin cells of the adrenal medulla, and parts of mesenchymal structures including cardiac muscle and cartilage. This diversity of cell fate is attributed to the multipotency and microenvironment sensing capabilities of NCCs, and they play important roles in various physiological phenomena such as tissue regeneration, damage recovery, and inflammation regulation.

[0073] The extracellular vesicles provided in the present invention are secreted and isolated from neural crest cells derived from UiPSCs (human urine-derived induced pluripotent stem cells). Such NCC-derived EVs reflect the physiological characteristics of the parent cell, NCC, and can possess the ability to induce neuronal regeneration, anti-inflammatory mediating functions, and target tissue specificity.

[0074] In the present invention, the phrase "extracellular vesicles are derived from neural crest cells" means that said extracellular vesicles are obtained by culturing neural crest cells and isolating and purifying them from the corresponding culture medium. That is, the extracellular vesicles are nano-sized vesicles released through the physiological secretion process of neural crest cells, and their constituent components (surface proteins, miRNA, lipids, etc.) are closely related to the biological characteristics of the parent cell.

[0075] In one embodiment of the present invention, the extracellular vesicle is derived from a neural crest cell.

[0076] NGF is a representative neurotrophic factor that plays a key role in inducing the survival, growth, differentiation, and regeneration of nerve cells. In the present invention, a gene introduction technique using a lentiviral vector was applied to overexpress NGF in neural crest cells, and as a result, it was confirmed that extracellular vesicles secreted from these cells exhibited enhanced physiological activity.

[0077] Specifically, according to the examples, NGF-overexpressing neural crest cell-derived EVs were proven to simultaneously exhibit anti-inflammatory effects and neuronal growth-promoting effects in an inflammatory environment. When the EVs of the present invention were treated under inflammatory conditions induced by LPS treatment of mouse microglia (BV2), the secretion of TNF-α and IL-6 was significantly reduced. This suggests that the EVs may contribute to the alleviation of inflammation by delivering anti-inflammatory cytokines or inhibiting inflammatory signaling pathways. Furthermore, when the EVs of the present invention were treated to SH-SY5y cells, a significant increase in neurite length was observed, which suggests that the NGF protein or NGF-related signaling factors contained in the EVs were involved in the differentiation of neurons and the promotion of axonal growth.

[0078] In one embodiment of the present invention, the neural crest cell is differentiated from a human adult stem cell (hASC), a human embryonic stem cell (hESC), or a human induced pluripotent stem cell.

[0079] In one embodiment of the present invention, the induced pluripotent stem cell is a human urine-derived induced pluripotent stem cell (hUiPSC).

[0080] The term "human urine cell-derived induced pluripotent stem cells" in this specification refers to stem cells derived from urine cells obtainable by non-invasive methods, which have acquired pluripotency through gene introduction. UiPSCs have the advantages of being easy to collect, having a low risk of immunological rejection, and being suitable for autologous cell-based regenerative medicine. By efficiently differentiating these UiPSCs into neural crest cells, NCCs with physiological functions can be stably obtained, and the extracellular vesicles derived therefrom simultaneously provide advantages such as high safety, reproducibility, and traceability of cell origin.

[0081] Consequently, the extracellular vesicles of the present invention are derived from neural crest cells with enhanced NGF expression, and since these neural crest cells are differentiated from human urine-derived iPSCs, they can be utilized as high-functional EVs capable of inducing various therapeutic effects such as nerve regeneration, anti-inflammation, and tissue repair. Furthermore, as such cell sources possess substantial advantages including the possibility of non-invasive acquisition and immunological safety, the EVs of the present invention can be considered next-generation extracellular vesicle-based therapeutics with very high potential for clinical application.

[0082] In one embodiment of the present invention, the extracellular vesicle is absorbed by microglia, neuroblastoma, or a combination thereof.

[0083] The absorption efficiency of extracellular vesicles (EVs) by specific recipient cells varies depending on their surface proteins, lipid composition, size, etc., and this is one of the key factors determining the biological activity of EVs. The extracellular vesicle of the present invention is derived from neural crest cells (NCCs), and p75 NTR It contains various neuro-related proteins such as SOX10 and NGF, and this composition can induce a high intracellular uptake rate for specific neuronal cells.

[0084] According to the examples, the EV of the present invention was fluorescently stained (DiL labeling) and treated with microglia (BV2) or neuroblastoma cell lines (SH-SY5y, etc.), after which the uptake rate was measured through fluorescence microscopy and flow cytometry (FACS). As a result, the EV of the present invention exhibited significantly higher intracellular fluorescence signal intensity in microglia and neuroblastoma cells compared to the control EV, suggesting that the cells effectively internalize the EV of the present invention.

[0085] These high uptake rates in target cells have technical and therapeutic implications, such as therapeutic targeting effects for neurological diseases, potential as a drug delivery platform, and improved therapeutic efficiency based on cell targeting:

[0086] Consequently, the EV of the present invention can provide significant technical advantages in precise cell-targeted therapeutic strategies for neuroinflammatory diseases, neurotumors, and the recovery of nervous system damage through interaction with microglia and neuroblastoma cells.

[0087]

[0088] In one aspect of the present invention, the present invention provides a method for producing an extracellular vesicle comprising the following steps:

[0089] (a) a step of dedifferentiating human induced pluripotent stem cells from urine stem cells;

[0090] (b) a step of differentiating neural crest cells from human induced pluripotent stem cells; and

[0091] (c) A step of obtaining extracellular vesicles from the culture medium.

[0092] Step (a) above is a process of inducing human induced pluripotent stem cells using urinary epithelial cells aseptically collected from a healthy donor. Specifically, urinary epithelial cells are obtained by centrifugation and washing, and then attached to a suitable medium (e.g., RE / MCDB medium) and cultured. Subsequently, a pluripotency factor expression plasmid containing Yamanaka factors (e.g., Oct4, Sox2, Klf4, c-Myc) is introduced using a method such as Sendai virus or an episomal vector, and iPSC colonies are induced by culturing for approximately 14 to 21 days. The induced iPSCs are verified to have undergone normal reprogramming by confirming the expression of pluripotency markers (e.g., Nanog, Oct4, SSEA4) through alkaline phosphatase (AP) staining, immunofluorescence (IHC) analysis, etc. However, this is not limited to any method, and any reprogramming method available to a person skilled in the art may be used.

[0093] Step (b) above is a step of differentiating induced iPSCs into neural crest cells. In this step, iPSCs can be induced into the neural crest lineage using, for example, an initial differentiation induction medium (e.g., neural induction medium). For differentiation induction, cells are cultured for about 5 to 7 days using a conditional medium containing a Wnt signaling pathway activator (e.g., CHIR99021), a BMP inhibitor (e.g., Noggin), and other growth factors (such as TGF-β inhibitors), and subsequently, changes in cell morphology and the expression of neural crest cell markers are observed. The cells take on a typical NCC morphology (bipolar, radial arrangement), and the completion of differentiation is determined by confirming the expression of NCC-specific markers such as SOX10, HNK1, and p75 NTR through immunofluorescence staining. However, this is not limited to this, and any differentiation method available to a person skilled in the art may be used.

[0094] Step (c) above is a step of obtaining extracellular vesicles from differentiated neural crest cells. Specifically, the supernatant is collected after culture and centrifuged at low speed (e.g., 300 xg, 10 min) and medium speed (2,000 xg, 20 min) to remove cell debris and foreign substances, and then centrifuged at high speed (25,000 rpm, 2 hours, 4°C) to precipitate the extracellular vesicles. This precipitate can be resuspended in PBS and purified through ultrafiltration or size exclusion chromatography, and the presence and characteristics of the extracellular vesicles can be confirmed through BCA assay, NTA (nanoparticle tracking analysis), TEM (transmission electron microscopy), Western blot (analysis of EV markers such as CD63, CD81, TSG101, etc.).

[0095] Therefore, the manufacturing method of the present invention is a process that uses urine-derived cells with high accessibility and safety as a starting point to induce neural crest cells in an efficient and reproducible manner, and can stably produce extracellular vesicles with enhanced physiological activity from them, and can be very usefully applied to the development of nerve regeneration and anti-inflammatory therapeutic agents.

[0096] In one embodiment of the present invention, the manufacturing method further comprises the step of transfecting to overexpress a neurotrophic factor selected from the group following step (a):

[0097] Nerve growth factor (NGF), Brain-Derived Neurotrophic Factor, Glial cell-the-line-Derived Neurotrophic Factor, Neurotrophin-3, Ciliary Neurotrophic Factor, and combinations thereof.

[0098] In this invention, to maximize the biological activity of NGF at the extracellular vesicle level, the NGF gene was introduced into iPSCs, which are the stage prior to differentiation into neural crest cells, thereby enhancing the function of downstream cell lineages and EVs derived therefrom.

[0099] At this time, the recombinant expression vector for introducing the above gene is not particularly limited, and various vectors commonly used in the art may be used. For example, the recombinant expression vector may be selected from the group consisting of non-viral vectors such as plasmid vectors, cosmid vectors, and bacteriophage vectors, or viral vectors such as adenovirus vectors, lentivirus vectors, retrovirus vectors, and adeno-associated virus (AAV) vectors. Specifically, the vector that can be used in the present invention may be constructed based on plasmids (e.g., FBPK plasmid, epizomyel plasmid, etc.) or viral vectors (e.g., adeno-associated virus vectors, lentivirus vectors, etc.) widely used in the art, but is not limited thereto.

[0100] Specifically, after human urine-derived iPSCs are induced, transfection is performed on the iPSCs using a lentiviral vector (LV-oe-NGF) in which the NGF gene is cloned. At this time, LV-oe-NGF or a control vector (LV-con) is co-infected with a packaging plasmid (gag, pol, rev, etc.) using PolyJet reagent (SignaGen Laboratories LLC). Infection is performed in serum-free medium or under conditions containing a transduction promoter, and stabilization culture is carried out for approximately 48 to 72 hours after infection. After the culture is complete, the virus is removed or diluted from the supernatant, and the iPSCs are induced to differentiate into neural crest cells while maintaining an overexpression of NGF.

[0101] In this way, by overexpressing NGF at the iPSC level, the subsequently induced neural crest cells also express high levels of NGF, and the extracellular vesicles secreted from them are characterized by being rich in NGF or containing large amounts of activating factors involved in NGF signaling.

[0102] In one embodiment of the present invention, the manufacturing method has an improved amount of extracellular vesicle secretion.

[0103] Generally, EVs are naturally generated by the secretory mechanism of cells, and their secretion amount can vary significantly depending on factors such as culture conditions, cell state, medium composition, and mechanical stimulation. In order to maximize the efficiency of obtaining extracellular vesicles, the present invention applies an advanced culture system in which human induced pluripotent stem cells are dedifferentiated from urine stem cells and neural crest cells are differentiated therefrom to obtain extracellular vesicles.

[0104] In other words, the method for manufacturing extracellular vesicles according to the present invention has technical significance in that it realizes a high-efficiency production platform capable of effectively producing EVs of high yield and quality by providing structural and environmental conditions that can significantly increase the secretion amount of extracellular vesicles.

[0105] In one embodiment of the present invention, the extracellular vesicle in the manufacturing method retains the characteristics of a neural crest cell.

[0106] In this specification, the term "maintains the characteristics of neural crest cells" means that the biological characteristics originally possessed by the said neural crest cells, such as immunomodulatory ability, regenerative ability, anti-inflammatory properties, and the ability to secrete various physiologically active factors, are reflected and delivered as is in the manufactured extracellular vesicles.

[0107] Therefore, the manufacturing method of the present invention possesses technical excellence in that it can maximize the extracellular vesicle secretion ability of neural crest cells while simultaneously obtaining EVs that reflect the unique therapeutic functions of neural crest cells. Through this, the extracellular vesicles of the present invention can be effectively applied as an alternative to neural crest cell therapy for the prevention or treatment of various diseases.

[0108]

[0109] In one aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of neuropathy comprising the extracellular vesicle.

[0110] In this specification, the term “prevention” means the prevention or protective treatment of a disease or diseased state. In this specification, the term “treatment” means the reduction, suppression, soothing, or eradication of a diseased state.

[0111] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0112] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0113] The pharmaceutical composition of the present invention may be administered orally or parenterally, for example by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, intratumoral injection, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0114] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the composition of the present invention is 0.0001-100 mg / kg.

[0115] The term "neuropathy" in this specification refers to a group of diseases encompassing various neurological syndromes resulting from structural damage or functional abnormalities of the peripheral or central nervous systems. These neuropathy is primarily caused by nerve damage, inflammation, compression, degenerative changes, or metabolic abnormalities, and representative symptoms include neuropathic pain, numbness, decreased sensation, abnormal sensation, muscle weakness, decreased reflexes, and autonomic dysfunction.

[0116] In particular, neuropathy is a disease in which the therapeutic effect of conventional analgesics or anti-inflammatory drugs is often limited, and depending on the cause, it tends to become chronic and can severely impair the quality of life. Therefore, a treatment strategy capable of inducing recovery at a more fundamental cellular level is required.

[0117] The extracellular vesicles of the present invention possess characteristics exhibiting various physiological functions, such as promoting neuronal differentiation, regulating inflammatory responses, inhibiting apoptosis, and promoting angiogenesis; these characteristics can contribute to the structural restoration and functional recovery of damaged nerves. Furthermore, the extracellular vesicles of the present invention can alleviate the chronic inflammatory state associated with neuropathy through immunomodulatory action and induce the survival and regeneration of nerve cells by delivering neurotrophic factors or neuroregeneration-related microRNAs.

[0118] Therefore, the extracellular vesicles of the present invention can be effectively applied to the prevention or treatment of neuropathy through pain relief, inhibition of inflammation, neuroprotection, and induction of regeneration, and based on such mechanisms, provide the potential to be utilized as a therapeutic agent in patients with various types of neuropathy.

[0119] In one embodiment of the present invention, the neuropathy is peripheral neuropathy, diabetic neuropathy, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, sciatic neuropathy, neuropathy due to cervical or lumbar disc herniation, entrapment neuropathy, inflammatory neuropathy, secondary neuropathy due to CNS injury, cancer-related neuropathy, or a combination thereof.

[0120] The pharmaceutical composition of the present invention can be used for the prevention or treatment of neuropathy caused by various causes as described above. In one embodiment of the present invention, the neuropathy is peripheral neuropathy caused by physical injury. The extracellular vesicles of the present invention exhibit excellent effects, particularly on diseases such as peripheral neuropathy caused by accidents, injuries, or physical compression, such as sciatic neuropathy.

[0121] These physical injury-based peripheral neuropathyes differ from other types of neuropathy in their causes and pathological mechanisms. For example, diabetic neuropathy involves the gradual damage of nerves caused by metabolic abnormalities and oxidative stress resulting from hyperglycemia, whereas chemotherapy-induced neuropathy (CIPN) is primarily caused by the direct damage to nerve cells caused by the toxicity of anticancer drugs. Additionally, in post-herpetic neuralgia, viral infection is the cause of nerve damage.

[0122] In contrast, neuropathy caused by physical injury, such as sciatic nerve compression, is characterized by the acute occurrence of direct axon destruction and myelin sheath loss due to mechanical stress applied to a specific area, which induces a strong inflammatory response and apoptosis at the site of injury. Conventional treatments have primarily focused on pain control and inflammation relief using non-steroidal anti-inflammatory drugs (NSAIDs), steroids, and anticonvulsants, which has clearly limited their ability to fundamentally induce structural regeneration or functional recovery of the damaged nerve.

[0123] However, the extracellular vesicles of the present invention, in particular the extracellular vesicles derived from neural crest cells overexpressing nerve growth factor (NGF) (NCCoe-NGF-EV), act comprehensively on the multifaceted pathological mechanisms of such physical injury-based neuropathy and exhibit excellent therapeutic effects. According to an embodiment of the present invention, in an animal model of sciatic nerve compression injury, the composition of the present invention was confirmed to exhibit complex effects such as promoting nerve regeneration and remyelination, strong anti-inflammatory effects, inhibition of neuronal cell death, and prevention of muscle atrophy.

[0124] In conclusion, the composition of the present invention enables the fundamental treatment of peripheral neuropathy caused by physical damage through multiple target mechanisms of regeneration of damaged nerves, regulation of inflammation, and cell protection, going beyond simple symptom relief, thereby providing an innovative treatment strategy that can overcome the limitations of existing treatments.

[0125]

[0126] In one aspect of the present invention, the present invention provides a method for preventing or treating neuropathy, comprising the step of administering the extracellular vesicle or a pharmaceutical composition comprising the extracellular vesicle to a subject requiring prevention or treatment.

[0127] In one aspect of the present invention, the present invention provides a use in the manufacture of a pharmaceutical composition comprising the extracellular vesicle or the extracellular body for the prevention or treatment of neuropathy.

[0128] The above-mentioned method for the prevention or treatment of neuropathy, or the use in the manufacture of a drug for the prevention or treatment of neuropathy, is common to the extracellular vesicles and compositions described above in that they include extracellular vesicles or compositions containing the same; therefore, to prevent excessive duplication of descriptions in the specification, descriptions of common contents are omitted.

[0129]

[0130] The features and advantages of the present invention are summarized as follows:

[0131] (a) The present invention provides an extracellular vesicle with enhanced expression of nerve growth factor (NGF).

[0132] (b) The present invention provides a method for manufacturing extracellular vesicles.

[0133] (c) Provides a pharmaceutical composition for the prevention or treatment of neuropathy comprising the above extracellular vesicles.

[0134] (d) When using the extracellular vesicles of the present invention, neuropathy can be effectively prevented or treated.

[0135] Figure 1 shows an overview of the process for producing extracellular vesicles.

[0136] Figures 2a and 2b show the results of the analysis of neural crest cell characteristics of extracellular vesicles.

[0137] Figure 3 shows the results of the analysis of neurotrophic factor expression levels in extracellular vesicles.

[0138] Figure 4 shows the results of the analysis of the physical properties of extracellular vesicles.

[0139] Figures 5 and 6 show the results of the analysis of labeled proteins in extracellular vesicles.

[0140] Figures 7 and 8 show the results of the analysis of the absorption rate of extracellular vesicles by microglia and the analysis of their anti-inflammatory effects.

[0141] Figures 9 and 10 show the results of the analysis of the absorption rate of extracellular vesicles in neuroblastoma and the analysis of the effect of promoting neuronal growth.

[0142] Figures 11 and 12 show the results of the analysis of the effect of extracellular vesicles on improving neuropathy caused by sciatic nerve injury.

[0143] Figures 13 and 14 show the results of the analysis of the neuroregeneration and remyelination effects of extracellular vesicles.

[0144] Figures 15 and 16 show the results of evaluating the anti-inflammatory and neuronal cell death inhibitory efficacy of extracellular vesicles.

[0145]

[0146]

[0147] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0148]

[0149] Examples

[0150] Example 1: Materials and Method

[0151] 1-1. Culture of neural crest cells differentiated from human urine-derived induced pluripotent stem cells overexpressing nerve growth factor for obtaining extracellular vesicles

[0152] Neural crest cells (NCCs) derived from human urine-derived induced pluripotent stem cells (UiPSCs) that overexpress Nerve Growth Factor (NGF) were prepared to obtain extracellular vesicles.

[0153] First, to construct the lentivirus vector, HEK293T cells were co-infected with the LV-con (control) or oe-NGF (NGF overexpression) lentivirus plasmid along with the packaging plasmid (containing gag, pol, and rev genes) using PolyJet reagent (Signagen Laboratories LLC). The supernatant was collected from cells cultured for 3 days after infection and concentrated to a total of 200 μL by centrifuging at 25,000 rpm for 2 hours at 4°C.

[0154] Subsequently, UiPSCs being cultured in a feeder-free state in a 24-well culture plate were infected using E-8 medium containing hexadimethrin bromide (2 μg / mL) along with concentrated virus particles. The medium was replaced daily for 4 days after infection, and subsequently, UiPSCs infected with LV-con or LV-oe-NGF viruses were isolated at the single-cell level. 500 isolated cells were inoculated into a 6-well plate and cultured for 7 to 10 days; afterward, the formed single-cell colonies were manually incised and transferred to a Matrigel-coated 12-well plate for further culture.

[0155] When the cell density of these urine-derived induced pluripotent stem cells (UiPSCs) reached 70% or higher, the cells were isolated using Accutase (Thermo Fisher Scientific) and centrifuged at 1,000 rpm for 3 minutes to obtain the cells. The obtained cells were inoculated into Matrigel-coated 60 mm culture dishes using E6 medium (added with Y-27632 10 μM) and cultured at 37°C under 5% CO2 conditions. The following day, the medium was replaced with Neural Crest Cell (NCC) induction medium consisting of TeSR-E6 medium supplemented with N-2 (Thermo Fisher Scientific), SB431542 (20 μM, MedChemExpress), and CHIR99021 (10 μM). This medium was replaced daily for 3 days to induce differentiation into NCCs.

[0156] After 3 days, when the cells were more than 90% dense, the cells were subcultured using Accutase, and the above process was repeated until passage 4 was reached. The NCC in Passage 4 was cultured for 3 days, with the medium replaced daily, and the culture medium containing extracellular vesicles was obtained from the supernatant.

[0157]

[0158] 1-2. Isolation and Purification of Extracellular Vesicles

[0159] To isolate extracellular vesicles from a culture medium containing extracellular vesicles while simultaneously concentrating the sample and exchanging the solvent, a tangential flow filtration (TFF) system filtration device (Repligen, USA) was used.

[0160] First, primary filtration of the culture medium was performed using a 0.2 μm filter. Subsequently, a hollow fiber column with a molecular weight cut-off of 100 kDa was applied to the TFF instrument for the isolation and purification of extracellular vesicles.

[0161] In the step for solvent exchange of extracellular vesicles, diafiltration was performed using sterile physiological saline (water for injection), thereby finally preparing a therapeutic composition in the form of isolated extracellular vesicles using sterile physiological saline as a substrate.

[0162]

[0163] 1-3. Analysis of Extracellular Vesicle Characteristics

[0164] Nanoparticle tracking analysis (NTA) was used to determine the number and concentration of extracellular vesicles obtained from the culture medium. The analysis was performed using a Zetaview (Particle Metrix) instrument on both the culture medium before concentration and the final extracellular vesicle samples after concentration and solvent exchange.

[0165] The morphology and structure of extracellular vesicles were observed using transmission electron microscopy (TEM; JEM-1010, Nippon Denshi, Tokyo, Japan) at 80 kV. Surface markers of extracellular vesicles were measured using a flow cytometer (Beckman Coulter, CytoFLEX) after capturing EVs using Exosome-Human CD9 Flow Detection Reagent (Invitrogen, 10620D), staining with CD63-PE (BD, 556020) and CD81-APC (miltenyi biotec, 130-119-787).

[0166] In addition, the expression patterns of extracellular vesicle proteins were confirmed through western blot analysis. CD9 (ab263023, Abcam), CD63 (ab59479, Abcam), and CD81 (ab109201, Abcam) were used as positive markers, while Calnexin (2679T, CST) and GM130 (12480, CST) were used as negative markers; each primary antibody was detected by reacting it with a secondary antibody appropriate for the corresponding species.

[0167]

[0168] 1-4. Analysis of Extracellular Vesicle Invagination into Target Cells

[0169] To confirm the uptake of extracellular vesicles into target cells such as microglia (BV2) or neuroblastoma (SHSY-5y), the extracellular vesicles were stained with 2 μg / mL of DiR (D12731, Invitrogen) reagent at room temperature for 1 hour. Subsequently, ultracentrifugation was performed at 178,000 g for 2 hours to remove free dye.

[0170] Afterwards, 1x10 8 Exosomes of the particles were treated to target cells for 6 hours, and the degree of intracellular invagination was measured using a flow cytometer and a super-resolution microscope. To use immunofluorescence staining, nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole; VECTASHIELD® Antifade Mounting Medium with DAPI, H-1200), and then visually confirmed through fluorescence imaging using confocal laser scanning microscopy (Carl Zeiss LSM 800).

[0171]

[0172] 1-5. Evaluation of the Efficacy of Neuroinflammation Treatment Using Microglia

[0173] BV2 microglia in 24-well culture dishes, 2 x 10 wells per well 5 Cells were inoculated and cultured for 24 hours. Afterwards, 1 x 10⁻⁶ 8 Extracellular vesicles were mixed with DMEM-low glucose medium at a concentration of particles / mL and pretreated for 6 hours, followed by treatment with lipopolysaccharide (LPS) at a concentration of 100 ng / mL for 24 hours to induce an inflammatory environment.

[0174] After 24 hours of treatment, the supernatant was collected and centrifuged at 3,000 rpm for 1 minute to remove cell debris. The production of nitric oxide (NO), an indicator of inflammatory response, was measured by mixing the supernatant with Griess reagent (a mixture of 5% phosphoric acid / 1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, 1:1 ratio) in a 1:1 ratio, reacting the mixture in a 96-well plate, and incubating at room temperature for 10 minutes. Absorbance was measured at 540 nm, and Bio-RAD xMark TM Quantified using a spectrophotometer.

[0175]

[0176] 1-6. Efficacy of Extracellular Vesicles on Neuroblastoma (SHSY-5y) Cells for Neurite Outgrowth

[0177] In this embodiment, an experiment using neuroblastoma (SHSY-5y) cells was performed to confirm the effect of extracellular vesicles derived from neural crest cells on neurite outgrowth.

[0178] First, undifferentiated SHSY-5y cells were inoculated onto Matrigel-coated culture slides and cultured until the cells occupied approximately 60–70% of the slide surface area. Subsequently, the culture medium was removed, and 1 x 10⁻⁶ 8After replacing the neural basal medium containing extracellular vesicles at a concentration of particles / mL with neural basal medium not containing extracellular vesicles, the culture was incubated for 6 hours under 5% CO2, 37℃ conditions.

[0179] After 6 hours of incubation, each medium was removed and replaced with fresh neural basal medium, followed by incubation for 3 days under 5% CO2 and 37°C conditions. After 3 days, cells were fixed with 4% paraformaldehyde (PFA) and neurocytes were stained using βIII-tubulin antibody.

[0180] Stained cells were imaged using a confocal microscope (LSM 800, Carl Zeiss), and the acquired images were used to measure and standardize the length of each neurite using the ImageJ program.

[0181]

[0182] 1-7. Evaluation of Efficacy in Improving Neuropathic Pain Caused by Sciatic Nerve Injury

[0183] A neuropathic pain animal model was induced using the right sciatic nerve of a rat to create a chronic neuropathic pain model. Specifically, nerve compression injury was induced by suturing the sciatic nerve at 2 to 4 sites at 1 mm intervals using 4-0 silk sutures. Subsequently, the muscles and skin were sutured and disinfected with povidone.

[0184] 3 days after induction, 1x10 9 Extracellular vesicles of the particles were administered alone, and improvements in motor function (Rotarod test) and pain heterogeneity were measured for 2 weeks. On day 14 of induction, the sciatic nerve and gastrocnemius muscle were collected, and nerve recovery, inflammation relief, and inhibition of muscle loss effects were evaluated through western blot and toluidine blue staining.

[0185]

[0186] Example 2: Construction of a platform for producing neural crest cell (NCC)-derived extracellular vesicles from human urine-derived induced pluripotent stem cells (hUiPSCs) overexpressing nerve growth factor (NGF) and confirmation of maintenance of cell characteristics

[0187] We established a mass production platform capable of inducing neural crest cells (NCCs) from human urine-derived induced pluripotent stem cells (hUiPSCs) overexpressing Nerve Growth Factor (NGF) and producing extracellular vesicles with well-maintained characteristics from them.

[0188] First, human urine was collected and centrifuged to isolate urine-derived stem cells (USCs). These were used to establish hUiPSCs, and subsequently, gene infection was performed using a lentivirus with the NGF gene introduced (oe-NGF) or a control lentivirus (LV-control). After selecting and establishing NGF-overexpressing UiPSC single-cell clones, differentiation into neural crest cells was induced.

[0189] An overview of the process for producing extracellular vesicles is shown in Figure 1. Specifically, the production method includes the steps of culturing urine-derived cells to induce hUiPSC, infecting LV-oe-NGF or LV-control, establishing a single-cell-derived clone, and inducing differentiation into neural crest cells (NCC differentiation).

[0190] As shown in Fig. 2a, it was confirmed that neural crest cells (NCCs) established in this manner stably overexpress the NGF gene while maintaining typical surface markers and phenotypes of neural crest cells. For example, in NGF-overexpressing cells, the neural crest cell marker p75 NTRIt was confirmed through flow cytometry that the expression of [the substance] was maintained at over 96%, which was similar to the level of the LV-control group. This means that NGF overexpression does not adversely affect the differentiation ability of cells or the expression of markers.

[0191] In addition, as shown in Figure 2b, immunofluorescence staining results confirmed that neural crest cell-specific proteins such as SOX10, HNK1, p75, PAX6, AP2α, and BAF1 were well expressed in both the LV-control and oe-NGF groups. This demonstrates that the extracellular vesicles produced in large quantities through this platform contain a large amount of NGF while maintaining the unique biological characteristics of neural crest cells.

[0192] Therefore, through this embodiment, a platform capable of mass-producing extracellular vesicles derived from neural crest cells that overexpress NGF was established, and it was confirmed that this is a production system with ensured stability and efficacy in that these cells maintain their inherent differentiation ability and characteristics despite genetic manipulation.

[0193]

[0194] Example 3: Analysis of NGF expression levels in extracellular vesicles derived from NGF-overexpressing neural crest cells

[0195] Established NGF-overexpressing neural crest cells (NCCs) oe-NGF We wanted to verify whether extracellular vesicles (EVs) produced from ) actually contain an abundance of nerve growth factor (NGF).

[0196] To this end, control neural crest cells (NCC-EV) and NGF-overexpressing neural crest cells (NCC oe-NGF The expression level of NGF in extracellular vesicles obtained from EV was quantified using ELISA (enzyme-linked immunosorbent assay).

[0197] The results are shown in Figure 3.

[0198] As shown in Figure 3, the concentration of NGF in the NCC-EV group was measured to be very low, less than 1 ng / ml, which corresponds to a background level. On the other hand, NCC oe-NGF -In the EV group, the NGF concentration was approximately 12 ng / ml, which was significantly increased by more than 10 times compared to the control group (p < 0.0001, ****).

[0199] These results demonstrate that EVs produced through the neural crest cell-derived extracellular vesicle production platform established in the present invention contain a large amount of NGF, and that if intracellular NGF expression is increased through genetic manipulation, the amount of NGF contained in the extracellular vesicles also increases significantly as a result.

[0200] This is important evidence demonstrating that extracellular vesicles function as biological carriers reflecting the physiological and genetic characteristics of parent cells, suggesting that the EV has very high potential for use as a bio-carrier for promoting nerve growth or treating neurological diseases.

[0201] Furthermore, since the concentration of NGF within the extracellular vesicles is maintained above the physiological concentration level at the time of therapeutic application, this suggests that the EV possesses value as a biotherapeutic candidate capable of functioning as an active factor that exerts a therapeutic effect on its own, rather than as a drug delivery vehicle.

[0202]

[0203] Example 4: Characterization of extracellular vesicles derived from NGF-overexpressing neural crest cells

[0204] In this embodiment, neural crest cells (NCC) induced to overexpress nerve growth factor (NGF) oe-NGF The physicochemical properties of extracellular vesicles derived from ) were analyzed by comparing them with extracellular vesicles derived from general neural crest cells (NCC-EV).

[0205] The particle size and distribution of extracellular vesicles were measured using nanoparticle tracking analysis with a Zetaview analyzer, and the results are shown in Figure 4. As shown in Figure 4, extracellular vesicles derived from NCC EVs had an average diameter of 123.6 ± 2.2 nm, and NCC oe-NGF The derived extracellular vesicles showed a similar size distribution of 124.5±1.3 nm, and the particle distribution curves between the two groups also showed an overlapping pattern. This indicates that NGF overexpression does not affect the production or size characteristics of extracellular vesicles.

[0206] Subsequently, extracellular vesicle marker protein expression was compared via Western blot analysis, and the results are shown in Figure 5. As shown in Figure 5, representative extracellular vesicle marker proteins such as CD9 and CD63 are NCC and NCC oe-NGF It was distinctly expressed in all derived extracellular vesicles, and GM130, Calnexin, and β-actin, which are used as intracellular contamination indicators, were not detected in the extracellular vesicle fractions. This indicates that the isolated extracellular vesicles are of high purity and suggest that they maintain the same protein expression pattern regardless of differences in cell origin or genetic manipulation.

[0207] In addition, CD63 and CD81 proteins expressed on the surface of extracellular vesicles were quantitatively analyzed using flow cytometry, and the results are shown in Figure 6. As shown in Figure 6, high expression rates of 93.3% and 96.8% were observed in extracellular vesicles derived from NCC-EV, and 95.8% and 98.4% were observed in extracellular vesicles derived from NCCoe-NGF, respectively, confirming that there was no significant difference between the two groups in the composition of surface markers.

[0208] Synthesizing the above results, extracellular vesicles derived from neural crest cells overexpressing NGF did not show significant differences in characteristics such as particle size, expression of typical extracellular vesicle marker proteins, and surface marker composition when compared to extracellular vesicles derived from normal neural crest cells, and it was confirmed that the overexpression of the NGF gene did not affect the basic structural and molecular characteristics of the extracellular vesicles. This example demonstrates that the extracellular vesicle production platform of the present invention can stably maintain the identity and quality of the extracellular vesicles while simultaneously enhancing function through genetic manipulation.

[0209]

[0210] Example 5: Effects of NGF-expressing neuro-derived exosomes on uptake and anti-inflammatory effects on microglia

[0211] To analyze the uptake and anti-inflammatory efficacy of exosomes on microglia, DiI-stained exosomes (NCC-derived EVs or NGF-overexpressing NCC-derived EVs: NCC oeNGF -EV) each 1×10 8 The cells were treated with particles / ml and cultured for 6 hours. Afterward, the absorption of exosomes into the cells was analyzed using confocal laser scanning microscopy and flow cytometry.

[0212] The results are shown in Figure 7.

[0213] As shown in Fig. 7, NCC oeNGF -EV showed a higher level of intracellular uptake than NCC-EV, and flow cytometry results showed a significant difference, with the uptake rate of NCC-EV being approximately 70.5% and the uptake rate of NCCoeNGF-EV being approximately 87.6%. This means that exosomes containing NGF are delivered more effectively to microglia.

[0214]

[0215] Next, to analyze the anti-inflammatory effect, BV2 cells were pretreated with exosomes for 6 hours, and then lipopolysaccharide (LPS) was added at a concentration of 100 ng / ml to induce an inflammatory response for 24 hours. The culture medium was collected, reacted with Griess reagent at a 1:1 ratio, and the amount of nitric oxide (NO) produced was measured.

[0216] The results are shown in Figure 8.

[0217] As shown in Figure 8, high levels of NO production were observed in the control group treated only with LPS, while NO production was significantly reduced in the group treated with exosomes. In particular, NCC oeNGF In the group treated with -EV, NO production decreased most significantly, confirming that NGF further enhances the anti-inflammatory effect.

[0218] In addition, analysis of the expression levels of inflammatory cytokine genes (mTNF-α, mCOX-2, mIL-1β, mIL-6) via RT-qPCR revealed that the inflammatory genes whose expression was induced by LPS were NCC-EV and NCC oeNGF -It decreased in all EV treatment groups, especially NCC oeNGF The most pronounced inhibitory effect was observed in the EV-treated group. This demonstrates that exosomes overexpressing NGF effectively suppress the inflammatory response of microglia in an inflammation-induced environment, suggesting potential as an anti-inflammatory therapeutic agent.

[0219]

[0220] Example 6: Absorption and Neuronal Growth Promotion Effects of NGF-Expressing Neuro-Derived Exosomes on SH-SY5Y Neuroblastoma Cells

[0221] To analyze the effects of neuro-derived exosomes containing NGF on neuronal uptake and growth, human neuroblastoma cell line SH-SY5Y cells were inoculated onto Matrigel-coated slides and cultured at 60–70% density, after which DiI-stained exosomes (NCC-EV or NCC oeNGF -EV) is 1X10 8 The cells were treated at a particle / ml concentration and cultured for 6 hours. Subsequently, the degree of intracellular uptake of exosomes was analyzed using confocal microscopy and flow cytometry, and the results are shown in Fig. 9. As shown in Fig. 9, NCC oeNGF The absorption rate of exosomes in the EV treatment group was approximately 66.8%, which was significantly higher than that of the NCC-EV treatment group (approximately 44.1%). This indicates that exosomes containing NGF are delivered to neurons more efficiently.

[0222] Next, to analyze the neuronal growth-inducing effect, SH-SY5Y cells were treated with the same exosomes and cultured for 72 hours. Subsequently, the cells were stained with an antibody against Tuj1 (Neuron-specific class III β-tubulin), a neuron-specific protein, and observed under a fluorescence microscope. The results are shown in Figure 10. As shown in Figure 10, analysis of the captured images revealed that NCC-EV and NCC compared to the control group without exosome treatment. oeNGF Neural growth significantly increased in the EV treatment group, especially NCC oeNGF - In the EV treatment group, it was confirmed that neurites were expressed more lengthily and clearly.

[0223] As a result of quantitatively analyzing the average length of the neurite using the Image J program, NCC oeNGF- It was observed that the longest neurites were formed in the EV-treated group, which was statistically significantly longer (p<0.01). These results suggest that neuronal-derived exosomes containing NGF have the function of promoting neuronal differentiation and growth of SH-SY5Y cells, demonstrating their potential for application as agents for neuroregeneration or the treatment of neurological diseases.

[0224]

[0225] Example 7: Evaluation of the effect of improving neuropathic pain caused by sciatic nerve injury and muscle protection

[0226] The therapeutic effects of neural crest cell-derived exosomes (NCC-EV) and NGF-overexpressing exosomes (NCCoe-NGF-EV) were compared and analyzed in a chronic neuropathic pain model induced by sciatic nerve injury, and pain improvement and muscle protective functions were evaluated.

[0227] A critical compression injury (CCI) model was used in the experiment. Injury was induced in experimental animals by tying the sciatic nerve with sutures 2 to 4 times, and then each EV was injected into the site of nerve injury. Subsequently, motor function, pain sensitivity, and recovery of histopathological structure were evaluated over time.

[0228] Motor function was evaluated using the Rotarod test, and the results are shown in Figure 11. As shown in Figure 11, motor ability rapidly declined in the Injury group, but recovery was observed in the EV treatment group, and the fastest and most significant recovery was confirmed in the NGF-overexpressing exosomes (NCCoe-NGF-EV).

[0229] Pain sensitivity was measured using the Von Frey test, and the results are shown in Figure 11. As shown in Figure 11, the response threshold to mechanical stimulation significantly increased in the EV treatment group, indicating improved pain sensitivity, and a more pronounced recovery effect was observed, particularly in the NGF-overexpressing exosomes (NCCoe-NGF-EV).

[0230] The recovery of muscle tissue damage was analyzed by obtaining gastrocnemius muscle samples and performing H&E staining and Masson's trichrome (MT) staining, and the results are shown in Figure 12. According to the tissue staining images and quantitative analysis graphs in Figure 12, increased collagen deposition and decreased muscle fiber area were observed in the injury group, whereas these injuries were inhibited in the EV-treated group. In particular, the inhibition of collagen accumulation and the increase in muscle fiber cross-sectional area were most significant in the NGF-overexpressing exosome (NCCoe-NGF-EV) treated group. Specifically, the muscle fiber cross-sectional area increased statistically significantly in the NGF-overexpressing exosome (NCCoe-NGF-EV) group compared to the injury group, and while the collagen volume ratio surged in the injury group, it recovered to near-sham levels in the NGF-overexpressing exosome (NCCoe-NGF-EV) group.

[0231] These results indicate that the EV of the present invention has the effect of inhibiting muscle fibrosis caused by nerve damage and protecting muscle structure, and further enhances the effects of restoring motor function and improving pain sensitivity.

[0232]

[0233] Example 8: Evaluation of the nerve regeneration and remyelination effects of NCCoe-NGF-EV

[0234] The effects of NCCoe-NGF-EV on nerve regeneration and remyelination were evaluated in a critical compression injury (CCI) model.

[0235] Three days after injury induction, NCC-EV or NCCoe-NGF-EV was injected, and on the 14th day, the sciatic nerve at the injury site was obtained and protein expression analysis (western blot) was performed.

[0236] The results are shown in Figs. 13 and 14.

[0237] As shown in Figure 13, Western blot analysis revealed that NCCoe-NGF-EV increased nNOS expression while simultaneously inducing ERK and AKT phosphorylation, thereby elevating GAP-43 and NF-H expression. Additionally, it increased the expression of p75NTR, a Schwann cell marker, demonstrating an effect of inducing nerve growth and axonal regeneration. Myelin Basic Protein (MBP) expression also significantly increased, indicating a remyelination-promoting effect. In particular, analysis of the MBP / NF-H ratio confirmed that axonal regeneration and myelination occurred simultaneously in the NCC-EV and NCCoe-NGF-EV groups compared to the Injury group.

[0238] As shown in Figure 14, the expression patterns of NF-H and MBP, a myelination marker that constitutes nerve fibers, were observed through immunofluorescence analysis. The results showed that both markers increased simultaneously in the NCCoe-NGF-EV group, which reflects the induction of expression of growth factors and neuroplasticity factors and the promotion of nerve regeneration in damaged peripheral nerves.

[0239] The above results demonstrate that NCCoe-NGF-EV, which overexpresses NGF, effectively promotes regeneration and remyelination of the nerve injury site by inducing the upregulation of BDNF and NT-3, activation of the NGF / nNOS / ERK / AKT pathway, promotion of GAP-43 and NF-H expression, and increased expression of MBP in the environment of sciatic nerve injury.

[0240] In summary, the NCCoe-NGF-EV of the present invention possesses a more potent neurorecovery-inducing function compared to conventional EVs and has demonstrated its potential as a therapeutic agent based on neuroregeneration and remyelination.

[0241]

[0242] Example 9: Evaluation of anti-inflammatory and neuronal cell death inhibition efficacy

[0243] To confirm the anti-inflammatory and neuronal death inhibitory effects of NGF-overexpressing neural crest cell-derived exosomes (NCCoe-NGF-EV), the inflammatory response and degree of neuronal death were analyzed in a mouse model of critical compression injury (CCI).

[0244] The results are shown in Figs. 15 and 16.

[0245] As shown in Figure 15, the cleaved caspase-3 immunohistochemistry results in the Injury group showed an increase in cell death, but in the groups administered NCC-EV or NCCoe-NGF-EV, the expression of cleaved caspase-3 was significantly reduced, confirming the inhibitory effect on cell death in damaged nerve tissue.

[0246] Furthermore, Western blot analysis revealed that while the expression of inflammation-related factors such as TNF-α, p-NF-κB, Iba-1, and COX-2 increased in the Injury group, the expression of all these factors was significantly reduced in the NCC-EV and NCCoe-NGF-EV administration groups. The inhibitory effect was particularly pronounced in the NCCoe-NGF-EV administration group.

[0247] As a result of measuring mRNA expression levels of TNF-α, IL-6, COX-2, IL-1β, etc. through RT-qPCR analysis, as shown in Figure 16, the expression of these genes was significantly increased in the Injury group, but the expression levels were significantly decreased in the NCC-EV and NCCoe-NGF-EV administration groups, and the inhibitory effect on TNF-α and IL-6 expression was particularly prominent.

[0248] These results indicate that NCCoe-NGF-EV effectively suppresses the expression of inflammatory cytokines in a neurological injury environment and blocks the activation of apoptotic pathways, thereby increasing neuronal survival rates and being effective in alleviating neuropathic pain. In other words, through this example, it was confirmed that NCCoe-NGF-EV possesses excellent anti-inflammatory and cytoprotective functions in a CCI injury environment.

Claims

1. Extracellular vesicle with enhanced expression of nerve growth factor (NGF).

2. The extracellular vesicle of claim 1, wherein the extracellular vesicle expresses a protein of SOX10, HNK1, PAX6, AP2α, BAF1, or a combination thereof.

3. The extracellular vesicle according to claim 1, wherein the average diameter of the extracellular vesicle is 50 to 300 nm.

4. The extracellular vesicle of claim 1, wherein the extracellular vesicle has the characteristics of promoting neuronal differentiation, regulating inflammatory response, inhibiting apoptosis, or a combination thereof.

5. In paragraph 1, the extracellular vesicle is an extracellular vesicle derived from a neural crest cell.

6. In paragraph 5, the neural crest cells are extracellular vesicles in which the expression of nerve growth factor (NGF) is enhanced.

7. In paragraph 5, the neural crest cells are extracellular vesicles differentiated from human adult stem cells (hASCs), human embryonic stem cells (hESCs), or human induced pluripotent stem cells.

8. In claim 7, the induced pluripotent stem cells are induced pluripotent stem cells derived from urine cells, and the extracellular vesicles.

9. The extracellular vesicle of claim 1, wherein the extracellular vesicle is absorbed by microglia, neuroblastoma, or a combination thereof.

10. A method for preparing an extracellular vesicle comprising the following steps: (a) a step of dedifferentiating human induced pluripotent stem cells from urine stem cells; (b) a step of differentiating neural crest cells from human induced pluripotent stem cells; and (c) A step of obtaining extracellular vesicles from the culture medium.

11. A method for preparing an extracellular vesicle according to claim 10, wherein, after step (a) above, human induced pluripotent stem cells are additionally transfected to overexpress a neurotrophic factor selected from the group consisting of the following: Nerve growth factor (NGF), Brain-Derived Neurotrophic Factor, Glial cell-the-line-Derived Neurotrophic Factor, Neurotrophin-3, Ciliary Neurotrophic Factor, and combinations thereof.

12. A method for manufacturing extracellular vesicles according to claim 10, wherein the above manufacturing method has an improved amount of extracellular vesicle secretion.

13. A method for manufacturing an extracellular vesicle according to claim 10, wherein the extracellular vesicle in the above manufacturing method retains the characteristics of a neural crest cell.

14. A pharmaceutical composition for the prevention or treatment of neuropathy comprising an extracellular vesicle of any one of claims 1 to 9.

15. A pharmaceutical composition for the prevention or treatment of neuropathy according to claim 14, wherein the neuropathy is peripheral neuropathy, diabetic neuropathy, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, sciatic neuropathy, neuropathy due to cervical or lumbar disc herniation, entrapment neuropathy, inflammatory neuropathy, secondary neuropathy due to central nervous system injury, cancer-related neuropathy, or a combination thereof.

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