Technology for degenerative brain disease-therapeutic protein delivery specifically to brain by using exosomes comprising degenerative brain disease-therapeutic protein

Abstract

The present invention relates to: a pharmaceutical composition for selectively preventing or treating neurodegenerative diseases including Parkinson's disease, comprising as an active ingredient exosomes into which has been delivered a degenerative brain disease-therapeutic protein via a photocleavable protein; and a method for preparing same. Parkinson's disease therapeutic proteins, such as Parkin protein, effectively inhibit the death of damaged dopaminergic neurons, and have protective effects against neurotoxic substances, and thus have an effect of preventing damage to neurons.

Description

Brain-specific degenerative brain disease therapeutic protein delivery technology using exosomes containing degenerative brain disease therapeutic proteins

[0001] This application claims priority to Korean Patent Application No. 10-2023-0122777, filed September 14, 2023, the entire disclosure of which is incorporated herein by reference. The present invention relates to a technique for delivering exosomes containing a protein for treating a degenerative brain disease to brain tissue specifically, and a pharmaceutical composition for treating a degenerative brain disease comprising the exosome as an active ingredient.

[0002] Parkinson's disease is a neurodegenerative brain disease caused by dopamine deficiency due to the specific loss of dopaminergic neurons in the substantia nigra and catecholaminergic neurons in the brainstem, resulting in motor dysfunction such as sedation and bradykinesia. Parkin, an E3 ubiquitin-ligase, is known to be an important protein that removes damaged mitochondria through mitophagy, and recessive mutations in PARK2, which encodes the parkin protein, are known to cause early-onset PD. In addition, it has been reported that the 'c-Abl' protein, one of the tyrosine kinases, is increased in Parkinson's disease patients, which causes phosphorylation of the tyrosine residue at position 143 (Y143) of the parkin protein, thereby inactivating the parkin protein.

[0003] Current treatment for Parkinson's disease primarily involves symptomatic medication and exercise therapy. Surgery is performed on advanced patients who fail to respond adequately to medication. Pharmacological treatments, such as dopamine precursors (levadopa) and dopamine agonists, can temporarily alleviate symptoms by replenishing dopamine deficiency and delaying dopamine loss in the brain. However, they do not halt the degeneration and loss of dopaminergic neurons, necessitating a more fundamental treatment.

[0004] Stem cell transplantation therapy is currently in the clinical stage, and it is known that injected stem cells differentiate into dopaminergic neurons, replacing damaged nerve cells or suppressing peripheral inflammation, resulting in therapeutic effects. However, while differentiation is possible in vitro, transplanted cells have low survival rates and differentiation efficiency. Furthermore, the invasive procedure associated with stem cell transplantation poses challenges, including the risk of permanent damage, cancerization of transplanted cells, and intractable immune responses. Furthermore, the risk of infection due to fetal bovine serum (FES) used in cell culture has led to limitations in clinical trials.

[0005] Furthermore, most existing protein therapies target cell membrane proteins. However, many disease-causing factors primarily exist within cells. To target these disease-causing factors, a technology for delivering therapeutic proteins into cells is needed. Therapeutic protein delivery technology using lipid nanoparticles, currently under development, has the problem that lipid nanoparticles cannot be effectively separated from therapeutic proteins. Furthermore, delivery technology utilizing protein transduction domains (PTDs), which play a key role in viral cell penetration, suffers from the problem of protein transporters degrading when exposed to bodily fluids such as blood or intestinal fluid. Therefore, to target specific disease-causing factors within cells, a technology for effectively delivering therapeutic proteins into cells is necessary.

[0006] Numerous references and citations are provided throughout this specification. The disclosures of these references are incorporated herein by reference in their entirety to further clarify the state of the art and the scope of the present invention.

[0007] The purpose of the present invention is to provide a technique for delivering exosomes specifically to brain tissue. More specifically, the goal is to develop a nanopharmaceutical that extracts exosomes by fusing a photocleavable protein, a brain-transmitting protein, and a protein for treating degenerative brain diseases, and efficiently delivers the protein for treating degenerative brain diseases into the brain, thereby improving neuroprotective and anti-apoptotic effects.

[0008] Therefore, the purpose of the present invention is to provide a recombinant exosome capable of expressing a fusion protein including a protein for treating a degenerative brain disease and a photocleavable protein.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing, improving, or treating a degenerative brain disease, comprising the exosome as an active ingredient, and a use of the exosome for preventing, improving, or treating a degenerative brain disease.

[0010] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0011]

[0012] One aspect of the present invention provides a recombinant exosome comprising a fusion protein including a protein for treating a degenerative brain disease and a photocleavable protein, wherein a brain delivery protein is expressed on the surface of the exosome.

[0013] The above-mentioned protein for treating degenerative brain diseases may be at least one selected from the group consisting of Parkin, Parkin Y143F mutant protein (Parkin Y143F), PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), and Nurr1 (Nuclear receptor-related transcription factor 1).

[0014] Parkin protein effectively inhibits the death of damaged dopaminergic neurons and has a protective effect against neurotoxic substances, thus preventing damage to neurons.

[0015] The Y143F mutant Parkin protein (Parkin Y143F) lacks the tyrosine residue at position 143, making it incapable of being phosphorylated by c-Abl, thus reducing tyrosine phosphorylation compared to wild-type Parkin. This suggests that the Y143F mutant retains E3 ubiquitin ligase activity even in the presence of c-Abl. The Y143F mutant Parkin, which is not phosphorylated by c-Abl, may provide protection against oxidative stress-induced dysfunction.

[0016] The therapeutic efficacy of PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) in treating Parkinson's disease is well known (Bin Zheng et al., PGC-1α, a potential therapeutic target for early intervention in Parkinson's disease, Published 6 October 2010, Sci. Transl. Med. 2, 52ra73 (2010)). Activation of PGC-1α increases the expression of nuclear-encoded subunits of the mitochondrial respiratory chain and blocks dopaminergic neuron loss induced by mutant α-synuclein or the pesticide rotenone in cellular disease models, suggesting its therapeutic efficacy in Parkinson's disease.

[0017] It is well known that decreased NURR1 expression can induce dopamine neuron dysfunction and promote degenerative changes leading to the development of Parkinson's disease (Mickael Decressac et al., NURR1 in Parkinson's disease—from pathogenesis to therapeutic potential, Nature Reviews Neurology volume 9, pages 629-636 (2013)). Nurr1 agonists may have therapeutic efficacy in enhancing function and improving behavioral deficits in patients with Parkinson's disease.

[0018] Degenerative brain disease refers to a degenerative disease that occurs in the brain due to aging, and includes, but is not limited to, Parkinson's disease, Alzheimer's disease, dementia, Huntington's disease, and Lou Gehrig's disease.

[0019] Intracellular delivery of the target protein, which is a protein for treating degenerative brain diseases, means transporting the target protein, which exists in a form bound to a photocleavable protein in an exosome, from the exosome into the interior of the target cell.

[0020] In the present invention, the photocleavable protein refers to a protein that is cleaved when exposed to light of a specific wavelength. In one embodiment, the photocleavable protein may be at least one selected from the group consisting of mMaple3 and PhoCl2c (photocleavable protein 2c).

[0021] The photocleavable protein can be cleaved by irradiation with light, and for example, the photocleavable protein can be cleaved within the exosome by irradiation with light having a wavelength of 401 nm to 480 nm. When the exosome containing the protein for treating Parkinson's disease, which is freed from the exosome-specific marker by cleavage of the photocleavable protein, is treated to dopaminergic neurons induced with Parkinson's disease, hyperphosphorylation of alpha-synuclein, a Parkinson's disease marker, and apoptosis can be suppressed.

[0022] In a preferred embodiment, the exosomes of the present invention are characterized in that they pass through the blood-brain barrier and specifically target brain tissue, and for this purpose, a brain transport protein is expressed on the surface of the exosomes.

[0023] Typically, less than 1% of systemically administered exosomes are delivered to the brain via the blood-brain barrier and the reticuloendothelial system (RES), significantly limiting the use of exosomes in the treatment of brain diseases. Therefore, efficient delivery of exosomes into the brain is crucial for effective treatment and minimizing adverse drug reactions. The present invention addresses this issue by modifying the surface of exosomes with various brain delivery proteins, enabling specific targeting of brain tissue.

[0024] In one embodiment, the brain transport protein is ApoB (3371-3409) (Apolipoprotein B (3371-3409) ), ApoE2 (141-149) (apolipoprotein E2 (141-149) ), Angiopep-2, Leptin 30, Rabies virus glycoprotein (RVG), and TAT (47-57) (trans-activator of transcription protein (47-57)) may be at least one selected from the group consisting of, and preferably, the brain transmission protein may be RVG.

[0025] In the present invention, a "fusion protein" is a protein produced by fusion of two or more proteins, and may include a protein for treating a degenerative brain disease, a photocleavable protein, and a brain transport protein, which are target proteins as described above, and may further include an exosome-specific marker protein. The exosome-specific marker proteins include CD9, CD63, Basigin, PTGFRN, etc., and preferably, the exosome-specific marker protein may be Basigin.

[0026] The above fusion protein may be a protein operably linked in the order of 'brain delivery protein-exosome-specific marker protein-photocleavable protein-target protein', and the fusion order may be arbitrary. In a preferred embodiment, the fusion protein may be a protein for treating a degenerative brain disease, Parkin or Parkin Y143F, a photocleavable protein, PhoCl2c or mMaple3, an exosome-specific marker protein, Basigin, and a brain delivery protein, RVG, fused in the order of RVG-Basigin-[PhoCl2c or mMaple3]-[Parkin or Parkin (Y143F)], and the RVG may be expressed on the surface of an exosome. In the present invention, “exosome” is a membrane vesicle with a lipid bilayer membrane structure that is secreted out of cells while encapsulating proteins, DNA, RNA, etc. for intercellular signal transmission, and exists in the body fluids of almost all eukaryotic organisms. The diameter of the exosomes can be 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, 10 nm to 250 nm, 10 nm to 200 nm, 10 nm to 150 nm, 50 nm to 350 nm, 50 nm to 300 nm, 50 nm to 250 nm, 50 nm to 200 nm, 50 nm to 150 nm, 100 nm to 300 nm, 100 nm to 200 nm, or 100 nm to 150 nm, and are released from the cell when the multivesicles fuse with the cell membrane or are released directly from the cell membrane.

[0027] The above exosomes can be prepared using an exosome extraction method known in the art, and there is no limitation on the extraction method.

[0028] Another aspect of the present invention is to provide a pharmaceutical composition for preventing, improving or treating a degenerative brain disease, comprising the exosome of the present invention as described above as an active ingredient.

[0029] The term “pharmaceutical composition” means a mixture of exosomes, which are the active ingredient of the present invention, with other ingredients such as a diluent or carrier.

[0030] The term "diluent" is defined as a compound that is diluted in a solvent that stabilizes the biologically active form of the active ingredient. Salts dissolved in buffer solutions are used as diluents in the field. A commonly used buffer solution is phosphate-buffered saline, as it mimics the salt content of human body fluids. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely alter the biological activity of a compound.

[0031] The term "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention 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, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0032] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses.

[0033] The pharmaceutical composition of the present invention may be administered to a subject via various routes. For example, various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, and intranasal. Among these, parenteral administration is preferred, and intravenous administration is more preferred.

[0034] For intravenous infusion, the pharmaceutical composition of the present invention may be formulated as a liquid solution, for example, in a pharmacologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer.

[0035] The pharmaceutical composition of the present invention may be administered to a patient after irradiating the exosomes with light prior to administration, for example, after irradiating them with light having a wavelength of 401 nm to 480 nm.

[0036] When the pharmaceutical composition of the present invention is administered to a patient, the exosome, which is an active ingredient, can pass through the blood-brain barrier and be specifically delivered to brain tissue.

[0037] The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0038] As one embodiment of the present invention, the present invention provides a method for preventing, treating, or improving a brain disease, comprising a step of administering the exosome or a composition comprising the exosome as an active ingredient to a subject in need thereof.

[0039] In another aspect of the present invention, the present invention provides a use of the exosome or a composition comprising the exosome for the prevention or treatment of brain diseases, preferably degenerative brain diseases, such as Parkinson's disease, Alzheimer's disease, dementia, Huntington's disease, or Lou Gehrig's disease.

[0040] In another aspect of the present invention, the present invention provides a use of the exosome or a composition comprising the exosome for the manufacture of a drug for treating a brain disease, preferably a degenerative brain disease, such as Parkinson's disease, Alzheimer's disease, dementia, Huntington's disease, or Lou Gehrig's disease.

[0041] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows, but is not limited thereto.

[0042] In the present invention, “administration” means providing an exosome of the present invention or a composition containing the same to a subject by any appropriate method.

[0043] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0044]

[0045] The exosome containing the protein for treating a degenerative brain disease of the present invention can specifically target brain tissue and effectively deliver the protein for treating a degenerative brain disease into nerve cells in which a degenerative brain disease has been induced.

[0046] One embodiment of the present invention provides a recombinant exosome comprising a fusion protein comprising a protein for treating a degenerative brain disease and a photocleavable protein, wherein a brain delivery protein is expressed on the surface of the exosome, and the exosome is used for the manufacture of a pharmaceutical product for preventing, improving, or treating a degenerative brain disease. The exosome can be used by being administered to a patient after being irradiated with light.

[0047] In another embodiment, the exosomes can be used in pharmaceuticals that are specifically delivered to brain tissue by crossing the blood-brain barrier.

[0048]

[0049] FIG. 1 is a diagram illustrating the structure and characteristics of a fusion protein that combines a target protein (Parkin or Parkin (Y143F)), a photocleavable protein, and an exosome-specific marker protein according to one embodiment of the present invention.

[0050] Figure 2 shows the results of confirming the cleavage effect of the fusion protein by 405 nm light after overexpressing the fusion protein in HEK293T cells through confocal microscopy imaging and fluorescence intensity measurement.

[0051] Figure 3 shows the results of confirming the cleavage effect of the fusion protein by Western blot and measuring the size and concentration of exosomes containing the fusion protein by NTA (Nanoparticle Tracking Assay).

[0052] Figure 4 shows the results of confirming the neuroprotective effect and mitochondrial protein recovery effect of Parkin-fusion exosomes in a CCCP-induced Parkinson's disease model using Tryphan blue assay and western blot.

[0053] Figure 5 shows the results of measuring the neuroprotective effect of Parkin fusion exosomes (Parkin Exo) or active Parkin fusion exosomes (Parkin (Y143F) Exo) in a 6-OHDA-induced Parkinson's disease model using the WST-8 assay.

[0054] Figure 6 shows the results of observing the inhibitory effect of alpha-synuclein hyperphosphorylation using confocal microscopy images after treating the alpha-synuclein precursor aggregate (PFF) model with Parkin fusion exosomes (Parkin Exo) or active Parkin fusion exosomes (Parkin (Y143F) Exo).

[0055] Figure 7 shows the results of measuring the in vivo distribution in the mouse brain for 24 hours by fusing various brain delivery proteins to the surface of exosomes, administering them after Cy5.5 fluorescent staining.

[0056] Figure 8 shows the results of measuring the biodistribution in the brain by administering various brain delivery proteins fused to the surface of exosomes and staining them with Cy5.5 fluorescence, sacrificing the mice 52 hours later, and extracting the brains.

[0057] Figure 9 shows the results of confirming the hyperphosphorylation inhibition effect and cell death inhibition effect of exosomes using confocal microscopy analysis.

[0058] Figure 10 shows that RVG-Basigin-Phocl2c-Parkin (CA) plasmid overexpression increases autophagy.

[0059] Figure 11 shows the results showing that RVG-Basigin-PhoCl2c-Parkin (CA) exosomes significantly improve motor ability in Parkinson's mice.

[0060]

[0061] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0062]

[0063] Example

[0064] Example 1. Preparation of exosomes containing parkin fusion proteins

[0065] 1-1. Manufacturing of Parkin fusion protein

[0066] A cDNA encoding a fusion protein (Parkin-mMaple3-CD9 or Parkin(Y143F)-mMaple3-CD9) was prepared by combining Parkin protein (Parkin) or active Parkin protein (Parkin(Y143F)) as a target protein, mMaple3 as a photocleavable protein, and CD9 as an exosome-specific marker protein, and the structure and characteristics of the fusion protein translated from the cDNA prepared as described above are schematically shown in Figure 1.

[0067] Specifically, first, cDNAs encoding Parkin or Parkin(Y143F), mMaple3, and CD9 were prepared and amplified by PCR, respectively, and the amplified cDNAs of mMaple3 and CD9 were combined into a single cDNA (mMaple3-CD9) by PCR. Then, cDNAs encoding fusion proteins were prepared by combining mMaple3-CD9 cDNA and Parkin or Parkin(Y143F) cDNA into a single cDNA (Parkin-mMaple3-CD9 or Parkin(Y143F)-mMaple3-CD9) by PCR.

[0068] Then, the Parkin fusion protein (Parkin-mMaple3-CD9) was transfected into HEK293T cells using PEI (polyethylenimine) so that the fusion protein could be expressed in mammalian cells. After the Parkin fusion protein (Parkin-mMaple3-CD9) was overexpressed in HEK293T cells, the cleavage effect of the fusion protein by light at 405 nm was confirmed through confocal microscopy imaging and fluorescence intensity measurement, and the results are shown in Fig. 2. As a result of the experiment, the expression and cleavage effect of the fusion protein were confirmed (Fig. 2).

[0069] The amino acid sequences of Parkin, Parkin(Y143F), mMaple3, and CD9 used above are shown in Table 1 below.

[0070]

[0071] Example 2. Exosome isolation and purification

[0072] In Example 1, exosomes were separated and purified from the medium of HEK293T cells overexpressing the fusion protein using a Tangential Fluid Filtration system.

[0073] Specifically, HEK293T cells were cultured and transfected with a vector for treating Parkinson's disease. The culture medium was replaced with serum-free medium 24 hours before extraction, and the culture was cultured for 24 hours. The cell culture supernatant was collected. The collected cell culture supernatant was centrifuged at 2,000 xg, 4℃ for 5 minutes, and filtered using a 2 ㎛ bottom filter to remove cell debris and waste. The solution collected after filtration was separated and purified using TFF (Tangential Flow Filtration) using a 300k membrane filter. The size and concentration of the exosomes containing the fusion protein were measured using NTA (Nanoparticle Tracking Assay) and BCA (Bicinchoninic Acid Assay), and the results are shown in Fig. 3B.

[0074] In addition, the cleavage effect of the exosome parkin fusion protein by 405 nm light was confirmed by Western blot in the above exosome. As shown in Fig. 3A, the amount of the uncleaved fusion protein decreased over time after irradiation with 405 nm light, while the amount of the cleaved fusion protein fragment increased, confirming the cleavage effect of the mMaple3 protein in the exosome.

[0075]

[0076] Example 3. Evaluation of the efficacy of exosomes for treating Parkinson's disease.

[0077] To evaluate whether exosomes for the treatment of Parkinson's disease are effective in treating Parkinson's disease, SH-SY5Y cells were treated with CCCP (Carbonyl cyanide m-chlorophenyl hydrazone) to induce Parkinson's disease. 5×10 Parkin-fused exosomes (Parkin Exo) isolated in Example 2 were added to the CCCP-induced Parkinson's disease model. 8 After treatment with particles / ml, the neuroprotective effects and mitochondrial protein recovery effects of Parkin-fused exosomes were confirmed using Tryphan blue assay and Western blot. The experimental results confirmed that Parkin-fused exosomes protect neurons and restore mitochondrial proteins in a CCCP-induced Parkinson's disease model (Fig. 4).

[0078] In addition, SH-SY5Y cells were treated with 6-hydroxydopamine (6-OHDA) to induce Parkinson's disease. 5×10 Parkin-fused exosomes (Parkin Exo) or active Parkin-fused exosomes (Parkin (Y143F) Exo) were added to the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease model. 8 particle / ml or 1×10 9 After treating with particles / ml, the WST-8 assay was performed to observe the neuroprotective effect. The experimental results showed that the delivery of the active form of Parkin protein (Parkin (Y143F)) was most effective (Fig. 5).

[0079] In addition, to evaluate whether exosomes for Parkinson's disease treatment are effective in treating Parkinson's disease, alpha-synuclein preformed-fibril (PFF) model was prepared by treating primary neuron cells with alpha-synuclein preformed-fibril to induce Parkinson's disease. Parkin (Parkin Exo) or active Parkin (Parkin (Y143F) Exo) fusion protein exosomes were added at a density of 5 × 10 in the alpha-synuclein preformed-fibril (PFF) model. 8After treatment with particles / ml, the inhibitory effect on alpha-synuclein hyperphosphorylation was observed using confocal microscopy images. The experimental results confirmed that both Parkin-fused exosomes (Parkin Exo) and activated Parkin-fused exosomes (Parkin (Y143F) Exo) significantly inhibited alpha-synuclein hyperphosphorylation (Fig. 6).

[0080]

[0081] Example 4. Preparation of exosomes for treating Parkinson's disease with brain transport proteins fused to the surface of the exosomes.

[0082] To treat Parkinson's disease, therapeutic proteins must be delivered to neurons or glial cells throughout the central nervous system (CNS). However, the presence of the blood-brain barrier hinders the delivery of these proteins from the blood. Since receptors on the blood-brain barrier bind to ligands and facilitate their transport into the CNS, we hypothesized that targeting these receptors could deliver target proteins to the CNS via brain transport proteins. To achieve brain targeting, we designed an exosome (Parkin (Y143F) Exo) that expresses various brain transport proteins (ApoB) on the surface of activated Parkin exosomes. (3371-3409) , ApoE2 (141-149) , Angiopep-2, Leptin 30, RVG (Rabies virus glycoprotein), TAT (47-57)} was attempted to be fused. For this purpose, Basigin was used as an exosome marker protein. In addition, PhoCl2c was used as a photocleavable protein.

[0083] Specifically, cDNA encoding each protein was prepared, amplified by PCR, and the amplified cDNAs were combined into a single cDNA (brain transfer protein-exosome specific marker protein PhoCl2c-Parkin (Y143F)) by PCR to produce a cDNA encoding a fusion protein. Then, the cDNA encoding the fusion protein was cloned into the pCMV14 vector using EcoR1 and Xba1 restriction enzymes. The plasmid was transfected into HEK293T cells and cultured to produce a cell culture solution.

[0084] The culture medium was replaced with serum-free medium 24 hours before extraction, and the cell culture supernatant was collected after culturing for 24 hours. The collected cell culture supernatant was centrifuged at 2,000 xg at 4°C for 5 minutes and filtered using a 2 μm bottom filter to remove cell debris and waste. The solution collected after filtration was separated and purified by TFF (Tangential Flow Filtration) using a 300k membrane filter.

[0085] Experimental results confirmed that brain transport proteins were successfully expressed on the surface of active Parkin exosomes (Parkin (Y143F) Exo) when Basigin protein was used as an exosome-specific marker protein. Therefore, subsequent experiments were performed using Basigin protein as an exosome-specific marker protein.

[0086]

[0087] Example 5. Evaluation of the intracerebral delivery efficiency of exosomes for treating Parkinson's disease, fused with various brain transport proteins.

[0088] Various brain transport proteins {ApoB} on the surface of activated Parkin exosomes {Parkin (Y143F) Exo} for brain targeting (3371-3409) , ApoE2 (141-149), Angiopep-2, Leptin 30, RVG (Rabies virus glycoprotein), TAT (47-57)} was administered after fusion with Cy5.5 fluorescent staining, and the biodistribution in the mouse brain was measured for 24 hours, and the results are shown in Figure 7.

[0089] Additionally, various brain transport proteins {ApoB} were detected on the surface of active Parkin exosomes {Parkin (Y143F) Exo}. (3371-3409) , ApoE2 (141-149) , Angiopep-2, Leptin 30, RVG (Rabies virus glycoprotein), TAT (47-57)} was administered after Cy5.5 fluorescent staining by fusing the mice, and the mice were sacrificed 52 hours later. The biodistribution in the mouse brain was measured, and the results are shown in Figure 8.

[0090] Experimental results showed that when fused with Rabies virus glycoprotein (RVG), among various brain delivery proteins, the brain delivery efficiency of exosomes significantly increased (Fig. 8). The amino acid sequences of RVG, Basigin, and PhoCl2c used above are shown in Table 2.

[0091] Amino acid sequence number

[0092] In order to evaluate whether the exosomes {RVG-Basigin-PhoCl2c-Parkin(Y143F) EVs} for treating Parkinson's disease, which were confirmed to have the best effect, are effective on Parkinson's disease, primary mouse neuron cells were treated with alpha-synuclein preformed-fibril to induce Parkinson's disease, and then the hyperphosphorylation inhibition effect and cell death inhibition effect of the exosomes were confirmed by confocal microscopy analysis. As a result of the experiment, it was confirmed that the brain-delivered exosomes for treating Parkinson's disease of the present invention significantly reduced alpha-synuclein hyperphosphorylation compared to the groups treated with PBS and control exosomes (Fig. 9).

[0093]

[0094] Example 6. Increased autophagy by overexpression of RVG-Basigin-Phocl2c-Parkin (CA) plasmid.

[0095] HEK293T cells were cultured and overexpressed (transfected) with the RVG-Basigin-PhoCl2c-Parkin (CA) vector. Autophagy activity was observed using Western blot and intracellular immunofluorescence staining (ICC).

[0096] As a result of the experiment, when RVG-Basigin-PhoCl2c-Parkin (CA) was overexpressed, the amount of Parkin and Tag protein Flag increased, and the amount of autophagy marker and lysosomal marker LC3-Ⅱ and LAMP-1 protein was confirmed to increase as a result of Western blot. In addition, co-localization of PhoCl2c fluorescence of the expressed vector and immunostained LAMP-1 protein confirmed that the RVG-Basigin-PhoCl2c-Parkin (CA) vector induces auto-lysosomes.

[0097]

[0098] Example 7. Improvement of motor ability in Parkinson's disease mice

[0099] To confirm the effect of RVG-Basigin-PhoCl2c-Parkin (CA) exosomes on improving motor ability in Parkinson's disease, 10 ug was administered to the left and right striatum of mice after 4 treatments with 20% amplitude, 1 s on / off cycles for 15 sec each. In the case of Sham mice, 2 ul of PBS was administered instead of PFF. Starting from 1 month after PFF administration, 50 ug of RVG-Basigin-PhoCl2c-Parkin (CA) exosomes were intravenously administered every other day for 2 months. In the Sham and PFF+Veh groups, exosomes that do not express the RVG-Basigin-PhoCl2c-Parkin (CA) vector were used as a control drug. After drug administration, the exercise ability of the mice was evaluated through grip strength test, pole test, and rotarod test, and the exercise ability was significantly improved.

[0100]

[0101] The present invention has the effect of treating, preventing, or improving brain diseases, preferably degenerative brain diseases. The present invention provides a composition or pharmaceutical for treating, preventing, or improving brain diseases, preferably degenerative brain diseases.

Claims

1. A recombinant exosome comprising a fusion protein including a protein for treating a degenerative brain disease and a photocleavable protein, characterized in that a brain transport protein is expressed on the surface of the exosome.

2. An exosome according to claim 1, characterized in that the protein for treating degenerative brain diseases is at least one selected from the group consisting of Parkin, Parkin Y143F mutant protein (Parkin Y143F), PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), and Nurr1 (Nuclear receptor-related transcription factor 1).

3. An exosome according to claim 1, characterized in that the photocleavable protein is at least one selected from the group consisting of mMaple3 and PhoCl2c (photocleavable protein 2c).

4. In the first paragraph, the brain transport protein is ApoB. (3371-3409) (Apolipoprotein B (3371-3409) ), ApoE2 (141-149) (apolipoprotein E2 (141-149) ), Angiopep-2, Leptin 30, Rabies virus glycoprotein (RVG), and TAT (47-57) (trans-activator of transcription protein (47-57) ) is characterized by at least one selected from the group consisting of.

5. An exosome according to claim 1, characterized in that the fusion protein further comprises an exosome-specific marker protein.

6. An exosome according to claim 5, characterized in that the exosome-specific marker protein is selected from the group consisting of CD9, CD63, Basigin, and PTGFRN.

7. In the first paragraph, the fusion protein is an exosome characterized in that Parkin or Parkin Y143F as a protein for treating a degenerative brain disease, PhoCl2c or mMaple3 as a photocleavable protein, Basigin as an exosome-specific marker protein, and RVG as a brain transport protein are fused in the order of RVG-Basigin-[PhoCl2c or mMaple3]-[Parkin or Parkin (Y143F)], and the RVG is expressed on the surface of the exosome.

8. In the first paragraph, the exosome is characterized in that it passes through the blood-brain barrier and specifically targets brain tissue.

9. A pharmaceutical composition for preventing, improving or treating a degenerative brain disease, comprising an exosome according to any one of claims 1 to 8 as an active ingredient.

10. A pharmaceutical composition characterized in that it is administered to a patient after irradiating the exosome with light in accordance with paragraph 9.

11. A pharmaceutical composition according to claim 10, characterized in that the light has a wavelength of 401 nm to 480 nm.

12. A pharmaceutical composition according to claim 10, characterized in that a photocleavable protein in an exosome is cleaved by irradiating the light.

13. A pharmaceutical composition according to claim 9, characterized in that the composition is for intravenous administration.

14. A pharmaceutical composition according to claim 9, characterized in that the exosome passes through the blood-brain barrier and is specifically delivered to brain tissue.

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