Pharmaceutical composition for preventing or treating brain diseases, comprising modified mitochondria

Abstract

The present invention relates to: a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a cerebrovascular endothelial cell surface protein binding site; modified mitochondria to which the fusion protein is bound; and a pharmaceutical composition comprising the modified mitochondria as an active ingredient. The modified mitochondria comprising the cerebrovascular endothelial cell surface protein binding site according to the present invention can pass through the blood-brain barrier at the cellular and animal level. In addition, administering the modified mitochondria to a Parkinson's disease mouse model can alleviate movement disorders. Therefore, the modified mitochondria according to the present invention can be used as a therapeutic agent for brain diseases caused by mitochondrial dysfunction.

Description

Pharmaceutical composition for preventing or treating brain diseases containing modified mitochondria

[0001] The present invention relates to a fusion protein capable of modifying mitochondria, mitochondria modified by the fusion protein, and a pharmaceutical composition comprising the same as an active ingredient.

[0002] Mitochondria are eukaryotic organelles involved in the synthesis and regulation of adenosine triphosphate (ATP), the cellular energy source. Mitochondria are involved in various metabolic pathways within the body, including cell signaling, cell differentiation, apoptosis, as well as the control of the cell cycle and growth. Mitochondria possess their own genome and are central to cellular energy metabolism. Mitochondria produce energy through electron transport and oxidative phosphorylation, and play a crucial role in apoptotic signaling pathways.

[0003] Recently, a correlation has been reported between mitochondrial dysfunction (e.g., disruption of the glycolytic process, decrease in mitochondrial enzymes, increase in reactive oxygen species, change in the number or size of mitochondria) and the development of neurological diseases such as Alzheimer's, Huntington's, Parkinson's, cerebral infarction, and dementia, raising the possibility of treating neurological diseases by improving mitochondrial function.

[0004] In particular, it is known that when mitochondria present within cells are isolated and processed ex vivo or injected into the body, the mitochondria enter the cells. Therefore, recent attempts have been made to directly administer mitochondria or develop them into drugs (Korean Patent No. 10-2111321).

[0005] Therefore, treating neurological diseases through the administration of mitochondria holds promise as a novel therapeutic approach. However, the blood-brain barrier (BBB) ​​poses a physical and functional obstacle to the delivery of mitochondria into the brain during pharmacological treatments. Therefore, strategies to overcome this obstacle are needed to utilize mitochondria as a therapeutic agent for neurological diseases.

[0006] Accordingly, the inventor of the present invention completed the present invention by studying a method for treating brain diseases using mitochondria and confirming that mitochondria containing a fusion protein in which a vascular endothelial cell surface protein binding site is linked to the mitochondrial outer membrane can improve the symptoms of brain diseases by passing through the BBB.

[0007] To achieve the above purpose, one aspect of the present invention provides a fusion protein comprising a mitochondrial outer membrane anchoring peptide and a cerebral vascular endothelial cell surface protein binding site.

[0008] Another aspect of the present invention provides a polynucleotide encoding the fusion protein.

[0009] Another aspect of the present invention provides a modified mitochondrion comprising the fusion protein.

[0010] Another aspect of the present invention provides a method for producing modified mitochondria, comprising the steps of: producing a transformed cell by introducing a polynucleotide encoding the fusion protein into the cell; and isolating modified mitochondria from the cell.

[0011] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating brain diseases, comprising the modified mitochondria as an active ingredient.

[0012] Another aspect of the present invention provides a use of the modified mitochondria for preventing or treating brain diseases.

[0013] Another aspect of the present invention provides a method for preventing or treating brain disease comprising administering the modified mitochondria to a subject.

[0014] Modified mitochondria containing a cerebrovascular endothelial cell surface protein binding site according to the present invention were confirmed to be capable of crossing the blood-brain barrier at both the cellular and animal levels. Furthermore, when the modified mitochondria were administered to a mouse model of Parkinson's disease, motor impairment was improved. Therefore, the modified mitochondria according to the present invention can be used as a therapeutic agent for brain diseases caused by mitochondrial dysfunction.

[0015] Figure 1a is a schematic diagram of an expression vector (pCMV-TOM20-GFP) loaded with a polynucleotide encoding a fusion protein (TOM20-GFP) containing TOM20 and GFP.

[0016] Figure 1b shows HEK293 cells containing mitochondria expressing TOM20-GFP fusion protein (HEK293-MT GFP ) is a diagram showing the results of confirming the expression of GFP using immunofluorescence staining. DAPI: Nuclear staining

[0017] Figure 2a is a schematic diagram of the expression vector of Figure 1a and an expression vector (pCMV-αIGF1RscFv-myc-TOM5) loaded with a polynucleotide encoding a fusion protein (α-IGF1RscFv-myc-TOM5) including an anti-IGF1R single chain antibody (α-IGF1RscFv) and TOM5.

[0018] Figure 2b shows HEK293 cells (HEK293-MT) containing mitochondria expressing α-IGF1RscFv-myc-TOM5 fusion protein. α-IGF1RscFv_GFP ) is a diagram showing the results of confirming the expression of flag and GFP using immunofluorescence staining. DAPI: Nuclear staining

[0019] Figure 3a is a schematic diagram of the expression vector of Figure 1a and an expression vector (pCMV-α-TfRscFv-flag-TOM5) loaded with a polynucleotide encoding a fusion protein (α-TfRscFv-flag-TOM5) including an anti-TfR single chain antibody (α-TfRscFv) and TOM5.

[0020] Figure 3b shows HEK293 cells (HEK293-MT) containing mitochondria expressing α-TfRscFv-flag-TOM5 fusion protein. α-TfRscFv_GFP ) is a diagram showing the results of confirming the expression of flag and GFP using immunofluorescence staining. DAPI: Nuclear staining

[0021] Figure 4a is a schematic diagram of the expression vector of Figure 1a and an expression vector (pCMV-Angiopep2-flag-TOM5) loaded with a polynucleotide encoding a fusion protein (Angiopep2-flag-TOM5) containing Angiopep2 and TOM5.

[0022] Figure 4b shows HEK293 cells containing mitochondria expressing Angiopep2-flag-TOM5 fusion protein (HEK293-MT Angiopep2_GFP ) is a diagram showing the results of confirming the expression of flag and GFP using immunofluorescence staining. DAPI: Nuclear staining

[0023] Figure 5 shows modified mitochondria (MT) derived from a human cell line (HEK293 cells) which is an embodiment of the present invention at the cellular level. GFP , MT α-IGF1RscFv_GFP , MT α-TfRscFv_GFP , MT Angiopep2_GFP )of This is a schematic diagram of a transwell assay to determine cell mobility. Mouse-derived brain endothelial cells (bEND.3 cells) were seeded in the upper well, and human skin fibroblasts were seeded in the lower well.

[0024] Figures 6a and 6b show modified mitochondria (MT) derived from a human cell line (HEK293 cells) which is an embodiment of the present invention at the cellular level. GFP , MT α-IGF1RscFv_GFP , MT α-TfRscFv_GFP , MT Angiopep2_GFP ) is a drawing showing the results of confirming the cell motility through microscopy (fluorescence microscopy and phase contrast microscopy). DAPI: Nuclear

[0025] Figure 7 shows modified mitochondria (MT) derived from a human cell line (HEK293 cells) which is an embodiment of the present invention at the cellular level. GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP )of This is a schematic diagram of a transwell assay to determine cell mobility. Mouse-derived brain endothelial cells (bEND.3 cells) were seeded in the upper well, and human neuroblastoma cells (SH-SY5Y cells) were seeded in the lower well.

[0026] Figure 8a shows modified mitochondria (MT) derived from a human cell line (HEK293 cells) as an example of the present invention at the cellular level. GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) is a drawing showing the results of confirming the cell motility through microscopy (fluorescence microscopy and phase contrast microscopy). DAPI: Nuclear

[0027] Figure 8b is a graph showing the results of measuring fluorescence by obtaining low well cells of Figure 8a.

[0028] Figure 9 shows modified mitochondria (MT) derived from human cell line (HEK293 cells) as an example of the present invention at the animal level. GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) is a schematic diagram of an experimental method to confirm the BBB permeability.

[0029] Figure 10 shows a modified mitochondrion (MT) derived from a human cell line (HEK293 cell) as an example of the present invention through the tail vein of a mouse. GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) is a diagram showing the results of immunohistochemical staining of the CA1 region of the hippocampus of mice 24 hours after administration. DAPI: nucleus, MTCO2: human mitochondria

[0030] Figure 11 shows a modified mitochondrion (MT) derived from a human cell line (HEK293 cell) as an example of the present invention through the tail vein of a mouse. GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) is a diagram showing the results of immunohistochemical staining of the CA3 region of the hippocampus of mice 24 hours after administration. DAPI: nucleus, MTCO2: human mitochondria

[0031] Figure 12 shows a modified mitochondrion (MT) derived from a human cell line (HEK293 cell) as one specific example of the present invention through the tail vein of a mouse. GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) was administered, and the results of immunohistochemical staining of the dentate gyrus region of the hippocampus of mice were observed 24 hours later. DAPI: nucleus, MTCO2: human mitochondria

[0032] Figure 13 shows a modified mitochondrion (MT) derived from a human cell line (HEK293 cell) as an example of the present invention through the tail vein of a mouse. GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) is a diagram showing the results of immunohistochemical staining of the substantia nigra region of the midbrain of mice 24 hours after administration. DAPI: nucleus, MTCO2: human-derived mitochondria

[0033] Figure 14 shows a specific example of the present invention, human pluripotent stem cell (iPSC)-derived modified mitochondria (iMT, iMT) in a Parkinson's disease mouse model induced by 6-OHDA treatment. α-IGF1RscFv ) is a schematic diagram of an experimental schedule to confirm the effect of improving motor dysfunction by administration.

[0034] Figure 15 shows a specific example of the present invention, human pluripotent stem cell (iPSC)-derived modified mitochondria (iMT, iMT) in a Parkinson's disease mouse model induced by 6-OHDA treatment. α-IGF1RscFv ) This is a graph showing the results of a pole test performed after administration, measuring the time it took for the pole to return from the top to the bottom.

[0035] Figure 16 shows a specific example of the present invention, human pluripotent stem cell (iPSC)-derived modified mitochondria (iMT, iMT) in a Parkinson's disease mouse model induced by 6-OHDA treatment. α-IGF1RscFv ) This is a graph showing the results of a pole test performed after administration, measuring the time it took for the mouse to land on the floor from the pole.

[0036] Figure 17 shows a specific example of the present invention, human pluripotent stem cell (iPSC)-derived modified mitochondria (iMT, iMT) in a Parkinson's disease mouse model induced by 6-OHDA treatment. α-IGF1RscFv ) This is a graph showing the results of the rotarod test performed after administration, measuring the delay time until the mouse fell from the rotarod.

[0037] Figure 18 shows a specific example of the present invention, human pluripotent stem cell (iPSC)-derived modified mitochondria (iMT, iMT) in a Parkinson's disease mouse model induced by 6-OHDA treatment. α-IGF1RscFv ) This is a graph showing the results of the rotarod test performed after administration, measuring the number of times the mouse fell from the rotarod.

[0038] Figure 19 shows a specific example of the present invention, human pluripotent stem cell (iPSC)-derived modified mitochondria (iMT, iMT) in a Parkinson's disease mouse model induced by 6-OHDA treatment. α-IGF1RscFv ) This is a graph showing the results of a cylinder test performed after administration, and it is the result of expressing the number of times the mouse used the forepaw on the contralateral side of the lesion as a percentage of the total number of times the mouse used the forepaw.

[0039] Figure 20a is a schematic diagram of an expression vector (pEF1α-αIGF1RscFv-myc-TOM5) loaded with a polynucleotide encoding a fusion protein (α-IGF1RscFv-myc-TOM5) comprising an anti-IGF1R single chain antibody (α-IGF1RscFv) and TOM5.

[0040] Figure 20b shows a human induced pluripotent stem cell line (iPSC-MT) containing mitochondria expressing α-IGF1RscFv-myc-TOM5 fusion protein. α-IGF1RscFv ) is a diagram showing the results of confirming the expression of α-IGF1RscFv-myc-TOM5 fusion protein through Western blot.

[0041] Figure 21a is a schematic diagram of an expression vector (pEF1α-Angiopep2-flag-TOM5) loaded with a polynucleotide encoding a fusion protein (Angiopep2-flag-TOM5) containing Angiopep2 and TOM5.

[0042] Figure 21b shows a human induced pluripotent stem cell line (iPSC-MT) containing mitochondria expressing the Angiopep2-flag-TOM5 fusion protein. Angiopep2 ) is a diagram showing the results of confirming the expression of Angiopep2-flag-TOM5 fusion protein through Western blot.

[0043] Definition of Terms

[0044] The term "antibody" as used herein refers to an immunoglobulin (Ig) molecule that immunologically reacts with a specific antigen, and a protein molecule that acts as a receptor that specifically recognizes the antigen, and is a concept that encompasses both whole antibodies and antibody fragments. The antibody fragment is a fragment of the overall immunoglobulin structure, and refers to a part of a polypeptide that contains a portion capable of binding to an antigen. In particular, an antibody fragment may have the same epitope determining region (CDR) as an antibody. The "antigen" is a structure capable of selectively binding to an antibody. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. Specifically, the antigen may be a polypeptide and a protein present on the cell surface or within a cell.

[0045] Specifically, the antibody or fragment thereof may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a single domain antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, an intrabody, a scFv, (scFv)2, an Fv, a Fab, a Fab', an F(ab')2, a diabody, a triabody, a tetrabody, a Bis-scFv, a nanobody (or VHH) and an epitope-binding fragment of any of the above. The antibody includes a heavy chain (HC) and a light chain (LC), and the heavy chain may include a variable region and a constant region.

[0046] The above "single-chain antibody (single-chain Fv, scFv)" refers to an antibody fragment that connects the variable regions of the light chain and the heavy chain. In some cases, it may include a linker composed of a peptide chain of about 15 amino acids. In this case, the scFv may have a structure of light chain variable region-linker-heavy chain variable region, or heavy chain variable region-linker-light chain variable region, and has an antigen specificity identical or similar to that of the entire antibody.

[0047] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, including a variable region (VH) and three constant regions, CH1, CH2, and CH3. Heavy chain constant regions (CH) exhibit different amino acid compositions and sequences, and thus possess different types of antigenicity. Accordingly, immunoglobulins can be classified into five categories and referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. In addition, depending on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same type of immunoglobulin can be classified into different subtypes. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4.

[0048] The term "light chain" as used herein refers to two types, λ and κ, consisting of approximately 211 to 217 amino acids. Each human antibody has exactly one such chain. The light chain is composed of a continuous variable region (VL) and a constant region (CL). The light chain may include both full-length light chains and fragments thereof.

[0049] The above "variable region" refers to the region of an antibody to which an antigen binds. The variable region includes three hypervariable regions called complementarity determining regions (CDRs) and four framework regions (FRs).

[0050] As used herein, the term "complementarity determining region" refers to a region among the variable regions of an antibody that confers binding specificity to an antigen. The CDRs primarily play a role in binding to epitopes of the antigen. The heavy and light chains each contain three complementarity determining regions. The CDRs of each chain are typically called CDR1, CDR2, and CDR3, sequentially starting from the N-terminus, and are identified by the chain on which the particular CDR is located. The FRs of each chain are typically called FR1, FR2, FR3, or FR4, sequentially starting from the N-terminus, and are identified by the chain on which the particular FR is located.

[0051] As used herein, the term "polynucleotide," also referred to as "nucleic acid," refers to a polymer of nucleotides of any length. Specifically, the polynucleotide may be DNA or RNA.

[0052] fusion protein

[0053] One aspect of the present invention provides a fusion protein comprising a mitochondrial outer membrane anchoring peptide; and a cerebrovascular endothelial cell surface protein binding site.

[0054] Mitochondrial outer membrane anchoring peptide

[0055] At this time, the mitochondrial outer membrane anchoring peptide may include an amino acid sequence that allows it to be located in the mitochondrial outer membrane. Therefore, the cerebrovascular endothelial cell surface protein binding site may be bound to the mitochondrial outer membrane by the mitochondrial anchoring peptide. The mitochondrial anchoring peptide may be a peptide that includes the N-terminal or C-terminal region of a protein present in a mitochondrial membrane protein, and the N-terminal or C-terminal region of a protein present in the mitochondrial outer membrane protein may be located in the mitochondrial outer membrane. At this time, the anchoring peptide may further include a mitochondrial signal sequence.

[0056] The protein present in the outer membrane of the above-mentioned mitochondria may be selected from proteins present in mitochondria present in eukaryotic cells. For example, it may be selected from proteins present in the outer membrane of mitochondria present in yeast, animal cells, or human cells, but is not limited thereto.

[0057] A specific example of a protein present in the membrane protein of the above mitochondrion may be any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1.

[0058] When the mitochondrial anchoring peptide is derived from any one selected from the group consisting of TOM20, TOM70 and OM45, the anchoring peptide may comprise the N-terminal region of TOM20, TOM70 or OM45.

[0059] In one specific embodiment, the mitochondrial anchoring peptide may comprise an N-terminal region of TOM20. More specifically, it may comprise an amino acid sequence of SEQ ID NO: 18 (human-derived TOM20), SEQ ID NO: 31 (human-derived TOM20), or SEQ ID NO: 32 (yeast-derived TOM20). In one specific embodiment, the mitochondrial anchoring peptide may comprise an N-terminal region of TOM70. More specifically, it may comprise an amino acid sequence of SEQ ID NO: 33 (human-derived TOM70) or SEQ ID NO: 34 (yeast-derived TOM70). In one specific embodiment, the mitochondrial anchoring peptide may comprise an N-terminal region of OM45. More specifically, it may comprise an amino acid sequence of SEQ ID NO: 35 (yeast-derived OM45).

[0060] Additionally, when the mitochondrial anchoring peptide is derived from any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1, it may include any one C-terminal region selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1.

[0061] In one embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of TOM5. More specifically, it may comprise the amino acid sequence of SEQ ID NO: 20 (human-derived TOM5), SEQ ID NO: 29 (human-derived TOM5), or SEQ ID NO: 30 (yeast-derived TOM5). In one embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of TOM6. In one embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of TOM7. More specifically, it may comprise the amino acid sequence of SEQ ID NO: 36 (human-derived TOM7) or SEQ ID NO: 37 (yeast-derived TOM7). In one embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of TOM22. More specifically, it may comprise the amino acid sequence of SEQ ID NO: 38 (human-derived TOM22) or SEQ ID NO: 39 (yeast-derived TOM22). In one specific embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of Fis1. More specifically, it may comprise the amino acid sequence of SEQ ID NO: 40 (human-derived Fis1) or SEQ ID NO: 41 (yeast-derived Fis1). In one specific embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of Bcl-2. More specifically, it may comprise the amino acid sequence of SEQ ID NO: 42 (human-derived Bcl-2 alpha). In one specific embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of Bcl-x. In one specific embodiment, the mitochondrial anchoring peptide may comprise the C-terminal region of VAMP1. More specifically, it may comprise the amino acid sequence of SEQ ID NO: 43 (human-derived VAMP1) or SEQ ID NO: 44 (yeast-derived VAMP1).

[0062] Cerebral vascular endothelial cell surface protein binding site

[0063] The term "cerebral vascular endothelial cell surface protein binding site" as used herein refers to a site that specifically binds to a protein expressed on the surface of cerebral vascular endothelial cells. The cerebral vascular endothelial cells are one of the cells that constitute the blood-brain barrier (BBB). Here, the "blood-brain barrier" refers to a structure centered on the cerebral blood vessels that strictly controls the movement of substances such as ions, molecules, and pathogens present in the blood into brain tissue, while enabling the selective absorption of essential components for sustaining life, such as amino acids and glucose. The blood-brain barrier is composed of cells such as blood-brain barrier endothelial cells, astrocytes, and pericytes. The cells are arranged in a form that shares a common basement membrane, and on one side of the blood-brain barrier, tightly connected blood-brain barrier endothelial cells are distributed, and on the other side, astrocytes that surround the blood vessels are distributed. Endothelial cells of the blood-brain barrier form the walls of capillaries and are connected by extremely strong and complex tight junctions. This structure forms a physical barrier, impeding the simple diffusion of most substances, including molecules of average to large size, such as insulin.

[0064] The above-mentioned cerebral vascular endothelial cell surface protein binding site may be a protein capable of binding to a receptor or ligand present on the cell membrane of cerebral vascular endothelial cells. Specifically, it may be selected from the group consisting of, but is not limited to, an antibody or a fragment thereof, a peptide or a fragment thereof, a receptor or a fragment thereof, a ligand or a fragment thereof, and an aptamer. In this case, the antibody fragment may be any one selected from the group consisting of, but is not limited to, a Fab, a Fab', a scFv, F(ab)2, a nanobody (VHH), and a combination thereof.

[0065] The above cerebrovascular endothelial cell surface protein is any one selected from the group consisting of transferrin receptor (TfR), insulin receptor (IR), insulin-like growth factor receptor (IGFR), low density lipoprotein receptor-related protein 8 (LRP8), low density lipoprotein receptor-related protein 1 (LRP1), heparin-binding epidermal growth factor-like growth factor (HB-EGF), leptin receptor, nicotinic acetylcholine receptors (nAChRs), glutathione transporter, calcium-activated potassium channel, and receptor for advanced glycation endproducts (RAGE). It may include, but is not limited to.

[0066] More specifically, the cerebrovascular endothelial cell surface protein binding site may be a site that specifically binds to transferrin receptor (TfR), insulin-like growth factor receptor (IGFR), or low-density lipoprotein receptor-related protein 1 (LRP1), but is not limited thereto.

[0067] The term "transferrin receptor (TfR)" used herein refers to a membrane glycoprotein expressed on the surface of cells that mediates the intracellular uptake of iron from transferrin, a plasma glycoprotein. It is widely distributed in normal cells of various tissues, and is also known to be expressed in large quantities in activated immune cells and tumor cells. The extracellular domain (ectodomain) of the transferrin receptor is divided into an apical domain, a helical domain, and a protease-like domain, and these are known to have different binding affinities for target substances during the receptor-mediated transcytosis process. The "transcytosis" is a type of transmembrane transport in which various types of macromolecules are transported through the cell as a cytopempsis system. At this time, the macromolecules enter a vesicle on one side of the cell and are then released to the other side.

[0068] The genetic information of the above transferrin receptor can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI). As a specific example, it may include the amino acid sequence of SEQ ID NO: 45.

[0069] As used herein, the term "insulin-like growth factor receptor (IGFR)" refers to a tyrosine kinase receptor activated by insulin-like growth factor (IGF). The insulin-like growth factor exists in two forms, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2), and binds to the insulin-like growth factor receptor (IGF1R or IGF2R). At this time, IGF-1 binds to IGF1R with a higher binding affinity, and IGF-2 binds to IGF2R with a higher binding affinity. The IGF is known to activate IGFR distributed in various tissue cell membranes to regulate cell division, cell differentiation, and cell survival. In the present invention, the IFGR may be IGF1R. The genetic information of the IGF1R can be obtained from a known database such as NCBI's GenBank. As a specific example, it may include the amino acid sequence of SEQ ID NO: 46.

[0070] As used herein, the term "low-density lipoprotein receptor-related protein 1 (LRP1)" refers to a low-density lipoprotein receptor (LDLR) family protein, also known as alpha-2-macroglobulin receptor (A2MR), apolipoprotein E receptor (APOER), or cluster of differentiation 91 (CD91). LRP1 is ubiquitously expressed in various tissues, and is known to be most highly expressed in vascular smooth muscle cells, hepatocytes, and neural cells. LRP1 plays an important role in intracellular signaling and endocytosis. In particular, it is known to regulate lipid and lipoprotein metabolism, proteolytic enzyme degradation, platelet-derived growth factor receptor regulation, integrin maturation and recycling, vascular tone, blood-brain barrier permeability regulation, cell growth, cell migration, inflammation, and apoptosis. The genetic information of the above LRP1 can be obtained from a known database such as NCBI's GenBank. As a specific example, it may include the amino acid sequence of SEQ ID NO: 47.

[0071] In one specific embodiment of the present invention, the cerebral vascular endothelial cell surface protein binding site may be a peptide or a fragment thereof.

[0072] In one specific example, the cerebral vascular endothelial cell surface protein binding site may include a peptide or fragment thereof that binds to low-density lipoprotein receptor-related protein 1 (LRP1). Specifically, it may include or consist of the amino acid sequence of SEQ ID NO: 16.

[0073] In addition, as a specific example of the present invention, the cerebral vascular endothelial cell surface protein binding site may be an antibody or a fragment thereof. In this case, the antibody fragment may be any one selected from the group consisting of Fab, Fab', scFv, F(ab)2, nanobody (VHH), and combinations thereof, but is not limited thereto.

[0074] As a specific example, the cerebral vascular endothelial cell surface protein binding site may be an antibody fragment that binds to the insulin-like growth factor 1 receptor (IGF1R).

[0075] As a specific example, the cerebral vascular endothelial cell surface protein binding site may be an antibody fragment that binds to the transferrin receptor (TfR).

[0076] In one embodiment of the present invention, the antibody fragment may be a scFv and may comprise or consist of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12.

[0077] Structure of fusion proteins

[0078] In the present invention, when the mitochondrial outer membrane anchoring peptide of the fusion protein includes the N-terminal region of TOM20, TOM70 or OM45, the mitochondrial outer membrane anchoring peptide and the cerebral vascular endothelial cell surface protein binding site can be combined from the N-terminus to the C-terminus.

[0079] Specifically, the fusion protein may be composed of the following structural formula (I):

[0080] N'-mitochondrial anchoring peptide-[L(1)]m-cerebrovascular endothelial cell surface protein binding site-C' (I)

[0081] In the above structural formula (I),

[0082] The above N' is the N-terminus,

[0083] The above C' is the C-terminal,

[0084] The above L(1) is a peptide linker,

[0085] The above m is 0 or 1.

[0086] At this time, the mitochondrial anchoring peptide may be the N-terminal region of TOM20, TOM70, or OM45. The cerebrovascular endothelial cell surface protein binding site may be a peptide, a ligand, a receptor, or a fragment thereof, or an aptamer.

[0087] In the present invention, when the mitochondrial outer membrane anchoring peptide of the fusion protein includes a C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1, the cerebrovascular endothelial cell surface protein binding site and the mitochondrial outer membrane anchoring peptide can be linked from the N-terminus to the C-terminus.

[0088] Specifically, the fusion protein may be composed of the following structural formula (II):

[0089] N'-cerebrovascular endothelial cell surface protein binding site-[L(2)]n-mitochondrial outer membrane anchoring peptide-C'(II)

[0090] In the above structural formula (II),

[0091] The above N' is the N-terminus of the fusion protein,

[0092] The above C' is the C-terminus of the fusion protein,

[0093] The above L(2) is a peptide linker,

[0094] The above n is 0 or 1.

[0095] At this time, the mitochondrial anchoring peptide may be a C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1. The cerebrovascular endothelial cell surface protein binding site may be an antibody, a peptide, a ligand, a receptor, or a fragment thereof, or an aptamer.

[0096] The above mitochondrial anchoring peptide and cerebral vascular endothelial cell surface protein binding sites are the same as described above.

[0097] The above linker (1) and linker (2) may be peptide linkers composed of about 1 to about 150 amino acids. The linker may be composed of about 1 to about 150, about 1 to about 100, about 1 to about 50, or about 1 to about 30 amino acids, but is not limited thereto. In addition, the linker may be formed by appropriately selecting from about 20 amino acids. In one specific example, the linker may be a polypeptide composed of 1 to 30 serine, glycine, or threonine, singly or in combination. In one specific example, the linker may be about 5 to about 50 amino acids composed of serine and glycine. For example, as (G4S)n, n is an integer from 1 to 10, and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker may comprise the amino acid sequence of SEQ ID NO: 26.

[0098] polynucleotide

[0099] Another aspect of the present invention provides a polynucleotide encoding the fusion protein. Specifically, the polynucleotide may comprise a nucleic acid sequence encoding the fusion protein of structural formula (I) or structural formula (II).

[0100] Additionally, the polynucleotide may include a sequence encoding a tag sequence. The tag may be, but is not limited to, flag, myc, a histidine tag, etc. In one embodiment of the present invention, the fusion protein may include flag (SEQ ID NO: 24) or myc (SEQ ID NO: 22), and in this case, the polynucleotide encoding the fusion protein may include a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0101] When the above polynucleotide sequence is manufactured by chemical synthesis, synthetic methods widely known in the art, for example, the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on solid supports, etc. can be used.

[0102] Additionally, the polynucleotide may comprise a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a nucleic acid sequence encoding a fusion protein of the structural formula (I) or structural formula (II).

[0103] vector loaded with polynucleotides

[0104] Another aspect of the present invention provides a vector loaded with the polynucleotide. The polynucleotide is the same as described above.

[0105] In one embodiment of the present invention, the vector may comprise a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0106] The above "vector" can be introduced into a host cell and recombined and integrated into the host cell genome. Alternatively, the vector is understood to be a nucleic acid vehicle containing a polynucleotide sequence capable of autonomously replicating as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0107] Specifically, the vector may be plasmid DNA, phage DNA, etc., and may be a commercially developed plasmid (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), yeast-derived plasmid (e.g., YEp13, YEp24, YCp50, etc.), phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.), insect virus vector (e.g., baculovirus, etc.). Since the protein expression amount and modification of the vector vary depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0108] As used herein, the term "gene expression" or "expression" of a target protein is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen) or a variant thereof. The expression vector may include a human cytomegalovirus (CMV) promoter to promote continuous transcription of the target gene in mammalian cells, and a polyadenylation signal sequence to increase the steady-state level of RNA after transcription.

[0109] Additionally, the vector may include a labeling factor such as a fluorescent marker protein. For example, it may be GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), BFP (blue fluorescent protein), RFP (red fluorescent protein), etc., but is not limited thereto. In one embodiment, the fluorescent marker protein may be GFP (SEQ ID NO: 14).

[0110] Transformed cells

[0111] Another aspect of the present invention provides a transformed cell into which the polynucleotide has been introduced.

[0112] As used herein, the term "transformed cell" refers to a eukaryotic cell into which the polynucleotide can be introduced. The transformed cell can be produced by introducing a vector loaded with the polynucleotide into a host cell and transforming the cell.

[0113] The above transformation can be performed using various methods. Specifically, the transformation methods include electroporation, calcium phosphate precipitation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation. Furthermore, the target product can be delivered into cells using viral particles via infection. Furthermore, the vector can be introduced into the host cell by gene bombardment, etc.

[0114] In addition, the host cell used to produce the transformed cell is not particularly limited, as long as it can produce the fusion protein of the present invention. For example, it may include a eukaryotic cell, mammalian cell, plant, insect, fungal, or cellular cell. Specifically, it may be a eukaryotic cell containing mitochondria or a cell of mammalian origin. Preferably, it may be a cell of human origin, but is not limited thereto. An example of the eukaryotic cell may be yeast. In addition, the mammalian cells may include CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, HEK293T cells, stem cells, platelets, etc., and all cells that can be used as mammalian host cells known to those skilled in the art may be used.

[0115] In addition, in order to optimize the characteristics of the fusion protein according to the present invention or for other purposes, the glycosylation-related genes of the host cell can be manipulated using methods known to those skilled in the art to adjust the pattern (e.g., sialic acid, fucosylation, glycosylation) of the fusion protein.

[0116] modified mitochondria

[0117] Another aspect of the present invention provides a mitochondrion comprising a fusion protein comprising a mitochondrial anchoring peptide and a vascular endothelial cell surface protein binding site. In this case, the fusion protein in the modified mitochondrion can bind to and be located in the mitochondrial outer membrane.

[0118] The term "mitochondria" used herein refers to eukaryotic organelles involved in the synthesis and regulation of adenosine triphosphate (ATP), the energy source within cells, and are associated with various metabolic pathways in the body, such as cell signaling, cell differentiation, and apoptosis, as well as the control of the cell cycle and cell growth. Therefore, it has been reported that mitochondrial dysfunction or dysfunction due to genetic, environmental, or unknown causes is related to the development of various diseases, such as mitochondrial-related genetic diseases, inflammatory diseases such as rheumatoid arthritis, ischemic diseases, infectious diseases, heart diseases, muscle diseases, degenerative diseases such as Parkinson's disease and Alzheimer's disease, and the occurrence and metastasis of various cancers.

[0119] The modified mitochondria may be obtained from a eukaryotic cell, or may be obtained from a mammal or a human. Specifically, the mitochondria may be isolated from a cell.

[0120] The term "cell" as used herein refers to a structural or functional unit that constitutes a living organism, consisting of cytoplasm surrounded by a cell membrane, and containing biomolecules such as proteins and nucleic acids. The cell refers to a cell that contains mitochondria within the cell membrane.

[0121] For example, the modified mitochondria may be obtained from somatic cells, germ cells, or stem cells, or may be isolated from blood cells or platelets. Furthermore, the modified mitochondria may be isolated after cell disruption and use, or may be isolated after disruption from a cell sample that has been frozen and then thawed. Furthermore, the modified mitochondria may be isolated from a cell sample that has been frozen and then thawed.

[0122] Specifically, the somatic cell may be a muscle cell, a hepatocyte, a nerve cell, a fibroblast, an epithelial cell, an adipocyte, an osteocyte, a leukocyte, a lymphocyte, a platelet, a germ cell, or a mucosal cell.

[0123] In addition, the stem cells are undifferentiated cells capable of differentiating into various types of tissue cells, and may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue-derived stem cells, but are not limited thereto. In this case, the mesenchymal stem cells may be obtained from any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, synovial fluid, testis, amniotic membrane, and placenta.

[0124] The modified mitochondria may be obtained from an autologous, allogenic, or xenogenic source. Specifically, autologous mitochondria refer to mitochondria obtained from tissues or cells of the same individual. Furthermore, allogeneic mitochondria refer to mitochondria obtained from an individual of the same species as the individual but with different genotypes for alleles. Furthermore, xenogenic mitochondria refer to mitochondria obtained from an individual of a different species from the individual.

[0125] At this time, the subject may be a mammal, and preferably a human.

[0126] Additionally, the modified mitochondria may be intact and have normal biological activity.

[0127] Meanwhile, when isolating the modified mitochondria from a specific cell, the mitochondria can be isolated using various known methods, including modified methods such as using a specific buffer solution or utilizing a potential difference and a magnetic field.

[0128] The above mitochondrial separation can be obtained by disrupting cells and centrifuging them in order to maintain mitochondrial activity.

[0129] In the present invention, the modified mitochondria include a binding site that binds to a surface protein of cerebral vascular endothelial cells, thereby binding to cerebral vascular endothelial cells of the blood-brain barrier and passing through the cerebral vascular endothelial cells through transcytosis to enter the blood-brain barrier.

[0130] Therefore, the modified mitochondria combined with the above fusion protein can be used to prevent or treat brain diseases caused by mitochondrial dysfunction.

[0131] Pharmaceutical composition comprising modified mitochondria

[0132] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating brain diseases, comprising the modified mitochondria. The modified mitochondria are the same as described above.

[0133] The above brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis (ALS), spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, mitochondrial encephalomyopathy, migraine, cerebral infarction or hypoxic encephalopathy. It may be selected from the group consisting of ischemic brain disorders such as cerebral arteriosclerosis, bipolar disorder, chronic fatigue syndrome, intracranial hypertension such as hydrocephalus or head trauma, normal pressure hydrocephalus, cerebral vasospasm after subarachnoid hemorrhage, cerebral ischemia during surgery and endovascular surgery, and frontotemporal dementia.

[0134] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the pharmaceutical composition. Furthermore, "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering the pharmaceutical composition.

[0135] In the pharmaceutical composition of the present invention, the modified mitochondria, which are the active ingredient, may be included in any amount (effective amount) depending on the intended use, formulation, compounding purpose, etc., as long as they can exhibit a disease-preventing or therapeutic effect. Here, the "effective amount" refers to the amount of the active ingredient that can induce a disease-preventing or therapeutic effect. Such an effective amount can be experimentally determined within the scope of a person skilled in the art's ordinary ability. With respect to the pharmaceutical composition, the modified mitochondria may be included in a concentration of 0.1 μg / ml to 500 μg / ml, 0.2 μg / ml to 450 μg / ml, or 0.5 μg / ml to 400 μg / ml, but is not limited thereto.

[0136] By including mitochondria within the above range, it is easy to control the mitochondrial dosage during administration, and the degree of improvement in the patient's symptoms can be improved. At this time, the mitochondrial dosage can be quantified by quantifying the membrane protein of the modified mitochondria. Specifically, the modified mitochondria can be quantified using the Bradford protein assay (paper written by James D. McCully (J Vis Exp. 2014; (91): 51682.).

[0137] In addition, for the pharmaceutical composition, the cerebrovascular endothelial cell surface protein binding site that binds to mitochondria may be included in a concentration of 0.1 μg / ml to 500 μg / ml, 0.2 μg / ml to 450 μg / ml, or 0.5 μg / ml to 400 μg / ml, but is not limited thereto. By including the cerebrovascular endothelial cell surface protein binding site in the above range, the influx of the mitochondria through the blood-brain barrier into the brain upon administration may be increased, thereby improving the degree of improvement in the patient's symptoms.

[0138] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Examples of such carriers include distilled water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0139] Specifically, the pharmaceutical composition may be formulated as a parenteral formulation, depending on the route of administration, using conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. When the pharmaceutical composition is formulated as a parenteral formulation, it may be formulated into injections, transdermal administration, and nasal inhalants using methods known in the art, together with a suitable carrier.

[0140] As a specific example, the pharmaceutical composition of the present invention may be an injectable formulation. Therefore, to ensure product stability during distribution of the injectable formulation, the pharmaceutical composition of the present invention can be manufactured into an injectable formulation that is extremely physically and chemically stable by adjusting the pH using a buffer solution, such as an acid solution or phosphate solution suitable for injectable formulations.

[0141] Specifically, the pharmaceutical composition of the present invention may include water for injection.

[0142] The above-mentioned water for injection is distilled water made for dissolving solid injections or diluting water-soluble injections, and may be glucose injection, xylitol injection, D-mannitol injection, fructose injection, physiological saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactated-Ringer's solution, or a phosphate buffer solution or sodium dihydrogen phosphate-citrate buffer solution having a pH ranging from about 3.5 to about 7.5.

[0143] The pharmaceutical composition of the present invention may further comprise a stabilizer or a solubilizer. For example, the stabilizer may be pyrosulfite or ethylene diaminetetraacetic acid, and the solubilizer may be hydrochloric acid, acetic acid, potassium phosphate hydroxide, potassium bicarbonate, potassium carbonate, or Tris. In one specific example, the pharmaceutical composition may comprise a mixed preservative solution such as a trehalose-tris-glycine (TTG) solution, which may be commonly used in pharmaceutically acceptable drug preparations.

[0144] Specifically, the pharmaceutical composition of the present invention may include an injectable liquid composition in addition to modified mitochondria. In this case, the modified mitochondria are the same as those described above. By including the injectable liquid composition, the pharmaceutical composition of the present invention is a composition for the prevention or treatment of diseases, and can suppress thrombosis that may be caused by mitochondrial aggregation, platelet reduction, and aggregation when administering mitochondria via injection, and can maintain and / or enhance mitochondrial stability and also stably maintain mitochondrial activity.

[0145] Here, the liquid composition may include glycine, sugar and a buffer.

[0146] The glycine may be present in the injectable liquid composition at a concentration of about 15 mM or more, but is not limited thereto, and specifically, may be present at a concentration of about 15 mM to about 150 mM, about 17 mM to about 130 mM, about 20 mM to about 120 mM, about 22 mM to about 110 mM, or about 25 mM to about 100 mM. In addition, the glycine may be used together with one or more amino acids selected from the group consisting of, but is not limited to, histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine.

[0147] In addition, the sugar included in the liquid composition for injection may be, but is not limited to, one or more selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin. In particular, the sugar may be trehalose, mannitol, or sucrose. Preferably, the sugar may be trehalose.

[0148] The buffer included in the above injectable liquid composition may be selected from the group consisting of, but not limited to, Tris buffer, HEPES buffer, MOPS buffer, and acetate or phosphate-containing buffer. Preferably, the buffer may be an injectable Tris buffer.

[0149] At this time, the pH of the buffer is not limited thereto, but may be in the range of about 7.0 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5.

[0150] Additionally, the buffer may be present in the injectable liquid composition at a concentration of, but not limited to, about 5 mM to about 50 mM, about 8 mM to about 40 mM, about 10 mM to about 35 mM, about 13 mM to about 30 mM, or about 15 mM to about 25 mM.

[0151] The above-described liquid composition for injection may have an osmolarity in the range of about 200 to about 400 mOsm, about 230 to about 380 mOsm, about 250 to about 350 mOsm, about 260 to about 320 mOsm, about 270 to about 330 mOsm, or about 280 to about 300 mOsm. In this case, the osmolarity in the above range facilitates long-term storage at a temperature of 2°C to 8°C or higher, while making the composition suitable for parenteral administration, for example, intravascular, intramuscular, or subcutaneous injection, without causing side effects to a subject.

[0152] As used herein, the term "osmolarity" refers to the moles of solute contributing to the osmotic pressure of a solution per kilogram of solvent, and osmolarity is determined by measuring the freezing point depression of a sample using an osmometer.

[0153] Additionally, the above injectable liquid composition may further comprise a chelating agent.

[0154] The chelating agent may be, but is not limited to, one or more selected from the group consisting of injectable grades of EGTA, EDTA, and BAPTA. The chelating agent may eliminate damage caused by ion outflow after obtaining mitochondria contained in the injectable liquid composition.

[0155] In addition, the pharmaceutical composition may include, as additives, antioxidants, ATP, magnesium, etc. that are effective in maintaining the function and activity of mitochondria.

[0156] The pharmaceutical composition of the present invention may be stored in a container selected from the group consisting of a vial, a cartridge, a syringe, and an autoinjector. Furthermore, the container containing the pharmaceutical composition may be stored at room temperature, a refrigerated temperature of about 2°C to about 8°C, or a temperature of about 25°C to about 40°C until administration to a subject in need of treatment.

[0157] Meanwhile, the pharmaceutical composition of the present invention can be administered to a subject in a pharmaceutically effective amount. The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount of an active ingredient effective in preventing or treating a target disease, which is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects. The level of the effective amount can be determined based on factors including the patient's health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, drugs used in combination or concurrently, and other factors well known in the medical field.

[0158] The term "administration" as used herein refers to introducing a given substance into a subject in an appropriate manner, and the route of administration of the composition may be any common route as long as it can reach the target tissue. Examples of such routes include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, and rectal administration.

[0159] The preferred dosage of the pharmaceutical composition of the present invention is, but is not limited to, about 0.01 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 4 mg / kg, or about 0.25 mg / kg to about 2.5 mg / kg of mitochondria per dose based on the body weight of the subject to be administered. That is, in terms of cell activity, it is most preferable that the modified mitochondria of the pharmaceutical composition be administered in the above range based on the body weight of the subject in which cancer tissue exists. In addition, the pharmaceutical composition may be administered once or multiple times. Specifically, it may be administered 1 to 10 times, 1 to 5 times, or 1 to 3 times, and preferably, it may be administered once. Such dosage should not be construed as limiting the scope of the present invention in any aspect.

[0160] The above "subject" refers to a subject to which the composition of the present invention can be applied (prescribed), and may be a subject suffering from a disease. In addition, the subject may be a mammal, including a human, and preferably a human.

[0161] The above pharmaceutical composition may additionally contain or be used in combination with an agent for preventing or treating brain diseases.

[0162] Another aspect of the present invention provides a method for preventing or treating a brain disease, comprising administering to a subject the modified mitochondria or a pharmaceutical composition comprising the same as an active ingredient for preventing or treating a brain disease.

[0163] Another aspect of the present invention provides a use of the modified mitochondria or a pharmaceutical composition comprising the same as an active ingredient for preventing or treating brain diseases.

[0164] At this time, the modified mitochondria, pharmaceutical composition, brain disease, prevention, treatment and subject are the same as described above.

[0165] In addition, the modified mitochondria or the pharmaceutical composition containing them as an active ingredient may additionally contain or be used in combination with a known agent for preventing or treating brain diseases.

[0166] Method for producing modified mitochondria

[0167] Another aspect of the present invention provides a method for producing modified mitochondria, comprising the steps of i) producing a transformed cell by introducing a polynucleotide encoding the fusion protein into the cell, and ii) isolating the modified mitochondria from the transformed cell.

[0168] At this time, the transformed cell may be a mammalian cell, and preferably a human cell. In addition, the polynucleotide may be loaded onto a vector and introduced into the cell. The polynucleotide encoding the fusion protein, the vector, the transformed cell, the modified mitochondria, and the human cell are the same as described above.

[0169] The above modified mitochondria can be separated by disrupting and centrifuging the transformed cells in order to maintain mitochondrial activity.

[0170] In one specific example, the method may be performed by culturing transformed cells, centrifuging a composition containing the cells a first time to produce a pellet, resuspending the pellet in a buffer solution and homogenizing it, centrifuging the homogenized solution a second time to produce a supernatant, and centrifuging the supernatant a third time to purify mitochondria. At this time, the speed may be increased from the first centrifugation to the third centrifugation.

[0171] Specifically, the first to third centrifugations may be performed at a temperature of about 0°C to about 10°C, preferably about 3°C ​​to about 5°C. In addition, the time for performing the centrifugation may be performed for about 1 minute to 50 minutes, and may be appropriately adjusted depending on the number of centrifugations and the content of the sample.

[0172] In addition, the first centrifugation may be performed at a speed of about 100xg to about 3,000xg, about 200xg to about 2,500xg, or about 300xg to about 2,000xg. In addition, the second centrifugation may be performed at a speed of about 1xg to about 15,000xg, about 25xg to about 13,000xg, or about 500xg to about 12,000xg. In addition, the third centrifugation may be performed at a speed of about 100xg to about 30,000xg, about 500xg to about 25,000xg, or about 800xg to about 20,000xg.

[0173] Pharmaceutically acceptable sugars may be used to stabilize the obtained mitochondria. Specifically, sugars such as sucrose, mannitol, and trehalose may be used, but are not limited thereto. Furthermore, pharmaceutically acceptable pH buffering agents such as tris, HEPES, and phosphate may be used, but are not limited thereto.

[0174] Meanwhile, after obtaining the modified mitochondria, additives such as chelators and antioxidants can be used to remove damage caused by ion outflow and to suppress oxidative stress. These additives can include, without limitation, reagents widely known in the art. Examples thereof include, but are not limited to, EDTA, EGTA, citrate, glycine, taurine, ATP, etc.

[0175] In addition, the above modified mitochondria can be separated by thawing the frozen transformed cells, crushing them, and centrifuging them. The method for obtaining the modified mitochondria can be performed by the steps of freezing the transformed cells, thawing the cells, and crushing the thawed cells.

[0176] The freezing may be, but is not limited to, at a temperature of -1°C or lower. Specifically, the freezing may be performed at a temperature range of about -5°C to about -200°C, about -15°C to about -180°C, about -25°C to about -160°C, about -40°C to about -140°C, about -55°C to about -120°C, about -60°C to about -100°C, or about -70°C to about -90°C.

[0177] The freezing can be performed using liquid nitrogen (LN2) or a freezing device. Specifically, the freezing or refrigeration process can be performed by rapid freezing using LN2, or by using a freezing device such as a deep freezer, a freezer, or a freezing container. The freezing temperature of the transformed cells is not particularly limited as long as the activity of the modified mitochondria can be maintained at normal levels. When the modified mitochondria are frozen using a freezing device, a freezing container can be used. The freezing container can freeze the cells by lowering the temperature by about 1°C per minute. Preferably, the freezing container can be used when a deep freezer is used. In addition, when the modified mitochondria are frozen using a freezing device, a freezing container may not be used.

[0178] The above freezing may be performed for about 24 hours or more, about 48 hours or more, or about 96 hours or more, but is not particularly limited as long as the frozen modified mitochondria have normal activity.

[0179] Mitochondria can be quantified by quantifying the membrane proteins of the modified mitochondria. Specifically, the modified mitochondria can be quantified using the BCA assay (bicinchoninic acid assay).

[0180] Additionally, the number of modified mitochondria can be measured using a particle counter (Multisizer 4e, Beckman Coulter).

[0181] The above modified mitochondria can be used immediately after being isolated from cells or tissues.

[0182] Additionally, the modified mitochondria can be separated from cells, frozen or lyophilized, and then used.

[0183] The above modified mitochondria can be mixed with a mitochondrial cryoprotectant containing glycine and frozen.

[0184] When freezing the modified mitochondria, the freezing step may be performed at a temperature of about -40°C or lower. Specifically, the freezing may be performed at a temperature of about -80°C to about -40°C, about -70°C to about -40°C, about -60°C to about -40°C, or about -50°C to about -40°C. At this time, the freezing may be performed for about 5 minutes to about 120 minutes, about 10 minutes to about 100 minutes, about 15 minutes to about 80 minutes, about 20 minutes to about 60 minutes, or about 25 minutes to about 40 minutes.

[0185] At this time, the cryoprotectant may additionally include any one selected from the group consisting of trehalose, tris, and combinations thereof. In the present invention, the cryoprotectant may be used in the form of an aqueous solution, and the aqueous solution may be water for injection, physiological saline, phosphate buffer solution, purified water, or deionized water.

[0186] The above cryoprotectant can maintain and / or enhance the stability of the mitochondria when the modified mitochondria are frozen, and can also stably maintain the activity of the modified mitochondria.

[0187] Specifically, the cryoprotectant of the present invention may include glycine. The glycine may be included in the protectant at a concentration of, but is not limited to, about 15 mM or more. Specifically, the glycine may be included at a concentration of, but is not limited to, about 15 mM to about 150 mM, about 17 mM to about 130 mM, about 20 mM to about 120 mM, about 22 mM to about 110 mM, or about 25 mM to about 100 mM. In addition, the glycine may be used together with one or more amino acids selected from the group consisting of, but not limited to, histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine.

[0188] The cryoprotectant may further comprise a sugar. In this case, the sugar may be at least one selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin. Preferably, the sugar may be trehalose. Specifically, trehalose in the cryoprotectant may be included at a concentration of about 40 mM to about 400 mM, about 50 mM to about 350 mM, about 60 mM to about 300 mM, about 70 mM to about 250 mM, or about 80 mM to about 200 mM.

[0189] The cryoprotectant may further comprise a buffer. The buffer included in the injectable liquid composition may be selected from the group consisting of, but is not limited to, Tris buffer, HEPES buffer, MOPS (3-(N-morpholino)propanesulfonic acid) buffer, and acetate or phosphate-containing buffer. At this time, the pH of the buffer may be, but is not limited to, about 7.0 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5. Preferably, the buffer in the present invention may be a Tris buffer. Specifically, the cryoprotectant may be included at a concentration of about 5 mM to about 50 mM, about 8 mM to about 40 mM, about 10 mM to about 35 mM, about 13 mM to about 30 mM, or about 15 mM to about 25 mM.

[0190] More specifically, the cryoprotectant of the present invention may include glycine, and may further include any one selected from the group consisting of trehalose, tris, and combinations thereof. In one specific example, the cryoprotectant may include glycine and trehalose. The cryoprotectant may include glycine and tris. In one specific example, the cryoprotectant may include glycine, trehalose, and tris.

[0191] Additionally, the cryoprotectant may further include a chelating agent.

[0192] The chelating agent may be, but is not limited to, one or more selected from the group consisting of injectable grades of EGTA, EDTA, and BAPTA. The chelating agent may inhibit damage caused by ion outflow after obtaining the modified mitochondria included in the injectable liquid composition.

[0193] In addition, the cryoprotectant may include, as an additive, an antioxidant, ATP, magnesium, etc., which are effective in maintaining the function and activity of mitochondria.

[0194] The freeze-drying process described above is not limited thereto, but can be divided into a step of freezing the modified mitochondria and a step of drying the mitochondria. The freeze-drying process can be performed under vacuum. The freeze-drying of the modified mitochondria can be performed by freezing the mitochondria under vacuum and then gradually increasing the temperature.

[0195] When freeze-drying modified mitochondria, the freezing step may be performed at a temperature of about -40°C or lower. Specifically, the freezing step may be performed at a temperature of about -80°C to about -40°C, about -70°C to about -40°C, about -60°C to about -40°C, or about -50°C to about -40°C. At this time, the freezing may be performed for about 5 minutes to about 120 minutes, about 10 minutes to about 100 minutes, about 15 minutes to about 80 minutes, about 20 minutes to about 60 minutes, or about 25 minutes to about 40 minutes.

[0196] When freeze-drying modified mitochondria, the drying step can be divided into three stages. The first stage drying can be performed at about -30°C to about 0°C. More specifically, it can be performed at about -40°C to about 0°C, about -30°C to about -5°C, or about -20°C to about -10°C. At this time, the first stage drying can be performed for about 1 hour to about 50 hours. Specifically, it can be performed for about 1 hour to about 50 hours, about 5 hours to about 50 hours, about 10 hours to about 50 hours, about 20 hours to about 50 hours, or about 30 hours to about 50 hours.

[0197] The second stage drying can be performed at a temperature of about 0°C to about 10°C or lower. More specifically, it can be performed at a temperature of about 0°C to about 10°C, about 2°C to about 9°C, about 4°C to about 8°C, or about 5°C to about 7°C. At this time, the second stage drying can be performed for about 1 hour to about 24 hours. Specifically, the second stage drying can be performed for about 1 hour to about 16 hours, about 2 hours to about 16 hours, about 4 hours to about 16 hours, about 6 hours to about 16 hours, or about 8 hours to about 16 hours.

[0198] The third stage drying can be performed at a temperature of about 11°C to about 30°C or less. More specifically, it can be performed at a temperature of about 11°C to about 30°C, about 15°C to about 30°C, about 20°C to about 30°C, or about 25°C to about 30°C. At this time, the third stage drying can be performed for about 1 hour to about 18 hours.

[0199] The frozen modified mitochondria can be thawed in a refrigerator. Specifically, they can be thawed at about 0°C to about 10°C. Furthermore, the thawed modified mitochondria can be stored in a refrigerator. Specifically, they can be stored at about 0°C to about 10°C.

[0200] The above-described freeze-dried modified mitochondria can be stored at room temperature or in a refrigerator. Specifically, the modified mitochondria can be stored at about 0°C to about 37°C. The modified mitochondria can be stored at about 0°C to about 8°C, about 9°C to about 15°C, about 16°C to about 22°C, about 23°C to about 29°C, or about 30°C to about 37°C.

[0201] Additionally, the above-described freeze-dried modified mitochondria can exhibit mitochondrial activity when recovered and used.

[0202] As used herein, the term "repair" refers to restoring lyophilized modified mitochondria to their original state, and can be accomplished by adding an aqueous solution to the lyophilized modified mitochondria. The aqueous solution may be water for injection, physiological saline, phosphate buffer, purified water, or deionized water. Deionized water is preferred.

[0203] At this time, the above-mentioned repair process can be performed at room temperature or refrigerated. Specifically, it can be performed at about 0°C to about 37°C. The mitochondria can be performed at about 0°C to about 8°C, about 9°C to about 15°C, about 16°C to about 22°C, about 23°C to about 29°C, or about 30°C to about 37°C. In addition, the above-mentioned repair process can be performed for about 1 minute to about 1 hour, about 1 minute to about 50 minutes, about 1 minute to about 40 minutes, about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 10 minutes, or about 1 minute to about 5 minutes. The above-mentioned repair process is not particularly limited as long as the freeze-dried mitochondria are repaired and not dried.

[0204] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the scope of the present invention is not limited to these examples.

[0205] I. Modified mitochondria derived from human cell lines

[0206] Example 1. Production of modified mitochondria derived from human cell lines

[0207] Example 1.1. Production of a human cell line containing mitochondria expressing GFP.

[0208] To express the fluorescent protein eGFP (enhanced green fluorescent protein) in mitochondria, the TOM20 gene was amplified using a human cDNA library. The primer sequences used are as follows. Forward primer: 5'-AAAAAACTCGAGATGGTGGGTCGGAACAGCGC-3' (SEQ ID NO: 27) Reverse primer: 5'-AAAAAAGTCGACTGTTCCACATCATCTTCAGCC-3' (SEQ ID NO: 28).

[0209] The amplified TOM20 polynucleotide was cleaved with restriction enzymes Xho1 and Sal1, and then loaded into the pEGFP-N1 vector cleaved with Xho1 and Sal1 using T4 DNA ligase to produce plasmid pCMV-TOM20-GFP (Fig. 1a). Through the above process, a polynucleotide (SEQ ID NO: 1) encoding TOM20-eGFP containing the amino acid sequence of SEQ ID NO: 2 was finally produced.

[0210] The above plasmid pCMV-TOM20-GFP was transfected into the human embryonic kidney cell line HEK293, and then the HER293 cell line (HEK293-MT) in which GFP is permanently expressed in mitochondria was cultured using a medium containing G418 antibiotic. GFP ) was produced. The expression of the fluorescent protein GFP fused to mitochondria was confirmed through immunohistochemical staining of the cell line (Fig. 1).

[0211] Example 1.2. Production of human cell lines containing mitochondria expressing anti-IGF1Rα single-chain antibodies.

[0212] In order to produce an α-IGF1RscFv-myc-TOM5 fusion protein in which TOM5 (SEQ ID NO: 20) is fused to a human antibody, anti-IGF1R single-chain antibody (α-IGF1RscFv) (SEQ ID NO: 10), gene synthesis was requested from Bionics Co., Ltd. to produce a polynucleotide (SEQ ID NO: 3) encoding α-IGF1RscFv-myc-TOM (SEQ ID NO: 4). The polynucleotide was loaded into the pUC57 vector (Bionics) to produce pUC57-α-IGF1RscFv-myc-TOM5.

[0213] The above pUC57-α-IGF1RscFv-myc-TOM5 was digested with restriction enzymes EcoRI and XhoI, and the digested polynucleotide fragment (approximately 912 bp) was loaded into the pCDNA3.1 Zeo(+) vector (Thermofisher) digested with restriction enzymes EcoRI and XhoI using T4 DNA ligase to produce plasmid pCMV-α-IGF1RscFv-myc-TOM5 (Fig. 2a).

[0214] The above pCMV-α-IGF1RscFv-myc-TOM5 was transfected into the HEK293 cell line in the same manner as in Example 1.1, and then cultured in a medium containing zeocin antibiotic to form HEK293-MT, a single cell line in which GFP and α-IGF1RscFv are permanently expressed in mitochondria. α-IGF1RscFv_GFP was produced.

[0215] The expression of the fluorescent proteins GFP and α-IGF1RscFv fused to mitochondria was confirmed in the above cell line using immunohistochemical staining (Fig. 1b). GFP expression was confirmed using an anti-GFP antibody (Invitrogen, A6455), and α-IGF1RscFv expression was confirmed using an anti-myc antibody (Cell signaling, 2278).

[0216] Example 1.3. Production of human cell lines containing mitochondria expressing anti-TfR single-chain antibodies.

[0217] In order to produce an α-hTfRscFv-flag-TOM5 fusion protein in which TOM5 (SEQ ID NO: 20) is fused to a human antibody, anti-TfR single-chain antibody (α-hTfRscFv) (SEQ ID NO: 12), gene synthesis was requested from Bionics Co., Ltd. to produce a polynucleotide (SEQ ID NO: 5) encoding α-hTfRscFv-flag-TOM5 (SEQ ID NO: 6). The polynucleotide was loaded into the pUC57 vector (Bionics) to obtain pUC57-α-hTfRscFv-flag-TOM5.

[0218] The above pUC57-α-hTfRscFv-flag-TOM5 was cleaved with restriction enzymes EcoRI and XhoI, and the cleaved polynucleotide fragment (approximately 912 bp) was loaded onto the pCDNA3.1 Zeo(+) vector (Thermofisher) cleaved with restriction enzymes EcoRI and XhoI using T4 DNA ligase to produce plasmid pCMV-α-hTfRscFv-flag-TOM5 (Fig. 3a).

[0219] The above pCMV-α-hTfRscFv-flag-TOM5 was transfected into the HEK293 cell line in the same manner as in Example 1.1, and then cultured in a medium containing zeocin antibiotic to produce a HER293 cell line (HEK293-MT) in which GFP and α-hTfRscFv are permanently expressed in the mitochondria. α-hTfRscFv_GFP ) was produced.

[0220] The expression of the fluorescent protein GFP and α-hTfRscFv fused to mitochondria was confirmed in the above cell line using immunohistochemical staining (Fig. 3b). GFP expression was confirmed using an anti-GFP antibody (Invitrogen, A6455), and α-hTfRscFv expression was confirmed using an anti-flag antibody (Invitrogen, F3165).

[0221] Example 1.4. Generation of human cell lines containing mitochondria expressing Angiopep2.

[0222] In order to produce an Angiopep2-flag-TOM5 fusion protein in which TOM5 (SEQ ID NO: 20) is fused to the cell-penetrating peptide Angiopep2 (SEQ ID NO: 16), gene synthesis was requested from Bionics Co., Ltd. to produce a polynucleotide (SEQ ID NO: 7) encoding Angiopep2-flag-TOM5 (SEQ ID NO: 8). The polynucleotide was loaded into the pUC57 vector (Bionics) to produce pUC57-Angiopep2-flag-TOM5.

[0223] The above pUC57-Angiopep2-flag-TOM5 was digested with restriction enzymes EcoRI and XhoI, and the digested polynucleotide fragment (approximately 237 bp) was loaded into the pCDNA3.1 Zeo(+) vector (Thermofisher) digested with restriction enzymes EcoRI and XhoI using T4 DNA ligase to produce plasmid pCMV-Angiopep2-flag-TOM5 (Fig. 4a).

[0224] The above pCMV-Angiopep2-flag-TOM5 was transfected into the HEK293 cell line in the same manner as in Example 1.1, and then cultured in a medium containing zeocin antibiotics to produce a HER293 cell line (HEK293-MT) in which GFP and Angiopep2 are permanently expressed in the mitochondria. Angiopep2_GFP ) was produced.

[0225] The expression of the fluorescent proteins GFP and Angiopep2, which are fused to mitochondria, was confirmed in the above cell line using immunohistochemical staining (Fig. 4b). GFP expression was confirmed using an anti-GFP antibody (Invitrogen, A6455), and Angiopep2 expression was confirmed using an anti-flag antibody (Invitrogen, F3165).

[0226] Example 2. Isolation of modified mitochondria from human cell lines

[0227] HEK293-MT produced in the same manner as in Example 1 above GFP , HEK293-MT α-IGF1RscFv_GFP , HEK293-MT α-hTfRscFv_GFP and HEK293-MT Angiopep2_GFP Mitochondria were isolated from .

[0228] Specifically, the above four types of cell lines were cultured in DMEM medium containing 10% FBS, washed twice with DPBS (Dulbecco's phosphate buffered saline, Gibco), and obtained by treatment with 0.25% (v / v) Trypsin-EDTA (TE, Gibco).

[0229] Each of the obtained cells was 1×10 7 After preparing the concentration of cells / ㎖, the first centrifugation was performed at 350×g for 10 minutes at 4℃. The pellet obtained through the above process was resuspended in homogenization buffer (250 mM sucrose, 20 mM HEPES, 2 mM EGTA, pH 7.4) and homogenized for 10 to 15 minutes. Thereafter, the suspension was centrifuged a second time at 2,000×g for 10 minutes at 4℃. After centrifugation, the supernatant was obtained and centrifuged a third time at 12,000×g for 15 minutes at 4℃. Then, the supernatant was removed, and the pellet was recovered and resuspended in the homogenization buffer. The suspension was centrifuged at 20,000×g for 10 minutes to obtain a pellet. The pellet was resuspended in a suspending agent (20 mM tris, 195 mM trehalose, 66.6 mM glycine, pH 7.4) and centrifuged at 20,000×g for 5 minutes to obtain a pellet. At this time, HEK293-MT GFP Cell line, HEK293-MT α-IGF1RscFv_GFP Cell line, HEK293-MT α-hTfRscFv_GFP cell lines and HEK293-MT Angiopep2_GFP Mitochondria isolated from each cell line were labeled "MT GFP ", "MT α-IGF1RscFv_GFP ", "MT α-hTfRscFv_GFP " and "MT Angiopep2_GFP " was named, and the separated mitochondria were suspended in a suspending agent and used in the following experiments after protein quantification using the BCA method.

[0230] Example 3. Confirmation of the cell motility of modified mitochondria derived from human cell lines at the cellular level.

[0231] Modified mitochondria (MT) obtained in the same manner as in Example 2 above GFP , MT α-IGF1RscFv_GFP , MT α-hTfRscFv_GFP , MT Angiopep2_GFP ) was confirmed in an in vitro model using a transwell assay. The in vitro model using the transwell assay mimics the blood surface in the upper well of the transwell and the brain tissue in the lower well.

[0232] Specifically, the upper well was cultured with bEND.3 cells (ATCC), which are mouse-derived brain endothelial cells, and the lower well was cultured with human-derived dermal fibroblasts (PromoCell) or neuroblastoma cells (SH-SY5Y cells, ATCC). bEND.3, human dermal fibroblasts, and SH-SY5Y cells were all cultured using DMEM containing 10% FBS. At this time, bEND.3 cells in the upper well were cultured at 0.8 × 10 5 Cells / well were seeded at a concentration of 1 × 10 , and dermal fibroblasts (Fig. 5) or SH-SY5Y cells (Fig. 7) in the lower well were seeded at a concentration of 1 × 10 5 After inoculating at a concentration of cells / well, it was prepared by culturing overnight.

[0233] The cells prepared as described above were treated with the modified mitochondria at a concentration of 20 μg / well, and mitochondria distributed in the cells of the lower well were observed using a fluorescence microscope after 2 and 8 hours. In addition, the cells of the lower well were harvested using trypsin-EDTA, and the fluorescence value was quantified using a fluorescence analyzer.

[0234] As a result of treating mitochondria in the upper well for 24 hours, MT GFP Compared to modified mitochondria (MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) was confirmed to be more delivered to dermal fibroblasts or SH-SY5Y cells in the lower well through bEND3 cells. In addition, MT was also delivered 2 or 8 hours after mitochondrial treatment. GFP Compared with the treatment group, modified mitochondria (MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) showed higher fluorescence values ​​than the treatment group. Through the above results, it was confirmed that more modified mitochondria were delivered to cells even in a short period of time (Fig. 6a, Fig. 6b, Fig. 8a and Fig. 8b).

[0235] Example 4. Confirmation of the blood-brain barrier permeability of modified mitochondria at the animal level.

[0236] Modified mitochondria (MT) obtained in the same manner as in Example 2 above GFP , MT α-IGF1RscFv_GFP , MT Angiopep2_GFP ) was confirmed to penetrate the blood-brain barrier (BBB) ​​at the animal level (in vivo).

[0237] Specifically, each of the four types of modified mitochondria was injected into the tail vein of 7-week-old C57BL / 6J male mice at a concentration of 20 μg / 100 μl per mouse. After 24 hours, the mice were anesthetized, brain tissues were extracted, and tissue staining for each tissue was performed to confirm the distribution of the injected modified mitochondria within the brain tissue (Fig. 9).

[0238] At this time, an anti-MTCO2 antibody (Abcam, ab79393) that specifically reacts with human mitochondria was used to distinguish between mouse-derived mitochondria and human cell line-derived mitochondria.

[0239] As a result, as shown in Figures 10 to 13, MT in the CA1 / 2 region (Figure 10), CA3 region (Figure 11), and DG region (Figure 12) of the hippocampus of the mouse brain GFP Modified mitochondria (MT) compared to the treatment group α-IGF1RscFv_GFP , MT Angiopep2_GFP ) treatment group, a greater number of mitochondria were observed. In addition, the same area as above was stained with an anti-MTCO2 antibody, a human cell line-derived mitochondrial antibody, to observe the distribution of human-derived mitochondria. As a result, it was confirmed that the mitochondria merged with the GFP fluorescent area, thereby reconfirming that the mitochondria were foreign mitochondria derived from the human cell line administered intravenously.

[0240] From the above results, modified mitochondria (MT) according to the present invention α-IGF1RscFv_GFP , MT Angiopep2_GFP ) BBB permeability of MT GFP It was confirmed that it was superior compared to .

[0241] Additionally, MT was detected near TH-labeled dopamine neurons in the substantia nigra region of the mouse midbrain. GFP Modified mitochondria (MT) compared to the treatment group α-IGF1RscFv_GFP , MT Angiopep2_GFP ) were observed more. This shows that modified mitochondria (MT) were observed when administered intravenously. α-IGF1RscFv_GFP , MT Angiopep2_GFP ) was confirmed to have a superior BBB penetration ability.

[0242] II. Modified mitochondria derived from human induced pluripotent stem cells

[0243] Example 5. Production of modified mitochondria derived from human induced pluripotent stem cells.

[0244] Example 5.1. Mitochondria containing anti-IFG1R single chain antibodies expressed Production of human induced pluripotent stem cells

[0245] A human induced pluripotent stem cell line was created in which the α-IGF1RscFv-myc-TOM5 fusion protein, in which TOM5 (SEQ ID NO: 20) is fused to the human antibody anti-IGF1R single-chain antibody (α-IGF1RscFv) (SEQ ID NO: 10), is expressed in the mitochondria.

[0246] Specifically, the plasmid pCMV-α-IGF1RscFv-myc-TOM5 used in Example 1.2 was cleaved with restriction enzymes BamH1 and Xba1, and then the cleaved polynucleotide fragment (approximately 912 bp) was loaded into the pEF1α vector cleaved with BamH1 and Xba1 using T4 DNA ligase to produce pEF1α-α-IGF1RscFv-myc-TOM5 (Fig. 20a).

[0247] The above plasmid pEF1α-α-IGF1RscFv-myc-TOM5 was transfected into human induced pluripotent stem cells (iPSCs) using lipofectamine (Invitrogen, Lipofectamine™ Stem Transfection Reagent). Then, the transfected iPSCs were cultured in a cell medium containing G418 antibiotics to generate an iPSC cell line (iPSC-MT) that permanently expresses α-IGF1RscFv-myc-TOM5 in the mitochondria. α-IGF1RscFv ) was produced.

[0248] Expression of α-IGF1RscFv-myc-TOM5 in the mitochondria of the above cell line was confirmed by Western blot (Fig. 20b). At this time, expression of α-IGF1RscFv was confirmed using an anti-myc antibody (Roche, 11667149001).

[0249] Example 5.2. Production of human induced pluripotent stem cells containing mitochondria expressing Angiopep2.

[0250] A human induced pluripotent stem cell line was created in which the Angiopep2-flag-TOM5 fusion protein, in which TOM5 (SEQ ID NO: 20) is fused to the cell-penetrating peptide Angiopep2 (SEQ ID NO: 16), is expressed in the mitochondria.

[0251] Specifically, the plasmid pCMV-Angiopep2-flag-TOM5 used in the above Example 1.4 was cleaved with restriction enzymes BamH1 and Xba1, and then the cleaved polynucleotide fragment (237 bp) was loaded into the pEF1α vector cleaved with BamH1 and Xba1 using T4 DNA ligase to produce pEF1α-Angiopep2-flag-TOM5 (Fig. 21a).

[0252] The above plasmid pEF1α-Angiopep2-flag-TOM5 was transfected into human induced pluripotent stem cells in the same manner as in Example 5.1 to produce an iPSC cell line (iPSC-MT) that permanently expresses Angiopep2-flag-TOM5. Angiopep2 ) was produced.

[0253] The expression of Angiopep2 in the mitochondria of the above cell lines was confirmed through Western blot (Fig. 21b). At this time, the expression of Angiopep2 was confirmed using an anti-flag antibody (Sigma, F3165).

[0254] Example 6. Isolation of modified mitochondria from human induced pluripotent stem cells.

[0255] Modified iPSC-MT, iPSC-MT produced in the same manner as in Example 5 above α-IGF1RscFv and iPSC-MT Angiopep2 Mitochondria were isolated from .

[0256] Specifically, the three types of cells were cultured in mTeSR Plus (Stemcell) medium containing G418 (400 μg / ml), washed twice with DPBS (Dulbecco's phosphate buffered saline, Gibco), and treated with Accutase (Stemcell).

[0257] Each of the obtained cells was 2×10 7 After preparing at a concentration of 10 cells / ml, mitochondria were isolated from the cells using the same method as in Example 2. The iPSC-MT, iPSC-MT α-IGF1RscFv and iPSC-MT Angiopep2 The mitochondria separated from each other were called “iMT” and “iMT α-IGF1RscFv " and "iMT Angiopep2 " was named, and the separated mitochondria were suspended in a suspending agent and used in the following experiments after protein quantification using the BCA method.

[0258] Example 7. Confirmation of the motor impairment improvement effect of iMT and iMTα-IGFRscFv in a Parkinson's disease mouse model.

[0259] Modified mitochondria (iMT) derived from human induced pluripotent stem cells obtained by the method of Example 6 were used in a Parkinson's disease mouse model induced by 6-OHDA treatment. α-IGF1RscFv ) was evaluated for its effect on improving motor impairment.

[0260] Specifically, the Parkinson's disease mouse model was prepared by directly injecting 6-OHDA (6-hydroxydopamine) into the right striatum of 6-week-old ICR mice at a concentration of 16 μg / 2 μl. Eight days after administration, human induced pluripotent stem cell-derived mitochondria (iPSC-MT) or human induced pluripotent stem cell-derived modified mitochondria (iMT) obtained by the method of Example 6 were injected into the right striatum of the mice. α-IGF1RscFv) was administered intravenously (iv) in two doses over two days at a concentration of 5 μg / 100 μl. Two days after mitochondria administration, the same amount of mitochondria was administered in the same manner, and behavioral experiments were performed for three days starting two days later. L-DOPA was used as a positive control and was orally administered daily at a concentration of 80 mg / kg once a day from the 8th day to the 15th day (end of experiment) after 6-OHDA administration (Fig. 14).

[0261] The above behavioral analysis assessed motor impairment through pole test, rotarod test, and cylinder test.

[0262] Specifically, the behavioral analysis method is as follows.

[0263] - Pole test

[0264] Mice were placed head-up on a 55 cm vertical pole (8 mm diameter), and the time it took for the entire body to turn downward (T-turn) and the time it took for the body to completely descend to the ground (T-LA) were measured. To ensure accurate measurements, the mice were acclimated to the vertical pole one day prior to the experiment.

[0265] - Rotarod test

[0266] Motor function was assessed by measuring the time it took the experimental animals to fall from the rotating shaft (latency to fall) and the total number of falls (number of falls) using a rotarod measuring device (Jungdo B&P) with a 3-cm diameter rotating shaft. To ensure accurate measurements, the animals were acclimated to the rotarod 1 day before the experiment (30 rpm, 3 min). In the measurement experiment, forced exercise was performed by rotating the shaft at 30 rpm for 3 min.

[0267] - Cylinder test

[0268] Mice were placed in a cylinder (diameter 110 mm, height 150 mm), and their spontaneous movements were recorded for 5 minutes to measure the number of forepaw uses. Specifically, the number of times the mice touched the cylinder wall with their lesioned and contralateral forepaws was measured, and the percentage of the total number of forepaw uses with the contralateral forepaw was calculated.

[0269] The results of the above behavioral analysis are shown in Figures 15 to 19.

[0270] As shown in Figures 15 and 16, the results of the pole test showed that T-turn and T-LA were significantly increased in the Parkinson's model mice induced by 6-OHDA administration compared to the normal mice (sham). On the other hand, iMT, iMT α-IGF1RscFv In the L-dopa-administered group, it was confirmed that T-turn and T-LA were reduced compared to the Parkinson's model mice. In particular, iMT α-IGF1RscFv In the administration group, the reduction of T-turn and T-LA was superior to that of iMT. Based on the above results, iMT or iMT α-IGF1RscFv It was confirmed that motor impairment was improved by iMT α-IGF1RscFv It was confirmed that the improvement rate was the best.

[0271] As shown in Figures 17 and 18, in the case of Parkinson's disease model mice induced by 6-OHDA administration, the time taken to fall from the rotation axis (latency to fall) decreased and the total number of falls (number of falls) increased compared to normal mice (sham). On the other hand, iMT, iMT α-IGF1RscFv In the L-dopa-administered group, latency increased and the number of falls decreased compared to Parkinson's disease model mice, confirming improvement in motor function impairment. In particular, iMT α-IGF1RscFvIn the administration group, the increase in latency and the decrease in the number of falls were superior to those of iMT.

[0272] As shown in Figure 19, the results of the cylinder test showed that the frequency of spontaneous use of the forepaws decreased in the Parkinson's model mice induced by 6-OHDA administration compared to the normal mice (sham). On the other hand, iMT, iMT α-IGF1RscFv In the L-dopa-administered group, the number of times the forelimbs were used increased compared to the Parkinson's model mice, and iMT α-IGF1RscFv It was confirmed that the treatment group showed the greatest improvement in motor impairment.

Claims

1. A fusion protein comprising a mitochondrial outer membrane anchoring peptide and a cerebrovascular endothelial cell surface protein binding site.

2. In paragraph 1, A fusion protein wherein the above mitochondrial outer membrane anchoring peptide comprises an N-terminal or C-terminal sequence of a protein present in the mitochondrial outer membrane.

3. In paragraph 1, A fusion protein, wherein the mitochondrial outer membrane anchoring peptide is any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1.

4. In paragraph 1, A fusion protein wherein the cerebral vascular endothelial cell surface protein binding site is selected from the group consisting of an antibody or a fragment thereof, a peptide or a fragment thereof, a receptor or a fragment thereof, a ligand or a fragment thereof, and an aptamer.

5. In paragraph 4, A fusion protein, wherein the antibody fragment is any one selected from the group consisting of Fab, Fab', scFv, F(ab)2, nanobody and combinations thereof.

6. In paragraph 1, A fusion protein wherein the cerebral vascular endothelial cell surface protein is any one selected from the group consisting of a transferrin receptor, an insulin receptor, an insulin-like growth factor receptor, a low density lipoprotein receptor-related protein 8, a low density lipoprotein receptor-related protein 1, a heparin-binding epidermal growth factor-like growth factor, a leptin receptor, a nicotinic acetylcholine receptor, a glutathione transporter, a calcium-activated potassium channel, and a receptor for advanced glycation endproducts (RAGE).

7. In paragraph 1, A fusion protein wherein the mitochondrial outer membrane anchoring peptide is TOM20, TOM70 or OM45, and the mitochondrial outer membrane anchoring peptide and the cerebral vascular endothelial cell surface protein binding site are linked from the N terminus to the C terminus.

8. In paragraph 7, The above fusion protein is a fusion protein having the following structural formula (I): N'-mitochondrial outer membrane anchoring peptide-[L(1)]m-cerebrovascular endothelial cell surface protein binding site-C' (I) In the above structural formula (I), The above N' is the N-terminus of the fusion protein, The above C' is the C-terminus of the fusion protein, The above L(1) is a peptide linker, The above m is 0 or 1.

9. In paragraph 1, A fusion protein in which the mitochondrial outer membrane anchoring peptide is any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1, and the cerebrovascular endothelial cell surface protein binding site and the mitochondrial outer membrane anchoring peptide are linked from the N-terminus to the C-terminus.

10. In paragraph 9, The above fusion protein is a fusion protein having the following structural formula (II): N'-cerebrovascular endothelial cell surface protein binding site-[L(2)]n-mitochondrial outer membrane anchoring peptide-C'(II) In the above structural formula (II), The above N' is the N-terminus of the fusion protein, The above C' is the C-terminus of the fusion protein, The above L(2) is a peptide linker, The above n is 0 or 1.

11. A polynucleotide encoding the fusion protein of paragraph 1.

12. Modified mitochondria containing the fusion protein of paragraph 1.

13. In paragraph 12, Modified mitochondria, wherein the above fusion protein is bound to the outer membrane of the mitochondria. 14.i) A step of producing a transformed cell by introducing the polynucleotide of clause 11 into the cell; and ii) A method for producing modified mitochondria, comprising the step of isolating modified mitochondria from the transformed cells.

15. A pharmaceutical composition for preventing or treating brain disease, comprising the modified mitochondria of Article 12 as an active ingredient.

16. In paragraph 15, The above brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis (ALS), spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, mitochondrial encephalomyopathy, migraine, cerebral infarction or hypoxic encephalopathy. A pharmaceutical composition for preventing or treating a brain disease, wherein the pharmaceutical composition is any one selected from the group consisting of ischemic brain disorders such as cerebral arteriosclerosis, bipolar disorder, chronic fatigue syndrome, intracranial hypertension such as hydrocephalus or head trauma, normal pressure hydrocephalus, cerebral vasospasm following subarachnoid hemorrhage, cerebral ischemia during surgery and endovascular surgery, and frontotemporal dementia.

17. Use of modified mitochondria containing the fusion protein of paragraph 1 for preventing or treating brain diseases.

18. A method for preventing or treating brain disease, comprising administering to a subject modified mitochondria containing the fusion protein of paragraph 1.

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