WO2024019489A1 - Aav vector and cell-penetrating peptide fusion platform and pharmaceutical composition for prevention or treatment of degenerative brain diseases including same - Google Patents
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
Definitions
- the present invention relates to an Adeno-Associate Virus (AAV) vector and cell-permeable peptide fusion platform and a pharmaceutical composition containing the same for preventing or treating degenerative brain diseases.
- AAV Adeno-Associate Virus
- Adeno-associated virus (AAV) vectors are the most commonly used delivery system for gene therapy drugs currently under development.
- the advantage of AAV vectors is that they are non-pathogenic and can be broadly targeted to various tissues. In addition, it can maintain long-term efficacy with a single administration, and because it does not have the ability to replicate on its own, it does not bind to the host gene, making it highly safe.
- delivery and expression of therapeutic substances may be difficult because continuous transmission between cells for delivery of therapeutic substances is not possible, and heterogeneity may occur when the genome size exceeds 4.7 kb. There is a possibility that immunogenicity may also occur. In some cases, there are limitations that make certain delivery (tropism) difficult.
- TSDT Pharmacological Substance Transduction Technology
- Parkinson's Disease is one of the degenerative brain diseases that affects approximately 7 to 10 million people worldwide.
- Parkinson's disease is a disease that causes movement disorders such as resting tremor and bradykinesia, which causes muscle rigidity and slow body movements. If Parkinson's disease (PD) does not receive appropriate treatment, movement disorder gradually progresses, making movement and walking difficult and making it impossible to carry out daily life.
- PD Parkinson's disease
- Parkinson's disease is caused by a combination of genetic and environmental factors and has a variety of causative factors. Brain neurons containing dopamine gradually die or degenerate in the substantia nigra (SN) area of the midbrain in patients with Parkinson's disease (PD). There is still no known exact cause, and the cause of Parkinson's disease (PD) is increasing. Among many factors, degeneration of the Parkin gene accounts for the high prevalence among genetic factors.
- the Parkin gene is an E3 ligase that is essential for the removal mechanism of various proteins, including ⁇ -Synuclein, and plays a role in reducing intracellular stress by removing damaged, oxidized, or abnormally structured proteins within the cell. Mutation of Parkin not only results in loss of the ubiquitin conjugating enzyme function that performs the role of E3 ligase, but also induces misfolding or aggregation of proteins, inhibits dopamine secretion, or causes death of dopaminergic neurons. It is known to be the main cause of genetic Parkinson's disease.
- Levodopa which is used as a treatment for Parkinson's disease (PD)
- PD Parkinson's disease
- ST striatum
- long-term administration causes side effects such as hepatotoxicity and gastrointestinal disorders, and although the dosage increases, the effectiveness of the drug decreases, so it is not a definite cure for Parkinson's disease (PD) but only serves to delay its progression.
- AD Alzheimer's disease dementia
- AD Alzheimer's disease dementia
- Various studies are being conducted to determine the causes of Alzheimer's disease dementia (AD), but not all definite causes have been identified.
- Clinical symptoms that appear in patients with Alzheimer's disease (AD) include decreased memory and cognitive ability, and are accompanied by psychiatric symptoms such as depression, hallucinations, delusions, and sleep disorders.
- Alzheimer's disease can be viewed as being caused by a complex pathogenic mechanism in which genetic risk factors and environmental risk factors are added as the aging phenomenon of the nervous system progresses rather than a specific single mechanism cause, and overall dementia (Dementia) 70 to 80% of patients fall into Alzheimer's disease dementia.
- the main mechanism of Alzheimer's disease dementia (AD) is damage to neuronal signaling in the brain, especially senile plaques in which amyloid beta (A ⁇ ) protein accumulates and tau protein tangles.
- Neurofibrillary Tangle (NFT) is a representative pathological phenomenon known to date. Amyloid beta (A ⁇ ) and Tau protein (Tau) accumulated in the brain gradually cause brain neurons to die or die. It degenerates.
- Amyloid beta (A ⁇ ) is produced by amyloid precursor protein and is normally cleaved by ⁇ -secretase. However, when cleaved by ⁇ -secretase, it produces toxic A ⁇ 1-42 peptide, and ultimately Due to its insoluble nature in water, it sticks together and accumulates to form Senile Plaque. Such plaque paralyzes or distorts the function of mitochondria within nerve cells, increasing reactive oxygen species (ROS) released from mitochondria. The increased reactive oxygen species cause fatal damage to proteins or DNA within cells, ultimately causing damage or damage to brain cells. It causes death (apoptosis). Tau protein is a protein that plays an important role in maintaining the skeleton of brain cells.
- ROS reactive oxygen species
- AD Alzheimer's disease
- brain neurons develop functional defects due to pathological protein aggregates and damage memory and judgment, leading to a decrease in cognitive function.
- AD Alzheimer's disease dementia
- drugs that allow acetylcholine to act effectively in the transmission of neurons
- various compounds that increase acetylcholine activity in patients with Alzheimer's disease (AD) are being used.
- AD Alzheimer's disease
- FDA U.S. Food and Drug Administration
- NDD neurodegenerative diseases
- PD Parkinson's disease
- AD Alzheimer's disease dementia
- AAV fusion Parkin protein such as improvement of motor function and cognitive function
- TSDT pharmacological substance transfer technology
- Patent Document 001 Republic of Korea Patent Publication No. 10-2018-0026565
- Patent Document 002 Republic of Korea Patent Publication No. 10-2022-0127877
- AAV adeno-associated virus
- TSDT in vivo delivery technology
- BBB blood-brain barrier
- an example of the present invention provides an AAV vector comprising an advanced macromolecule transduction domain (aMTD) coding polynucleotide sequence and a Parkin protein coding polynucleotide sequence.
- aMTD advanced macromolecule transduction domain
- Another example of the present invention provides a host cell transformed with the vector.
- Another example of the present invention provides a pharmaceutical composition for treating degenerative brain diseases containing the vector.
- Another example of the present invention provides a method for preventing or treating degenerative brain diseases comprising administering the vector, host cell, and/or pharmaceutical composition to a subject.
- the present invention overcomes the limitations of the adeno-associated virus (AAV) vector through the fusion of a cell-permeable Parkin recombinant protein and is delivered into dopaminergic neurons in an in vivo environment to inhibit apoptosis or increase dopamine secretion.
- AAV adeno-associated virus
- Figure 1a is a diagram showing the transfer plasmid backbone structure ( Figure 1a) of adeno-associated virus (AAV).
- AAV adeno-associated virus
- Figure 1b is a diagram showing the structure of Parkin protein.
- Figure 1c is a diagram showing the structure of pAAV9-Parkin ( ⁇ Ubl)-SDB.
- Figure 1d is a diagram showing the structure of pAAV9-aMTD524-Parkin ( ⁇ Ubl)-SDB.
- Figure 2a is a diagram showing the process of gene therapy and exercise testing using adeno-associated virus (AAV) in a 6-OHDA-induced Parkinson's disease animal model.
- AAV adeno-associated virus
- Figure 2b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 2c is a photograph of dopaminergic neurons in the striatum and substantia nigra in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 2d is a photograph showing that AAV-aMTD-Parkin is transmitted in the striatum and substantia nigra of a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 2e is a photograph showing the expression level of tyrosine hydroxylase in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
- Figure 2f is a graph showing the expression level of tyrosine hydroxylase in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
- Figure 3a is a diagram showing the process of gene therapy and cognitive function testing using adeno-associated virus (AAV) in an fA ⁇ -induced Alzheimer's disease animal model.
- AAV adeno-associated virus
- Figure 3b is a graph showing the effect of improving cognitive function in an Alzheimer's disease (AD) animal model after administration of AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 3c is a photograph showing that pathological A ⁇ was removed and Parkin was expressed in the hippocampus region of an Alzheimer's disease (AD) animal model after administration of AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 3d is a photograph showing a decrease in pathological A ⁇ plaques in an Alzheimer's disease (AD) animal model after administration of AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 4a shows an experimental process to compare the therapeutic material delivery ability of the AAV-TSDT fusion platform (AAV-aMTD-Parkin) and the existing gene therapy (AAV-Parkin) according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model. It's a picture.
- Figure 4b is a photograph of the striatum region of the brain in a Parkinson's disease (PD) animal model after administration of AAV-GFP-aMTD-Parkin-mCherry.
- PD Parkinson's disease
- Figure 4c is a photograph of neurons and glial cells in the striatal region of the brain in a Parkinson's disease (PD) animal model after administration of AAV-GFP-aMTD-Parkin-mCherry.
- PD Parkinson's disease
- Figure 4d is a diagram showing the experimental process for comparing the therapeutic substance delivery ability of AAV-aMTD-Parkin according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
- PD Parkinson's disease
- Figure 4e is a diagram showing the ICM administration location and tissue acquisition location of AAV-aMTD-Parkin according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
- PD Parkinson's disease
- Figure 4f is a graph showing the amount of exogenous Parkin in the substantia nigra region after administration of AAV-aMTD-Parkin in a Parkinson's disease (PD) animal model.
- Figure 4g is a graph showing the amount of exogenous Parkin in the striatum region after administration of AAV-aMTD-Parkin in a Parkinson's disease (PD) animal model.
- Figure 4h is a graph showing the amount of exogenous Parkin in the cerebral cortex area after administration of AAV-aMTD-Parkin in a Parkinson's disease (PD) animal model.
- Figure 5a is a diagram showing the process of low-dose gene therapy and motor function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
- AAV adeno-associated virus
- Figure 5b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 5c is a graph showing the change in the amount of dopamine in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 5d is a photograph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- TH tyrosine hydroxylase
- Figure 5e is a graph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- TH tyrosine hydroxylase
- Figure 5f is a photograph of the expression of tyrosine hydroxylase (TH) in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- TH tyrosine hydroxylase
- Figure 5g is a graph showing the amount of exogenous Parkin in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 6a is a diagram showing the process of high-dose gene therapy and motor function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
- AAV adeno-associated virus
- Figure 6b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 6c is a graph showing the change in the amount of dopamine in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 6d is a photograph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- TH tyrosine hydroxylase
- Figure 6e is a photograph of the expression of tyrosine hydroxylase (TH) in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- TH tyrosine hydroxylase
- Figure 6f is a graph showing the amount of exogenous Parkin in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 7a is a diagram showing the process of low-dose gene therapy and motor function testing using adeno-associated virus (AAV) to confirm the therapeutic effect depending on the presence or absence of cell permeability in a chronic Parkinson's disease (PD) animal model.
- AAV adeno-associated virus
- Figure 7b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 7c is a graph showing the change in the amount of dopamine in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 7d is a photograph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- TH tyrosine hydroxylase
- Figure 7e is a photograph of the expression of tyrosine hydroxylase (TH) in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- TH tyrosine hydroxylase
- Figure 7f is a photograph showing the expression level of phosphorylated ⁇ -syunclein in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- PD Parkinson's disease
- Figure 8a is a diagram showing the process of low-dose gene therapy and cognitive function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in an Alzheimer's disease (AD) animal model.
- AAV adeno-associated virus
- Figure 8b is a graph showing the effect of improving cognitive function in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 8c is a photograph showing the amount of pathological A ⁇ plaques in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 8d is a photograph showing the amount of pathological A ⁇ plaques in the hippocampus area of an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 9a is a diagram showing the process of extremely low-dose gene therapy and cognitive function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in an Alzheimer's disease (AD) animal model.
- AAV adeno-associated virus
- Figure 9b is a graph showing the effect of improving cognitive function in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 9c is a photograph showing the amount of pathological A ⁇ plaques in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- AD Alzheimer's disease
- Figure 9d is a photograph showing the amount of pathological A ⁇ plaques in the hippocampus area of an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
- AD Alzheimer's disease
- One example of the present invention provides an AAV vector comprising an advanced macromolecule transduction domain (aMTD) coding polynucleotide sequence and a Parkin protein coding polynucleotide sequence.
- aMTD advanced macromolecule transduction domain
- the aMTD coding polynucleotide sequence may be a polynucleotide sequence of SEQ ID NO: 3 to 242, preferably a polynucleotide sequence of SEQ ID NO: 125, but is not limited thereto.
- the vector may additionally include one or more solubilization domain (SD)(s).
- SD solubilization domain
- the SD(s) may have the polynucleotide sequence of SEQ ID NO: 243.
- the promoter has SEQ ID NO:
- the recombinant protein is expressed in one of the following structural formulas,
- the Parkin protein-encoding polynucleotide sequence may be the polynucleotide sequence of SEQ ID NO: 245.
- the vector may include the CMV promoter of SEQ ID NO: 244 and/or the BGH poly A sequence of SEQ ID NO: 246.
- the vector may contain the polynucleotide sequence of SEQ ID NO: 2.
- One example of the present invention provides host cells transformed with the vector.
- Another example of the present invention provides a pharmaceutical composition for treating degenerative brain diseases containing the vector.
- the vector is used in the treatment of degenerative brain diseases,
- the degenerative brain diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann- Pick's disease, senile dementia, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph disease -Joseph's disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy, and Frontotemporal Dementia, preferably Parkinson's disease and Alzheimer's disease. .
- the pharmaceutical composition may include a peptide compound, a stereoisomer thereof, a solvate, or a pharmaceutically acceptable salt as an active ingredient.
- the pharmaceutical composition may further include known active ingredients that have preventive or therapeutic activity for degenerative brain diseases.
- the pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier.
- the diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof.
- the carrier may be an excipient, disintegrant, binder, lubricant, or a combination thereof.
- the excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof.
- the disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate anhydride, or a combination thereof.
- the binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof.
- the lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
- the pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
- the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule.
- the solid preparation may further include excipients. Excipients may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, the solid preparation may further include a lubricant such as magnesium stearate or talc.
- the oral liquid preparation may be a suspension, oral solution, emulsion, or syrup.
- the liquid formulation may contain water or liquid paraffin.
- the liquid formulation may contain excipients such as wetting agents, sweeteners, flavoring agents, or preservatives.
- preparations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried products, and suppositories.
- Non-aqueous solvents or suspensions may contain vegetable oil or ester.
- the vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil.
- the ester may be, for example, ethyl oleate.
- the base of the suppository may be witepsol, macrogol, tween 61, cacao, laurel, or glycerogelatin.
- the preferred dosage of the pharmaceutical composition varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art.
- the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt may be used in an amount of, for example, about 0.0001 mg/kg to about 100 mg/kg, or about 0.001 mg/kg to about 100 mg/kg.
- the amount can be divided and administered 1 to 24 times per day, 1 to 7 times per 2 days to 1 week, or 1 to 24 times per month to 12 months.
- the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt is present in an amount of about 0.0001% to about 10% by weight, or about 0.001% to about 1% by weight, based on the total weight of the composition. may be included.
- the degenerative brain diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann- Pick's disease, senile dementia, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph disease -Joseph's disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy, and Frontotemporal Dementia, preferably Parkinson's disease and Alzheimer's disease. .
- Another example of the present invention provides a method for preventing or treating a degenerative brain disease comprising administering the vector, the host cell, and/or the pharmaceutical composition to a subject.
- the administration may be administered intravenous, parenteral, transdermal, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, or intraventricular. (intracerebroventricular), intracerebral injection, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, intrarectal, intravaginal ( It can be administered to humans and animals by intravaginal, spraying or oral administration, preferably parenterally such as intravenously, subcutaneously, intranasally or intraperitoneally. Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection, and intravenous injection.
- Another example of the present invention provides a use for preventing or treating degenerative brain diseases by administering the vector, the host cell, and/or the pharmaceutical composition to a subject.
- amino acid in its broadest sense includes naturally occurring L ⁇ -amino acids or residues thereof as well as D-amino acids and chemically modified amino acids.
- amino acids may include mimetics and analogs of the amino acids described above.
- mimetics and analogs may include functional equivalents.
- prevention used in the present invention refers to all actions that inhibit or delay the occurrence of degenerative brain disease by administering a composition containing the Adeno-Associate Virus (AAV) vector according to the present invention.
- treatment refers to all actions in which the symptoms of a degenerative brain disease are improved or advantageously modified by administering a composition containing the adeno-associated viral vector according to the present invention.
- administering means providing a predetermined pharmaceutical composition of the invention to a subject in any suitable manner.
- subject refers to all animals, including humans, that have developed or are likely to develop a degenerative brain disease.
- Animals may include, but are not limited to, humans as well as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, or cats that require treatment for similar symptoms.
- composition according to the invention can be formulated in various oral or parenteral dosage forms, preferably parenteral dosage forms.
- a representative formulation for parenteral administration is an injectable formulation, and an isotonic aqueous solution or suspension is preferred.
- injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline or buffer solution and formulated for injection.
- the route of administration of the composition according to the present invention includes intravenous (IV), intrahippocampal (IH), intracerebral, intracranial, intraspinal, and intracerebrospinal fluid (intracerebrospinal). ), intracerebroventricular (ICV), intrathecal, subcutaneous, intranasal or intraperitoneal, etc. can be administered to humans and animals.
- Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection, and intravenous injection.
- composition of the present invention may further contain auxiliaries such as preservatives, wetting agents, emulsification accelerators, salts and/or buffers for osmotic pressure adjustment, and other therapeutically useful substances, and may be formulated according to conventional methods.
- auxiliaries such as preservatives, wetting agents, emulsification accelerators, salts and/or buffers for osmotic pressure adjustment, and other therapeutically useful substances, and may be formulated according to conventional methods.
- the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier and/or additive.
- a pharmaceutically acceptable carrier and/or additive for example, it contains sterile water, physiological saline, common buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do.
- the pharmaceutical composition according to one embodiment is prepared in a formulation suitable for injection, the recombinant protein may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state.
- AAV-aMTD-Parkin was prepared with the structure of 5' ITR - CMV promoter - Kozak - aMTD - Parkin - SDB - BGH pA - 3' ITR, and the aMTD sequence was changed according to the aMTD sequence below.
- a cell-permeable transfer plasmid of the Parkin protein was created by including Parkin, SDB, and aMTD in the transfer plasmid backbone ( Figure 1a).
- Figure 1b shows the structure of the protein containing Parkin, SDB, and aMTD, the structure of the transfer plasmid containing Parkin and SDB ( Figure 1c), and the structure of the transfer plasmid containing Parkin, SDB, and aMTD ( Figure 1d).
- the location and size of the structure for the virus are shown in Table 2 (pAAV9-Parkin ( ⁇ Ubl)-SDB) and Table 3 (pAAV9-aMTD524-Parkin ( ⁇ Ubl)-SDB) below.
- Virus name designation location Size (bp) pAAV9-Parkin( ⁇ Ubl)-SDB 5'ITR 1-141 141 CMV 169-757 589 Kozak 782-787 6 Parkin ( ⁇ UBL)-SDB 788-2254 1467 BGH pA 2309-2516 208 3'ITR 2524-2664 141
- Virus name designation location Size pAAV9-aMTD524-Parkin ( ⁇ Ubl)-SDB 5'ITR 1-141 141 CMV 169-757 589 Kozak 782-787 6 aMTD524-Parkin ( ⁇ UBL)-SDB 788-2290 1503 BGH pA 2345-2552 208 3'ITR 2560-2700 141
- PD Parkinson's disease
- AAV adeno-associated virus
- AAV-aMTD-Parkin a cell-permeable fusion of Parkin protein
- Example 3 Confirmation of Alzheimer's disease (AD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin)
- AD Alzheimer's disease
- AAV adeno-associated virus
- AAV-aMTD-Parkin a fusion of the cell-permeable Parkin protein
- fA ⁇ Fibril Amyloid-beta
- Example 4 Confirmation of therapeutic substance delivery ability of AAV-TSDT fusion platform (AAV-aMTD-Parkin) depending on the presence or absence of cell permeability
- AAV-aMTD-Parkin the cell-permeable AAV-TSDT fusion platform
- Parkin therapeutic substances
- AAV-Parkin the cell-permeable AAV-TSDT fusion platform
- AAV-aMTD-Parkin-mCherry was used to confirm the excellent delivery ability of protein
- the adeno-associated virus (AAV) vector emits green fluorescence (GFP)
- the aMTD-Parkin protein emits red fluorescence (mCherry).
- AAV-GFP-aMTD-Parkin-mCherry was injected into the striatum region of the Parkinson's disease model brain, and immunofluorescence (IF) analysis was performed (FIG. 4b).
- Cells labeled with AAV-GFP contain the viral vector, and cells labeled with aMTD-Parkin-mCherry contain the aMTD-Parkin protein.
- AAV-Parkin co-expressed AAV and Parkin (white arrows)
- AAV-aMTD-Parkin co-expressed cells as well as cells in which only Parkin was delivered (yellow arrows). It was confirmed that the cells to which only Parkin was delivered were capable of delivering the drug not only to neurons (NeuN) but also to glial cells (GFAP) ( Figure 4c).
- AAV-aMTD-Parkin delivers a larger amount of Parkin to various brain regions such as the substantia nigra (Figure 4f), striatum (Figure 4g), and cerebral cortex (Figure 4h), and increases the expression of Parkin until the 6th week. It was confirmed that this was maintained.
- AAV-aMTD-Parkin increased by 2.7-fold, 1.7-fold, 1.7-fold, and 2.1-fold in the substantia nigra by parking, and by 6.2-fold, 1.5-fold, 1.7-fold, and 2.6-fold in the striatum. This result shows that more Parkin is delivered to dopaminergic neurons through cell-to-cell transfer of aMTD.
- Example 5 Confirmation of Parkinson's disease (PD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) (3 x 10 6 GC/Brain, ICV Route)
- AAV-Parkin / AAV-aMTD-Parkin was administered ICV at a low dose ( 3 ).
- motor function improved by 49% in the AAV-Parkin administration group and by 105% in the AAV-aMTD-Parkin administration group ( Figure 5b).
- dopamine was measured in the blood (plasma) of Parkinson's disease animals, it increased to 38% in the AAV-Parkin administered group and 106% in the AAV-aMTD-Parkin administered group ( Figure 5c). This result shows that aMTD shows a clear Add-On Effect depending on the presence or absence of cell permeability when administered at low doses.
- Example 6 Confirmation of Parkinson's disease (PD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) (3 x 10 10 GC/Brain, ICV Route)
- AAV-Parkin / AAV-aMTD-Parkin was administered by ICV at a high dose (3 x 10 10 GC/Brain) to a 6-OHDA-induced Parkinson's disease animal model ( Figure 6a) ).
- Parkinson's disease model motor function improved by 37% in the AAV-Parkin administration group and 99% in the AAV-aMTD-Parkin administration group ( Figure 6b).
- dopamine was measured in the blood (plasma) of Parkinson's disease animals, it increased to 65% in the AAV-Parkin administered group and to 164% in the AAV-aMTD-Parkin administered group ( Figure 6c).
- Example 7 Confirmation of Parkinson's disease (PD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) in a-Synuclein-induced Parkinson's disease (PD) model (3 x 10 6 GC/Brain, ICV Route)
- AAV-a-Syn induced Parkinson's disease animal model that can induce chronic Parkinson's disease (PD)
- AAV-Parkin / AAV-aMTD-Parkin was administered as low dose ( 3
- the treatment effect was confirmed depending on the presence or absence ( Figure 7a).
- the AAV-Parkin administration group showed improved motor function by 12% and the AAV-aMTD-Parkin administration group by 68% ( Figure 7b).
- the amount of dopamine was measured in the blood (plasma) of Parkinson's disease animals, the amount of dopamine increased to 56% in the AAV-Parkin administered group and 95% in the AAV-aMTD-Parkin administered group ( Figure 7c).
- Example 8 Confirmation of Alzheimer's disease (AD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) in Alzheimer's disease (AD) dementia model (3 x 10 6 GC/Brain, ICV Route)
- Example 9 Confirmation of Alzheimer's disease (AD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) in Alzheimer's disease (AD) dementia model (3 x 10 4 GC/Brain, ICV Route)
- AAV-Parkin / AAV-aMTD-Parkin was administered by ICV at extremely low doses (3 x 10 4 GC/Brain) to an fA ⁇ -induced Alzheimer's disease animal model ( Figure 9a).
- cognitive function was improved by 13% in the AAV-Parkin administration group and 57% in the AAV-aMTD-Parkin administration group ( Figure 9b). This result shows that aMTD shows a clear Add-On Effect depending on the presence or absence of cell permeability when administered at extremely low doses.
- CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT GGGAAGAGAA TAGCAGGCAT GCTGGGGA
Abstract
The present invention relates to an AAV vector and cell-penetrating peptide fusion platform and a pharmaceutical composition for the prevention or treatment of degenerative brain diseases, including same. The platform overcomes the limitation of the adeno-associated virus (AAV) vector-employing gene therapy by the cell penetration based on the fusion with therapeutic molecule systemic delivery technology (TSDT) and can be utilized in next-generation gene therapy applications through fusion with Parkin, a therapeutic protein for degenerative brain diseases.
Description
본 발명은 아데노-부속 바이러스 (Adeno-Associate Virus: AAV) 벡터와 세포투과성 펩티드 융합 플랫폼 및 이를 포함하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to an Adeno-Associate Virus (AAV) vector and cell-permeable peptide fusion platform and a pharmaceutical composition containing the same for preventing or treating degenerative brain diseases.
아데노-부속 바이러스 (AAV) 벡터는 현재까지 개발 중인 유전자치료제에 가장 보편적으로 사용하는 전달 시스템이다. AAV 벡터의 장점은 비병원성으로 다양한 조직에 광범위하게 특정하여 표적화 할 수 있다. 또한 1회 투여로 장기간 효능을 유지할 수 있으며, 스스로 복제할 수 있는 기능이 없기 때문에 숙주유전자에 결합하지 않아 안전성이 높다. 다만 AAV 벡터를 이용하였을 때 치료물질 전달을 위한 세포간 연속전송이 되지 못하기 때문에 치료물질의 전달, 발현이 어려울 수 있고, 지놈 (Genome) 사이즈가 4.7 kb 초과하였을 때 이질성 (Heterogeneity)이 발생할 가능성이 있으며, 면역원성 (Immunogenecity) 역시 나타날 가능성이 있다. 경우에 따라서는 특정한 전달 (Tropism)이 어려운 한계점이 있다.Adeno-associated virus (AAV) vectors are the most commonly used delivery system for gene therapy drugs currently under development. The advantage of AAV vectors is that they are non-pathogenic and can be broadly targeted to various tissues. In addition, it can maintain long-term efficacy with a single administration, and because it does not have the ability to replicate on its own, it does not bind to the host gene, making it highly safe. However, when using AAV vectors, delivery and expression of therapeutic substances may be difficult because continuous transmission between cells for delivery of therapeutic substances is not possible, and heterogeneity may occur when the genome size exceeds 4.7 kb. There is a possibility that immunogenicity may also occur. In some cases, there are limitations that make certain delivery (tropism) difficult.
약리물질 생체 내 전송기술 (TSDT)은 치료기능이 있는 약리물질 (Cargo)을 소수성 세포막 투과 펩티드 (Hydrophobic Cell-Penetrating Peptide: CPP)인 aMTD (advanced Macromolecule Transduction Domain)와 융합 (Fusion) 또는 접합 (Conjugation)을 통해 세포와 조직에 직접 전달하는 기술이며, 치료 효능이 있는 약리물질들을 세포 조직에 전달하여 신약물을 개발할 수 있는 장점이 있다. 소수성(Hydrophobic) CPP 서열을 분석하여 높은 세포투과능과 구조적 안정성을 부여하는 6가지 Critical Factors를 도출하였고, 정량적인 평가로 보다 진보된 소수성(Hydrophobic) CPP인 3세대 aMTD 및 Customized Solubilization Domain (SD)를 이용하여 단백질의 구조적 안정성 (Structurally Stability) 및 세포/조직 투과성(Cell-/Tissue-Permeability)을 개선하였다.Pharmacological Substance Transduction Technology (TSDT) is the fusion or conjugation of a pharmacological substance (Cargo) with a therapeutic function with aMTD (advanced Macromolecule Transduction Domain), a hydrophobic cell-penetrating peptide (CPP). ), it is a technology that delivers drugs directly to cells and tissues, and has the advantage of developing new drugs by delivering pharmacological substances with therapeutic efficacy to cells and tissues. By analyzing the hydrophobic CPP sequence, we derived six critical factors that provide high cell penetration ability and structural stability, and through quantitative evaluation, we developed the 3rd generation aMTD and Customized Solubilization Domain (SD), which are more advanced hydrophobic CPPs. was used to improve the structural stability and cell/tissue-permeability of the protein.
파킨슨병 (Parkinson's Disease: PD), 알츠하이머병 치매 (Alzheimer's Disease: AD) 등과 같은 퇴행성 뇌질환의 경우 명확한 발병요인이 밝혀지지 않았지만 포괄적으로 ① 환경적인 요인, ② 유전적인 요인 등이 있지만 대부분은 불명확하여 명확한 기전이 파악되어야 치료제로 개발될 수 있다. 뇌에는 혈관을 통해 신경세포에 다른 물질이 쉽게 전달되지 못하도록 하는 혈뇌장벽 (Blood-Brain Barrier: BBB)이 존재하여 주사나 약물투여 같은 일반적인 치료법을 활용한다면 약리물질이 혈뇌장벽 (BBB) 때문에 뇌의 신경세포에 직접적으로 약리물질을 전달하지 못한다. 파킨슨병 (PD) 환자는 전 세계적으로 약 700만 명에서 많게는 1천만 명이 앓고 있는 퇴행성 뇌질환 중 하나이며, 대부분 노인계층에게 발병하는 질환으로 인구의 고령화와 함께 환자수도 크게 증가하고 있으며, 최근에는 40대 젊은 나이에서도 유전적인 원인에 의해 발생하는 경우도 있다. 파킨슨병은 몸이 떨리는 진전운동 (Resting Tremor), 근육의 경직 (Rigidity)이 생기면서 몸동작이 느려지는 서동 (Bradykinesia) 등의 운동장애 (Movement Disorder)가 나타나는 질환이다. 파킨슨병 (PD)은 적절한 치료를 받지 못하면 운동장애 (Movement Disorder)가 점점 진행되어 움직임과 걷기가 어렵게 되며 일상생활을 수행할 수 없게 된다.In the case of degenerative brain diseases such as Parkinson's Disease (PD), Alzheimer's Disease (AD), etc., the exact cause of the disease has not been revealed, but comprehensively, there are ① environmental factors and ② genetic factors, but most of them are unclear. Only when a clear mechanism is identified can a treatment be developed. There is a blood-brain barrier (BBB) in the brain that prevents other substances from being easily transmitted to nerve cells through blood vessels. Therefore, if general treatments such as injections or drug administration are used, pharmacological substances may enter the brain due to the blood-brain barrier (BBB). Pharmacological substances cannot be delivered directly to nerve cells. Parkinson's disease (PD) is one of the degenerative brain diseases that affects approximately 7 to 10 million people worldwide. It is a disease that mostly affects the elderly, and the number of patients is increasing significantly with the aging of the population. Recently, Even in people in their 40s, it can occur due to genetic causes. Parkinson's disease is a disease that causes movement disorders such as resting tremor and bradykinesia, which causes muscle rigidity and slow body movements. If Parkinson's disease (PD) does not receive appropriate treatment, movement disorder gradually progresses, making movement and walking difficult and making it impossible to carry out daily life.
파킨슨병 (PD)은 유전적 인자와 환경적 인자가 복합적으로 작용하여 발생하는 것으로 발병요인이 다양하다. 파킨슨병 (PD) 환자의 중뇌 흑질 (Substantia Nigra: SN) 영역에서 도파민 (Dopamine)을 함유한 뇌 신경세포가 점점 사멸 또는 퇴화하는데, 아직까지 확실한 원인에 대해서는 알려진 것은 없으며, 파킨슨병 (PD)의 여러 요인 중 Parkin 유전자의 변성이 유전적 인자 중 높은 유병률을 차치한다.Parkinson's disease (PD) is caused by a combination of genetic and environmental factors and has a variety of causative factors. Brain neurons containing dopamine gradually die or degenerate in the substantia nigra (SN) area of the midbrain in patients with Parkinson's disease (PD). There is still no known exact cause, and the cause of Parkinson's disease (PD) is increasing. Among many factors, degeneration of the Parkin gene accounts for the high prevalence among genetic factors.
Parkin 유전자는 α-Synuclein을 포함한 여러가지 단백질들을 제거하는 기전에 필수적인 E3 ligase로써 세포 내에서 손상되었거나, 산화된 단백질, 또는 비정상적 구조를 이룬 단백질들을 제거하여 세포 내 스트레스를 감소시켜주는 역할을 한다. Parkin의 변이는 E3 ligase의 역할을 수행하는 유비퀴틴 접합효소 기능을 상실할 뿐만 아니라, 단백질의 잘못된 접힘 (Misfolded)이나 응집 (Aggregation)을 유도하며, 도파민 (Dopamine) 분비 저해 또는 도파민 신경세포의 사멸을 일으키는 등 유전적 파킨슨 질환의 주요원인으로 알려져 있다.The Parkin gene is an E3 ligase that is essential for the removal mechanism of various proteins, including α-Synuclein, and plays a role in reducing intracellular stress by removing damaged, oxidized, or abnormally structured proteins within the cell. Mutation of Parkin not only results in loss of the ubiquitin conjugating enzyme function that performs the role of E3 ligase, but also induces misfolding or aggregation of proteins, inhibits dopamine secretion, or causes death of dopaminergic neurons. It is known to be the main cause of genetic Parkinson's disease.
파킨슨병 (PD)의 치료제로 사용중인 레보도파 (Levodopa)는 도파민 (Dopamine)의 전구체로 뇌의 선조체 (Striatum: ST) 에서 도파민 (Dopamine)의 결핍을 보충하고자 도파민 (Dopamine)의 농도를 높여주는 역할을 한다. 그러나 장기간 투여시 간독성이나 위장장애 등 부작용이 발생하며, 복용량은 증가하지만 약물의 효과가 낮아지게 되어 확실한 파킨슨병 (PD)의 치료제가 아닌 진행을 지연시키는 역할을 할 뿐이다.Levodopa, which is used as a treatment for Parkinson's disease (PD), is a precursor of dopamine and plays a role in increasing the concentration of dopamine to compensate for the deficiency of dopamine in the striatum (ST) of the brain. Do it. However, long-term administration causes side effects such as hepatotoxicity and gastrointestinal disorders, and although the dosage increases, the effectiveness of the drug decreases, so it is not a definite cure for Parkinson's disease (PD) but only serves to delay its progression.
알츠하이머병 치매 (AD) 환자는 고령화가 급속도로 가속화되면서 2050년에는 전 세계적으로 약 1억 5000만 명을 넘어설 것으로 추정할 정도로 퇴행성 뇌질환 중 가장 환자 수가 많은 질환이다. 알츠하이머병 치매 (AD)의 원인을 밝히기 위한 다양한 연구가 진행되고 있지만 확실한 원인들이 모두 밝혀진 것은 아니다. 알츠하이머병 (AD) 환자에게서 나타나는 임상적 증상으로는 기억 (Memory)과 인지능력 (cognitive ability)의 감소가 있으며, 우울증 (Depression), 환각, 망상, 수면장애 등의 정신 의학적 증세가 동반된다. 이와 같은 알츠하이머병 (AD)은 특정한 단일 기전 원인보다 신경계의 노화 현상이 진행되는 상태에서 유전적 위험 요소 및 환경적 위험 인자가 더해져 복합적인 발병 기전에 의해 나타난다고 볼 수 있으며, 전체 치매 (Dementia) 환자의 70 ~ 80%가 알츠하이머병 치매에 속한다. 알츠하이머병 치매 (AD)의 주요 메커니즘은 뇌 신경세포의 신호전달 (Neuronal Signaling) 손상으로 이루어지며 특히, 아밀로이드 베타 (Aβ) 단백질이 축적된 노인성 플라크 (Senile Plaque) 및 타우 (Tau) 단백질의 엉킴현상으로 생기는 신경섬유매듭 (Neurofibrillary Tangle: NFT)이 현재까지 알려진 대표적인 병리학적인 (Pathological) 현상으로 뇌 속에 축적된 아밀로이드 베타 단백질 (Amyloid beta: Aβ)과 타우 단백질 (Tau)이 뇌 신경세포를 점점 사멸 또는 퇴화시킨다. 아밀로이드 베타 (Aβ)는 아밀로이드 전구단백질 (Amyloid precursor Protein)에 의해 만들어지는 것으로 β-secretase에 정상적으로 Cleavage되지만, β-secretase에 의해 Cleavage가 되면 독성을 가지는 Aβ 1-42의 peptide를 생성하게 되고, 최종적으로 물에 잘 녹지 않는 성질로 인해 서로 엉겨 붙고, 축적되어 Senile Plaque를 형성한다. 이와 같은 Plaque는 신경세포 내 미토콘드리아의 기능을 마비시키거나 왜곡시켜 미토콘드리아에서 배출되는 활성산소 (ROS)를 증가시키고, 증가된 활성산소는 세포 내 단백질이나 DNA에 치명적인 상처를 입혀 결국 뇌세포의 손상 또는 사망 (Apoptosis)을 초래한다. Tau 단백질은 뇌 세포의 골격 유지에 중요한 역할을 하는 단백질로써 Tau 단백질의 과도한 인산화(Phosphorylation), 염증반응과 같은 생화학적 반응을 거쳐 신경세포 파괴를 촉진함으로써 알츠하이머병 치매 (AD)의 발병에 기여하며, 환자 뇌의 기억 및 인지기능을 관장하는 영역에서 뇌 신경세포들은 병리학적 단백질 응집체들에 의해 기능적인 결함이 발생하고 기억 및 판단에 대한 손상을 주어 인지기능이 감소하게 된다.Alzheimer's disease dementia (AD) is the most prevalent of the degenerative brain diseases, with the number of patients estimated to exceed 150 million worldwide by 2050 as the aging population rapidly accelerates. Various studies are being conducted to determine the causes of Alzheimer's disease dementia (AD), but not all definite causes have been identified. Clinical symptoms that appear in patients with Alzheimer's disease (AD) include decreased memory and cognitive ability, and are accompanied by psychiatric symptoms such as depression, hallucinations, delusions, and sleep disorders. Alzheimer's disease (AD) can be viewed as being caused by a complex pathogenic mechanism in which genetic risk factors and environmental risk factors are added as the aging phenomenon of the nervous system progresses rather than a specific single mechanism cause, and overall dementia (Dementia) 70 to 80% of patients fall into Alzheimer's disease dementia. The main mechanism of Alzheimer's disease dementia (AD) is damage to neuronal signaling in the brain, especially senile plaques in which amyloid beta (Aβ) protein accumulates and tau protein tangles. Neurofibrillary Tangle (NFT) is a representative pathological phenomenon known to date. Amyloid beta (Aβ) and Tau protein (Tau) accumulated in the brain gradually cause brain neurons to die or die. It degenerates. Amyloid beta (Aβ) is produced by amyloid precursor protein and is normally cleaved by β-secretase. However, when cleaved by β-secretase, it produces toxic Aβ 1-42 peptide, and ultimately Due to its insoluble nature in water, it sticks together and accumulates to form Senile Plaque. Such plaque paralyzes or distorts the function of mitochondria within nerve cells, increasing reactive oxygen species (ROS) released from mitochondria. The increased reactive oxygen species cause fatal damage to proteins or DNA within cells, ultimately causing damage or damage to brain cells. It causes death (apoptosis). Tau protein is a protein that plays an important role in maintaining the skeleton of brain cells. It contributes to the development of Alzheimer's disease (AD) by promoting neuronal destruction through biochemical reactions such as excessive phosphorylation of Tau protein and inflammation. , In the area that governs memory and cognitive functions of the patient's brain, brain neurons develop functional defects due to pathological protein aggregates and damage memory and judgment, leading to a decrease in cognitive function.
알츠하이머병 치매 (AD)의 치료제로는 콜린성 (Choline) 신경 전달의 손상을 억제할 수 있도록 아세틸콜린 (Acetylcholine)의 양을 증가시키거나, 아세틸콜린 (Acetylcholine)이 장기간 존재할 수 있도록 하거나, 신경세포 (Neuron)의 전달에 아세틸콜린 (Acetylcholine)이 효과적으로 작용하도록 하는 약물이 있으며, 알츠하이머병 치매 (AD) 환자들의 아세틸콜린 (Acetylcholine) 활성도를 높이는 다양한 화합물들이 사용되고 있다. 그러나, 미국 식품의약국 (Food and Drug Administration: FDA)에서 알츠하이머병 치매 (AD) 치료제로 유통되고 있지만, 확실한 치료제가 아닌 질환의 진행을 완화시키는 역할밖에 하지 못한다.Treatments for Alzheimer's disease dementia (AD) include increasing the amount of acetylcholine to suppress damage to choline neurotransmission, allowing acetylcholine to exist for a long period of time, or increasing the amount of acetylcholine in nerve cells ( There are drugs that allow acetylcholine to act effectively in the transmission of neurons, and various compounds that increase acetylcholine activity in patients with Alzheimer's disease (AD) are being used. However, although it is being distributed as a treatment for Alzheimer's disease (AD) by the U.S. Food and Drug Administration (FDA), it is not a definitive treatment and only serves to alleviate the progression of the disease.
본 발명에서는 파킨슨병 (PD) 및 알츠하이머병 치매 (AD) 등과 같은 퇴행성 뇌질환 (neurodegenerative disease, NDD)에서 AAV 융합 Parkin 단백질을 적은 투여 횟수에 비해 장기간 지속적으로 운동기능 및 인지기능 개선 등 치료효능을 갖는 것과 환자 친화적으로 투여가 가능하고 부작용이 없다. 이러한 본 발명은 AAV 벡터와 약리물질 생체 내 전송기술 (TSDT)의 융합 플랫폼기술로 안전하고 효율적인 방법으로 기존 유전자치료제의 단점을 극복하여 차세대 퇴행성 뇌질환 치료제로 사용이 가능하다.In the present invention, in neurodegenerative diseases (NDD) such as Parkinson's disease (PD) and Alzheimer's disease dementia (AD), the therapeutic efficacy of AAV fusion Parkin protein, such as improvement of motor function and cognitive function, continuously over a long period of time compared to a small number of administrations. It can be administered in a patient-friendly manner and has no side effects. This invention is a fusion platform technology of AAV vector and pharmacological substance transfer technology (TSDT), which overcomes the shortcomings of existing gene therapy in a safe and efficient manner and can be used as a next-generation treatment for degenerative brain diseases.
[선행기술문헌][Prior art literature]
(특허문헌 001) 대한민국 공개특허 제10-2018-0026565호(Patent Document 001) Republic of Korea Patent Publication No. 10-2018-0026565
(특허문헌 002) 대한민국 공개특허 제10-2022-0127877호(Patent Document 002) Republic of Korea Patent Publication No. 10-2022-0127877
아데노-부속 바이러스 (AAV) 벡터와 약리물질 생체 내 전송기술 (TSDT)의 장점을 융합하면 치료물질의 전달, 발현 및 면역반응이 없으며, 투여 용량이 낮고 혈뇌장벽 (BBB)처럼 치료물질 전송이 큰 장벽을 쉽고 효과적으로 전송할 수 있는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물의 제공이 목적이다.Combining the advantages of adeno-associated virus (AAV) vectors and in vivo delivery technology (TSDT) allows for the delivery, expression, and immune response of therapeutic substances, low administration dose, and large transfer of therapeutic substances across the blood-brain barrier (BBB). The purpose is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases that can easily and effectively transmit the barrier.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 aMTD(advanced macromolecule transduction domain) 코딩 폴리뉴클레오티드 서열, Parkin 단백질 코딩 폴리뉴클레오티드 서열을 포함하는 것인, AAV 벡터를 제공한다.To achieve the above object, an example of the present invention provides an AAV vector comprising an advanced macromolecule transduction domain (aMTD) coding polynucleotide sequence and a Parkin protein coding polynucleotide sequence.
본 발명의 다른 일 예는 상기 벡터가 형질전환된 숙주세포를 제공한다.Another example of the present invention provides a host cell transformed with the vector.
본 발명의 다른 일 예는 상기 벡터를 포함하는 퇴행성 뇌질환 치료용 약학적 조성물을 제공한다.Another example of the present invention provides a pharmaceutical composition for treating degenerative brain diseases containing the vector.
본 발명의 또다른 일 예는 상기 벡터, 숙주세포 및/또는 약학적 조성물을 대상체에게 투여하는 단계를 포함하는 퇴행성 뇌질환의 예방 또는 치료 방법을 제공한다.Another example of the present invention provides a method for preventing or treating degenerative brain diseases comprising administering the vector, host cell, and/or pharmaceutical composition to a subject.
본 발명은 아데노-부속 바이러스 (AAV) 벡터의 한계를 세포투과성을 갖는 Parkin 재조합 단백질의 융합을 통해 극복하고 생체 내 (In Vivo) 환경에서 도파민 신경세포 내로 전달되어 세포사멸을 억제하거나, 도파민 분비를 증가 효능을 통해 파킨슨병 및 알츠하이머병 치매와 같은 퇴행성 뇌질환 치료 목적 연구에 사용될 수 있으며, 궁극적으로 인간에게 퇴행성 뇌질환 치료제를 위한 차세대 유전자 치료제로 유용하게 사용될 수 있을 것으로 기대된다.The present invention overcomes the limitations of the adeno-associated virus (AAV) vector through the fusion of a cell-permeable Parkin recombinant protein and is delivered into dopaminergic neurons in an in vivo environment to inhibit apoptosis or increase dopamine secretion. Through its increased efficacy, it can be used in research for the treatment of degenerative brain diseases such as Parkinson's disease, Alzheimer's disease, and dementia, and is ultimately expected to be useful as a next-generation gene therapy for the treatment of degenerative brain diseases in humans.
도 1a는 아데노-부속 바이러스 (AAV)의 전이 플라스미드 골격(Transfer Plasmid Backbone) 구조(도 1a)를 나타낸 그림이다. Figure 1a is a diagram showing the transfer plasmid backbone structure (Figure 1a) of adeno-associated virus (AAV).
도 1b는 Parkin 단백질의 구조를 나타낸 그림이다.Figure 1b is a diagram showing the structure of Parkin protein.
도 1c는 pAAV9-Parkin (△Ubl)-SDB의 구조를 나타낸 그림이다. Figure 1c is a diagram showing the structure of pAAV9-Parkin (ΔUbl)-SDB.
도 1d는 pAAV9-aMTD524-Parkin (△Ubl)-SDB의 구조를 나타낸 그림이다.Figure 1d is a diagram showing the structure of pAAV9-aMTD524-Parkin (ΔUbl)-SDB.
도 2a는 6-OHDA 유도 파킨슨병 동물모델에 아데노-부속 바이러스 (AAV)를 이용한 유전자치료 및 운동시험 과정을 나타낸 그림이다.Figure 2a is a diagram showing the process of gene therapy and exercise testing using adeno-associated virus (AAV) in a 6-OHDA-induced Parkinson's disease animal model.
도 2b는 AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 운동기능 개선효과를 나타낸 그래프이다.Figure 2b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
도 2c는 AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 선조체 및 흑질에서의 도파민 신경세포를 촬영한 사진이다.Figure 2c is a photograph of dopaminergic neurons in the striatum and substantia nigra in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
도 2d는 AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 선조체 및 흑질에서 AAV-aMTD-Parkin이 전송되었음을 나타낸 사진이다.Figure 2d is a photograph showing that AAV-aMTD-Parkin is transmitted in the striatum and substantia nigra of a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
도 2e는 AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 티로신 수산화효소의 발현량을 나타낸 사진이다.Figure 2e is a photograph showing the expression level of tyrosine hydroxylase in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
도 2f는 AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 티로신 수산화효소의 발현량을 나타낸 그래프이다.Figure 2f is a graph showing the expression level of tyrosine hydroxylase in a Parkinson's disease (PD) animal model after administration of AAV-aMTD-Parkin.
도 3a는 fAβ 유도 알츠하이머병 동물모델에 아데노-부속 바이러스 (AAV)를 이용한 유전자치료 및 인지기능 시험 과정을 나타낸 그림이다.Figure 3a is a diagram showing the process of gene therapy and cognitive function testing using adeno-associated virus (AAV) in an fAβ-induced Alzheimer's disease animal model.
도 3b는 AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델에서의 인지기능 개선효과를 나타낸 그래프이다.Figure 3b is a graph showing the effect of improving cognitive function in an Alzheimer's disease (AD) animal model after administration of AAV-aMTD-Parkin.
도 3c는 AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델의 해마 영역에서 병리학적 Aβ가 제거되고, Parkin이 발현되었음을 나타내는 사진이다.Figure 3c is a photograph showing that pathological Aβ was removed and Parkin was expressed in the hippocampus region of an Alzheimer's disease (AD) animal model after administration of AAV-aMTD-Parkin.
도 3d는 AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델에서 병리학적 Aβ plaque가 감소하였음을 나타낸 사진이다.Figure 3d is a photograph showing a decrease in pathological Aβ plaques in an Alzheimer's disease (AD) animal model after administration of AAV-aMTD-Parkin.
도 4a는 파킨슨병(PD) 동물모델에서 세포투과성 유무에 따른 AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)과 기존 유전자치료법 (AAV-Parkin)의 치료물질전달 능력을 비교하기 위한 실험과정을 나타낸 그림이다.Figure 4a shows an experimental process to compare the therapeutic material delivery ability of the AAV-TSDT fusion platform (AAV-aMTD-Parkin) and the existing gene therapy (AAV-Parkin) according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model. It's a picture.
도 4b는 AAV-GFP-aMTD-Parkin-mCherry의 투여 후 파킨슨병(PD) 동물모델에서 뇌의 선조체 영역을 촬영한 사진이다.Figure 4b is a photograph of the striatum region of the brain in a Parkinson's disease (PD) animal model after administration of AAV-GFP-aMTD-Parkin-mCherry.
도 4c는 AAV-GFP-aMTD-Parkin-mCherry의 투여 후 파킨슨병(PD) 동물모델에서 뇌의 선조체 영역에서 신경세포 및 신경교세포를 촬영한 사진이다.Figure 4c is a photograph of neurons and glial cells in the striatal region of the brain in a Parkinson's disease (PD) animal model after administration of AAV-GFP-aMTD-Parkin-mCherry.
도 4d는 파킨슨병(PD) 동물모델에서 세포투과성 유무에 따른 AAV-aMTD-Parkin의 치료물질전달 능력을 비교하기 위한 실험과정을 나타낸 그림이다.Figure 4d is a diagram showing the experimental process for comparing the therapeutic substance delivery ability of AAV-aMTD-Parkin according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
도 4e는 파킨슨병(PD) 동물모델에서 세포투과성 유무에 따른 AAV-aMTD-Parkin의 ICM 투여 위치 및 조직 획득 위치를 나타낸 그림이다.Figure 4e is a diagram showing the ICM administration location and tissue acquisition location of AAV-aMTD-Parkin according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
도 4f는 파킨슨병(PD) 동물모델에서 AAV-aMTD-Parkin의 투여 후 흑질 영역에서의 외인성(exogenous) Parkin의 양을 나타낸 그래프이다.Figure 4f is a graph showing the amount of exogenous Parkin in the substantia nigra region after administration of AAV-aMTD-Parkin in a Parkinson's disease (PD) animal model.
도 4g는 파킨슨병(PD) 동물모델에서 AAV-aMTD-Parkin의 투여 후 선조체 영역에서의 외인성(exogenous) Parkin의 양을 나타낸 그래프이다.Figure 4g is a graph showing the amount of exogenous Parkin in the striatum region after administration of AAV-aMTD-Parkin in a Parkinson's disease (PD) animal model.
도 4h는 파킨슨병(PD) 동물모델에서 AAV-aMTD-Parkin의 투여 후 대뇌피질 영역에서의 외인성(exogenous) Parkin의 양을 나타낸 그래프이다.Figure 4h is a graph showing the amount of exogenous Parkin in the cerebral cortex area after administration of AAV-aMTD-Parkin in a Parkinson's disease (PD) animal model.
도 5a는 파킨슨병(PD) 동물모델에서 세포투과성 유무에 따른 치료효과를 확인하기 위하여 아데노-부속 바이러스 (AAV)를 이용한 저용량 유전자치료 및 운동기능 시험 과정을 나타낸 그림이다.Figure 5a is a diagram showing the process of low-dose gene therapy and motor function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
도 5b는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 운동기능 개선효과를 나타낸 그래프이다.Figure 5b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 5c는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 도파민 양의 변화를 나타낸 그래프이다.Figure 5c is a graph showing the change in the amount of dopamine in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 5d는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 티로신 수산화효소(TH)의 발현량을 나타낸 사진이다.Figure 5d is a photograph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 5e는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 티로신 수산화효소(TH)의 발현량을 나타낸 그래프이다.Figure 5e is a graph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 5f는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 흑질 영역에서 티로신 수산화효소(TH)의 발현을 촬영한 사진이다.Figure 5f is a photograph of the expression of tyrosine hydroxylase (TH) in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 5g는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 외인성(exogenous) Parkin의 양을 나타낸 그래프이다.Figure 5g is a graph showing the amount of exogenous Parkin in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 6a는 파킨슨병(PD) 동물모델에서 세포투과성 유무에 따른 치료효과를 확인하기 위하여 아데노-부속 바이러스 (AAV)를 이용한 고용량 유전자치료 및 운동기능 시험 과정을 나타낸 그림이다.Figure 6a is a diagram showing the process of high-dose gene therapy and motor function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in a Parkinson's disease (PD) animal model.
도 6b는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 운동기능 개선효과를 나타낸 그래프이다.Figure 6b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 6c는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 도파민 양의 변화를 나타낸 그래프이다.Figure 6c is a graph showing the change in the amount of dopamine in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 6d는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 티로신 수산화효소(TH)의 발현량을 나타낸 사진이다.Figure 6d is a photograph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 6e는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 흑질 영역에서 티로신 수산화효소(TH)의 발현을 촬영한 사진이다.Figure 6e is a photograph of the expression of tyrosine hydroxylase (TH) in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 6f는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 외인성(exogenous) Parkin의 양을 나타낸 그래프이다.Figure 6f is a graph showing the amount of exogenous Parkin in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 7a는 만성 파킨슨병(PD) 동물모델에서 세포투과성 유무에 따른 치료효과를 확인하기 위하여 아데노-부속 바이러스 (AAV)를 이용한 저용량 유전자치료 및 운동기능 시험 과정을 나타낸 그림이다.Figure 7a is a diagram showing the process of low-dose gene therapy and motor function testing using adeno-associated virus (AAV) to confirm the therapeutic effect depending on the presence or absence of cell permeability in a chronic Parkinson's disease (PD) animal model.
도 7b는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 운동기능 개선효과를 나타낸 그래프이다.Figure 7b is a graph showing the effect of improving motor function in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 7c는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델에서의 도파민 양의 변화를 나타낸 그래프이다.Figure 7c is a graph showing the change in the amount of dopamine in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 7d는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 티로신 수산화효소(TH)의 발현량을 나타낸 사진이다.Figure 7d is a photograph showing the expression level of tyrosine hydroxylase (TH) in a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 7e는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 흑질 영역에서 티로신 수산화효소(TH)의 발현을 촬영한 사진이다.Figure 7e is a photograph of the expression of tyrosine hydroxylase (TH) in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 7f는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 파킨슨병(PD) 동물모델의 흑질 영역에서 인산화된 α-syunclein의 발현량을 나타낸 사진이다.Figure 7f is a photograph showing the expression level of phosphorylated α-syunclein in the substantia nigra region of a Parkinson's disease (PD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 8a는 알츠하이머병(AD) 동물모델에서 세포투과성 유무에 따른 치료효과를 확인하기 위하여 아데노-부속 바이러스 (AAV)를 이용한 저용량 유전자치료 및 인지기능 시험 과정을 나타낸 그림이다.Figure 8a is a diagram showing the process of low-dose gene therapy and cognitive function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in an Alzheimer's disease (AD) animal model.
도 8b는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델에서의 인지기능 개선효과를 나타낸 그래프이다.Figure 8b is a graph showing the effect of improving cognitive function in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 8c는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델에서의 병리학적 Aβ Plaque 양을 나타낸 사진이다.Figure 8c is a photograph showing the amount of pathological Aβ plaques in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 8d는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델의 해마영역에서 병리학적 Aβ Plaque 양을 나타낸 사진이다.Figure 8d is a photograph showing the amount of pathological Aβ plaques in the hippocampus area of an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 9a는 알츠하이머병(AD) 동물모델에서 세포투과성 유무에 따른 치료효과를 확인하기 위하여 아데노-부속 바이러스 (AAV)를 이용한 극저용량 유전자치료 및 인지기능 시험 과정을 나타낸 그림이다.Figure 9a is a diagram showing the process of extremely low-dose gene therapy and cognitive function testing using adeno-associated virus (AAV) to confirm the therapeutic effect according to the presence or absence of cell permeability in an Alzheimer's disease (AD) animal model.
도 9b는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델에서의 인지기능 개선효과를 나타낸 그래프이다.Figure 9b is a graph showing the effect of improving cognitive function in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 9c는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델에서의 병리학적 Aβ Plaque 양을 나타낸 사진이다.Figure 9c is a photograph showing the amount of pathological Aβ plaques in an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
도 9d는 AAV-Parkin / AAV-aMTD-Parkin의 투여 후 알츠하이머병(AD) 동물모델의 해마영역에서 병리학적 Aβ Plaque 양을 나타낸 사진이다.Figure 9d is a photograph showing the amount of pathological Aβ plaques in the hippocampus area of an Alzheimer's disease (AD) animal model after administration of AAV-Parkin / AAV-aMTD-Parkin.
본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 달리 정의되지 않는 한, 본 발명이 속하는 기술분야의 전문가에 의해 통상적으로 이해되는 것과 동일한 의미를 가진다. 본 명세서에서 언급된 모든 간행물, 특허 및 기타 다른 참고문헌은 전체가 참고로 포함된다.All technical and scientific terms used in this specification, unless otherwise defined, have the same meaning as commonly understood by experts in the technical field to which the present invention pertains. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.
본 명세서 전체에서 특별한 언급이 없는 한 "포함" 또는 "함유"라 함은 어떤 구성 요소(또는 구성 성분)를 별다른 제한 없이 포함함을 지칭하며, 다른 구성 요소(또는 구성 성분)의 부가를 제외하는 것으로 해석될 수 없다.Throughout this specification, unless otherwise specified, “include” or “contains” refers to the inclusion of a certain component (or component) without particular limitation, excluding the addition of other components (or components). cannot be interpreted as
본 발명의 일 예는 aMTD(advanced macromolecule transduction domain) 코딩 폴리뉴클레오티드 서열, Parkin 단백질 코딩 폴리뉴클레오티드 서열을 포함하는 것인, AAV 벡터를 제공한다.One example of the present invention provides an AAV vector comprising an advanced macromolecule transduction domain (aMTD) coding polynucleotide sequence and a Parkin protein coding polynucleotide sequence.
상기 aMTD 코딩 폴리뉴클레오티드 서열은 서열번호 3 내지 242의 폴리뉴클레오티드 서열, 바람직하게는 서열번호 125의 폴리뉴클레오티드 서열일 수 있으나 이에 제한되지 않는다.The aMTD coding polynucleotide sequence may be a polynucleotide sequence of SEQ ID NO: 3 to 242, preferably a polynucleotide sequence of SEQ ID NO: 125, but is not limited thereto.
상기 벡터는 하나 또는 그 이상의 가용화 도메인(solubilization domain; SD)(들)을 추가로 포함하는 것일 수 있다.The vector may additionally include one or more solubilization domain (SD)(s).
상기 SD(들)는 서열번호: 243의 폴리뉴클레오티드 서열을 가질 수 있다.The SD(s) may have the polynucleotide sequence of SEQ ID NO: 243.
상기 프로모터는 서열번호 재조합 단백질은 하기의 구조식 중 어느 하나로 표현되며,The promoter has SEQ ID NO: The recombinant protein is expressed in one of the following structural formulas,
상기 Parkin 단백질 코딩 폴리뉴클레오티드 서열은 서열번호 245의 폴리뉴클레오티드 서열일 수 있다.The Parkin protein-encoding polynucleotide sequence may be the polynucleotide sequence of SEQ ID NO: 245.
상기 벡터는 서열번호 244의 CMV 프로모터 및/또는 서열번호 246의 BGH poly A 서열을 포함할 수 있다.The vector may include the CMV promoter of SEQ ID NO: 244 and/or the BGH poly A sequence of SEQ ID NO: 246.
상기 벡터는 서열번호 2의 폴리뉴클레오티드 서열을 포함할 수 있다.The vector may contain the polynucleotide sequence of SEQ ID NO: 2.
본 발명의 일 예는 상기 벡터가 형질전환된 숙주세포를 제공한다.One example of the present invention provides host cells transformed with the vector.
본 발명의 다른 일 예는 상기 벡터를 포함하는 퇴행성 뇌질환 치료용 약학적 조성물을 제공한다.Another example of the present invention provides a pharmaceutical composition for treating degenerative brain diseases containing the vector.
상기 벡터는 퇴행성 뇌질환의 치료에 사용되고,The vector is used in the treatment of degenerative brain diseases,
상기 퇴행성 뇌질환은 파킨슨병, 알츠하이머병, 헌팅턴병, 경도인지장애(mild cognitive impairment), 대뇌 아밀로이드 맥관병중, 다운증후군, 아밀로이드성 뇌졸증(stroke), 전신성 아밀로이드병, 더취(Dutch)형 아밀로이드증, 니만-픽병, 노인성 치매, 근위축성 측삭 경화증 (amyotrophic lateral sclerosis), 척수소뇌성 운동실조증(Spinocerebellar Atrophy), 뚜렛 증후군 (Tourette`s Syndrome), 프리드리히 보행실조 (Friedrich`s Ataxia), 마차도-조셉 병 (Machado-Joseph`s disease), 루이 소체 치매 (Lewy Body Dementia), 근육긴장이상 (Dystonia), 진행성 핵상 마비(Progressive Supranuclear Palsy) 및 전두측두엽 치매(Frontotemporal Dementia), 바람직하게는 파킨슨병 및 알츠하이머병일 수 있다.The degenerative brain diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann- Pick's disease, senile dementia, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph disease -Joseph's disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy, and Frontotemporal Dementia, preferably Parkinson's disease and Alzheimer's disease. .
상기 약학적 조성물은 펩티드 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 유효 성분으로 포함할 수 있다. 상기 약학적 조성물은 퇴행성 뇌질환의 예방 또는 치료 활성을 갖는 공지의 유효 성분을 더 포함할 수 있다.The pharmaceutical composition may include a peptide compound, a stereoisomer thereof, a solvate, or a pharmaceutically acceptable salt as an active ingredient. The pharmaceutical composition may further include known active ingredients that have preventive or therapeutic activity for degenerative brain diseases.
상기 약학적 조성물은 약제학적으로 허용가능한 희석제 또는 담체를 추가적으로 포함할 수 있다. 상기 희석제는 유당, 옥수수 전분, 대두유, 미정질 셀룰로오스, 또는 만니톨, 활택제로는 스테아린산 마그네슘, 탈크, 또는 그 조합일 수 있다. 상기 담체는 부형제, 붕해제, 결합제, 활택제, 또는 그 조합일 수 있다. 상기 부형제는 미결정 셀룰로오즈, 유당, 저치환도 히드록시셀룰로오즈, 또는 그 조합일 수 있다. 상기 붕해제는 카르복시메틸셀룰로오스 칼슘, 전분글리콜산 나트륨, 무수인산일수소 칼슘, 또는 그 조합일 수 있다. 상기 결합제는 폴리비닐피롤리돈, 저치환도 히드록시프로필셀룰로오즈, 히드록시프로필셀룰로오즈, 또는 그 조합일 수 있다. 상기 활택제는 스테아린산 마그네슘, 이산화규소, 탈크, 또는 그 조합일 수 있다.The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, disintegrant, binder, lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate anhydride, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
상기 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화될 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다.The pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
상기 약학적 조성물에 있어서, 경구 투여를 위한 고형 제제는 정제, 환제, 산제, 과립제, 또는 캡슐제일 수 있다. 상기 고형 제제는 부형제를 더 포함할 수 있다. 부형제는 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose), 락토오스(lactose), 또는 젤라틴일 수 있다. 또한, 상기 고형 제제는 마그네슘 스테아레이트, 또는 탈크와 같은 윤활제를 더 포함할 수 있다. 상기 약학적 조성물에 있어서, 경구를 위한 액상제제는 현탁제, 내용액제, 유제, 또는 시럽제일 수 있다. 상기 액상 제제는 물, 또는 리퀴드 파라핀을 포함할 수 있다. 상기 액상 제제는 부형제, 예를 들면 습윤제, 감미제, 방향제, 또는 보존제를 포함할 수 있다. 상기 약학적 조성물에 있어서, 비경구 투여를 위한 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 또는 및 좌제일 수 있다. 비수성용제 또는 현탁제는 식물성 기름 또는 에스테르를 포함할 수 있다. 식물성 기름은 예를 들면, 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 또는 올리브 오일일 수 있다. 에스테르는 예를 들면 에틸올레이트일 수 있다. 좌제의 기제는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 또는 글리세로젤라틴일 수 있다. 상기 약학적 조성물의 바람직한 투여량은 개체의 상태 및 체중, 질병의 정도, 약물 형태, 투여 경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 상기 화합물, 이의 이성질체, 유도체, 용매화물, 또는 약학적으로 허용가능한 염은 예를 들면, 약 0.0001 ㎎/㎏ 내지 약 100 ㎎/㎏, 또는 약 0.001 ㎎/㎏ 내지 약 100 ㎎/㎏의 양을 일일 1회 내지 24회, 2일 내지 1주에 1 내지 7회, 또는 1개월 내지 12개월에 1 내지 24회로 나누어 투여할 수 있다. 상기 약학적 조성물에서 화합물, 이의 이성질체, 유도체, 용매화물, 또는 약학적으로 허용가능한 염은 전체 조성물 총 중량에 대하여 약 0.0001 중량% 내지 약 10 중량%, 또는 약 0.001 중량% 내지 약 1 중량%로 포함될 수 있다.In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid preparation may further include excipients. Excipients may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid preparation may be a suspension, oral solution, emulsion, or syrup. The liquid formulation may contain water or liquid paraffin. The liquid formulation may contain excipients such as wetting agents, sweeteners, flavoring agents, or preservatives. In the pharmaceutical composition, preparations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried products, and suppositories. Non-aqueous solvents or suspensions may contain vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao, laurel, or glycerogelatin. The preferred dosage of the pharmaceutical composition varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt may be used in an amount of, for example, about 0.0001 mg/kg to about 100 mg/kg, or about 0.001 mg/kg to about 100 mg/kg. The amount can be divided and administered 1 to 24 times per day, 1 to 7 times per 2 days to 1 week, or 1 to 24 times per month to 12 months. In the pharmaceutical composition, the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt is present in an amount of about 0.0001% to about 10% by weight, or about 0.001% to about 1% by weight, based on the total weight of the composition. may be included.
상기 퇴행성 뇌질환은 파킨슨병, 알츠하이머병, 헌팅턴병, 경도인지장애(mild cognitive impairment), 대뇌 아밀로이드 맥관병중, 다운증후군, 아밀로이드성 뇌졸증(stroke), 전신성 아밀로이드병, 더취(Dutch)형 아밀로이드증, 니만-픽병, 노인성 치매, 근위축성 측삭 경화증 (amyotrophic lateral sclerosis), 척수소뇌성 운동실조증(Spinocerebellar Atrophy), 뚜렛 증후군 (Tourette`s Syndrome), 프리드리히 보행실조 (Friedrich`s Ataxia), 마차도-조셉 병 (Machado-Joseph`s disease), 루이 소체 치매 (Lewy Body Dementia), 근육긴장이상 (Dystonia), 진행성 핵상 마비(Progressive Supranuclear Palsy) 및 전두측두엽 치매(Frontotemporal Dementia), 바람직하게는 파킨슨병 및 알츠하이머병일 수 있다.The degenerative brain diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann- Pick's disease, senile dementia, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph disease -Joseph's disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy, and Frontotemporal Dementia, preferably Parkinson's disease and Alzheimer's disease. .
본 발명의 또 다른 일 예는 상기 벡터, 상기 숙주세포 및/또는 상기 약학적 조성물을 대상체에게 투여하는 단계를 포함하는 퇴행성 뇌질환의 예방 또는 치료방법을 제공한다.Another example of the present invention provides a method for preventing or treating a degenerative brain disease comprising administering the vector, the host cell, and/or the pharmaceutical composition to a subject.
상기 투여는 정맥내(intravenous), 비경구(parenteral), 경피(transdermal), 피하(subcutaneous), 근육내(intramuscular), 두개내(intracranial), 안와내(intraorbital), 안내(intraocular), 뇌실내(intracerebroventricular), 뇌실질내 주사(intracerabral injection), 피막내(intracapsular), 척수강내(intrathecal), 수조내(intracisternal), 복강내(intraperitoneal), 비강내(intranasal), 직장내(intrarectal), 질내(intravaginal), 분무(spraying) 또는 경구(oral) 투여, 바람직하게는 정맥내, 피하, 비강내 또는 복강내 등과 같은 비경구적으로 사람과 동물에게 투여될 수 있다. 비경구적 투여는 피하주사, 근육내 주사 및 정맥 주사와 같은 주사법 및 점적법을 포함한다.The administration may be administered intravenous, parenteral, transdermal, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, or intraventricular. (intracerebroventricular), intracerebral injection, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, intrarectal, intravaginal ( It can be administered to humans and animals by intravaginal, spraying or oral administration, preferably parenterally such as intravenously, subcutaneously, intranasally or intraperitoneally. Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection, and intravenous injection.
본 발명의 또다른 일 예는 상기 벡터, 상기 숙주세포 및/또는 상기 약학적 조성물을 대상체에게 투여하여 퇴행성 뇌질환을 예방 또는 치료하는 용도를 제공한다.Another example of the present invention provides a use for preventing or treating degenerative brain diseases by administering the vector, the host cell, and/or the pharmaceutical composition to a subject.
이하 본 발명을 실험예 및 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실험예 및 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실험예 및 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through experimental examples and examples. However, these experimental examples and examples are for illustrative purposes only and the scope of the present invention is not limited to these experimental examples and examples.
본 발명에 사용된 용어 "아미노산"은 가장 넓은 의미에서, 자연 발생한 L α-아미노산 또는 이의 잔기뿐만 아니라 D-아미노산 및 화학적으로 변형된 아미노산을 포함한다. 예를 들어, 아미노산은 전술한 아미노산의 모방체 및 유사체를 포함할 수 있다. 본 개시에서, 모방체 및 유사체는 기능적 등가물을 포함할 수 있다.As used herein, the term “amino acid” in its broadest sense includes naturally occurring L α-amino acids or residues thereof as well as D-amino acids and chemically modified amino acids. For example, amino acids may include mimetics and analogs of the amino acids described above. In the present disclosure, mimetics and analogs may include functional equivalents.
본 발명에 사용된 "예방"이라는 용어는 본 발명에 따른 아데노-부속 바이러스 (Adeno-Associate Virus: AAV) 벡터를 포함하는 조성물을 투여함으로써 퇴행성 뇌질환의 발생을 억제 또는 지연시키는 모든 작용을 의미하며, 그리고 "치료"라는 용어는 본 발명에 따른 아데노-부속 바이러스 벡터를 포함하는 조성물을 투여함으로써 퇴행성 뇌질환의 증상이 호전되거나 유리하게 변형되는 모든 작용을 의미한다.The term "prevention" used in the present invention refers to all actions that inhibit or delay the occurrence of degenerative brain disease by administering a composition containing the Adeno-Associate Virus (AAV) vector according to the present invention. , and the term “treatment” refers to all actions in which the symptoms of a degenerative brain disease are improved or advantageously modified by administering a composition containing the adeno-associated viral vector according to the present invention.
본 발명에 사용된 "투여"는 임의의 적절한 방식으로 대상체에게 본 발명의 미리 결정된 약학적 조성물을 제공하는 것을 의미한다.As used herein, “administering” means providing a predetermined pharmaceutical composition of the invention to a subject in any suitable manner.
본 발명에 사용된 용어 "대상체"는 퇴행성 뇌질환이 발병했거나 발병할 가능성이 있는 인간을 포함하는 모든 동물을 의미한다. 동물은 인간뿐만 아니라 유사한 증상의 치료가 필요한 소, 말, 양, 돼지, 염소, 낙타, 영양, 개 또는 고양이를 포함할 수 있으나, 이에 제한되지 않는다.As used herein, the term “subject” refers to all animals, including humans, that have developed or are likely to develop a degenerative brain disease. Animals may include, but are not limited to, humans as well as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, or cats that require treatment for similar symptoms.
본 발명에 따른 조성물은 다양한 경구 또는 비경구 투여 형태, 바람직하게는 비경구 투여 형태로 제형화 될 수 있다. 비경구 투여용 제형의 대표적인 것은 주사용 제형으로 등장성 수용액 또는 현탁액이 바람직하다. 주사용 제형은 적합한 분산제 또는 습윤제 및 현탁화제를 사용하여 당업계에 공지된 기술에 따라 제조할 수 있다. 예를 들면, 각 성분을 식염수 또는 완충액에 용해시켜 주사용으로 제형화할 수 있다.The composition according to the invention can be formulated in various oral or parenteral dosage forms, preferably parenteral dosage forms. A representative formulation for parenteral administration is an injectable formulation, and an isotonic aqueous solution or suspension is preferred. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline or buffer solution and formulated for injection.
또한, 본 발명에 따른 조성물의 투여 경로로는 정맥 내 (intravenous; IV), 해마 내 (intrahippocampal; IH), 뇌 내 (intracerebral), 두개 내 (intracranial)， 척추 내 (intraspinal), 뇌척수액 내(intracerebrospinal), 뇌실 내 (intracerebroventricular; ICV), 척수강 내(intrathecal), 피하, 비강내 또는 복강내 등과 같은 비경구적으로 사람과 동물에게 투여될 수 있다. 비경구적 투여는 피하주사, 근육내 주사 및 정맥 주사와 같은 주사법 및 점적법을 포함한다.In addition, the route of administration of the composition according to the present invention includes intravenous (IV), intrahippocampal (IH), intracerebral, intracranial, intraspinal, and intracerebrospinal fluid (intracerebrospinal). ), intracerebroventricular (ICV), intrathecal, subcutaneous, intranasal or intraperitoneal, etc. can be administered to humans and animals. Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection, and intravenous injection.
본 발명의 조성물은 방부제, 수화제, 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제와 같은 보조제와 기타 치료적으로 유용한 물질을 추가로 포함할 수 있으며, 통상적인 방법에 따라 제제화될 수 있다.The composition of the present invention may further contain auxiliaries such as preservatives, wetting agents, emulsification accelerators, salts and/or buffers for osmotic pressure adjustment, and other therapeutically useful substances, and may be formulated according to conventional methods.
본 발명의 약학적 조성물은 약학적 조성물은 약학적으로 허용가능한 담체 및/또는 첨가물을 포함할 수 있다. 예를 들어, 멸균수, 생리식염수, 관용의 완충제(인산, 구연산, 그 밖의 유기산 등), 안정제, 염, 산화방지제(아스코르브산 등), 계면활성제, 현탁제, 등장화제, 또는 보존제 등을 포함할 수 있다. 일 구체예에 따른 약학적 조성물이 주사에 적당한 제형으로 조제되는 경우에는 재조합 단백질이 약학적으로 허용가능한 담체 중에 용해되어 있거나 또는 용해되어 있는 용액상태로 동결된 것일 수 있다.The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier and/or additive. For example, it contains sterile water, physiological saline, common buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do. When the pharmaceutical composition according to one embodiment is prepared in a formulation suitable for injection, the recombinant protein may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state.
제조예. AAV 벡터 제조Manufacturing example. AAV vector preparation
AAV-aMTD-Parkin은 5' ITR - CMV promoter - Kozak - aMTD - Parkin - SDB - BGH pA - 3' ITR의 구조로 제조하였고, 이때, aMTD 서열은 하기의 aMTD 서열에 따라 바꾸어 제작하였다.AAV-aMTD-Parkin was prepared with the structure of 5' ITR - CMV promoter - Kozak - aMTD - Parkin - SDB - BGH pA - 3' ITR, and the aMTD sequence was changed according to the aMTD sequence below.
| SEQ ID No.SEQ ID No. | SequenceSequence |
| 33 | GCGGCGGCGCTGGCGCCGGTGGTGCTGGCGCTGCCGGCGGCGGCGCTGGCGCCGGTGGTGCTGGCGCTGCCG |
| 44 | GCGGCGGCGGTGCCGCTGCTGGCGGTGGTGGTGCCGGCGGCGGCGGTGCCGCTGCTGGCGGTGGTGGTGCCG |
| 55 | GCGGCGCTGCTGGTGCCGGCGGCGGTGCTGGCGCCGGCGGCGCTGCTGGTGCCGGCGGCGGTGCTGGCGCCG |
| 66 | GCGCTGGCGCTGCTGCCGGTGGCGGCGCTGGCGCCGGCGCTGGCGCTGCTGCCGGTGGCGGCGCTGGCGCCG |
| 77 | GCGGCGGCGCTGCTGCCGGTGGCGCTGGTGGCGCCGGCGGCGGCGCTGCTGCCGGTGGCGCTGGTGGCGCCG |
| 88 | GTGGTGGCGCTGGCGCCGGCGCTGGCGGCGCTGCCGGTGGTGGCGCTGGCGCCGGCGCTGGCGGCGCTGCCG |
| 99 | CTGCTGGCGGCGGTGCCGGCGGTGCTGCTGGCGCCGCTGCTGGCGGCGGTGCCGGCGGTGCTGCTGGCGCCG |
| 1010 | GCGGCGGCGCTGGTGCCGGTGGTGGCGCTGCTGCCGGCGGCGGCGCTGGTGCCGGTGGTGGCGCTGCTGCCG |
| 1111 | GCGGTGGCGCTGCTGCCGGCGCTGCTGGCGGTGCCGGCGGTGGCGCTGCTGCCGGCGCTGCTGGCGGTGCCG |
| 1212 | GCGGTGGTGCTGGTGCCGGTGCTGGCGGCGGCGCCGGCGGTGGTGCTGGTGCCGGTGCTGGCGGCGGGCCCG |
| 1313 | GTGGTGCTGGTGCTGCCGGCGGCGGCGGCGGTGCCGGTGGTGCTGGTGCTGCCGGCGGCGGCGGCGGTGCCG |
| 1414 | ATTGCGCTGGCGGCGCCGGCGCTGATTGTGGCGCCGATTGCGCTGGCGGGCGCCGGCGCTGATTGTGGGCCCG |
| 1515 | ATTGTGGCGGTGGCGCCGGCGCTGGTGGCGCTGCCGATTGTGGCGGTGGCGCCGGCGCTGGTGGCGCTGCCG |
| 1616 | GTGGCGGCGCTGCCGGTGGTGGCGGTGGTGGCGCCGGTGGCGGCGCTGCCGGTGGTGGCGGTGGTGGCGCCG |
| 1717 | CTGCTGGCGGCGCCGCTGGTGGTGGCGGCGGTGCCGCTGCTGGCGGCGCCGCTGGTGGTGGCGGCGGTGCCG |
| 1818 | GCGCTGGCGGTGCCGGTGGCGCTGCTGGTGGCGCCGGCGCTGGCGGTGCCGGTGGCGCTGCTGGTGGCGCCG |
| 1919 | GTGGCGGCGCTGCCGGTGCTGCTGGCGGCGCTGCCGGTGGCGGCGCTGCCGGTGCTGCTGGCGGCGCTGCCG |
| 2020 | GTGGCGCTGCTGGCGCCGGTGGCGCTGGCGGTGCCGGTGGCGCTGCTGGCGCCGGTGGCGCTGGCGGTGCCG |
| 2121 | GCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGTGCCGGCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGTGCCG |
| 2222 | GCGATTGTGGCGCTGCCGGTGGCGGTGCTGGCGCCGGCGATTGTGGCGCTGCCGGTGGCGGTGCTGGCGCCG |
| 2323 | ATTGCGATTGTGGCGCCGGTGGTGGCGCTGGCGCCGATTGCGATTGTGGGCCCGGTGTGGGCGCTGGGCCCG |
| 2424 | GCGGCGCTGCTGCCGGCGCTGGCGGCGCTGCTGCCGGCGGCGCTGCTGCCGGCGCTGGCGGCGCTGCTGCCG |
| 2525 | GCGGTGGTGCTGGCGCCGGTGGCGGCGGTGCTGCCGGCGGTGGTGCTGGCGCCGGTGGCGGCGGTGCTGCCG |
| 2626 | CTGGCGGTGGCGGCGCCGCTGGCGCTGGCGCTGCCGCTGGCGGTGGCGGCGCCGCTGGCGCTGGCGCTGCCG |
| 2727 | GCGGCGGTGGCGGCGCCGCTGCTGCTGGCGCTGCCGGCGGCGGTGGCGGCGCCGCTGCTGCTGGCGCTGCCG |
| 2828 | CTGCTGGTGCTGCCGGCGGCGGCGCTGGCGGCGCCGCTGCTGGTGCTGCCGGCGGCGGCGCTGGCGGCGCCG |
| 2929 | CTGGTGGCGCTGGCGCCGGTGGCGGCGGTGCTGCCGCTGGTGGCGCTGGCGCCGGTGGCGGCGGTGCTGCCG |
| 3030 | CTGGCGCTGGCGCCGGCGGCGCTGGCGCTGCTGCCGCTGGCGCTGGCGCCGGCGGCGCTGGCGCTGCTGCCG |
| 3131 | GCGCTGATTGCGGCGCCGATTCTGGCGCTGGCGCCGGCGCTGATTGCGGGCCCGATTCTGGGCCTGGGCCCG |
| 3232 | GCGGTGGTGGCGGCGCCGCTGGTGCTGGCGCTGCCGGCGGTGGTGGCGGCGCCGCTGGTGCTGGCGCTGCCG |
| 3333 | CTGCTGGCGCTGGCGCCGGCGGCGCTGCTGGCGCCGCTGCTGGCGCTGGCGCCGGCGGCGCTGCTGGCGCCG |
| 3434 | GCGATTGTGGCGCTGCCGGCGCTGGCGCTGGCGCCGGCGATTGTGGCGCTGCCGGCGCTGGCGCTGGCGCCG |
| 3535 | GCGGCGATTATTGTGCCGGCGGCGCTGCTGGCGCCGGCGGCGATTATTGTGCCGGCGGCGCTGCTGGCGCCG |
| 3636 | ATTGCGGTGGCGCTGCCGGCGCTGATTGCGGCGCCGATTGCGGTGGCGCTGGCCGGCGCTGATTGCGGGCCCG |
| 3737 | GCGGTGATTGTGCTGCCGGCGCTGGCGGTGGCGCCGGCGGTGATTGTGCTGCCGGGCGCTGGCGGTGGCGCCG |
| 3838 | GCGGTGCTGGCGGTGCCGGCGGTGCTGGTGGCGCCGGCGGTGCTGGCGGTGCCGGCGGTGCTGGTGGCGCCG |
| 3939 | GTGCTGGCGATTGTGCCGGCGGTGGCGCTGGCGCCGGTGCTGGCGATTGTGCCGGCGGTGGCGCTGGCGCCG |
| 4040 | CTGCTGGCGGTGGTGCCGGCGGTGGCGCTGGCGCCGCTGCTGGCGGTGGTGCCGGCGGTGGCGCTGGCGCCG |
| 4141 | GCGGTGATTGCGCTGCCGGCGCTGATTGCGGCGCCGGCGGTGATTGCGCTGCCGGGCGCTGATTGCGGGCCCG |
| 4242 | GCGGTGGTGGCGCTGCCGGCGGCGCTGATTGTGCCGGCGGTGGTGGCGCTGCCGGCGGCGCTGATTGTGCCG |
| 4343 | CTGGCGCTGGTGCTGCCGGCGGCGCTGGCGGCGCCGCTGGCGCTGGTGCTGCCGGCGGCGCTGGCGGCGCCG |
| 4444 | CTGGCGGCGGTGCTGCCGGCGCTGCTGGCGGCGCCGCTGGCGGCGGTGCTGCCGGCGCTGCTGGCGGCGCCG |
| 4545 | GCGCTGGCGGTGCCGGTGGCGCTGGCGATTGTGCCGGCGCTGGCGGTGCCGGTGGCGCTGGCGATTGTGCCG |
| 4646 | GCGCTGATTGCGCCGGTGGTGGCGCTGGTGGCGCCGGCGCTGATTGCGCCGTGGTGGCGCTGGTGGCGCCG |
| 4747 | CTGCTGGCGGCGCCGGTGGTGATTGCGCTGGCGCCGCTGCTGGCGGCGCCGGTGGTGATTGCGCTGGCGCCG |
| 4848 | CTGGCGGCGATTGTGCCGGCGATTATTGCGGTGCCGCTGGCGGCGATTGTGCCGGGCGATTATTGCGGTGCCG |
| 4949 | GCGGCGCTGGTGCTGCCGCTGATTATTGCGGCGCCGGCGGCGCTGGTGCTGCCGCTGATTATTGCGGGCCCG |
| 5050 | CTGGCGCTGGCGGTGCCGGCGCTGGCGGCGCTGCCGCTGGCGCTGGCGGTGCCGGCGCTGGCGGCGCTGCCG |
| 5151 | CTGATTGCGGCGCTGCCGGCGGTGGCGGCGCTGCCGCTGATTGCGGCGCTGCCGGCGGTGGCGGCGCTGCCG |
| 5252 | GCGCTGGCGCTGGTGCCGGCGATTGCGGCGCTGCCGGCGCTGGCGCTGGTGCCGGCGATTGCGGCGCTGCCG |
| 5353 | GCGGCGATTCTGGCGCCGATTGTGGCGCTGGCGCCGGCGGCGATTCTGGCGCCGATTGTGGCCTGGGCCCG |
| 5454 | GCGCTGCTGATTGCGCCGGCGGCGGTGATTGCGCCGGCGCTGCTGATTGCGCCGGGCGGCGGTGATTGCGCCG |
| 5555 | GCGATTCTGGCGGTGCCGATTGCGGTGGTGGCGCCGGCGATTCTGGCGGTGCCGATTGCGGTGGTGGGCCCG |
| 5656 | ATTCTGGCGGCGGTGCCGATTGCGCTGGCGGCGCCGATTGCTGGCGGCGGTGCCGATTGCGCTGGCGGGCCCG |
| 5757 | GTGGCGGCGCTGCTGCCGGCGGCGGCGGTGCTGCCGGTGGCGGCGCTGCTGCCGGCGGCGGCGGTGCTGCCG |
| 5858 | GCGGCGGCGGTGGTGCCGGTGCTGCTGGTGGCGCCGGCGGCGGCGGTGGTGCCGGTGCTGCTGGTGGCGCCG |
| 5959 | GCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGCGCCGGCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGGCCCG |
| 6060 | GCGGCGGTGCTGCTGCCGGTGGCGCTGGCGGCGCCGGCGGGCGGTGCTGCTGCCGGTGGCGCTGGCGGGCCCG |
| 6161 | GCGGCGGCGCTGGCGCCGGTGCTGGCGCTGGTGCCGGCGGGCGGCGCTGGCGCCGGTGCTGGCGCTGGTGCCG |
| 6262 | CTGGTGCTGGTGCCGCTGCTGGCGGCGGCGGCGCCGCTGGTGCTGGTGCCGCTGCTGGCGGCGGCGGCGCCG |
| 6363 | GCGCTGATTGCGGTGCCGGCGATTATTGTGGCGCCGGCGCTGATTGCGGTGCCGGCGATTATTGTGGGCCCG |
| 6464 | GCGCTGGCGGTGATTCCGGCGGCGGCGATTCTGCCGGCGCTGGCGGTGATTCCGGCGGCGGCGATTCTGCCG |
| 6565 | CTGGCGGCGGCGCCGGTGGTGATTGTGATTGCGCCGCTGGCGGCGGCGCCGGTGGTGATTGTGATTGCGCCG |
| 6666 | GTGCTGGCGATTGCGCCGCTGCTGGCGGCGGTGCCGGTGCTGGCGATTGCGCCGCTGCTGGCGGCGGTGCCG |
| 6767 | GCGCTGATTGTGCTGCCGGCGGCGGTGGCGGTGCCGGCGCTGATTGTGCTGCCGGCGGCGGTGGCGGTGCCG |
| 6868 | GTGCTGGCGGTGGCGCCGGCGCTGATTGTGGCGCCGGTGCTGGCGGTGGCGCCGGCGCTGATTGTGGGCCCG |
| 6969 | GCGGCGCTGCTGGCGCCGGCGCTGATTGTGGCGCCGGGCGGCGCTGCTGGCGCCGGCGCTGATTGTGGCGCCG |
| 7070 | GCGCTGATTGCGCCGGCGGTGGCGCTGATTGTGCCGGCGCTGATTGCGCCGGCGGTGGCGCTGATTGTGCCG |
| 7171 | GCGATTGTGCTGCTGCCGGCGGCGGTGGTGGCGCCGGCGATTGTGCTGCTGCCGGCGGCGGTGGTGGCGCCG |
| 7272 | GTGATTGCGGCGCCGGTGCTGGCGGTGCTGGCGCCGGTGATTGCGGCGCCGGTGCTGGCGGTGCTGGCGCCG |
| 7373 | CTGGCGCTGGCGCCGGCGCTGGCGCTGCTGGCGCCGCTGGCGCTGGCGCCGGCGCTGGCGCTGCTGGCGCCG |
| 7474 | GCGATTATTCTGGCGCCGATTGCGGCGATTGCGCCGGCGATTATTCTGGCGCCGATTGCGGCGATTGCGCCG |
| 7575 | ATTGCGCTGGCGGCGCCGATTCTGCTGGCGGCGCCGATTGCGCTGGGCGGCGCCGATTCTGCTGGCGGCGCCG |
| 7676 | ATTGTGGCGGTGGCGCTGCCGGCGCTGGCGGTGCCGATTGTGGCGGTGGCGCTGCCGGCGCTGGCGGTGCCG |
| 7777 | GTGGTGGCGATTGTGCTGCCGGCGCTGGCGGCGCCGGTGGTGGCGATTGTGCTGCCGGCGCTGGCGGCGCCG |
| 7878 | ATTGTGGCGGTGGCGCTGCCGGTGGCGCTGGCGCCGATTGTGGCGGTGGCGCTGCCGGTGGCGCTGGCGCCG |
| 7979 | ATTGTGGCGGTGGCGCTGCCGGCGGCGCTGGTGCCGATTGTGGCGGTGGCGCTGCCGGCGGCGCTGGTGCCG |
| 8080 | ATTGTGGCGGTGGCGCTGCCGGCGGTGGCGCTGCCGATTGTGGCGGTGGCGCTGCCGGCGGTGGCGCTGCCG |
| 8181 | ATTGTGGCGGTGGCGCTGCCGGCGGTGCTGGCGCCGATTGTGGCGGTGGCGCTGCCGGCGGTGCTGGCGCCG |
| 8282 | GTGATTGTGGCGCTGGCGCCGGCGGTGCTGGCGCCGGTGATTGTGGCGCTGGCGCCGGCGGTGCTGGCGCCG |
| 8383 | ATTGTGGCGGTGGCGCTGCCGGCGCTGGTGGCGCCGATTGTGGCGGTGGCGCTGCCGGCGCTGGTGGCGCCG |
| 8484 | GCGCTGCTGATTGTGGCGCCGGTGGCGGTGGCGCCGGCGCTGCTGATTGTGGCGCCGGTGGCGGTGGCGCCG |
| 8585 | GCGGTGGTGATTGTGGCGCCGGCGGTGATTGCGCCGGCGGTGGTGATTGTGGGCCCGGGCGGTGATTGCGCCG |
| 8686 | GCGGTGCTGGCGGTGGCGCCGGCGCTGATTGTGCCGGCGGTGCTGGCGGTGGCGCCGGCGCTGATTGTGCCG |
| 8787 | CTGGTGGCGGCGGTGGCGCCGGCGCTGATTGTGCCGCTGGTGGCGGGCGGTGGCGCCGGCGCTGATTGTGCCG |
| 8888 | GCGGTGATTGTGGTGGCGCCGGCGCTGCTGGCGCCGGCGGTGATTGTGGTGGCGCCGGCGCTGCTGGGCCCG |
| 8989 | GTGGTGGCGATTGTGCTGCCGGCGGTGGCGGCGCCGGTGGTGGCGATTGTGCTGCCGGCGGTGGCGGCGCCG |
| 9090 | GCGGCGGCGCTGGTGATTCCGGCGATTCTGGCGCCGGCGGGCGGCGCTGGTGATTCCGGCGATTCTGGCGCCG |
| 9191 | GTGATTGTGGCGCTGGCGCCGGCGCTGCTGGCGCCGGTGATTGTGGCGCTGGCGCCGGCGCTGCTGGCGCCG |
| 9292 | GTGATTGTGGCGATTGCGCCGGCGCTGCTGGCGCCGGTGATTGTGGCGATTGCGCCGGCGCTGCTGGGCCCG |
| 9393 | ATTGTGGCGATTGCGGTGCCGGCGCTGGTGGCGCCGATTGTGGCGATTGCGGTGCCGGCGCTGGTGGCGCCG |
| 9494 | GCGGCGCTGGCGGTGATTCCGGCGGCGATTCTGCCGGCGGCGCTGGCGGTGATTCCGGGCGGCGATTCTGCCG |
| 9595 | GCGCTGGCGGCGGTGATTCCGGCGGCGATTCTGCCGGCGCTGGCGGCGGTGATTCCGGGCGGCGATTCTGCCG |
| 9696 | GCGGCGGCGCTGGTGATTCCGGCGGCGATTCTGCCGGCGGCGGCGCTGGTGATTCCGGGCGGCGATTCTGCCG |
| 9797 | CTGGCGGCGGCGGTGATTCCGGCGGCGATTCTGCCGCTGGCGGCGGGCGGTGATTCCGGGCGGCGATTCTGCCG |
| 9898 | CTGGCGGCGGCGGTGATTCCGGTGGCGATTCTGCCGCTGGCGGCGGGCGGTGATTCCGGTGGCGATTCTGCCG |
| 9999 | GCGGCGATTCTGGCGGCGCCGCTGATTGCGGTGCCGGCGGCGATTCTGGCGGCGCGCTGATTGCGGTGCCG |
| 100100 | GTGGTGGCGATTCTGGCGCCGCTGCTGGCGGCGCCGGTGGTGGCGATTCTGGCGCCGCTGCTGGCGGCGCCG |
| 101101 | GCGGTGGTGGTGGCGGCGCCGGTGCTGGCGCTGCCGGCGGTGGTGGTGGCGGCGCCGGTGCTGGCGCTGCCG |
| 102102 | GCGGTGGTGGCGATTGCGCCGGTGCTGGCGCTGCCGGCGGTGGTGGCGATTGCGCCGTGCTGGCGCTGCCG |
| 103103 | GCGCTGGCGGCGCTGGTGCCGGCGGTGCTGGTGCCGGCGCTGGCGGCGCTGGTGCCGGCGGTGCTGGTGCCG |
| 104104 | GCGCTGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCGGCGCTGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCG |
| 105105 | CTGGCGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCGCTGGCGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCG |
| 106106 | GCGCTGGCGGCGCTGGTGCCGGCGCTGGTGGTGCCGGCGCTGGCGGCGCTGGTGCCGGCGCTGGTGGTGCCG |
| 107107 | ATTGCGGCGGTGATTGTGCCGGCGGTGGCGCTGCCGATTGCGGGCGGTGATTGTGCCGGCGGTGGCGCTGCCG |
| 108108 | ATTGCGGCGGTGCTGGTGCCGGCGGTGGCGCTGCCGATTGCGGGCGGTGCTGGTGCCGGCGGTGGCGCTGCCG |
| 109109 | GCGGTGGCGATTCTGGTGCCGCTGCTGGCGGCGCCGGCGGTGGCGATTCTGGTGCCGCTGCTGGCGGCGCCG |
| 110110 | GCGGTGGTGATTCTGGTGCCGCTGGCGGCGGCGCCGGCGGTGGTGATTCTGGTGCCGCTGGCGGCGGCGCCG |
| 111111 | ATTGCGGCGGTGATTGTGCCGGTGGCGGCGCTGCCGATTGCGGCGGTGATTGTGCCGTGGCGGCGCTGCCG |
| 112112 | GCGATTGCGATTGCGATTGTGCCGGTGGCGCTGCCGGCGATTGCGATTGCGATTGTGCCGGTGGCGCTGCCG |
| 113113 | ATTCTGGCGGTGGCGGCGATTCCGGTGGCGGTGCCGATTCCTGGCGGTGGCGGCGATTCCGGTGGCGGTGCCG |
| 114114 | ATTCTGGCGGCGGCGATTATTCCGGCGGCGCTGCCGATTCTGGCGGCGGCGATTATTCCGGCGGCGCTGCCG |
| 115115 | CTGGCGGTGGTGCTGGCGGCGCCGGCGATTGTGCCGCTGGGCGGTGGTGCTGGCGGCGCCGGCGATTGTGCCG |
| 116116 | GCGATTCTGGCGGCGATTGTGCCGCTGGCGGTGCCGGCGATTCTGGGCGGCGATTGTGCCGCTGGCGGTGCCG |
| 117117 | GTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCGGTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCG |
| 118118 | GCGATTGTGGCGCTGGCGGTGCCGGTGCTGGCGCCGGCGATTGTGGCGCTGGCGGTGCCGGTGCTGGCGCCG |
| 119119 | GCGGCGATTATTATTGTGCTGCCGGCGGCGCTGCCGGCGGCGATTATTATTGTGCTGCCGGCGGCGCTGCCG |
| 120120 | CTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCGCTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCG |
| 121121 | GCGATTATTATTGTGATTGCGCCGGCGGCGGCGCCGGCGATTATTATTGTGATTGCGCCGGCGGCGGCGCCG |
| 122122 | CTGGCGGCGCTGATTGTGGTGCCGGCGGTGGCGCCGCTGGCGGCGCTGATTGTGGTGCCGGGCGGTGGCGCCG |
| 123123 | GCGCTGCTGGTGATTGCGGTGCCGGCGGTGGCGCCGGCGCTGCTGGTGATTGCGGTGCCGGGCGGTGGCGCCG |
| 124124 | GCGGTGGCGCTGATTGTGGTGCCGGCGCTGGCGCCGGCGGTGGCGCTGATTGTGGTGCCGGGCCTGGGCCCG |
| 125125 | GCGGTTGCGCTGATTGTGGTTCCGGCGCTGGCGCCGGCGGTTGCGCTGATTGTGGTTCCGGGCCTGGGCCCG |
| 126126 | CTGCTGGCGCTGATTATTGCGCCGGCGGCGGCGCCGCTGCTGGCGCTGATTATTGCGCCGGCGGCGGCGCCG |
| 127127 | GCGCTGGCGCTGATTATTGTGCCGGCGGTGGCGCCGGCGCTGGCGCTGATTATTGTGCCGGCGGTGGCGCCG |
| 128128 | CTGCTGGCGGCGCTGATTGCGCCGGCGGCGCTGCCGCTGCTGGCGGGCGCTGATTGCGCCGGCGGCGCTGCCG |
| 129129 | ATTGTGGCGCTGATTGTGGCGCCGGCGGCGGTGCCGATTGTGGCGCTGATTGTGGCGCCGGCGGCGGTGCCG |
| 130130 | GTGGTGCTGGTGCTGGCGGCGCCGGCGGCGGTGCCGGTGGTGCTGGTGCTGGCGGCGCCGGCGGCGGTGCCG |
| 131131 | GCGGCGGTGGCGATTGTGCTGCCGGCGGTGGTGCCGGGCGGCGGTGGCGATTGTGCTGCCGGCGGTGGTGCCG |
| 132132 | GCGCTGATTGCGGCGATTGTGCCGGCGCTGGTGCCGGCGCTGATTGCGGCGATTGTGCCGGCGCTGGTGCCG |
| 133133 | GCGCTGGCGGTGATTGTGGTGCCGGCGCTGGCGCCGGCGCTGGCGGTGATTGTGGTGCCGGGCCTGGGCCCG |
| 134134 | GTGGCGATTGCGCTGATTGTGCCGGCGCTGGCGCCGGTGGCGATTGCGCTGATTGTGCCGGCGCTGGCGCCG |
| 135135 | GTGGCGATTGTGCTGGTGGCGCCGGCGGTGGCGCCGGTGGCGATTGTGCTGGGTGGCGCCGGCGGTGGCGCCG |
| 136136 | GTGGCGGTGGCGCTGATTGTGCCGGCGCTGGCGCCGGTGGGCGGTGGCGCTGATTGTGCCGGCGCTGGCGCCG |
| 137137 | GCGGTGATTCTGGCGCTGGCGCCGATTGTGGCGCCGGCGGTGATTCTGGCGCTGGCGCCGATTGTGGCCCG |
| 138138 | GCGCTGATTGTGGCGATTGCGCCGGCGCTGGTGCCGGCGCTGATTGTGGCGATTGCGCCGGCGCTGGTGCCG |
| 139139 | GCGGCGATTCTGATTGCGGTGCCGATTGCGGCGCCGGCGGCGATTCTGATTGCGGTGCCGATTGCGGGCCCG |
| 140140 | GTGATTGTGGCGCTGGCGGCGCCGGTGCTGGCGCCGGTGATTGTGGCGCTGGCGGCGCCGGTGCTGGCGCCG |
| 141141 | GTGCTGGTGGCGCTGGCGGCGCCGGTGATTGCGCCGGTGCTGTGGCGCTGGCGGCGCCGGTGATTGCGCCG |
| 142142 | GTGGCGCTGATTGCGGTGGCGCCGGCGGTGGTGCCGGTGGGCCTGATTGCGGTGGCGCCGGCGGTGGTGCCG |
| 143143 | GTGATTGCGGCGGTGCTGGCGCCGGTGGCGGTGCCGGTGATTGCGGCGGTGCTGGCGCCGGTGGCGGTGCCG |
| 144144 | GCGCTGATTGTGCTGGCGGCGCCGGTGGCGGTGCCGGCGCTGATTGTGCTGGCGGCGCCGGTGGCGGTGCCG |
| 145145 | GTGGCGGCGGCGATTGCGCTGCCGGCGATTGTGCCGGTGGCGGCGGCGATTGCGCTGCCGGCGATTGTGCCG |
| 146146 | ATTCTGGCGGCGGCGGCGGCGCCGCTGATTGTGCCGATTCTGGCGGCGGCGGCGGCGCCGCTGATTGTGCCG |
| 147147 | CTGGCGCTGGTGCTGGCGGCGCCGGCGATTGTGCCGCTGGCGCTGGTGCTGGGCGGCGCCGGCGATTGTGCCG |
| 148148 | GCGCTGGCGGTGGTGGCGCTGCCGGCGATTGTGCCGGCGCTGGCGGTGGTGGCGCTGCCGGCGATTGTGCCG |
| 149149 | GCGGCGATTCTGGCGCCGATTGTGGCGGCGCTGCCGGCGGCGATTCTGGCGCCGATTGTGGCGGCGCTGCCG |
| 150150 | ATTCTGATTGCGATTGCGATTCCGGCGGCGGCGCCGATTCTGATTGCGATTGCGATTCCGGCGGCGGGCCCG |
| 151151 | CTGGCGATTGTGCTGGCGGCGCCGGTGGCGGTGCCGCTGGCGATTGTGCTGGCGGCGCCGGTGGCGGTGCCG |
| 152152 | GCGGCGATTGCGATTATTGCGCCGGCGATTGTGCCGGCGGCGATTGCGATTATTGCGCCGGGCGATTGTGCCG |
| 153153 | CTGGCGGTGGCGATTGTGGCGCCGGCGCTGGTGCCGCTGGCGGTGGCGATTGTGGCGCCGGGCCTGGTGCCG |
| 154154 | CTGGCGATTGTGCTGGCGGCGCCGGCGGTGCTGCCGCTGGCGATTGTGCTGGCGGCGCCGGGCGGTGCTGCCG |
| 155155 | GCGGCGATTGTGCTGGCGCTGCCGGCGGTGCTGCCGGCGGCGATTGTGCTGGCGCTGCCGGGCGGTGCTGCCG |
| 156156 | GCGCTGCTGGTGGCGGTGCTGCCGGCGGCGCTGCCGGCGCTGCTGGTGGCGGTGCTGCCGGCGGCGCTGCCG |
| 157157 | GCGGCGCTGGTGGCGGTGCTGCCGGTGGCGCTGCCGGCGGCGCTGGTGGCGGTGCTGCCGGTGGCGCTGCCG |
| 158158 | GCGATTCTGGCGGTGGCGCTGCCGCTGCTGGCGCCGGCGATTCTGGCGGTGGCGCTGCCGCTGCTGGCGCCG |
| 159159 | ATTGTGGCGGTGGCGCTGGTGCCGGCGCTGGCGCCGATTGTGGCGGTGGCGCTGGTGCCGGCGCTGGCGCCG |
| 160160 | ATTGTGGCGGTGGCGCTGCTGCCGGCGCTGGCGCCGATTGTGGCGGTGGCGCTGCTGCCGGCGCTGGCGCCG |
| 161161 | ATTGTGGCGGTGGCGCTGCTGCCGGCGGTGGCGCCGATTGTGGCGGTGGCGCTGCTGCCGGCGGTGGCGCCG |
| 162162 | ATTGTGGCGCTGGCGGTGCTGCCGGCGGTGGCGCCGATTGTGGCGCTGGCGGTGCTGCCGGCGGTGGCGCCG |
| 163163 | GTGGCGGTGCTGGCGGTGCTGCCGGCGCTGGCGCCGGTGGCGGTGCTGGCGGTGCTGCCGGCGCTGGCGCCG |
| 164164 | ATTGCGGTGCTGGCGGTGGCGCCGGCGGTGCTGCCGATTGCGGGTGCTGGCGGTGGCGCCGGCGGTGCTGCCG |
| 165165 | CTGGCGGTGGCGATTATTGCGCCGGCGGTGGCGCCGCTGGCGGTGGCGATTATTGCGCCGGCGGTGGCGCCG |
| 166166 | GTGGCGCTGGCGATTGCGCTGCCGGCGGTGCTGCCGGTGGCGCTGGCGATTGCGCTGCCGGGCGGTGCTGCCG |
| 167167 | GCGATTGCGATTGCGCTGGTGCCGGTGGCGCTGCCGGCGATTGCGATTGCGCTGGTGCCGGTGGCGCTGCCG |
| 168168 | GCGGCGGTGGTGATTGTGGCGCCGGTGGCGCTGCCGGGCGGCGGTGGTGATTGTGGCGCCGGTGGCGCTGCCG |
| 169169 | GTGGCGATTATTGTGGTGGCGCCGGCGCTGGCGCCGGTGGCGATTATTGTGGTGGCGCCGGCGCTGGCGCCG |
| 170170 | GTGGCGCTGCTGGCGATTGCGCCGGCGCTGGCGCCGGTGGGCCTGCTGGGCGATTGCGCCGGCGCTGGCGCCG |
| 171171 | GTGGCGGTGCTGATTGCGGTGCCGGCGCTGGCGCCGGTGGCGGTGCTGATTGCGGTGCCGGCGCTGGCGCCG |
| 172172 | GCGGTGGCGCTGGCGGTGCTGCCGGCGGTGGTGCCGGCGGTGGCGCTGGCGGTGCTGCCGGCGGTGGTGCCG |
| 173173 | GCGGTGGCGCTGGCGGTGGTGCCGGCGGTGCTGCCGGCGGTGGCGCTGGCGGTGGTGCCGGCGGTGCTGCCG |
| 174174 | ATTGTGGTGATTGCGGTGGCGCCGGCGGTGGCGCCGATTGTGGTGATTGCGGTGGCGCCGGCGGTGGCGCCG |
| 175175 | ATTGTGGTGGCGGCGGTGGTGCCGGCGCTGGCGCCGATTGTGGTGGCGGCGGTGGTGCCGGCGCTGGCGCCG |
| 176176 | ATTGTGGCGCTGGTGCCGGCGGTGGCGATTGCGCCGATTGTGGCGCTGGTGCCGGCGGTGGCGATTGCGCCG |
| 177177 | GTGGCGGCGCTGCCGGCGGTGGCGCTGGTGGTGCCGGTGGCGGCGCTGCCGGCGGTGGCGCTGGTGGTGCCG |
| 178178 | CTGGTGGCGATTGCGCCGCTGGCGGTGCTGGCGCCGCTGGTGGCGATTGCGCCGCTGGCGGTGCTGGCGCCG |
| 179179 | GCGGTGGCGCTGGTGCCGGTGATTGTGGCGGCGCCGGCGGTGGCGCTGGTGCCGGTGATTGTGGCGGCGCCG |
| 180180 | GCGATTGCGGTGGCGATTGCGCCGGTGGCGCTGCCGGCGATTGCGGTGGCGATTGCGCCGGTGGCGCTGCCG |
| 181181 | GCGATTGCGCTGGCGGTGCCGGTGCTGGCGCTGCCGGCGATTGCGCTGGCGGTGCCGGTGCTGGCGCTGCCG |
| 182182 | CTGGTGCTGATTGCGGCGGCGCCGATTGCGCTGCCGCTGGTGCTGATTGCGGCGGCGCCGATTGCGCTGCCG |
| 183183 | CTGGTGGCGCTGGCGGTGCCGGCGGCGGTGCTGCCGCTGGTGGCGCTGGCGGTGCCGGCGGCGGTGCTGCCG |
| 184184 | GCGGTGGCGCTGGCGGTGCCGGCGCTGGTGCTGCCGGCGGTGGCGCTGGCGGTGCCGGCGCTGGTGCTGCCG |
| 185185 | CTGGTGGTGCTGGCGGCGGCGCCGCTGGCGGTGCCGCTGGTGGTGCTGGCGGCGGCGCCGCTGGCGGTGCCG |
| 186186 | CTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCGCTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCG |
| 187187 | GTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCGGTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCG |
| 188188 | GCGGTGGTGCTGGCGGTGCCGGCGCTGGCGGTGCCGGCGGTGGTGCTGGCGGTGCCGGCGCTGGCGGTGCCG |
| 189189 | CTGATTATTGTGGCGGCGGCGCCGGCGGTGGCGCCGCTGATTATTGTGGCGGCGGCGCCGGGCGGTGGCGCCG |
| 190190 | ATTGTGGCGGTGATTGTGGCGCCGGCGGTGGCGCCGATTGTGGCGGTGATTGTGGGCCCGGGCGGTGGCGCCG |
| 191191 | CTGGTGGCGCTGGCGGCGCCGATTATTGCGGTGCCGCTGGTGGCGCTGGCGGGCCCGATTATTGCGGTGCCG |
| 192192 | ATTGCGGCGGTGCTGGCGGCGCCGGCGCTGGTGCCGATTGCGGGCGGTGCTGGCGGCGCCGGCGCTGGTGCCG |
| 193193 | ATTGCGCTGCTGGCGGCGCCGATTATTGCGGTGCCGATTGCGCTGCTGGCGGCGCCGATTATTGCGGTGCCG |
| 194194 | GCGGCGCTGGCGCTGGTGGCGCCGGTGATTGTGCCGGCGGCGCTGGCGCTGGTGGCGCCGGTGATTGTGCCG |
| 195195 | ATTGCGCTGGTGGCGGCGCCGGTGGCGCTGGTGCCGATTGCGCTGGGTGGCGGCGCCGGTGGCGCTGGTGCCG |
| 196196 | ATTATTGTGGCGGTGGCGCCGGCGGCGATTGTGCCGATTATTGTGGGCGGTGGCGCCGGCGGCGATTGTGCCG |
| 197197 | GCGGTGGCGGCGATTGTGCCGGTGATTGTGGCGCCGGCGGTGGCGGCGATTGTGCCGTGATTGTGGGCCCG |
| 198198 | GCGGTGCTGGTGCTGGTGGCGCCGGCGGCGGCGCCGGCGGTGCTGGTGCTGGTGGCGCCGGCGGCGGCGCCG |
| 199199 | GTGGTGGCGCTGCTGGCGCCGCTGATTGCGGCGCCGGTGGTGGCGCTGCTGGCGCCGCTGATTGCGGGCCCG |
| 200200 | GCGGCGGTGGTGATTGCGCCGCTGCTGGCGGTGCCGGGCGGCGGTGGTGATTGCGCCGCTGCTGGCGGTGCCG |
| 201201 | ATTGCGGTGGCGGTGGCGGCGCCGCTGCTGGTGCCGATTGCGGGTGGCGGTGGCGGCGCCGCTGCTGGTGCCG |
| 202202 | CTGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCGCTGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCG |
| 203203 | GCGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCGGCGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCG |
| 204204 | GCGGTGATTCTGCTGGCGCCGCTGATTGCGGCGCCGGCGGTGATTCTGCTGGCGCGCTGATTGCGGGCCCG |
| 205205 | CTGGTGATTGCGCTGGCGGCGCCGGTGGCGCTGCCGCTGGTGATTGCGCTGGCGGCGCCGGTGGCGCTGCCG |
| 206206 | GTGCTGGCGGTGGTGCTGCCGGCGGTGGCGCTGCCGGTGCTGGCGGTGGTGCTGCCGGCGGTGGCGCTGCCG |
| 207207 | GTGCTGGCGGTGGCGGCGCCGGCGGTGCTGCTGCCGGTGCTGGCGGTGGCGGCGCCGGCGGTGCTGCTGCCG |
| 208208 | GCGGCGGTGGTGCTGCTGCCGATTATTGCGGCGCCGGCGGCGGTGGTGCTGCTGCCGATTATTGCGGGCCCG |
| 209209 | GCGCTGCTGGTGATTGCGCCGGCGATTGCGGTGCCGGCGCTGCTGGTGATTGCGCCGGGCGATTGCGGTGCCG |
| 210210 | GCGGTGCTGGTGATTGCGGTGCCGGCGATTGCGCCGGCGGTGCTGGTGATTGCGGTGCCGGCGATTGCGCCG |
| 211211 | GCGCTGCTGGTGGTGATTGCGCCGCTGGCGGCGCCGGCGCTGCTGGTGGTGATTGCGCCGCTGGCGGCGCCG |
| 212212 | GTGCTGGTGGCGGCGATTCTGCCGGCGGCGATTCCGGTGCTGTGGCGGCGATTCTGCCGGCGGCGATTCCG |
| 213213 | GTGCTGGTGGCGGCGGTGCTGCCGATTGCGGCGCCGGTGCTGGTGGCGGCGGTGCTGCCGATTGCGGGCCCG |
| 214214 | GTGCTGGCGGCGGCGGTGCTGCCGCTGGTGGTGCCGGTGCTGGCGGCGGCGGTGCTGCCGCTGGTGGTGCCG |
| 215215 | GCGATTGCGATTGTGGTGCCGGCGGTGGCGGTGCCGGCGATTGCGATTGTGGTGCCGGCGGTGGCGGTGCCG |
| 216216 | GTGGCGATTATTGCGGTGCCGGCGGTGGTGGCGCCGGTGGCGATTATTGCGGTGCCGGCGGTGGTGGCGCCG |
| 217217 | ATTGTGGCGCTGGTGGCGCCGGCGGCGGTGGTGCCGATTGTGGCGCTGGTGGCGCCGGCGGCGGTGGTGCCG |
| 218218 | GCGGCGATTGTGCTGCTGCCGGCGGTGGTGGTGCCGGCGGCGATTGTGCTGCTGCCGGCGGTGGTGGTGCCG |
| 219219 | GCGGCGCTGATTGTGGTGCCGGCGGTGGCGGTGCCGGCGGCGCTGATTGTGGTGCCGGCGGTGGCGGTGCCG |
| 220220 | GCGATTGCGCTGGTGGTGCCGGCGGTGGCGGTGCCGGCGATTGCGCTGGTGGTGCCGGCGGTGGCGGTGCCG |
| 221221 | CTGGCGATTGTGCCGGCGGCGATTGCGGCGCTGCCGCTGGCGATTGTGCCGGCGGCGATTGCGGCGCTGCCG |
| 222222 | CTGGTGGCGATTGCGCCGGCGGTGGCGGTGCTGCCGCTGGTGGCGATTGCGCCGGCGGTGGCGGTGCTGCCG |
| 223223 | GTGCTGGCGGTGGCGCCGGCGGTGGCGGTGCTGCCGGTGCTGGCGGTGGCGCCGGCGGTGGCGGTGCTGCCG |
| 224224 | ATTCTGGCGGTGGTGGCGATTCCGGCGGCGGCGCCGATTCCTGGCGGTGGTGGCGATTCCGGCGGCGGGCCCG |
| 225225 | ATTCTGGTGGCGGCGGCGCCGATTGCGGCGCTGCCGATTCTGGTGGCGGCGGCGCCGATTGCGGCGCTGCCG |
| 226226 | ATTCTGGCGGTGGCGGCGATTCCGGCGGCGCTGCCGATTCCTGGCGGTGGCGGCGATTCCGGCGGCGCTGCCG |
| 227227 | GTGATTGCGATTCCGGCGATTCTGGCGGCGGCGCCGGTGATTGCGATTCCGGCGATTCTGGCGGCGGGCCCG |
| 228228 | GCGATTATTATTGTGGTGCCGGCGATTGCGGCGCCGGCGATTATTATTGTGGTGCCGGCGATTGCGGGCCCG |
| 229229 | GCGATTCTGATTGTGGTGGCGCCGATTGCGGCGCCGGCGATTCTGATTGTGGTGGCGCCGATTGCGGGCCCG |
| 230230 | GCGGTGATTGTGCCGGTGGCGATTATTGCGGCGCCGGCGGTGATTGTGCCGGTGGCGATTATTGCGGGCCCG |
| 231231 | GCGGTGGTGATTGCGCTGCCGGCGGTGGTGGCGCCGGCGGTGGTGATTGCGCTGCCGGCGGTGGTGGCGCCG |
| 232232 | GCGCTGGTGGCGGTGATTGCGCCGGTGGTGGCGCCGGCGCTGGTGGCGGTGATTGCGCCGGTGGTGGCGCCG |
| 233233 | GCGCTGGTGGCGGTGCTGCCGGCGGTGGCGGTGCCGGCGCTGGTGGCGGTGCTGCCGGCGGTGGCGGTGCCG |
| 234234 | GCGCTGGTGGCGCCGCTGCTGGCGGTGGCGGTGCCGGCGCTGGTGGCGCCGCTGCTGGCGGTGGCGGTGCCG |
| 235235 | GCGGTGCTGGCGGTGGTGGCGCCGGTGGTGGCGCCGGCGGTGCTGGCGGTGGTGGCGCCGGTGGTGGCGCCG |
| 236236 | GCGGTGATTGCGGTGGCGCCGCTGGTGGTGGCGCCGGCGGTGATTGCGGTGGCGCGCTGGTGGTGGCGCCG |
| 237237 | GCGGTGATTGCGCTGGCGCCGGTGGTGGTGGCGCCGGCGGTGATTGCGCTGGCCGCCGGTGGTGGTGGCGCCG |
| 238238 | GTGGCGATTGCGCTGGCGCCGGTGGTGGTGGCGCCGGTGGCGATTGCGCTGGCGCCGTGGTGGTGGCGCCG |
| 239239 | GTGGCGCTGGCGCTGGCGCCGGTGGTGGTGGCGCCGGTGGCGCTGGCGCTGGCGCCGGTGGTGGTGGCGCCG |
| 240240 | GTGGCGGCGCTGCTGCCGGCGGTGGTGGTGGCGCCGGTGGCGGCGCTGCTGCCGGCGGTGGTGGTGGCGCCG |
| 241241 | GTGGCGCTGGCGCTGCCGGCGGTGGTGGTGGCGCCGGTGGCGCTGGCGCTGCCGGCGGTGGTGGTGGCGCCG |
| 242242 | GTGGCGCTGCTGGCGCCGGCGGTGGTGGTGGCGCCGGTGGCGCTGCTGGCGCCGGCGGTGGTGGTGGCGCCG |
실시예 1. AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin) 개발Example 1. Development of AAV-TSDT convergence platform (AAV-aMTD-Parkin)
아데노-부속 바이러스(AAV)-TSDT 융합 플랫폼을 제조하기 위하여, 전이 플라스미드 골격(transfer plasmid backbone, 도 1a)에 Parkin, SDB 및 aMTD를 포함함으로써 세포투과성을 갖는 Parkin 단백질의 전이 플라스미드를 만들었다. 도 1b는 Parkin, SDB 및 aMTD를 포함하는 단백질의 구조를 나타내고, Parkin 및 SDB를 포함하는 전이 플라스미드의 구조(도 1c)와 Parkin, SDB 및 aMTD를 포함하는 전이 플라스미드의 구조(도 1d)를 나타내었으며, 상기 바이러스에 대한 구조의 위치 및 크기는 하기의 표 2(pAAV9-Parkin (△Ubl)-SDB) 및 표 3(pAAV9-aMTD524-Parkin (△Ubl)-SDB)에 나타내었다.To prepare the adeno-associated virus (AAV)-TSDT fusion platform, a cell-permeable transfer plasmid of the Parkin protein was created by including Parkin, SDB, and aMTD in the transfer plasmid backbone (Figure 1a). Figure 1b shows the structure of the protein containing Parkin, SDB, and aMTD, the structure of the transfer plasmid containing Parkin and SDB (Figure 1c), and the structure of the transfer plasmid containing Parkin, SDB, and aMTD (Figure 1d). The location and size of the structure for the virus are shown in Table 2 (pAAV9-Parkin (△Ubl)-SDB) and Table 3 (pAAV9-aMTD524-Parkin (△Ubl)-SDB) below.
| 바이러스명Virus name | 명칭designation | 위치location | 크기 (bp)Size (bp) |
| pAAV9-Parkin (△Ubl)-SDBpAAV9-Parkin(△Ubl)-SDB | 5' ITR5'ITR | 1-1411-141 | 141141 |
| CMVCMV | 169-757169-757 | 589589 | |
| KozakKozak | 782-787782-787 | 66 | |
| Parkin (△UBL)-SDBParkin (△UBL)-SDB | 788-2254788-2254 | 14671467 | |
| BGH pABGH pA | 2309-25162309-2516 | 208208 | |
| 3' ITR3'ITR | 2524-26642524-2664 | 141141 |
| 바이러스명Virus name | 명칭designation | 위치location | 크기 (bp)Size (bp) |
| pAAV9-aMTD524-Parkin (△Ubl)-SDBpAAV9-aMTD524-Parkin (△Ubl)-SDB | 5' ITR5'ITR | 1-1411-141 | 141141 |
| CMVCMV | 169-757169-757 | 589589 | |
| KozakKozak | 782-787782-787 | 66 | |
| aMTD524-Parkin (△UBL)-SDBaMTD524-Parkin (△UBL)-SDB | 788-2290788-2290 | 15031503 | |
| BGH pABGH pA | 2345-25522345-2552 | 208208 | |
| 3' ITR3'ITR | 2560-27002560-2700 | 141141 |
실시예2. AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)을 이용한 파킨슨병(PD) 치료효과 확인Example 2. Confirmation of Parkinson's disease (PD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin)
파킨슨병(PD)의 치료효과를 확인하기 위해 아데노-부속 바이러스 (AAV)를 이용한 유전자치료 전달법과 세포투과성이 있는 Parkin 단백질의 융합 (AAV-aMTD-Parkin)을 6-OHDA로 유도한 파킨슨병(PD) 동물모델에 기간을 두어 투여한 뒤 결과를 확인하였다(도 2a). To confirm the therapeutic effect of Parkinson's disease (PD), a gene therapy delivery method using adeno-associated virus (AAV) and a cell-permeable fusion of Parkin protein (AAV-aMTD-Parkin) were used to induce Parkinson's disease (PD) with 6-OHDA. PD) animal model was administered over a period of time and the results were confirmed (Figure 2a).
파킨슨병 동물모델에 투여한지 6주후에 시험한 결과에서 운동기능은 102% 개선되었으며(도 2b), 파킨슨병 동물모델의 뇌 중 선조체 (Striatum: ST) 및 흑질 (Substantia Nigra: SN)에서 AAV-aMTD-Parkin 투여로 인해 도파민 신경세포가 회복되었다(도 2c). 또한, 약리물질 생체 내 전송기술 (TSDT)에 의해 뇌의 투여 부위 (선조체) 이외의 영역 (흑질)으로 AAV-aMTD-Parkin이 전송되었음을 확인할 수 있었으며(도 2d), AAV-aMTD-Parkin에 의해 파킨슨병 동물 뇌에서 도파민을 분비하는 신경세포의 티로신 수산화효소(Tyrosine Hydroxylase, TH)의 발현이 정상 수준의 77%까지 회복되었다(도 2e, 도 2f).As a result of testing 6 weeks after administration to the Parkinson's disease animal model, motor function was improved by 102% (Figure 2b), and AAV- Dopaminergic neurons were recovered due to aMTD-Parkin administration (Figure 2c). In addition, it was confirmed that AAV-aMTD-Parkin was transmitted to a region (substantia nigra) other than the administration site (striatum) of the brain by in vivo pharmacological transfer technology (TSDT) (Figure 2d), and it was confirmed that AAV-aMTD-Parkin In the brains of Parkinson's disease animals, the expression of tyrosine hydroxylase (TH) in neurons secreting dopamine was restored to 77% of the normal level (Figures 2e, 2f).
실시예3. AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)을 이용한 알츠하이머병(AD) 치료효과 확인Example 3. Confirmation of Alzheimer's disease (AD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin)
이번에는 알츠하이머병(AD)의 치료효과를 확인하기 위해 아데노-부속 바이러스 (AAV)를 이용한 유전자치료 전달법과 세포투과성이 있는 Parkin 단백질의 융합 (AAV-aMTD-Parkin)을 Fibril Amyloid-beta (fAβ) 유도 알츠하이머병 치매(AD) 동물모델에 기간을 두어 투여한 뒤 결과를 확인하였다(도 3a).This time, to confirm the therapeutic effect of Alzheimer's disease (AD), a gene therapy delivery method using adeno-associated virus (AAV) and a fusion of the cell-permeable Parkin protein (AAV-aMTD-Parkin) were combined with Fibril Amyloid-beta (fAβ). The results were confirmed after administration over a period of time to an induced Alzheimer's disease dementia (AD) animal model (Figure 3a).
알츠하이머병 치매 동물모델에 투여한지 4주후에 시험한 결과에서 인지기능은 64% 개선되었다(도 3b). 알츠하이머병 치매 동물의 뇌 영역 중 해마 (Hippocampus) 영역에서 병리학적 Aβ가 제거되었으며, 조직면역학 분석 (IF)에서는 투여한 해마 영역의 반대편 해마에서도 Parkin이 발현되었음을 확인하였다(도 3c). 알츠하이머병 치매 동물의 뇌에서 병리학적 Aβ Plaque가 AAV-aMTD-Parkin을 투여한 경우에 감소되었다(도 3d).As a result of testing 4 weeks after administration to an Alzheimer's disease dementia animal model, cognitive function was improved by 64% (Figure 3b). Among the brain regions of Alzheimer's disease dementia animals, pathological Aβ was removed from the hippocampus region, and tissue immunology analysis (IF) confirmed that Parkin was also expressed in the hippocampus opposite to the administered hippocampus region (Figure 3c). In the brains of Alzheimer's disease dementia animals, pathological Aβ plaques were reduced when AAV-aMTD-Parkin was administered (Figure 3d).
실시예4. 세포투과성 유무에 따른 AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)의 치료물질 전달능력 확인Example 4. Confirmation of therapeutic substance delivery ability of AAV-TSDT fusion platform (AAV-aMTD-Parkin) depending on the presence or absence of cell permeability
파킨슨병(PD) 동물 모델에서 세포투과성이 있는 AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)이 세포투과성이 없는 기존 유전자치료법 (AAV-Parkin)과 비교하여 뇌 세포와 뇌 영역으로 치료물질(Parkin 단백질)의 전달능력이 우수한지를 확인하기 위하여 AAV-GFP-aMTD-Parkin-mCherry를 이용했으며, 아데노-부속 바이러스 (AAV) 벡터는 녹색 형광(GFP)을, aMTD-Parkin 단백질은 빨간색 형광(mCherry)이 발현되도록 실험을 설계하였다(도 4a).In an animal model of Parkinson's disease (PD), the cell-permeable AAV-TSDT fusion platform (AAV-aMTD-Parkin) was used to deliver therapeutic substances (Parkin) to brain cells and brain regions compared to the non-cell-permeable conventional gene therapy (AAV-Parkin). AAV-GFP-aMTD-Parkin-mCherry was used to confirm the excellent delivery ability of protein), the adeno-associated virus (AAV) vector emits green fluorescence (GFP), and the aMTD-Parkin protein emits red fluorescence (mCherry). An experiment was designed to achieve this (Figure 4a).
AAV-GFP-aMTD-Parkin-mCherry를 파킨슨병 모델 뇌안의 선조체 영역에 주입 후 조직면역학 분석 (Immunofluorescence: IF)을 진행하였다(도 4b). AAV-GFP로 표시된 세포는 바이러스 벡터가 존재하며, aMTD-Parkin-mCherry로 표시된 세포는 aMTD-Parkin 단백질이 존재한다. AAV-Parkin은 AAV와 Parkin이 동시에 발현되지만 (흰색 화살표), AAV-aMTD-Parkin은 동시에 발현되는 세포뿐만 아니라 Parkin 만 전달된 세포 (노란색 화살표)가 나타났다. Parkin만 전달된 세포는 신경세포(NeuN) 뿐만 아니라 신경교세포(GFAP)까지 약물의 전달이 가능한 것을 확인하였다(도 4c). AAV-GFP-aMTD-Parkin-mCherry was injected into the striatum region of the Parkinson's disease model brain, and immunofluorescence (IF) analysis was performed (FIG. 4b). Cells labeled with AAV-GFP contain the viral vector, and cells labeled with aMTD-Parkin-mCherry contain the aMTD-Parkin protein. AAV-Parkin co-expressed AAV and Parkin (white arrows), whereas AAV-aMTD-Parkin co-expressed cells as well as cells in which only Parkin was delivered (yellow arrows). It was confirmed that the cells to which only Parkin was delivered were capable of delivering the drug not only to neurons (NeuN) but also to glial cells (GFAP) (Figure 4c).
이번에는 파킨슨병 모델에서 AAV-aMTD-Parkin의 투여경로를 ICM으로 변경하여 약물전달 효능을 확인하였다(도 4d). ICM 투여 위치는 도 4e에 나타내었다. AAV-aMTD-Parkin은 AAV-Parkin에 비해 흑질(도 4f), 선조체(도 4g), 대뇌피질(도 4h)과 같이 다양한 뇌 영역으로 보다 더 많은 양의 Parkin이 전달되고 6주차까지 Parkin의 발현이 유지되는 것을 확인하였다. AAV-Parkin 대비 AAV-aMTD-Parkin이 주차별로 흑질에서 2.7배, 1.7배, 1.7배, 2.1배 증가되었고, 선조체에서 6.2배, 1.5배, 1.7배 2.6배 증가되었다. 이는 도파민성 뉴런에 aMTD의 세포간 전달(cell-to-cell transfer)에 의해 더 많은 Parkin이 전달되는 것을 보여주는 결과이다.This time, the drug delivery efficacy was confirmed by changing the administration route of AAV-aMTD-Parkin to ICM in the Parkinson's disease model (Figure 4d). ICM administration locations are shown in Figure 4e. Compared to AAV-Parkin, AAV-aMTD-Parkin delivers a larger amount of Parkin to various brain regions such as the substantia nigra (Figure 4f), striatum (Figure 4g), and cerebral cortex (Figure 4h), and increases the expression of Parkin until the 6th week. It was confirmed that this was maintained. Compared to AAV-Parkin, AAV-aMTD-Parkin increased by 2.7-fold, 1.7-fold, 1.7-fold, and 2.1-fold in the substantia nigra by parking, and by 6.2-fold, 1.5-fold, 1.7-fold, and 2.6-fold in the striatum. This result shows that more Parkin is delivered to dopaminergic neurons through cell-to-cell transfer of aMTD.
실시예5. AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)을 이용한 파킨슨병(PD) 치료효과 확인(3 x 10Example 5. Confirmation of Parkinson's disease (PD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) (3 x 10
66
GC/Brain, ICV Route) GC/Brain, ICV Route)
세포투과성 유무에 따른 Add-On Effect를 확인하기 위해 6-OHDA 유도 파킨슨병 동물모델에 AAV-Parkin / AAV-aMTD-Parkin을 저용량(3 x 106 GC/Brain)의 ICV로 투여하였다(도 5a). 파킨슨병 동물모델에서 AAV-Parkin 투여군은 49%, AAV-aMTD-Parkin 투여군은 105%로 운동기능이 개선되었다(도 5b). 또한, 파킨슨병 동물의 혈액 (Plasma)에서 도파민 (Dopamine) 측정하였을 때, AAV-Parkin 투여군은 38%, AAV-aMTD-Parkin 투여군은 106%로 증가하였다(도 5c). 이는 aMTD가 저용량 투여시 세포투과성 유무에 따라 확연한 Add-On Effect를 나타내고 있음을 보여주는 결과이다. 뇌에서 티로신 수산화효소(TH)의 발현을 측정했을 때(도 5d 및 도 5e), 파킨슨병 모델과 AAV-Parkin 투여군의 단백질 양은 차이가 없었으나, AAV-aMTD-Parkin 투여군에서는 TH의 발현이 회복되었음을 확인하였다. 마찬가지로, 뇌의 흑질 (SN) 영역에서 TH 발현을 조직면역학 분석을 통해 검증하였을 때 AAV-Parkin 투여군은 파킨슨병 동물모델(대조군)과 비교했을 때 차이가 없으나, AAV-aMTD-Parkin 투여군에서는 TH의 발현이 회복되었음을 확인하였다(도 5f). AAV에 의해 발현되는 외인성(exogenous) Parkin 단백질의 양은 AAV-Parkin과 AAV-aMTD-Parkin간에 차이가 없었다(도 5g).To confirm the Add-On Effect depending on the presence or absence of cell permeability, AAV-Parkin / AAV-aMTD-Parkin was administered ICV at a low dose ( 3 ). In the Parkinson's disease animal model, motor function improved by 49% in the AAV-Parkin administration group and by 105% in the AAV-aMTD-Parkin administration group (Figure 5b). In addition, when dopamine was measured in the blood (plasma) of Parkinson's disease animals, it increased to 38% in the AAV-Parkin administered group and 106% in the AAV-aMTD-Parkin administered group (Figure 5c). This result shows that aMTD shows a clear Add-On Effect depending on the presence or absence of cell permeability when administered at low doses. When measuring the expression of tyrosine hydroxylase (TH) in the brain (Figures 5d and 5e), there was no difference in the amount of protein between the Parkinson's disease model and the AAV-Parkin administered group, but the expression of TH recovered in the AAV-aMTD-Parkin administered group. It was confirmed that it was done. Similarly, when TH expression in the substantia nigra (SN) region of the brain was verified through tissue immunology analysis, there was no difference in the AAV-Parkin administered group compared to the Parkinson's disease animal model (control group), but in the AAV-aMTD-Parkin administered group, there was no difference in TH. It was confirmed that expression was restored (Figure 5f). The amount of exogenous Parkin protein expressed by AAV did not differ between AAV-Parkin and AAV-aMTD-Parkin (Figure 5g).
실시예6. AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)을 이용한 파킨슨병(PD) 치료효과 확인(3 x 10Example 6. Confirmation of Parkinson's disease (PD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) (3 x 10
1010
GC/Brain, ICV Route) GC/Brain, ICV Route)
세포투과성 유무에 따른 Add-On Effect를 확인하기 위해 6-OHDA 유도 파킨슨병 동물모델에 AAV-Parkin / AAV-aMTD-Parkin을 고용량(3 x 1010 GC/Brain)의 ICV로 투여하였다(도 6a). 파킨슨병 모델에서 AAV-Parkin 투여군은 37%, AAV-aMTD-Parkin 투여군은 99%로 운동기능이 개선되었다(도 6b). 또한, 파킨슨병 동물의 혈액 (Plasma)에서 도파민 (Dopamine) 측정하였을 때, AAV-Parkin 투여군은 65%, AAV-aMTD-Parkin 투여군은 164%로 증가하였다(도 6c). 이는 aMTD가 고용량 투여시 세포투과성 유무에 따라 확연한 Add-On Effect를 나타내고 있음을 보여주는 결과이다. 뇌에서 티로신 수산화효소(TH)의 발현을 측정했을 때(도 6d), 파킨슨병 모델과 AAV-Parkin 투여군의 단백질 양은 차이가 없었으나, AAV-aMTD-Parkin 투여군에서는 TH의 발현이 회복되었음을 확인하였다. 마찬가지로, 뇌의 흑질 (SN) 영역에서 TH 발현을 조직면역학 분석을 통해 검증하였을 때 AAV-Parkin 투여군은 파킨슨병 동물모델(대조군)과 비교했을 때 차이가 없으나, AAV-aMTD-Parkin 투여군에서는 TH의 발현이 회복되었음을 확인하였다(도 6e). AAV에 의해 발현되는 외인성(exogenous) Parkin 단백질의 양은 AAV-Parkin과 AAV-aMTD-Parkin간에 차이가 없었다(도 6f).To confirm the Add-On Effect depending on the presence or absence of cell permeability, AAV-Parkin / AAV-aMTD-Parkin was administered by ICV at a high dose (3 x 10 10 GC/Brain) to a 6-OHDA-induced Parkinson's disease animal model (Figure 6a) ). In the Parkinson's disease model, motor function improved by 37% in the AAV-Parkin administration group and 99% in the AAV-aMTD-Parkin administration group (Figure 6b). In addition, when dopamine was measured in the blood (plasma) of Parkinson's disease animals, it increased to 65% in the AAV-Parkin administered group and to 164% in the AAV-aMTD-Parkin administered group (Figure 6c). This result shows that aMTD shows a clear Add-On Effect depending on the presence or absence of cell permeability when administered in high doses. When measuring the expression of tyrosine hydroxylase (TH) in the brain (Figure 6d), there was no difference in protein amount between the Parkinson's disease model and the AAV-Parkin administration group, but it was confirmed that the expression of TH was recovered in the AAV-aMTD-Parkin administration group. . Similarly, when TH expression in the substantia nigra (SN) region of the brain was verified through tissue immunology analysis, there was no difference in the AAV-Parkin administered group compared to the Parkinson's disease animal model (control group), but in the AAV-aMTD-Parkin administered group, there was no difference in TH. It was confirmed that expression was restored (Figure 6e). The amount of exogenous Parkin protein expressed by AAV did not differ between AAV-Parkin and AAV-aMTD-Parkin (Figure 6f).
실시예7. a-Synuclein 유도 파킨슨병 (PD) 모델에서 AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)을 이용한 파킨슨병(PD) 치료효과 확인(3 x 10Example 7. Confirmation of Parkinson's disease (PD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) in a-Synuclein-induced Parkinson's disease (PD) model (3 x 10
66
GC/Brain, ICV Route) GC/Brain, ICV Route)
만성 파킨슨병(PD)을 유도할 수 있는 AAV-a-Syn 유도 파킨슨병 동물모델에서 AAV-Parkin / AAV-aMTD-Parkin을 각각 저용량(3 x 106 GC/Brain)의 ICV로 투여하여 세포투과성 유무에 따른 치료효과를 확인하였다(도 7a). 파킨슨병 모델에서 AAV-Parkin 투여군은 12%, AAV-aMTD-Parkin 투여군은 68%으로 개선된 운동기능을 나타냈다(도 7b). 또한, 파킨슨병 동물의 혈액 (Plasma)에서 도파민 (Dopamine) 양을 측정하였을 때, AAV-Parkin 투여군은 56%, AAV-aMTD-Parkin 투여군은 95%로 도파민 양이 증가하였다(도 7c). 뇌에서 TH의 발현을 단백질 양 측정 및 조직면역학 분석을 통해 확인한 결과, AAV-Parkin 투여군은 대조군과 비교했을 때 차이가 없으나, AAV-aMTD-Parkin 투여군에서는 TH 발현이 회복되었다(도 7d, 도 7e). 뇌에서 pS129-a-Synuclein 단백질 축적량은 대조군과 AAV-Parkin 투여군은 차이가 없으나, AAV-aMTD-Parkin 투여군에서는 감소된 것을 확인하였다(도 7f).In the AAV-a-Syn induced Parkinson's disease animal model that can induce chronic Parkinson's disease (PD), AAV-Parkin / AAV-aMTD-Parkin was administered as low dose ( 3 The treatment effect was confirmed depending on the presence or absence (Figure 7a). In the Parkinson's disease model, the AAV-Parkin administration group showed improved motor function by 12% and the AAV-aMTD-Parkin administration group by 68% (Figure 7b). In addition, when the amount of dopamine was measured in the blood (plasma) of Parkinson's disease animals, the amount of dopamine increased to 56% in the AAV-Parkin administered group and 95% in the AAV-aMTD-Parkin administered group (Figure 7c). As a result of confirming the expression of TH in the brain through protein measurement and tissue immunology analysis, there was no difference in the AAV-Parkin administered group compared to the control group, but TH expression was recovered in the AAV-aMTD-Parkin administered group (Figures 7d, 7e) ). There was no difference in the amount of pS129-a-Synuclein protein accumulation in the brain between the control group and the AAV-Parkin administration group, but it was confirmed to be reduced in the AAV-aMTD-Parkin administration group (Figure 7f).
실시예8. 알츠하이머병 (AD) 치매모델에서 AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)을 이용한 알츠하이머병(AD) 치료효과 확인(3 x 10Example 8. Confirmation of Alzheimer's disease (AD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) in Alzheimer's disease (AD) dementia model (3 x 10
66
GC/Brain, ICV Route) GC/Brain, ICV Route)
세포투과성 유무에 따른 Add-On Effect를 확인하기 위해 fAβ 유도 알츠하이머병 동물모델에 AAV-Parkin / AAV-aMTD-Parkin을 각각 저용량(3 x 106 GC/Brain)의 ICV로 투여하였다(도 8a). 알츠하이머병 치매모델에서 AAV-Parkin 투여군은 39%, AAV-aMTD-Parkin 투여군은 87%로 개선된 운동기능을 나타냈다(도 8b). 이는 aMTD가 저용량 투여시에도 세포투과성 유무에 따라 확연한 Add-On Effect를 나타내고 있음을 보여주는 결과이다. 뇌에서 병리학적 Aβ Plaque의 축적량을 단백질 양 및 조직면역학 분석을 통해 확인하였을 때, Aβ Plaque는 AAV-Parkin 투여군과 대조군 간에 차이가 없으나, AAV-aMTD-Parkin 투여군에서는 감소된 것을 확인하였다.To confirm the Add-On Effect depending on the presence or absence of cell permeability, low doses (3 x 10 6 GC/Brain) of AAV-Parkin / AAV-aMTD-Parkin were administered by ICV to an fAβ-induced Alzheimer's disease animal model, respectively (Figure 8a). . In the Alzheimer's disease dementia model, the AAV-Parkin administered group showed improved motor function by 39% and the AAV-aMTD-Parkin administered group showed improved motor function by 87% (Figure 8b). This result shows that aMTD shows a clear Add-On Effect depending on the presence or absence of cell permeability even when administered at low doses. When the accumulation of pathological Aβ plaques in the brain was confirmed through protein quantity and tissue immunology analysis, there was no difference in Aβ plaques between the AAV-Parkin administered group and the control group, but it was confirmed that it was reduced in the AAV-aMTD-Parkin administered group.
실시예9. 알츠하이머병 (AD) 치매모델에서 AAV-TSDT 융합 플랫폼 (AAV-aMTD-Parkin)을 이용한 알츠하이머병(AD) 치료효과 확인(3 x 10Example 9. Confirmation of Alzheimer's disease (AD) treatment effect using AAV-TSDT fusion platform (AAV-aMTD-Parkin) in Alzheimer's disease (AD) dementia model (3 x 10
44
GC/Brain, ICV Route) GC/Brain, ICV Route)
세포투과성 유무에 따른 Add-On Effect를 확인하기 위해 fAβ 유도 알츠하이머병 동물모델에 AAV-Parkin / AAV-aMTD-Parkin을 각각 극저용량(3 x 104 GC/Brain)의 ICV로 투여하였다(도 9a). 알츠하이머병 치매모델에서 AAV-Parkin 투여군은 13%, AAV-aMTD-Parkin 투여군은 57%로 개선된 인지기능을 나타냈다(도 9b). 이는 aMTD가 극저용량 투여시 세포투과성 유무에 따라 확연한 Add-On Effect를 나타내고 있음을 보여주는 결과이다. 뇌에서 병리학적 Aβ Plaque의 축적 정도를 단백질 양과 조직면역학 분석을 통해 확인하였을 때, 축적된 Aβ Plaque의 양은 AAV-Parkin 투여군과 대조군 간에 차이가 없으나, AAV-aMTD-Parkin 투여군에서는 감소된 것을 확인하였다(도 9c, 도 9d).To confirm the Add-On Effect depending on the presence or absence of cell permeability, AAV-Parkin / AAV-aMTD-Parkin was administered by ICV at extremely low doses (3 x 10 4 GC/Brain) to an fAβ-induced Alzheimer's disease animal model (Figure 9a). ). In the Alzheimer's disease dementia model, cognitive function was improved by 13% in the AAV-Parkin administration group and 57% in the AAV-aMTD-Parkin administration group (Figure 9b). This result shows that aMTD shows a clear Add-On Effect depending on the presence or absence of cell permeability when administered at extremely low doses. When the degree of accumulation of pathological Aβ plaques in the brain was confirmed through protein amount and tissue immunology analysis, there was no difference in the amount of accumulated Aβ plaques between the AAV-Parkin administered group and the control group, but it was confirmed to be reduced in the AAV-aMTD-Parkin administered group. (Figure 9c, Figure 9d).
1. pAAV9-Parkin (ΔUbl)-SDB1. pAAV9-Parkin(ΔUbl)-SDB
CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TTCTAGACAA CTTTGTATAG AAAAGTTG/TA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTGGTT TAGTGAACCG TCAGATCC/AA GTTTGTACAA AAAAGCAGGC TGCCACCATG CAAGAAATGA ACGCGACCGG TGGCGACGAT CCGCGTAACG CGGCGGGTGG CTGCGAGCGT GAACCGCAGA GCCTGACCCG TGTGGACCTG AGCAGCAGCG TGCTGCCGGG TGATAGCGTG GGCCTGGCGG TTATCCTGCA CACCGACAGC CGTAAGGATA GCCCGCCGGC GGGTAGCCCG GCGGGCCGTA GCATTTATAA CAGCTTTTAC GTGTATTGCA AGGGTCCGTG CCAGCGTGTG CAACCGGGTA AACTGCGTGT TCAGTGCAGC ACCTGCCGTC AAGCGACCCT GACCCTGACC CAGGGTCCGA GCTGCTGGGA CGATGTTCTG ATCCCGAACC GTATGAGCGG CGAGTGCCAA AGCCCGCACT GTCCGGGCAC CAGCGCGGAG TTCTTCTTCA AGTGCGGTGC GCACCCGACC AGCGACAAAG AAACCAGCGT GGCGCTGCAC CTGATCGCGA CCAACAGCCG TAACATCACC TGCATTACCT GCACCGACGT TCGTAGCCCG GTGCTGGTTT TTCAGTGCAA CAGCCGTCAC GTGATTTGCC TGGATTGCTT CCACCTGTAT TGCGTGACCC GTCTGAACGA CCGTCAGTTT GTTCACGATC CGCAACTGGG TTATAGCCTG CCGTGCGTTG CGGGTTGCCC GAACAGCCTG ATCAAAGAAC TGCACCACTT CCGTATTCTG GGCGAGGAAC AGTACAACCG TTATCAGCAA TACGGCGCGG AGGAATGCGT GCTGCAAATG GGTGGCGTTC TGTGCCCGCG TCCGGGTTGC GGTGCGGGTC TGCTGCCGGA GCCGGACCAG CGTAAGGTGA CCTGCGAAGG TGGCAACGGC CTGGGTTGCG GCTTTGCGTT CTGCCGTGAG TGCAAAGAAG CGTATCACGA GGGTGAATGC AGCGCGGTTT TTGAGGCGAG CGGCACCACC ACCCAGGCGT ACCGTGTGGA TGAGCGTGCG GCGGAACAAG CGCGTTGGGA GGCGGCGAGC AAGGAAACCA TCAAGAAAAC CACCAAGCCG TGCCCGCGTT GCCATGTGCC GGTTGAGAAG AACGGTGGCT GCATGCACAT GAAATGCCCG CAGCCGCAAT GCCGTCTGGA GTGGTGCTGG AACTGCGGTT GCGAATGGAA CCGTGTGTGC ATGGGCGACC ACTGGTTCGA TGTTATGGCG GAACAGAGCG ACAAGGATGT GAAATACTAT ACCCTGGAGG AAATCCAAAA GCACAAAGAC AGCAAGAGCA CCTGGCTGAT TCTGCACCAC AAGGTTTATG ATCTGACCAA ATTCCTGGAG GAACATCCGG GTGGTGAGGA AGTGCTGGGT GAACAAGCGG GTGGCGACGC GACCGAGAAC TTTGAAGACG TTGGCCACAG CACCGATGCG CGTGAGCTGA GCAAAACCTA CATCATTGGT GAACTGCACC CGGACGATCG TAGCAAGATT GCGAAACCGA GCGAAACCCT GTAAACCCAG CTTTCTTGTA CAAAGTGGGA ATTCCTAGAG CTCGCTGATC AGCCTCGACT GTGCCTTCTA GTTGCCAGCC ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT CCTTTCCTAA TAAAATGAGG AAATTGCATC GCATTGTCTG AGTAGGTGTC ATTCTATTCT GGGGGGTGGG GTGGGGCAGG ACAGCAAGGG GGAGGATTGG GAAGAGAATA GCAGGCATGC TGGGGAGGGC CGCAGGAACC CCTAGTGATG GAGTTGGCCA CTCCCTCTCT GCGCGCTCGC TCGCTCACTG AGGCCGGGCG ACCAAAGGTC GCCCGACGCC CGGGCTTTGC CCGGGCGGCC TCAGTGAGCG AGCGAGCGCG CAGCTGCCTG CAGGCCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TTCTAGACAA CTTTGTATAG AAAAGTTG/TA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCG TTAC ATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TG ACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTGGTT TAGTGAACCG TCAGATCC/AA GTTTGTACAA AAAAGCAGGC TGCCACCATG CAAGAAATGA ACGCGACCGG TGGCGACGAT CCGCGTAACG CGGCGGGTGG CTGCGAGCGT GAACCGCAGA GCCTGACCCG TGTGGACCTG AGCAGCAGCG TGCTGCCGGG TGATAGCGTG GGCCTGGCGG TTATCCTGCA CACCGACAGC CGTAAGGATA GCCCGCCGGC GGGTAGCCCG GCGGGCCGTA GCATTTATAA CAGCTTTTAC GTGTATTGCA AGGGTCCGTG CCAGCGTGTG CAACCGGGTA AACTGCGTGT TCAGTGCAGC ACCTGCCGTC AAGCGACCCT GACCCTGACC CAGGGTCCGA GCTGCTGGGA CGATGTTCTG ATCCCGAACC GTATGAGCGG CGAGTGCCAA AGCCCGCACT GTCCGGGCAC CAGCGCGGAG TTCTTCTTCA AGTGCG GTGC GCACCCGACC AGCGACAAAG AAACCAGCGT GGCGCTGCAC CTGATCGCGA CCAACAGCCG TAACATCACC TGCATTACCT GCACCGACGT TCGTAGCCCG GTGCTGGTTT TTCAGTGCAA CAGCCGTCAC GTGATTTGCC TGGATTGCTT CCACCTGTAT TGCGTGACCC GTCTGAACGA CCGTCAGTTT GTTCACGATC CGCAACTGGG TTATAGCCTG CCGTGCGTTG CGGGTTGCCC GAACAGCCTG ATCAAAGAAC TGCACCACTT CCGTATTCTG GGCGAGGAAC AGTACAACCG TTATCAGCAA TACGGCGCGG AGGAATGCGT GCTGCAAATG GGTGGCGTTC TGTGCCCGCG TCCGGGTTGC GGTGCGGGTC TGCTGCCGGA GCCGGACCAG CGTAAGGTGA CCTGCGAAGG TGGCAACGGC CTGGGTTGCG GCTTTGCGTT CTGCCGTGAG TGCAAAGAAG CGTATCACGA GGGT GAATGC AGCGCGGTTT TTGAGGCGAG CGGCACCACC ACCCAGGCGT ACCGTGTGGA TGAGCGTGCG GCGGAACAAG CGCGTTGGGA GGCGGCGAGC AAGGAAACCA TCAAGAAAAC CACCAAGCCG TGCCCGCGTT GCCATGTGCC GGTTGAGAAG AACGGTGGCT GCATGCACAT GAAATGCCCG CAGCCGCAAT GCCGTCTGGA GTGGTGCTGG AACTGCGGTT GCGAATGG AA CCGTGTGTGC ATGGGCGACC ACTGGTTCGA TGTTATGGCG GAACAGAGCG ACAAGGATGT GAAATACTAT ACCCTGGAGG AAATCCAAAA GCACAAAGAC AGCAAGAGCA CCTGGCTGAT TCTGCACCAC AAGGTTTATG ATCTGACCAA ATTCCTGGAG GAACATCCGG GTGGTGAGGA AGTGCTGGGT GAACAAGCGG GTGGCGACGC GACCGAGAAC TTTGAAGACG TTGGCCACAG CACCGATGCG CGTGAGCTGA GCAAAACCTA CATCATTGGT GA ACTGCACC CGGACGATCG TAGCAAGATT GCGAAACCGA GCGAAACCCT GTAAACCCAG CTTTCTTGTA CAAAGTGGGA ATTCCTAGAG CTCGCTGATC AGCCTCGACT GTGCCTTCTA GTTGCCAGCC ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT CCTTTCCTAA TAAAATGAGG AAATTGCATC GCATTGTCTG AGTAGGTGTC ATTCTATT CTGGGGGGTGGG GTGGGGCAGG ACAGCAAGGG GGAGGATTGG GAAGAGAATA GCAGGCATGC TGGGGAGGGC CGCAGGAACC CCTAGTGATG GAGTTGGCCA CTCCCTCTCT GCGCGCTCGC TCGCTCACTG AGGCCGGGCG ACCAAAGGTC GCCCGACGCC CGGGCTTTGC CCGGGCGGCC TCAGTGAGCG AGCGAGCGCG CAGCTGCCTG CAGG
2. pAAV9-aMTD524-Parkin (ΔUbl)-SDB2. pAAV9-aMTD524-Parkin(ΔUbl)-SDB
CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TTCTAGACAA CTTTGTATAG AAAAGTTGTA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTGGTT TAGTGAACCG TCAGATCCAA GTTTGTACAA AAAAGCAGGC TGCCACCATG GCGGTTGCGC TGATTGTGGT TCCGGCGCTG GCGCCGCAAG AAATGAACGC GACCGGTGGC GACGATCCGC GTAACGCGGC GGGTGGCTGC GAGCGTGAAC CGCAGAGCCT GACCCGTGTG GACCTGAGCA GCAGCGTGCT GCCGGGTGAT AGCGTGGGCC TGGCGGTTAT CCTGCACACC GACAGCCGTA AGGATAGCCC GCCGGCGGGT AGCCCGGCGG GCCGTAGCAT TTATAACAGC TTTTACGTGT ATTGCAAGGG TCCGTGCCAG CGTGTGCAAC CGGGTAAACT GCGTGTTCAG TGCAGCACCT GCCGTCAAGC GACCCTGACC CTGACCCAGG GTCCGAGCTG CTGGGACGAT GTTCTGATCC CGAACCGTAT GAGCGGCGAG TGCCAAAGCC CGCACTGTCC GGGCACCAGC GCGGAGTTCT TCTTCAAGTG CGGTGCGCAC CCGACCAGCG ACAAAGAAAC CAGCGTGGCG CTGCACCTGA TCGCGACCAA CAGCCGTAAC ATCACCTGCA TTACCTGCAC CGACGTTCGT AGCCCGGTGC TGGTTTTTCA GTGCAACAGC CGTCACGTGA TTTGCCTGGA TTGCTTCCAC CTGTATTGCG TGACCCGTCT GAACGACCGT CAGTTTGTTC ACGATCCGCA ACTGGGTTAT AGCCTGCCGT GCGTTGCGGG TTGCCCGAAC AGCCTGATCA AAGAACTGCA CCACTTCCGT ATTCTGGGCG AGGAACAGTA CAACCGTTAT CAGCAATACG GCGCGGAGGA ATGCGTGCTG CAAATGGGTG GCGTTCTGTG CCCGCGTCCG GGTTGCGGTG CGGGTCTGCT GCCGGAGCCG GACCAGCGTA AGGTGACCTG CGAAGGTGGC AACGGCCTGG GTTGCGGCTT TGCGTTCTGC CGTGAGTGCA AAGAAGCGTA TCACGAGGGT GAATGCAGCG CGGTTTTTGA GGCGAGCGGC ACCACCACCC AGGCGTACCG TGTGGATGAG CGTGCGGCGG AACAAGCGCG TTGGGAGGCG GCGAGCAAGG AAACCATCAA GAAAACCACC AAGCCGTGCC CGCGTTGCCA TGTGCCGGTT GAGAAGAACG GTGGCTGCAT GCACATGAAA TGCCCGCAGC CGCAATGCCG TCTGGAGTGG TGCTGGAACT GCGGTTGCGA ATGGAACCGT GTGTGCATGG GCGACCACTG GTTCGATGTT ATGGCGGAAC AGAGCGACAA GGATGTGAAA TACTATACCC TGGAGGAAAT CCAAAAGCAC AAAGACAGCA AGAGCACCTG GCTGATTCTG CACCACAAGG TTTATGATCT GACCAAATTC CTGGAGGAAC ATCCGGGTGG TGAGGAAGTG CTGGGTGAAC AAGCGGGTGG CGACGCGACC GAGAACTTTG AAGACGTTGG CCACAGCACC GATGCGCGTG AGCTGAGCAA AACCTACATC ATTGGTGAAC TGCACCCGGA CGATCGTAGC AAGATTGCGA AACCGAGCGA AACCCTGTAA ACCCAGCTTT CTTGTACAAA GTGGGAATTC CTAGAGCTCG CTGATCAGCC TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGT GCCTTCCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAAT TGCATCGCAT TGTCTGAGTA GGTGTCATTC TATTCTGGGG GGTGGGGTGG GGCAGGACAG CAAGGGGGAG GATTGGGAAG AGAATAGCAG GCATGCTGGG GAGGGCCGCA GGAACCCCTA GTGATGGAGT TGGCCACTCC CTCTCTGCGC GCTCGCTCGC TCACTGAGGC CGGGCGACCA AAGGTCGCCC GACGCCCGGG CTTTGCCCGG GCGGCCTCAG TGAGCGAGCG AGCGCGCAGC TGCCTGCAGGCCTGCAGGCA GCTGCCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC TTCTAGACAA CTTTGTATAG AAAAGTTGTA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATA ACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGAC CTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTGGTT TA GTGAACCG TCAGATCCAA GTTTGTACAA AAAAGCAGGC TGCCACCATG GCGGTTGCGC TGATTGTGGT TCCGGCGCTG GCGCCGC AAG AAATGAACGC GACCGGTGGC GACGATCCGC GTAACGCGGC GGGTGGCTGC GAGCGTGAAC CGCAGAGCCT GACCCGTGTG GACCTGAGCA GCAGCGTGCT GCCGGGTGAT AGCGTGGGCC TGGCGGTTAT CCTGCACACC GACA GCCGTAAGGATAGCCC GCCGGCGGGT AGCCCGGCGG GCCGTAGCAT TTATAACAGC TTTTACGTGT ATTGCAAGGG TCCGTGCCAG CGTGTGCAAC CGGGTAAACT GCGTGTTCAG TGCAGCACCT GCCGTCAAGC GACCCTGACC CTGACCCAGG GTCCGAGCTG CTGGGACGAT GTTCTGATCC CGAACCGTAT GAGCGGCGAG TGCCAAAGCC CGCACTGTCC GGGCACCAGC GCGGAGTTCT TCTTCAAGTG CGGTGCGCAC CCGACCAGCG ACAAAGAAAC CAGCGTGGCG CTGCACCTGA TCGCGACCAA CAGCCGTAAC ATCACCTGCA TTACCTGCAC CGACGTTCGT AGCCCGGTGC TGGTTTTTCA GTGCAACAGC CGTCACGTGA TTTGCCTGGA TTGCTTCCAC CTGTATTGCG TGACCCGTCT GAACGACCGT CAGTTTGTTC ACGATCCGCA ACTGGGTTAT AG CCTGCCGT GCGTTGCGGG TTGCCCGAAC AGCCTGATCA AAGAACTGCA CCACTTCCGT ATTCTGGGCG AGGAACAGTA CAACCGTTAT CAGCAATACG GCGCGGAGGA ATGCGTGCTG CAAATGGGTG GCGTTCTGTG CCCGCGTCCG GGTTGCGGTG CGGGTCTGCT GCCGGAGCCG GACCAGCGTA AGGTGACCTG CGAAGGTGGC AACGGCCTGG GTTGCGGCTT TGCGTTCTGC CGTGAGTGCA AAGAAGCGTA TCACGAGGGT GAATGCAGCG CGGTTTTTGA GGCGAGCGGC ACCACCACCC AGGCGTACCG TGTGGATGAG CGTGCGGCGG AACAAGCGCG TTGGGAGGCG GCGAGCAAGG AAACCATCAA GAAAACCACC AAGCCGTGCC CGCGTTGCCA TGTGCCGGTT GAGAAGAACG GTGGCTGCAT GCACATGAAA TGCCCGCAGC CGCAATGCCG TCTGGAGTGG TGCTGGAACT GCGGTTGCGA ATGG AACCGT GTGTGCATGG GCGACCACTG GTTCGATGTT ATGGCGGAAC AGAGCGACAA GGATGTGAAA TACTATACCC TGGAGGAAAT CCAAAAGCAC AAAGACAGCA AGAGCACCTG GCTGATTCTG CACCACAAGG TTTATGATCT GACCAAATTC CTGGAGGAAC ATCCGGGTGG TGAGGAAGTG CTGGGTGAAC AAGCGGGTGG CGACGCGACC GAGAACTTTG AAGACGTTGG CCACAGCACC GATGCGCGTG AGCTGAGCAA AACCTACATC ATTGGTGAAC TGCACCCGGA CGATCGTAGC AAGATTGCGA AACCGAGCGA AACCCTGTAA ACCCAGCTTT CTTGTACAAA GTGGGAATTC CTAGAGCTCG CTGATCAGCC TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGT GCCTTCCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAAT TGCATCGCAT TGTCTGAGTA G GTGTCATTC TATTCTGGGG GGTGGGGTGG GGCAGGACAG CAAGGGGGAG GATTGGGAAG AGAATAGCAG GCATGCTGGG GAGGGCCGCA GGAACCCCTA GTGATGGAGT TGGCCACTCC CTCTCTGCGC GCTCGCTCGC TCACTGAGGC CGGGCGACCA AAGGTCGCCC GACGCCCGGG CTTTGCCCGG GCGGCCTCAG TGAGCGAGCG AGCGCGCAGC TGCCTGCAGG
3. aMTD 서열3. aMTD sequence
| SEQ ID No.SEQ ID No. | SequenceSequence |
| 33 | GCGGCGGCGCTGGCGCCGGTGGTGCTGGCGCTGCCGGCGGCGGCGCTGGCGCCGGTGGTGCTGGCGCTGCCG |
| 44 | GCGGCGGCGGTGCCGCTGCTGGCGGTGGTGGTGCCGGCGGCGGCGGTGCCGCTGCTGGCGGTGGTGGTGCCG |
| 55 | GCGGCGCTGCTGGTGCCGGCGGCGGTGCTGGCGCCGGCGGCGCTGCTGGTGCCGGCGGCGGTGCTGGCGCCG |
| 66 | GCGCTGGCGCTGCTGCCGGTGGCGGCGCTGGCGCCGGCGCTGGCGCTGCTGCCGGTGGCGGCGCTGGCGCCG |
| 77 | GCGGCGGCGCTGCTGCCGGTGGCGCTGGTGGCGCCGGCGGCGGCGCTGCTGCCGGTGGCGCTGGTGGCGCCG |
| 88 | GTGGTGGCGCTGGCGCCGGCGCTGGCGGCGCTGCCGGTGGTGGCGCTGGCGCCGGCGCTGGCGGCGCTGCCG |
| 99 | CTGCTGGCGGCGGTGCCGGCGGTGCTGCTGGCGCCGCTGCTGGCGGCGGTGCCGGCGGTGCTGCTGGCGCCG |
| 1010 | GCGGCGGCGCTGGTGCCGGTGGTGGCGCTGCTGCCGGCGGCGGCGCTGGTGCCGGTGGTGGCGCTGCTGCCG |
| 1111 | GCGGTGGCGCTGCTGCCGGCGCTGCTGGCGGTGCCGGCGGTGGCGCTGCTGCCGGCGCTGCTGGCGGTGCCG |
| 1212 | GCGGTGGTGCTGGTGCCGGTGCTGGCGGCGGCGCCGGCGGTGGTGCTGGTGCCGGTGCTGGCGGCGGGCCCG |
| 1313 | GTGGTGCTGGTGCTGCCGGCGGCGGCGGCGGTGCCGGTGGTGCTGGTGCTGCCGGCGGCGGCGGCGGTGCCG |
| 1414 | ATTGCGCTGGCGGCGCCGGCGCTGATTGTGGCGCCGATTGCGCTGGCGGGCGCCGGCGCTGATTGTGGGCCCG |
| 1515 | ATTGTGGCGGTGGCGCCGGCGCTGGTGGCGCTGCCGATTGTGGCGGTGGCGCCGGCGCTGGTGGCGCTGCCG |
| 1616 | GTGGCGGCGCTGCCGGTGGTGGCGGTGGTGGCGCCGGTGGCGGCGCTGCCGGTGGTGGCGGTGGTGGCGCCG |
| 1717 | CTGCTGGCGGCGCCGCTGGTGGTGGCGGCGGTGCCGCTGCTGGCGGCGCCGCTGGTGGTGGCGGCGGTGCCG |
| 1818 | GCGCTGGCGGTGCCGGTGGCGCTGCTGGTGGCGCCGGCGCTGGCGGTGCCGGTGGCGCTGCTGGTGGCGCCG |
| 1919 | GTGGCGGCGCTGCCGGTGCTGCTGGCGGCGCTGCCGGTGGCGGCGCTGCCGGTGCTGCTGGCGGCGCTGCCG |
| 2020 | GTGGCGCTGCTGGCGCCGGTGGCGCTGGCGGTGCCGGTGGCGCTGCTGGCGCCGGTGGCGCTGGCGGTGCCG |
| 2121 | GCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGTGCCGGCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGTGCCG |
| 2222 | GCGATTGTGGCGCTGCCGGTGGCGGTGCTGGCGCCGGCGATTGTGGCGCTGCCGGTGGCGGTGCTGGCGCCG |
| 2323 | ATTGCGATTGTGGCGCCGGTGGTGGCGCTGGCGCCGATTGCGATTGTGGGCCCGGTGTGGGCGCTGGGCCCG |
| 2424 | GCGGCGCTGCTGCCGGCGCTGGCGGCGCTGCTGCCGGCGGCGCTGCTGCCGGCGCTGGCGGCGCTGCTGCCG |
| 2525 | GCGGTGGTGCTGGCGCCGGTGGCGGCGGTGCTGCCGGCGGTGGTGCTGGCGCCGGTGGCGGCGGTGCTGCCG |
| 2626 | CTGGCGGTGGCGGCGCCGCTGGCGCTGGCGCTGCCGCTGGCGGTGGCGGCGCCGCTGGCGCTGGCGCTGCCG |
| 2727 | GCGGCGGTGGCGGCGCCGCTGCTGCTGGCGCTGCCGGCGGCGGTGGCGGCGCCGCTGCTGCTGGCGCTGCCG |
| 2828 | CTGCTGGTGCTGCCGGCGGCGGCGCTGGCGGCGCCGCTGCTGGTGCTGCCGGCGGCGGCGCTGGCGGCGCCG |
| 2929 | CTGGTGGCGCTGGCGCCGGTGGCGGCGGTGCTGCCGCTGGTGGCGCTGGCGCCGGTGGCGGCGGTGCTGCCG |
| 3030 | CTGGCGCTGGCGCCGGCGGCGCTGGCGCTGCTGCCGCTGGCGCTGGCGCCGGCGGCGCTGGCGCTGCTGCCG |
| 3131 | GCGCTGATTGCGGCGCCGATTCTGGCGCTGGCGCCGGCGCTGATTGCGGGCCCGATTCTGGGCCTGGGCCCG |
| 3232 | GCGGTGGTGGCGGCGCCGCTGGTGCTGGCGCTGCCGGCGGTGGTGGCGGCGCCGCTGGTGCTGGCGCTGCCG |
| 3333 | CTGCTGGCGCTGGCGCCGGCGGCGCTGCTGGCGCCGCTGCTGGCGCTGGCGCCGGCGGCGCTGCTGGCGCCG |
| 3434 | GCGATTGTGGCGCTGCCGGCGCTGGCGCTGGCGCCGGCGATTGTGGCGCTGCCGGCGCTGGCGCTGGCGCCG |
| 3535 | GCGGCGATTATTGTGCCGGCGGCGCTGCTGGCGCCGGCGGCGATTATTGTGCCGGCGGCGCTGCTGGCGCCG |
| 3636 | ATTGCGGTGGCGCTGCCGGCGCTGATTGCGGCGCCGATTGCGGTGGCGCTGGCCGGCGCTGATTGCGGGCCCG |
| 3737 | GCGGTGATTGTGCTGCCGGCGCTGGCGGTGGCGCCGGCGGTGATTGTGCTGCCGGGCGCTGGCGGTGGCGCCG |
| 3838 | GCGGTGCTGGCGGTGCCGGCGGTGCTGGTGGCGCCGGCGGTGCTGGCGGTGCCGGCGGTGCTGGTGGCGCCG |
| 3939 | GTGCTGGCGATTGTGCCGGCGGTGGCGCTGGCGCCGGTGCTGGCGATTGTGCCGGCGGTGGCGCTGGCGCCG |
| 4040 | CTGCTGGCGGTGGTGCCGGCGGTGGCGCTGGCGCCGCTGCTGGCGGTGGTGCCGGCGGTGGCGCTGGCGCCG |
| 4141 | GCGGTGATTGCGCTGCCGGCGCTGATTGCGGCGCCGGCGGTGATTGCGCTGCCGGGCGCTGATTGCGGGCCCG |
| 4242 | GCGGTGGTGGCGCTGCCGGCGGCGCTGATTGTGCCGGCGGTGGTGGCGCTGCCGGCGGCGCTGATTGTGCCG |
| 4343 | CTGGCGCTGGTGCTGCCGGCGGCGCTGGCGGCGCCGCTGGCGCTGGTGCTGCCGGCGGCGCTGGCGGCGCCG |
| 4444 | CTGGCGGCGGTGCTGCCGGCGCTGCTGGCGGCGCCGCTGGCGGCGGTGCTGCCGGCGCTGCTGGCGGCGCCG |
| 4545 | GCGCTGGCGGTGCCGGTGGCGCTGGCGATTGTGCCGGCGCTGGCGGTGCCGGTGGCGCTGGCGATTGTGCCG |
| 4646 | GCGCTGATTGCGCCGGTGGTGGCGCTGGTGGCGCCGGCGCTGATTGCGCCGTGGTGGCGCTGGTGGCGCCG |
| 4747 | CTGCTGGCGGCGCCGGTGGTGATTGCGCTGGCGCCGCTGCTGGCGGCGCCGGTGGTGATTGCGCTGGCGCCG |
| 4848 | CTGGCGGCGATTGTGCCGGCGATTATTGCGGTGCCGCTGGCGGCGATTGTGCCGGGCGATTATTGCGGTGCCG |
| 4949 | GCGGCGCTGGTGCTGCCGCTGATTATTGCGGCGCCGGCGGCGCTGGTGCTGCCGCTGATTATTGCGGGCCCG |
| 5050 | CTGGCGCTGGCGGTGCCGGCGCTGGCGGCGCTGCCGCTGGCGCTGGCGGTGCCGGCGCTGGCGGCGCTGCCG |
| 5151 | CTGATTGCGGCGCTGCCGGCGGTGGCGGCGCTGCCGCTGATTGCGGCGCTGCCGGCGGTGGCGGCGCTGCCG |
| 5252 | GCGCTGGCGCTGGTGCCGGCGATTGCGGCGCTGCCGGCGCTGGCGCTGGTGCCGGCGATTGCGGCGCTGCCG |
| 5353 | GCGGCGATTCTGGCGCCGATTGTGGCGCTGGCGCCGGCGGCGATTCTGGCGCCGATTGTGGCCTGGGCCCG |
| 5454 | GCGCTGCTGATTGCGCCGGCGGCGGTGATTGCGCCGGCGCTGCTGATTGCGCCGGGCGGCGGTGATTGCGCCG |
| 5555 | GCGATTCTGGCGGTGCCGATTGCGGTGGTGGCGCCGGCGATTCTGGCGGTGCCGATTGCGGTGGTGGGCCCG |
| 5656 | ATTCTGGCGGCGGTGCCGATTGCGCTGGCGGCGCCGATTGCTGGCGGCGGTGCCGATTGCGCTGGCGGGCCCG |
| 5757 | GTGGCGGCGCTGCTGCCGGCGGCGGCGGTGCTGCCGGTGGCGGCGCTGCTGCCGGCGGCGGCGGTGCTGCCG |
| 5858 | GCGGCGGCGGTGGTGCCGGTGCTGCTGGTGGCGCCGGCGGCGGCGGTGGTGCCGGTGCTGCTGGTGGCGCCG |
| 5959 | GCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGCGCCGGCGGCGCTGCTGGTGCCGGCGCTGGTGGCGGGCCCG |
| 6060 | GCGGCGGTGCTGCTGCCGGTGGCGCTGGCGGCGCCGGCGGGCGGTGCTGCTGCCGGTGGCGCTGGCGGGCCCG |
| 6161 | GCGGCGGCGCTGGCGCCGGTGCTGGCGCTGGTGCCGGCGGGCGGCGCTGGCGCCGGTGCTGGCGCTGGTGCCG |
| 6262 | CTGGTGCTGGTGCCGCTGCTGGCGGCGGCGGCGCCGCTGGTGCTGGTGCCGCTGCTGGCGGCGGCGGCGCCG |
| 6363 | GCGCTGATTGCGGTGCCGGCGATTATTGTGGCGCCGGCGCTGATTGCGGTGCCGGCGATTATTGTGGGCCCG |
| 6464 | GCGCTGGCGGTGATTCCGGCGGCGGCGATTCTGCCGGCGCTGGCGGTGATTCCGGCGGCGGCGATTCTGCCG |
| 6565 | CTGGCGGCGGCGCCGGTGGTGATTGTGATTGCGCCGCTGGCGGCGGCGCCGGTGGTGATTGTGATTGCGCCG |
| 6666 | GTGCTGGCGATTGCGCCGCTGCTGGCGGCGGTGCCGGTGCTGGCGATTGCGCCGCTGCTGGCGGCGGTGCCG |
| 6767 | GCGCTGATTGTGCTGCCGGCGGCGGTGGCGGTGCCGGCGCTGATTGTGCTGCCGGCGGCGGTGGCGGTGCCG |
| 6868 | GTGCTGGCGGTGGCGCCGGCGCTGATTGTGGCGCCGGTGCTGGCGGTGGCGCCGGCGCTGATTGTGGGCCCG |
| 6969 | GCGGCGCTGCTGGCGCCGGCGCTGATTGTGGCGCCGGGCGGCGCTGCTGGCGCCGGCGCTGATTGTGGCGCCG |
| 7070 | GCGCTGATTGCGCCGGCGGTGGCGCTGATTGTGCCGGCGCTGATTGCGCCGGCGGTGGCGCTGATTGTGCCG |
| 7171 | GCGATTGTGCTGCTGCCGGCGGCGGTGGTGGCGCCGGCGATTGTGCTGCTGCCGGCGGCGGTGGTGGCGCCG |
| 7272 | GTGATTGCGGCGCCGGTGCTGGCGGTGCTGGCGCCGGTGATTGCGGCGCCGGTGCTGGCGGTGCTGGCGCCG |
| 7373 | CTGGCGCTGGCGCCGGCGCTGGCGCTGCTGGCGCCGCTGGCGCTGGCGCCGGCGCTGGCGCTGCTGGCGCCG |
| 7474 | GCGATTATTCTGGCGCCGATTGCGGCGATTGCGCCGGCGATTATTCTGGCGCCGATTGCGGCGATTGCGCCG |
| 7575 | ATTGCGCTGGCGGCGCCGATTCTGCTGGCGGCGCCGATTGCGCTGGGCGGCGCCGATTCTGCTGGCGGCGCCG |
| 7676 | ATTGTGGCGGTGGCGCTGCCGGCGCTGGCGGTGCCGATTGTGGCGGTGGCGCTGCCGGCGCTGGCGGTGCCG |
| 7777 | GTGGTGGCGATTGTGCTGCCGGCGCTGGCGGCGCCGGTGGTGGCGATTGTGCTGCCGGCGCTGGCGGCGCCG |
| 7878 | ATTGTGGCGGTGGCGCTGCCGGTGGCGCTGGCGCCGATTGTGGCGGTGGCGCTGCCGGTGGCGCTGGCGCCG |
| 7979 | ATTGTGGCGGTGGCGCTGCCGGCGGCGCTGGTGCCGATTGTGGCGGTGGCGCTGCCGGCGGCGCTGGTGCCG |
| 8080 | ATTGTGGCGGTGGCGCTGCCGGCGGTGGCGCTGCCGATTGTGGCGGTGGCGCTGCCGGCGGTGGCGCTGCCG |
| 8181 | ATTGTGGCGGTGGCGCTGCCGGCGGTGCTGGCGCCGATTGTGGCGGTGGCGCTGCCGGCGGTGCTGGCGCCG |
| 8282 | GTGATTGTGGCGCTGGCGCCGGCGGTGCTGGCGCCGGTGATTGTGGCGCTGGCGCCGGCGGTGCTGGCGCCG |
| 8383 | ATTGTGGCGGTGGCGCTGCCGGCGCTGGTGGCGCCGATTGTGGCGGTGGCGCTGCCGGCGCTGGTGGCGCCG |
| 8484 | GCGCTGCTGATTGTGGCGCCGGTGGCGGTGGCGCCGGCGCTGCTGATTGTGGCGCCGGTGGCGGTGGCGCCG |
| 8585 | GCGGTGGTGATTGTGGCGCCGGCGGTGATTGCGCCGGCGGTGGTGATTGTGGGCCCGGGCGGTGATTGCGCCG |
| 8686 | GCGGTGCTGGCGGTGGCGCCGGCGCTGATTGTGCCGGCGGTGCTGGCGGTGGCGCCGGCGCTGATTGTGCCG |
| 8787 | CTGGTGGCGGCGGTGGCGCCGGCGCTGATTGTGCCGCTGGTGGCGGGCGGTGGCGCCGGCGCTGATTGTGCCG |
| 8888 | GCGGTGATTGTGGTGGCGCCGGCGCTGCTGGCGCCGGCGGTGATTGTGGTGGCGCCGGCGCTGCTGGGCCCG |
| 8989 | GTGGTGGCGATTGTGCTGCCGGCGGTGGCGGCGCCGGTGGTGGCGATTGTGCTGCCGGCGGTGGCGGCGCCG |
| 9090 | GCGGCGGCGCTGGTGATTCCGGCGATTCTGGCGCCGGCGGGCGGCGCTGGTGATTCCGGCGATTCTGGCGCCG |
| 9191 | GTGATTGTGGCGCTGGCGCCGGCGCTGCTGGCGCCGGTGATTGTGGCGCTGGCGCCGGCGCTGCTGGCGCCG |
| 9292 | GTGATTGTGGCGATTGCGCCGGCGCTGCTGGCGCCGGTGATTGTGGCGATTGCGCCGGCGCTGCTGGGCCCG |
| 9393 | ATTGTGGCGATTGCGGTGCCGGCGCTGGTGGCGCCGATTGTGGCGATTGCGGTGCCGGCGCTGGTGGCGCCG |
| 9494 | GCGGCGCTGGCGGTGATTCCGGCGGCGATTCTGCCGGCGGCGCTGGCGGTGATTCCGGGCGGCGATTCTGCCG |
| 9595 | GCGCTGGCGGCGGTGATTCCGGCGGCGATTCTGCCGGCGCTGGCGGCGGTGATTCCGGGCGGCGATTCTGCCG |
| 9696 | GCGGCGGCGCTGGTGATTCCGGCGGCGATTCTGCCGGCGGCGGCGCTGGTGATTCCGGGCGGCGATTCTGCCG |
| 9797 | CTGGCGGCGGCGGTGATTCCGGCGGCGATTCTGCCGCTGGCGGCGGGCGGTGATTCCGGGCGGCGATTCTGCCG |
| 9898 | CTGGCGGCGGCGGTGATTCCGGTGGCGATTCTGCCGCTGGCGGCGGGCGGTGATTCCGGTGGCGATTCTGCCG |
| 9999 | GCGGCGATTCTGGCGGCGCCGCTGATTGCGGTGCCGGCGGCGATTCTGGCGGCGCGCTGATTGCGGTGCCG |
| 100100 | GTGGTGGCGATTCTGGCGCCGCTGCTGGCGGCGCCGGTGGTGGCGATTCTGGCGCCGCTGCTGGCGGCGCCG |
| 101101 | GCGGTGGTGGTGGCGGCGCCGGTGCTGGCGCTGCCGGCGGTGGTGGTGGCGGCGCCGGTGCTGGCGCTGCCG |
| 102102 | GCGGTGGTGGCGATTGCGCCGGTGCTGGCGCTGCCGGCGGTGGTGGCGATTGCGCCGTGCTGGCGCTGCCG |
| 103103 | GCGCTGGCGGCGCTGGTGCCGGCGGTGCTGGTGCCGGCGCTGGCGGCGCTGGTGCCGGCGGTGCTGGTGCCG |
| 104104 | GCGCTGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCGGCGCTGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCG |
| 105105 | CTGGCGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCGCTGGCGGCGGCGCTGGTGCCGGTGGCGCTGGTGCCG |
| 106106 | GCGCTGGCGGCGCTGGTGCCGGCGCTGGTGGTGCCGGCGCTGGCGGCGCTGGTGCCGGCGCTGGTGGTGCCG |
| 107107 | ATTGCGGCGGTGATTGTGCCGGCGGTGGCGCTGCCGATTGCGGGCGGTGATTGTGCCGGCGGTGGCGCTGCCG |
| 108108 | ATTGCGGCGGTGCTGGTGCCGGCGGTGGCGCTGCCGATTGCGGGCGGTGCTGGTGCCGGCGGTGGCGCTGCCG |
| 109109 | GCGGTGGCGATTCTGGTGCCGCTGCTGGCGGCGCCGGCGGTGGCGATTCTGGTGCCGCTGCTGGCGGCGCCG |
| 110110 | GCGGTGGTGATTCTGGTGCCGCTGGCGGCGGCGCCGGCGGTGGTGATTCTGGTGCCGCTGGCGGCGGCGCCG |
| 111111 | ATTGCGGCGGTGATTGTGCCGGTGGCGGCGCTGCCGATTGCGGCGGTGATTGTGCCGTGGCGGCGCTGCCG |
| 112112 | GCGATTGCGATTGCGATTGTGCCGGTGGCGCTGCCGGCGATTGCGATTGCGATTGTGCCGGTGGCGCTGCCG |
| 113113 | ATTCTGGCGGTGGCGGCGATTCCGGTGGCGGTGCCGATTCCTGGCGGTGGCGGCGATTCCGGTGGCGGTGCCG |
| 114114 | ATTCTGGCGGCGGCGATTATTCCGGCGGCGCTGCCGATTCTGGCGGCGGCGATTATTCCGGCGGCGCTGCCG |
| 115115 | CTGGCGGTGGTGCTGGCGGCGCCGGCGATTGTGCCGCTGGGCGGTGGTGCTGGCGGCGCCGGCGATTGTGCCG |
| 116116 | GCGATTCTGGCGGCGATTGTGCCGCTGGCGGTGCCGGCGATTCTGGGCGGCGATTGTGCCGCTGGCGGTGCCG |
| 117117 | GTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCGGTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCG |
| 118118 | GCGATTGTGGCGCTGGCGGTGCCGGTGCTGGCGCCGGCGATTGTGGCGCTGGCGGTGCCGGTGCTGGCGCCG |
| 119119 | GCGGCGATTATTATTGTGCTGCCGGCGGCGCTGCCGGCGGCGATTATTATTGTGCTGCCGGCGGCGCTGCCG |
| 120120 | CTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCGCTGATTGTGGCGCTGGCGGTGCCGGCGCTGGCGCCG |
| 121121 | GCGATTATTATTGTGATTGCGCCGGCGGCGGCGCCGGCGATTATTATTGTGATTGCGCCGGCGGCGGCGCCG |
| 122122 | CTGGCGGCGCTGATTGTGGTGCCGGCGGTGGCGCCGCTGGCGGCGCTGATTGTGGTGCCGGGCGGTGGCGCCG |
| 123123 | GCGCTGCTGGTGATTGCGGTGCCGGCGGTGGCGCCGGCGCTGCTGGTGATTGCGGTGCCGGGCGGTGGCGCCG |
| 124124 | GCGGTGGCGCTGATTGTGGTGCCGGCGCTGGCGCCGGCGGTGGCGCTGATTGTGGTGCCGGGCCTGGGCCCG |
| 125125 | GCGGTTGCGCTGATTGTGGTTCCGGCGCTGGCGCCGGCGGTTGCGCTGATTGTGGTTCCGGGCCTGGGCCCG |
| 126126 | CTGCTGGCGCTGATTATTGCGCCGGCGGCGGCGCCGCTGCTGGCGCTGATTATTGCGCCGGCGGCGGCGCCG |
| 127127 | GCGCTGGCGCTGATTATTGTGCCGGCGGTGGCGCCGGCGCTGGCGCTGATTATTGTGCCGGCGGTGGCGCCG |
| 128128 | CTGCTGGCGGCGCTGATTGCGCCGGCGGCGCTGCCGCTGCTGGCGGGCGCTGATTGCGCCGGCGGCGCTGCCG |
| 129129 | ATTGTGGCGCTGATTGTGGCGCCGGCGGCGGTGCCGATTGTGGCGCTGATTGTGGCGCCGGCGGCGGTGCCG |
| 130130 | GTGGTGCTGGTGCTGGCGGCGCCGGCGGCGGTGCCGGTGGTGCTGGTGCTGGCGGCGCCGGCGGCGGTGCCG |
| 131131 | GCGGCGGTGGCGATTGTGCTGCCGGCGGTGGTGCCGGGCGGCGGTGGCGATTGTGCTGCCGGCGGTGGTGCCG |
| 132132 | GCGCTGATTGCGGCGATTGTGCCGGCGCTGGTGCCGGCGCTGATTGCGGCGATTGTGCCGGCGCTGGTGCCG |
| 133133 | GCGCTGGCGGTGATTGTGGTGCCGGCGCTGGCGCCGGCGCTGGCGGTGATTGTGGTGCCGGGCCTGGGCCCG |
| 134134 | GTGGCGATTGCGCTGATTGTGCCGGCGCTGGCGCCGGTGGCGATTGCGCTGATTGTGCCGGCGCTGGCGCCG |
| 135135 | GTGGCGATTGTGCTGGTGGCGCCGGCGGTGGCGCCGGTGGCGATTGTGCTGGGTGGCGCCGGCGGTGGCGCCG |
| 136136 | GTGGCGGTGGCGCTGATTGTGCCGGCGCTGGCGCCGGTGGGCGGTGGCGCTGATTGTGCCGGCGCTGGCGCCG |
| 137137 | GCGGTGATTCTGGCGCTGGCGCCGATTGTGGCGCCGGCGGTGATTCTGGCGCTGGCGCCGATTGTGGCCCG |
| 138138 | GCGCTGATTGTGGCGATTGCGCCGGCGCTGGTGCCGGCGCTGATTGTGGCGATTGCGCCGGCGCTGGTGCCG |
| 139139 | GCGGCGATTCTGATTGCGGTGCCGATTGCGGCGCCGGCGGCGATTCTGATTGCGGTGCCGATTGCGGGCCCG |
| 140140 | GTGATTGTGGCGCTGGCGGCGCCGGTGCTGGCGCCGGTGATTGTGGCGCTGGCGGCGCCGGTGCTGGCGCCG |
| 141141 | GTGCTGGTGGCGCTGGCGGCGCCGGTGATTGCGCCGGTGCTGTGGCGCTGGCGGCGCCGGTGATTGCGCCG |
| 142142 | GTGGCGCTGATTGCGGTGGCGCCGGCGGTGGTGCCGGTGGGCCTGATTGCGGTGGCGCCGGCGGTGGTGCCG |
| 143143 | GTGATTGCGGCGGTGCTGGCGCCGGTGGCGGTGCCGGTGATTGCGGCGGTGCTGGCGCCGGTGGCGGTGCCG |
| 144144 | GCGCTGATTGTGCTGGCGGCGCCGGTGGCGGTGCCGGCGCTGATTGTGCTGGCGGCGCCGGTGGCGGTGCCG |
| 145145 | GTGGCGGCGGCGATTGCGCTGCCGGCGATTGTGCCGGTGGCGGCGGCGATTGCGCTGCCGGCGATTGTGCCG |
| 146146 | ATTCTGGCGGCGGCGGCGGCGCCGCTGATTGTGCCGATTCTGGCGGCGGCGGCGGCGCCGCTGATTGTGCCG |
| 147147 | CTGGCGCTGGTGCTGGCGGCGCCGGCGATTGTGCCGCTGGCGCTGGTGCTGGGCGGCGCCGGCGATTGTGCCG |
| 148148 | GCGCTGGCGGTGGTGGCGCTGCCGGCGATTGTGCCGGCGCTGGCGGTGGTGGCGCTGCCGGCGATTGTGCCG |
| 149149 | GCGGCGATTCTGGCGCCGATTGTGGCGGCGCTGCCGGCGGCGATTCTGGCGCCGATTGTGGCGGCGCTGCCG |
| 150150 | ATTCTGATTGCGATTGCGATTCCGGCGGCGGCGCCGATTCTGATTGCGATTGCGATTCCGGCGGCGGGCCCG |
| 151151 | CTGGCGATTGTGCTGGCGGCGCCGGTGGCGGTGCCGCTGGCGATTGTGCTGGCGGCGCCGGTGGCGGTGCCG |
| 152152 | GCGGCGATTGCGATTATTGCGCCGGCGATTGTGCCGGCGGCGATTGCGATTATTGCGCCGGGCGATTGTGCCG |
| 153153 | CTGGCGGTGGCGATTGTGGCGCCGGCGCTGGTGCCGCTGGCGGTGGCGATTGTGGCGCCGGGCCTGGTGCCG |
| 154154 | CTGGCGATTGTGCTGGCGGCGCCGGCGGTGCTGCCGCTGGCGATTGTGCTGGCGGCGCCGGGCGGTGCTGCCG |
| 155155 | GCGGCGATTGTGCTGGCGCTGCCGGCGGTGCTGCCGGCGGCGATTGTGCTGGCGCTGCCGGGCGGTGCTGCCG |
| 156156 | GCGCTGCTGGTGGCGGTGCTGCCGGCGGCGCTGCCGGCGCTGCTGGTGGCGGTGCTGCCGGCGGCGCTGCCG |
| 157157 | GCGGCGCTGGTGGCGGTGCTGCCGGTGGCGCTGCCGGCGGCGCTGGTGGCGGTGCTGCCGGTGGCGCTGCCG |
| 158158 | GCGATTCTGGCGGTGGCGCTGCCGCTGCTGGCGCCGGCGATTCTGGCGGTGGCGCTGCCGCTGCTGGCGCCG |
| 159159 | ATTGTGGCGGTGGCGCTGGTGCCGGCGCTGGCGCCGATTGTGGCGGTGGCGCTGGTGCCGGCGCTGGCGCCG |
| 160160 | ATTGTGGCGGTGGCGCTGCTGCCGGCGCTGGCGCCGATTGTGGCGGTGGCGCTGCTGCCGGCGCTGGCGCCG |
| 161161 | ATTGTGGCGGTGGCGCTGCTGCCGGCGGTGGCGCCGATTGTGGCGGTGGCGCTGCTGCCGGCGGTGGCGCCG |
| 162162 | ATTGTGGCGCTGGCGGTGCTGCCGGCGGTGGCGCCGATTGTGGCGCTGGCGGTGCTGCCGGCGGTGGCGCCG |
| 163163 | GTGGCGGTGCTGGCGGTGCTGCCGGCGCTGGCGCCGGTGGCGGTGCTGGCGGTGCTGCCGGCGCTGGCGCCG |
| 164164 | ATTGCGGTGCTGGCGGTGGCGCCGGCGGTGCTGCCGATTGCGGGTGCTGGCGGTGGCGCCGGCGGTGCTGCCG |
| 165165 | CTGGCGGTGGCGATTATTGCGCCGGCGGTGGCGCCGCTGGCGGTGGCGATTATTGCGCCGGCGGTGGCGCCG |
| 166166 | GTGGCGCTGGCGATTGCGCTGCCGGCGGTGCTGCCGGTGGCGCTGGCGATTGCGCTGCCGGGCGGTGCTGCCG |
| 167167 | GCGATTGCGATTGCGCTGGTGCCGGTGGCGCTGCCGGCGATTGCGATTGCGCTGGTGCCGGTGGCGCTGCCG |
| 168168 | GCGGCGGTGGTGATTGTGGCGCCGGTGGCGCTGCCGGGCGGCGGTGGTGATTGTGGCGCCGGTGGCGCTGCCG |
| 169169 | GTGGCGATTATTGTGGTGGCGCCGGCGCTGGCGCCGGTGGCGATTATTGTGGTGGCGCCGGCGCTGGCGCCG |
| 170170 | GTGGCGCTGCTGGCGATTGCGCCGGCGCTGGCGCCGGTGGGCCTGCTGGGCGATTGCGCCGGCGCTGGCGCCG |
| 171171 | GTGGCGGTGCTGATTGCGGTGCCGGCGCTGGCGCCGGTGGCGGTGCTGATTGCGGTGCCGGCGCTGGCGCCG |
| 172172 | GCGGTGGCGCTGGCGGTGCTGCCGGCGGTGGTGCCGGCGGTGGCGCTGGCGGTGCTGCCGGCGGTGGTGCCG |
| 173173 | GCGGTGGCGCTGGCGGTGGTGCCGGCGGTGCTGCCGGCGGTGGCGCTGGCGGTGGTGCCGGCGGTGCTGCCG |
| 174174 | ATTGTGGTGATTGCGGTGGCGCCGGCGGTGGCGCCGATTGTGGTGATTGCGGTGGCGCCGGCGGTGGCGCCG |
| 175175 | ATTGTGGTGGCGGCGGTGGTGCCGGCGCTGGCGCCGATTGTGGTGGCGGCGGTGGTGCCGGCGCTGGCGCCG |
| 176176 | ATTGTGGCGCTGGTGCCGGCGGTGGCGATTGCGCCGATTGTGGCGCTGGTGCCGGCGGTGGCGATTGCGCCG |
| 177177 | GTGGCGGCGCTGCCGGCGGTGGCGCTGGTGGTGCCGGTGGCGGCGCTGCCGGCGGTGGCGCTGGTGGTGCCG |
| 178178 | CTGGTGGCGATTGCGCCGCTGGCGGTGCTGGCGCCGCTGGTGGCGATTGCGCCGCTGGCGGTGCTGGCGCCG |
| 179179 | GCGGTGGCGCTGGTGCCGGTGATTGTGGCGGCGCCGGCGGTGGCGCTGGTGCCGGTGATTGTGGCGGCGCCG |
| 180180 | GCGATTGCGGTGGCGATTGCGCCGGTGGCGCTGCCGGCGATTGCGGTGGCGATTGCGCCGGTGGCGCTGCCG |
| 181181 | GCGATTGCGCTGGCGGTGCCGGTGCTGGCGCTGCCGGCGATTGCGCTGGCGGTGCCGGTGCTGGCGCTGCCG |
| 182182 | CTGGTGCTGATTGCGGCGGCGCCGATTGCGCTGCCGCTGGTGCTGATTGCGGCGGCGCCGATTGCGCTGCCG |
| 183183 | CTGGTGGCGCTGGCGGTGCCGGCGGCGGTGCTGCCGCTGGTGGCGCTGGCGGTGCCGGCGGCGGTGCTGCCG |
| 184184 | GCGGTGGCGCTGGCGGTGCCGGCGCTGGTGCTGCCGGCGGTGGCGCTGGCGGTGCCGGCGCTGGTGCTGCCG |
| 185185 | CTGGTGGTGCTGGCGGCGGCGCCGCTGGCGGTGCCGCTGGTGGTGCTGGCGGCGGCGCCGCTGGCGGTGCCG |
| 186186 | CTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCGCTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCG |
| 187187 | GTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCGGTGATTGTGCTGGCGGCGCCGGCGCTGGCGGCGCCG |
| 188188 | GCGGTGGTGCTGGCGGTGCCGGCGCTGGCGGTGCCGGCGGTGGTGCTGGCGGTGCCGGCGCTGGCGGTGCCG |
| 189189 | CTGATTATTGTGGCGGCGGCGCCGGCGGTGGCGCCGCTGATTATTGTGGCGGCGGCGCCGGGCGGTGGCGCCG |
| 190190 | ATTGTGGCGGTGATTGTGGCGCCGGCGGTGGCGCCGATTGTGGCGGTGATTGTGGGCCCGGGCGGTGGCGCCG |
| 191191 | CTGGTGGCGCTGGCGGCGCCGATTATTGCGGTGCCGCTGGTGGCGCTGGCGGGCCCGATTATTGCGGTGCCG |
| 192192 | ATTGCGGCGGTGCTGGCGGCGCCGGCGCTGGTGCCGATTGCGGGCGGTGCTGGCGGCGCCGGCGCTGGTGCCG |
| 193193 | ATTGCGCTGCTGGCGGCGCCGATTATTGCGGTGCCGATTGCGCTGCTGGCGGCGCCGATTATTGCGGTGCCG |
| 194194 | GCGGCGCTGGCGCTGGTGGCGCCGGTGATTGTGCCGGCGGCGCTGGCGCTGGTGGCGCCGGTGATTGTGCCG |
| 195195 | ATTGCGCTGGTGGCGGCGCCGGTGGCGCTGGTGCCGATTGCGCTGGGTGGCGGCGCCGGTGGCGCTGGTGCCG |
| 196196 | ATTATTGTGGCGGTGGCGCCGGCGGCGATTGTGCCGATTATTGTGGGCGGTGGCGCCGGCGGCGATTGTGCCG |
| 197197 | GCGGTGGCGGCGATTGTGCCGGTGATTGTGGCGCCGGCGGTGGCGGCGATTGTGCCGTGATTGTGGGCCCG |
| 198198 | GCGGTGCTGGTGCTGGTGGCGCCGGCGGCGGCGCCGGCGGTGCTGGTGCTGGTGGCGCCGGCGGCGGCGCCG |
| 199199 | GTGGTGGCGCTGCTGGCGCCGCTGATTGCGGCGCCGGTGGTGGCGCTGCTGGCGCCGCTGATTGCGGGCCCG |
| 200200 | GCGGCGGTGGTGATTGCGCCGCTGCTGGCGGTGCCGGGCGGCGGTGGTGATTGCGCCGCTGCTGGCGGTGCCG |
| 201201 | ATTGCGGTGGCGGTGGCGGCGCCGCTGCTGGTGCCGATTGCGGGTGGCGGTGGCGGCGCCGCTGCTGGTGCCG |
| 202202 | CTGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCGCTGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCG |
| 203203 | GCGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCGGCGGTGGCGATTGTGGTGCTGCCGGCGGTGGCGCCG |
| 204204 | GCGGTGATTCTGCTGGCGCCGCTGATTGCGGCGCCGGCGGTGATTCTGCTGGCGCGCTGATTGCGGGCCCG |
| 205205 | CTGGTGATTGCGCTGGCGGCGCCGGTGGCGCTGCCGCTGGTGATTGCGCTGGCGGCGCCGGTGGCGCTGCCG |
| 206206 | GTGCTGGCGGTGGTGCTGCCGGCGGTGGCGCTGCCGGTGCTGGCGGTGGTGCTGCCGGCGGTGGCGCTGCCG |
| 207207 | GTGCTGGCGGTGGCGGCGCCGGCGGTGCTGCTGCCGGTGCTGGCGGTGGCGGCGCCGGCGGTGCTGCTGCCG |
| 208208 | GCGGCGGTGGTGCTGCTGCCGATTATTGCGGCGCCGGCGGCGGTGGTGCTGCTGCCGATTATTGCGGGCCCG |
| 209209 | GCGCTGCTGGTGATTGCGCCGGCGATTGCGGTGCCGGCGCTGCTGGTGATTGCGCCGGGCGATTGCGGTGCCG |
| 210210 | GCGGTGCTGGTGATTGCGGTGCCGGCGATTGCGCCGGCGGTGCTGGTGATTGCGGTGCCGGCGATTGCGCCG |
| 211211 | GCGCTGCTGGTGGTGATTGCGCCGCTGGCGGCGCCGGCGCTGCTGGTGGTGATTGCGCCGCTGGCGGCGCCG |
| 212212 | GTGCTGGTGGCGGCGATTCTGCCGGCGGCGATTCCGGTGCTGTGGCGGCGATTCTGCCGGCGGCGATTCCG |
| 213213 | GTGCTGGTGGCGGCGGTGCTGCCGATTGCGGCGCCGGTGCTGGTGGCGGCGGTGCTGCCGATTGCGGGCCCG |
| 214214 | GTGCTGGCGGCGGCGGTGCTGCCGCTGGTGGTGCCGGTGCTGGCGGCGGCGGTGCTGCCGCTGGTGGTGCCG |
| 215215 | GCGATTGCGATTGTGGTGCCGGCGGTGGCGGTGCCGGCGATTGCGATTGTGGTGCCGGCGGTGGCGGTGCCG |
| 216216 | GTGGCGATTATTGCGGTGCCGGCGGTGGTGGCGCCGGTGGCGATTATTGCGGTGCCGGCGGTGGTGGCGCCG |
| 217217 | ATTGTGGCGCTGGTGGCGCCGGCGGCGGTGGTGCCGATTGTGGCGCTGGTGGCGCCGGCGGCGGTGGTGCCG |
| 218218 | GCGGCGATTGTGCTGCTGCCGGCGGTGGTGGTGCCGGCGGCGATTGTGCTGCTGCCGGCGGTGGTGGTGCCG |
| 219219 | GCGGCGCTGATTGTGGTGCCGGCGGTGGCGGTGCCGGCGGCGCTGATTGTGGTGCCGGCGGTGGCGGTGCCG |
| 220220 | GCGATTGCGCTGGTGGTGCCGGCGGTGGCGGTGCCGGCGATTGCGCTGGTGGTGCCGGCGGTGGCGGTGCCG |
| 221221 | CTGGCGATTGTGCCGGCGGCGATTGCGGCGCTGCCGCTGGCGATTGTGCCGGCGGCGATTGCGGCGCTGCCG |
| 222222 | CTGGTGGCGATTGCGCCGGCGGTGGCGGTGCTGCCGCTGGTGGCGATTGCGCCGGCGGTGGCGGTGCTGCCG |
| 223223 | GTGCTGGCGGTGGCGCCGGCGGTGGCGGTGCTGCCGGTGCTGGCGGTGGCGCCGGCGGTGGCGGTGCTGCCG |
| 224224 | ATTCTGGCGGTGGTGGCGATTCCGGCGGCGGCGCCGATTCCTGGCGGTGGTGGCGATTCCGGCGGCGGGCCCG |
| 225225 | ATTCTGGTGGCGGCGGCGCCGATTGCGGCGCTGCCGATTCTGGTGGCGGCGGCGCCGATTGCGGCGCTGCCG |
| 226226 | ATTCTGGCGGTGGCGGCGATTCCGGCGGCGCTGCCGATTCCTGGCGGTGGCGGCGATTCCGGCGGCGCTGCCG |
| 227227 | GTGATTGCGATTCCGGCGATTCTGGCGGCGGCGCCGGTGATTGCGATTCCGGCGATTCTGGCGGCGGGCCCG |
| 228228 | GCGATTATTATTGTGGTGCCGGCGATTGCGGCGCCGGCGATTATTATTGTGGTGCCGGCGATTGCGGGCCCG |
| 229229 | GCGATTCTGATTGTGGTGGCGCCGATTGCGGCGCCGGCGATTCTGATTGTGGTGGCGCCGATTGCGGGCCCG |
| 230230 | GCGGTGATTGTGCCGGTGGCGATTATTGCGGCGCCGGCGGTGATTGTGCCGGTGGCGATTATTGCGGGCCCG |
| 231231 | GCGGTGGTGATTGCGCTGCCGGCGGTGGTGGCGCCGGCGGTGGTGATTGCGCTGCCGGCGGTGGTGGCGCCG |
| 232232 | GCGCTGGTGGCGGTGATTGCGCCGGTGGTGGCGCCGGCGCTGGTGGCGGTGATTGCGCCGGTGGTGGCGCCG |
| 233233 | GCGCTGGTGGCGGTGCTGCCGGCGGTGGCGGTGCCGGCGCTGGTGGCGGTGCTGCCGGCGGTGGCGGTGCCG |
| 234234 | GCGCTGGTGGCGCCGCTGCTGGCGGTGGCGGTGCCGGCGCTGGTGGCGCCGCTGCTGGCGGTGGCGGTGCCG |
| 235235 | GCGGTGCTGGCGGTGGTGGCGCCGGTGGTGGCGCCGGCGGTGCTGGCGGTGGTGGCGCCGGTGGTGGCGCCG |
| 236236 | GCGGTGATTGCGGTGGCGCCGCTGGTGGTGGCGCCGGCGGTGATTGCGGTGGCGCGCTGGTGGTGGCGCCG |
| 237237 | GCGGTGATTGCGCTGGCGCCGGTGGTGGTGGCGCCGGCGGTGATTGCGCTGGCCGCCGGTGGTGGTGGCGCCG |
| 238238 | GTGGCGATTGCGCTGGCGCCGGTGGTGGTGGCGCCGGTGGCGATTGCGCTGGCGCCGTGGTGGTGGCGCCG |
| 239239 | GTGGCGCTGGCGCTGGCGCCGGTGGTGGTGGCGCCGGTGGCGCTGGCGCTGGCGCCGGTGGTGGTGGCGCCG |
| 240240 | GTGGCGGCGCTGCTGCCGGCGGTGGTGGTGGCGCCGGTGGCGGCGCTGCTGCCGGCGGTGGTGGTGGCGCCG |
| 241241 | GTGGCGCTGGCGCTGCCGGCGGTGGTGGTGGCGCCGGTGGCGCTGGCGCTGCCGGCGGTGGTGGTGGCGCCG |
| 242242 | GTGGCGCTGCTGGCGCCGGCGGTGGTGGTGGCGCCGGTGGCGCTGCTGGCGCCGGCGGTGGTGGTGGCGCCG |
4. SDB 서열 (SEQ ID: 243)4. SDB sequence (SEQ ID: 243)
ATGGCGGAAC AGAGCGACAA GGATGTGAAA TACTATACCC TGGAGGAAAT CCAAAAGCAC AAAGACAGCA AGAGCACCTG GctgATTCTG CACCACAAGG TTTATGATCT GACCAAATTC CTGGAGGAAC ATCCGGGTGG TGAGGAAGTG CTGGGTGAAC AAGCGGGTGG CGACGCGACC GAGAACTTTG AAGACGTTGG CCACAGCACC GATGCGCGTG AGCTGAGCAA AACCTACATC ATTGGTGAAC TGCACCCGGA CGATCGTAGC AAGATTGCGA AACCGAGCGA AACCCTGATGGCGGAAC AGAGCGACAA GGATGTGAAA TACTATACCC TGGAGGAAAT CCAAAAGCAC AAAGACAGCA AGAGCACCTG GctgATTCTG CACCACAAGG TTTATGATCT GACCAAATTC CTGGAGGAAC ATCCGGGTGG TGAGGAAGTG CTGGGTGAAC AAGCGGGTGG CGACGCGACC GAGAACTTTG AAGACGTTGG CCACAGCACC GATGCGCGTG AGCTGAGCAA AACCTACATC ATTGGTGAAC TGCACCCGGA CGATCGTAGC AAGATTGCGA AACCGAGCGA AACCCTG
5. CMV 서열 (SEQ ID: 244)5. CMV sequence (SEQ ID: 244)
TA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTGGTT TAGTGAACCG TCAGATCTA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGC CAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAA CGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTGGTT TAGTGAACCG TCAGATC
6. Parkin 코딩 서열 (SEQ ID: 245)6. Parkin coding sequence (SEQ ID: 245)
CAAGAAATGA ACGCGACCGG TGGCGACGAT CCGCGTAACG CGGCGGGTGG CTGCGAGCGT GAACCGCAGA GCCTGACCCG TGTGGACCTG AGCAGCAGCG TGCTGCCGGG TGATAGCGTG GGCCTGGCGG TTATCCTGCA CACCGACAGC CGTAAGGATA GCCCGCCGGC GGGTAGCCCG GCGGGCCGTA GCATTTATAA CAGCTTTTAC GTGTATTGCA AGGGTCCGTG CCAGCGTGTG CAACCGGGTA AACTGCGTGT TCAGTGCAGC ACCTGCCGTC AAGCGACCCT GACCCTGACC CAGGGTCCGA GCTGCTGGGA CGATGTTCTG ATCCCGAACC GTATGAGCGG CGAGTGCCAA AGCCCGCACT GTCCGGGCAC CAGCGCGGAG TTCTTCTTCA AGTGCGGTGC GCACCCGACC AGCGACAAAG AAACCAGCGT GGCGCTGCAC CTGATCGCGA CCAACAGCCG TAACATCACC TGCATTACCT GCACCGACGT TCGTAGCCCG GTGCTGGTTT TTCAGTGCAA CAGCCGTCAC GTGATTTGCC TGGATTGCTT CCACCTGTAT TGCGTGACCC GTCTGAACGA CCGTCAGTTT GTTCACGATC CGCAACTGGG TTATAGCCTG CCGTGCGTTG CGGGTTGCCC GAACAGCCTG ATCAAAGAAC TGCACCACTT CCGTATTCTG GGCGAGGAAC AGTACAACCG TTATCAGCAA TACGGCGCGG AGGAATGCGT GCTGCAAATG GGTGGCGTTC TGTGCCCGCG TCCGGGTTGC GGTGCGGGTC TGCTGCCGGA GCCGGACCAG CGTAAGGTGA CCTGCGAAGG TGGCAACGGC CTGGGTTGCG GCTTTGCGTT CTGCCGTGAG TGCAAAGAAG CGTATCACGA GGGTGAATGC AGCGCGGTTT TTGAGGCGAG CGGCACCACC ACCCAGGCGT ACCGTGTGGA TGAGCGTGCG GCGGAACAAG CGCGTTGGGA GGCGGCGAGC AAGGAAACCA TCAAGAAAAC CACCAAGCCG TGCCCGCGTT GCCATGTGCC GGTTGAGAAG AACGGTGGCT GCATGCACAT GAAATGCCCG CAGCCGCAAT GCCGTCTGGA GTGGTGCTGG AACTGCGGTT GCGAATGGAA CCGTGTGTGC ATGGGCGACC ACTGGTTCGA TGTTCAAGAAATGA ACGCGACCGG TGGCGACGAT CCGCGTAACG CGGCGGGTGG CTGCGAGCGT GAACCGCAGA GCCTGACCCG TGTGGACCTG AGCAGCAGCG TGCTGCCGGG TGATAGCGTG GGCCTGGCGG TTATCCTGCA CACCGACAGC CGTAAGGATA GCCCGCCGGC GGGTAGCCCG GCGGGCCGTA GCATTTATAA CAGCTTTTAC GTGTATTGCA AGGGTC CGTG CCAGCGTGTG CAACCGGGTA AACTGCGTGT TCAGTGCAGC ACCTGCCGTC AAGCGACCCT GACCCTGACC CAGGGTCCGA GCTGCTGGGA CGATGTTCTG ATCCCGAACC GTATGAGCGG CGAGTGCCAA AGCCCGCACT GTCCGGGCAC CAGCGCGGAG TTCTTCTTCA AGTGCGGTGC GCACCCGACC AGCGACAAAG AAACCAGCGT GGCGCTGCAC CTGATCGCGA CCAA CAGCCG TAACATCACC TGCATTACCT GCACCGACGT TCGTAGCCCG GTGCTGGTTT TTCAGTGCAA CAGCCGTCAC GTGATTTGCC TGGATTGCTT CCACCTGTAT TGCGTGACCC GTCTGAACGA CCGTCAGTTT GTTCACGATC CGCAACTGGG TTATAGCCTG CCGTGCGTTG CGGGTTGCCC GAACAGCCTG ATCAAAGAAC TGCACCACTT CCGTATTCTG GGCGAGGAAC AGTACAACCG TTATCAGCAA TACGGCGCGG AGGA ATGCGT GCTGCAAATG GGTGGCGTTC TGTGCCCGCG TCCGGGTTGC GGTGCGGGTC TGCTGCCGGA GCCGGACCAG CGTAAGGTGA CCTGCGAAGG TGGCAACGGC CTGGGTTGCG GCTTTGCGTT CTGCCGTGAG TGCAAAGAAG CGTATCACGA GGGTGAATGC AGCGCGGTTT TTGAGGCGAG CGGCACCACC ACCCAGGCGT ACCGTGTGGA TGAGCGTGCGG GCGGAACAAG CGCGTTGGGA GGCGGCGAGC AAGGAAACCA TCAAGAAAAC CACCAAGCCG TGCCCGCGTT GCCATGTGCC GGTTGAGAAG AACGGTGGCT GCATGCACAT GAAATGCCCG CAGCCGCAAT GCCGTCTGGA GTGGTGCTGG AACTGCGGTT GCGAATGGAA CCGTGTGTGC ATGGGCGACC ACTGGTTCGA TGTT
7. BGH poly A 서열 (SEQ ID: 246)7. BGH poly A sequence (SEQ ID: 246)
CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT GGGAAGAGAA TAGCAGGCAT GCTGGGGACTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT GGGAAGAGAA TAGCAGGCAT GCTGGGGA
Claims (13)
- aMTD(advanced macromolecule transduction domain) 코딩 폴리뉴클레오티드 서열, Parkin 단백질 코딩 폴리뉴클레오티드 서열을 포함하는 것인, AAV 벡터.An AAV vector comprising an advanced macromolecule transduction domain (aMTD) coding polynucleotide sequence and a Parkin protein coding polynucleotide sequence.
- 제1항에 있어서, According to paragraph 1,상기 aMTD 코딩 폴리뉴클레오티드 서열은 서열번호 3 내지 242의 폴리뉴클레오티드 서열인 것인, AAV 벡터.The aMTD coding polynucleotide sequence is a polynucleotide sequence of SEQ ID NOs: 3 to 242.
- 제1항에 있어서, According to paragraph 1,상기 벡터는 하나 이상의 가용화 도메인(solubilization domain; SD)의 폴리뉴클레오티드 서열을 추가로 포함하는 것인, AAV 벡터.An AAV vector, wherein the vector further includes a polynucleotide sequence of one or more solubilization domains (SD).
- 제3항에 있어서,According to paragraph 3,상기 SD의 폴리뉴클레오티드 서열은 서열번호 243의 폴리뉴클레오티드 서열인 것인, AAV 벡터.An AAV vector, wherein the polynucleotide sequence of the SD is the polynucleotide sequence of SEQ ID NO: 243.
- 제1항에 있어서, According to paragraph 1,상기 Parkin 단백질 코딩 폴리뉴클레오티드 서열은 서열번호 245의 폴리뉴클레오티드 서열인 것인, AAV 벡터.An AAV vector, wherein the Parkin protein-encoding polynucleotide sequence is the polynucleotide sequence of SEQ ID NO: 245.
- 제1항에 있어서,According to paragraph 1,상기 벡터는 서열번호 244의 CMV 프로모터를 포함하는 것인, AAV 벡터.The vector is an AAV vector containing the CMV promoter of SEQ ID NO: 244.
- 제1항에 있어서,According to paragraph 1,상기 벡터는 서열번호 246의 BGH(bovine growth hormone) poly A 서열을 포함하는 것인, AAV 벡터.The vector is an AAV vector containing the BGH (bovine growth hormone) poly A sequence of SEQ ID NO: 246.
- 제1항에 있어서,According to paragraph 1,상기 벡터는 서열번호 2의 폴리뉴클레오티드 서열을 포함하는 AAV 벡터.The vector is an AAV vector containing the polynucleotide sequence of SEQ ID NO: 2.
- 제1항 내지 제8항 중 어느 한 항의 벡터가 형질전환된 숙주세포.A host cell transformed with the vector of any one of claims 1 to 8.
- 제1항 내지 제8항의 벡터를 포함하는 퇴행성 뇌질환 치료용 약학적 조성물.A pharmaceutical composition for treating degenerative brain diseases comprising the vector of claims 1 to 8.
- 제10항에 있어서, According to clause 10,상기 퇴행성 뇌질환은 파킨슨병, 알츠하이머병, 헌팅턴병, 경도인지장애(mild cognitive impairment), 대뇌 아밀로이드 맥관병중, 다운증후군, 아밀로이드성 뇌졸증(stroke), 전신성 아밀로이드병, 더취(Dutch)형 아밀로이드증, 니만-픽병, 노인성 치매, 근위축성 측삭 경화증 (amyotrophic lateral sclerosis), 척수소뇌성 운동실조증(Spinocerebellar Atrophy), 뚜렛 증후군 (Tourette`s Syndrome), 프리드리히 보행실조 (Friedrich`s Ataxia), 마차도-조셉 병 (Machado-Joseph`s disease), 루이 소체 치매 (Lewy Body Dementia), 근육긴장이상 (Dystonia), 진행성 핵상 마비(Progressive Supranuclear Palsy) 및 전두측두엽 치매(Frontotemporal Dementia)를 포함하는 것인, 약학적 조성물.The degenerative brain diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann- Pick's disease, senile dementia, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph disease A pharmaceutical composition comprising -Joseph's disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy, and Frontotemporal Dementia.
- 제1항 내지 제8항의 벡터, 제9항의 숙주세포 및/또는 제10항의 약학적 조성물을 대상체에게 투여하는 단계를 포함하는 퇴행성 뇌질환의 예방 또는 치료 방법.A method for preventing or treating a degenerative brain disease comprising administering the vector of claims 1 to 8, the host cell of claim 9, and/or the pharmaceutical composition of claim 10 to a subject.
- 제12항에 있어서, According to clause 12,상기 투여는 정맥내(intravenous), 비경구(parenteral), 경피(transdermal), 피하(subcutaneous), 근육내(intramuscular), 두개내(intracranial), 안와내(intraorbital), 안내(intraocular), 뇌실내(intracerebroventricular), 뇌실질내 주사(intracerabral injection), 피막내(intracapsular), 척수강내(intrathecal), 수조내(intracisternal), 복강내(intraperitoneal), 비강내(intranasal), 직장내(intrarectal), 질내(intravaginal), 분무(spraying) 또는 경구(oral) 투여를 포함하는 것인, 방법.The administration may be administered intravenous, parenteral, transdermal, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, or intraventricular. (intracerebroventricular), intracerebral injection, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, intrarectal, intravaginal ( A method comprising intravaginal, spraying, or oral administration.
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| WO2012050402A2 (en) * | 2010-10-14 | 2012-04-19 | 주식회사 프로셀제약 | Cell-permeable recombinant parkin protein and a pharmaceutical composition for treating degenerative brain diseases containing the same |
| KR102132311B1 (en) * | 2015-07-27 | 2020-07-13 | (주)셀리버리 | Improved cell permeability (ICP) parkin recombinant proteins and uses thereof |
| WO2022050778A1 (en) * | 2020-09-04 | 2022-03-10 | Cellivery Therapeutics, Inc. | Improved cell-permeable modified parkin recombinant protein for treatment of neurodegenerative diseases and use thereof |
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| WO2012050402A2 (en) * | 2010-10-14 | 2012-04-19 | 주식회사 프로셀제약 | Cell-permeable recombinant parkin protein and a pharmaceutical composition for treating degenerative brain diseases containing the same |
| KR102132311B1 (en) * | 2015-07-27 | 2020-07-13 | (주)셀리버리 | Improved cell permeability (ICP) parkin recombinant proteins and uses thereof |
| WO2022050778A1 (en) * | 2020-09-04 | 2022-03-10 | Cellivery Therapeutics, Inc. | Improved cell-permeable modified parkin recombinant protein for treatment of neurodegenerative diseases and use thereof |
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