Composition for the prevention, improvement, or treatment of mitochondrial complex 1 disorder, comprising a recombinant adeno-associated virus vector carrying HYDRA NDI1.

Abstract

This invention relates to a composition for the prevention, improvement, or treatment of mitochondrial complex I disorder, comprising a recombinant adeno-associated virus vector carrying Hydra NDi1. The inventors have confirmed that scAAV-hydra NDi1 of this invention has the effect of restoring the decrease in cellular respiration caused by rotenone and the effect of suppressing the death of LHON patient-derived cells caused by rotenone. It is expected that this can be used not only for LHON but also for the prevention, improvement, or treatment of diseases caused by mitochondrial complex I disorder.

Description

Technical Field

[0001] The present invention relates to a composition for preventing, ameliorating or treating mitochondrial complex I abnormal diseases, including a recombinant adeno-associated virus vector carrying Hydra NDi1.

Background Art

[0002] Mitochondrial genetic diseases caused by mutations in the mitochondrial genome were first reported about 30 years ago. Since then, understanding of the numerous diseases caused by mutations in the mitochondrial genome and mitochondrial genetics involved in the transmission of the mitochondrial genome has advanced. There are about several thousand mitochondrial genomes in a cell, and most of the mutated mitochondrial genomes coexist with normal genomes in the cell, which is called heteroplasmy. The symptoms of the disease vary in severity depending on the ratio of the mutated genome. The higher the ratio of the mutated genome, that is, the higher the heteroplasmy, the more severe the symptoms appear. Similarly induced mutations in the mitochondrial genome have very diverse heteroplasmy in each tissue and individual of the human body, and many studies are still needed to elucidate the mechanism for controlling heteroplasmy.

[0003] LHON (Leber's hereditary optic neuropathy) is a mitochondrial genetic disorder characterized by sudden, painless loss of vision in both eyes due to optic nerve damage in adolescence. It was first described in 1871 by the German ophthalmologist Theodore Leber. It primarily affects males (approximately 80%), with females experiencing later onset and more severe effects. While the onset usually occurs between the ages of 18 and 30, it can appear without warning at any age, and can cause acute or subacute simultaneous or sequential loss of vision in both eyes. The incidence is not yet precisely known. LHON was the first genetic disorder discovered that is inherited through mutations in mitochondrial genes (mitochondrial DNA, mtDNA), and it is inherited only through the maternal line. Most patients have a family history from the maternal side, but 40% are sporadic.

[0004] Mitochondria are vital intracellular organelles that produce energy (ATP) used for biological activity, and unlike nuclear genes, they have independent genes. Many genetic disorders are caused by defects in nuclear genes, but some can be caused by abnormalities in mitochondrial genes. While a pair of genes located in the cell nucleus are inherited from both parents, mitochondrial genes are inherited only from the mother because mitochondria containing paternal genes, located in the tail of the sperm, are lost during fertilization and cannot enter the egg. LHON accounts for 30-50% of bilateral optic nerve disorders of unknown cause and 2% of legal blindness. Approximately 90% of LHON cases carry one of three point mutations: G11778A (approximately 70% worldwide), G3460A, or T14484C (most common in people of French-Canadian descent), and several other point mutation genes have also been identified. Most have one mutant gene (homoplasic), but rarely, some may have two or more mutant genes (heteroplasic). Although the exact mechanism of disease onset is not yet fully understood, it is believed that defective mitochondrial genes are involved in normal cell division and growth processes, leading to problems in energy production in optic nerve cells and ultimately cell destruction.

[0005] Drug treatments include vitamin C and vitamin B 12 While the administration of idebenone may mitigate retinal ganglion cell damage in patients who have developed the disease, its effect is minimal, leading to limited clinical use and the inability to provide fundamental prevention. Therefore, to date, there has been no appropriate treatment, and gene therapy is the only potential fundamental cure.

[0006] Therefore, the inventors have completed the present invention by creating a recombinant viral vector containing hydra NDi1 to effectively treat mitochondrial disorders, and by experimentally demonstrating that this can be used to prevent, improve, and treat mitochondrial disorders. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of mitochondrial disorders, comprising a recombinant viral vector carrying the hydra NDi1 gene represented by Sequence ID No. 1.

[0008] Furthermore, an object of the present invention is to provide a method for preventing or treating mitochondrial disorders, comprising the step of administering to an individual a pharmaceutical composition for the prevention or treatment of mitochondrial disorders, which includes a recombinant viral vector carrying the hydra NDi1 gene represented by Sequence ID No. 1.

[0009] However, the technical problems that the present invention aims to solve are not limited to those described above, and other problems not mentioned will be clearly understood by those with ordinary skill in the art from the following description. [Means for solving the problem]

[0010] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of mitochondrial disorders, comprising a recombinant viral vector carrying the hydra NDi1 gene represented by Sequence ID No. 1.

[0011] Furthermore, the present invention provides a method for preventing or treating mitochondrial disorders, comprising the step of administering a pharmaceutical composition containing the vector of the present invention as an active ingredient to an individual in need.

[0012] Furthermore, the present invention provides a pharmaceutical composition containing the vector of the present invention as an active ingredient for the prevention or treatment of mitochondrial disorders.

[0013] Furthermore, the present invention provides applications for the vector of the present invention for producing agents for the prevention or treatment of mitochondrial abnormalities. [Effects of the Invention]

[0014] The inventors have confirmed that the scAAV-hydra NDi1 of the present invention has the effect of restoring the decrease in cellular respiration caused by rotenone, and the effect of suppressing the death of cells derived from LHON patients caused by rotenone. It is expected that this can be used not only for LHON, but also for the prevention, improvement, or treatment of diseases caused by mitochondrial complex 1 abnormalities. [Brief explanation of the drawing]

[0015] [Figure 1] This diagram schematically illustrates the production of recombinant adeno-associated viruses such as ssAAV-2 / DJ-GFP, ssAAV-2 / DJ-IRES-GFP, or scAAV-2 / DJ-GFP. [Figure 2] This diagram illustrates the process for producing recombinant adeno-associated viruses ssAAV-2 / DJ-yeast NDi1, ssAAV-2 / DJ-hydra NDi1, and scAAV-2 / DJ-hydra NDi1. [Figure 3a] This figure shows the cleavage maps of recombinant adeno-associated virus vectors used in experiments to confirm the gene expression intensity and stability of ssAAV and scAAV. [Figure 3b] This figure shows the results of checking the degree of green fluorescence expression of ssAAV-GFP and scAAV-GFP. [Figure 4] This figure shows the results of measuring the oxygen consumption rate (OCR) after adding rotenone to dermal fibroblasts derived from LHON patients infected with ssAAV-IRES-GFP, ssAAV-yeast NDi1, or scAAV-hydra NDi1. [Figure 5a] This figure shows the results of adding rotenone to dermal fibroblasts derived from LHON patients infected with ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1, and observing the degree of cell death 1 to 4 days after rotenone treatment. [Figure 5b]This figure shows the results of adding rotenone to dermal fibroblasts derived from LHON patients infected with ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1, and observing the degree of cell death 1 to 4 days after rotenone treatment. [Figure 5c] This figure shows the results of adding rotenone to dermal fibroblasts derived from LHON patients infected with ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1, and observing the degree of cell death 1 to 4 days after rotenone treatment. [Figure 5d] This figure shows the results of adding rotenone to dermal fibroblasts derived from LHON patients infected with ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1, and observing the degree of cell death 1 to 4 days after rotenone treatment. [Best Mode for Carrying Out the Invention]

[0016] Terms used herein are provided solely for illustrative purposes and should not be construed as restrictive. Singular expressions include plural expressions unless the context clearly indicates otherwise. Terms such as “includes” or “has” herein should be understood as specifying the presence of features, figures, stages, actions, components, parts, or combinations thereof as described in the specification, without prejudice to the possibility of the presence or addition of one or more other features, figures, stages, actions, components, parts, or combinations thereof.

[0017] Unless otherwise specified, all terms used in this specification, including technical or scientific terms, shall have the same meaning as commonly understood by those of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries shall be interpreted as having a meaning consistent with the context of the relevant art and shall not be interpreted in an idealized or overly formal sense unless clearly defined in this application.

[0018] Hereinafter, the present invention will be described in more detail.

[0019] The present invention provides a pharmaceutical composition for preventing or treating mitochondrial abnormal diseases, comprising a recombinant viral vector carrying the hydra NDi1 gene represented by SEQ ID NO: 1.

[0020] In the present invention, "NDi1 (NADH dehydrogenase or NADH-ubiquinone oxidoreductase)" is a NADH dehydrogenase present in the mitochondria of organisms that do not have a large enzyme complex such as mammalian complex 1, and is responsible for the function of mitochondrial complex 1. The NDi1 transfers electrons from NADH to ubiquinone. In one embodiment of the present invention, NDi1 derived from Hydra vulgaris is used, and the NDi1 derived from Hydra vulgaris has a smaller molecular weight than NDi1 derived from Saccharomyces cerevisiae. In one embodiment of the present invention, the hydra NDi1 gene represented by SEQ ID NO: 1 below is used.

[0021] SEQ ID NO: 1: agttttagaa tttataccag cagctagtta taagcgcaaa atgccattct ctttatcatc 60 tgttctgtat cggaaaagac ttgttttatt aggaacagga tggggatgtt atagtgtgtt 120 aaaaaatatt aacaagaagc tttatgatgt gattgttgtt agtccacgaa atcattttct 180 ttttacaccc ttgttaaata gcacaactgt tgggacttta gaatttcgaa gcataattga 240 accgattcga aacactaagt ttcgggatga tcatcatttt caattggctg aagcaattca 300 cttaaatcca catgataaat tagttgtttg taaagcagtc tcatctgata aagaatatac 360 tttatcatac gataaagttg taattggtgt tggagctgtt agtaatactt ttggaattcc 420 tggtgtccca aagtatgcat attttttgaa agaaatagca gatgctagaa aaatacgtaa 480 tcaaatcata tctaattttg agcaaagttt gtttccat gtaaatgaag aggaaaggtt 540 atcactgttg cattttgtca ttgttggtgg tggtccaact ggtattgaat ttggagctga 600 gttgtatgac tttataacac atgatgttgc aagacttttt ccaggagaga agaatgatgt 660 tcatgtaaca ttagttgaag gagattcaat attaccttca tttgaccagc gattacgtaa 720 atttgctgag agaaaaaatta cacaaagaga aaatttccac ttaatcaaag attttgttgt 780 ggaagttggt gaaaactatg tgaagttgaa atcaggaaaa gttcttttaa ctaagcttgt 840 agtgtggtca accggtcttg gtcccagaaa gtttatagaa agtttagacc tacctaaagg 900 taaatcaaaa caactcaagg tagatgatca tcttcgtgta gttggttatg acagtatatt 960 tgcaattggt gattgttcat acatagatgg atctccttta ccaagcacgg ctcaagtggc 1020 tgaaagacaa ggtcgatatg ttgcacagta cttatcctta ttagaaacaa attctaaaac 1080 agacagtaaa ccatttatgt ggtccaatgc tggtatgctt gcatatattg gtggatataa 1140 agctgttgca gatcttccaa caaaagctgg gaagataact ggatttaaaa gttggttaat 1200 atggcgatcg gtttatctta ctcgcttagg gagctggaga aatcgaatgc aagttccatt 1260 tgactgggct aggacttttt ttttggcag ggatatatct aggttttaaa ttacaaactt 1320 tatttaatat ttagtatttt ttgtatctta ttaatttgtt tttaa

[0022] In the present invention, a "recombinant vector" means a gene construct containing essential regulatory elements operably linked to express a gene insert, and can be a plasmid vector, cosmid vector, bacteriophage vector, or viral vector. According to one embodiment of the present invention, it may be a viral vector, specifically an adenovirus vector, an adeno-associated virus vector, or a retrovirus vector, and more specifically an adeno-associated virus vector, but is not limited thereto.

[0023] In the present invention, the recombinant viral vector contains the hydra NDi1 gene represented by Sequence ID No. 1. The hydra NDi1 gene sequence is preferably present in a suitable expression construct. The hydra NDi1 gene sequence in the expression construct is preferably operatively linked to a promoter.

[0024] In the present invention, "operably bound" means a functional binding between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby the regulatory sequence regulating the transcription and / or translation of the other nucleic acid sequence.

[0025] In the present invention, any promoter capable of initiating the expression of hydra NDi1 can be used as the promoter bound to the hydra NDi1 gene sequence, but it is preferable to select one that operates in animal cells, more specifically mammalian cells, and can regulate the transcription of the hydra NDi1 gene sequence. The promoter includes, but is not limited to, promoters derived from mammalian viruses and promoters derived from the genome of mammalian cells, and includes, for example, the U6 promoter, T7 promoter, H1 promoter, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothione promoter, β-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter, inducible promoter, or tissue-specific promoter (e.g., albumin promoter). The vector can be manufactured using genetic engineering techniques well known in the art, and enzymes and the like, which are generally known in the art, can be used for site-specific DNA cleavage and ligation.

[0026] In the present invention, the adeno-associated virus (AAV) is a single-stranded DNA virus belonging to the parvovirus family. It cannot replicate on its own, but is a deficient virus that can replicate and proliferate when infected together with helper viruses such as adenovirus, vaccinia, or herpesvirus. The adeno-associated virus can infect both dividing and non-dividing cells. In one embodiment of the present invention, the adeno-associated virus may be a self-complementary AAV (scAAV), which has high expression stability and intensity, and low immunogenicity.

[0027] In the present invention, the recombinant viral vector can suppress the decrease in cellular respiration or cell death caused by mitochondrial complex 1 abnormalities, but is not limited thereto. The mitochondrial complex 1 abnormalities can be caused by ND1, ND4, or ND6 gene mutations, but is not limited thereto.

[0028] In the present invention, "mitochondrial disorder" refers to a disease caused by mutations in mitochondrial DNA (mtDNA, mitochondrial genes), resulting in various symptoms due to morphological and biochemical changes in mitochondria in various tissues. The mitochondrial disorder may be caused by abnormalities in mitochondrial complex 1, but is not limited thereto. Furthermore, the mitochondrial disorder may be caused by mutations in one or more genes selected from the group consisting of ND1 (NADH dehydrogenase subunit 1), ND4 (NADH dehydrogenase subunit 4), and ND6 (NADH dehydrogenase subunit 6), but is not limited thereto. Furthermore, the aforementioned mitochondrial disorders include Leber's hereditary optic neuropathy (LHON), MELAS (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episode) syndrome, neurogenic muscle weakness, MERRF (Myoclonic Epilepsy with Ragged Red Fiber) syndrome, mitochondrial encephalomyopathy, Leigh syndrome, and Kearns-Sayre syndrome. The following conditions can be selected from the group consisting of Leber's hereditary optic neuropathy, multiple sclerosis, encephalomyelitis, neuromyositis, peripheral neuropathy, Friedreich's ataxia, Alpers' disease, stroke, migraine, psychosis, depression, seizures, dementia, stroke, optic nerve atrophy, optic neuropathy, glaucoma, retinitis pigmentosa, cataracts, hyperaldosteronism, hypoparathyroidism, muscle disease, muscle atrophy, myoglobinuria, hypotonia, muscle pain, decreased exercise tolerance, renal tubulopathy, renal insufficiency, hepatic failure, hepatomegaly, sideroblastic anemia, neutropenia, thrombocytopenia, diarrhea, chorionic atrophy, multiple vomiting, dysphagia, constipation, sensorineural hearing loss, intellectual disability, epilepsy, Alzheimer's disease, Parkinson's disease, and Huntington's disease, preferably Leber's hereditary optic neuropathy.The following conditions can be selected from the group consisting of optic neuropathy (LHON), MELAS (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episode) syndrome, neurogenic muscle weakness, MERRF (Myoclonic Epilepsy with Ragged Red Fiber) syndrome, mitochondrial encephalomyopathy, Leigh syndrome, and Kearns-Sayre syndrome. In one embodiment of the present invention, Leber's hereditary optic neuropathy (LHON) was used, but the invention is not limited thereto.

[0029] In the present invention, Leber's hereditary optic neuropathy (LHON), also known as Leber's optic nerve atrophy, refers to a disease in which mitochondrial complex 1 does not function properly due to mutations in mitochondrial ND1, ND4, or ND6 mtDNA (mitochondria-DNA), resulting in abnormalities in the optic nerve and blindness.

[0030] The content of the vector of the present invention in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the progression of the symptoms, the patient's condition, etc. For example, it may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the weight of the total composition, but is not limited thereto. The content ratio is a value based on the dry weight after removing the solvent.

[0031] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film coating substances, and release control additives.

[0032] The pharmaceutical compositions according to the present invention can be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, spirits, lozenges, aromatic perfumes, lemonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dried extracts, liquid extracts, injections, capsules, perfusion solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, respectively, according to conventional methods. The external preparations may have formulation forms such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasms.

[0033] Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0034] When formulating the product, it can be prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, or other diluents or excipients.

[0035] Additives for tablets, powders, granules, capsules, pills, and lozenges according to the present invention include corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, monocalcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, excipients such as propylene glycol, casein, calcium lactate, and Primogel; gelatin, acacia gum, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, carboxymethylcellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used as binders; hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum (Guar Disintegrants such as gum, baking soda, polyvinylpyrrolidone, calcium phosphate, pregelatinized starch, acacia gum, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium powder, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, polyethylene glycol 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid esters, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

[0036] Possible additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose fatty acid esters, polyoxyethylene sorbitan fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamin, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, and the like.

[0037] The syrup according to the present invention may contain a sucrose solution, other sugars, or sweeteners, and may also contain fragrances, colorants, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc., as needed.

[0038] Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc., can be used as needed.

[0039] The suspending agent according to the present invention may use suspending agents such as acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0040] The injectable preparation according to the present invention contains solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's solution, glucose injection, glucose + sodium chloride injection, polyethylene glycol (PEG), Ringer's lactate injection, ethanol, propylene glycol, non-volatile oils (sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil), ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidinedione, propylene glycol, twins, nicotinamide, hexamine, and dimethylacetamide; weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate) It may contain buffering agents such as ammonium, organic compounds, proteins, albumin, peptone, and gums; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium pyrosulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; antioxidants such as 0.1% sodium bisulfite, formaldehyde sodium sulfite, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Twin 80, and aluminum monostearate.

[0041] The suppositories according to the present invention include cocoa butter, lanolin, vitepsol, polyethylene glycol, glycerol gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, twin or span, Imhausen, monolen (propylene glycol monostearate), glycerin, Adeps solidus, Butyrum Tego-G, and Cebes Pharma 16. 16) Hexalide Base 95, Cotomar, Hydrocoat SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocoat 25, Hydrocoat 711, Idropostal, Massa Estralium Bases such as estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Maspol, Maspol-15, Neospostal-N, Paramount-B, Sposyl (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0042] Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0043] Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are frequently used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0044] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined according to factors including the type and severity of the patient’s disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and elimination rate, the duration of treatment, drugs used concurrently, and other factors known in the pharmaceutical field.

[0045] The pharmaceutical compositions according to the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that provides the greatest effect with the minimum amount without side effects, which can be easily determined by a person of the art to which the present invention belongs.

[0046] The pharmaceutical composition of the present invention can be administered to an individual by a variety of routes. Any form of administration is conceivable, for example, by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ophthalmic administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

[0047] The pharmaceutical composition of the present invention is determined by the type of drug that is the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

[0048] In this invention, "individual" means an animal that requires treatment for a disease, and more specifically, it means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0049] In this invention, "administration" means providing a predetermined composition of the present invention to an individual by any suitable method.

[0050] In the present invention, "prevention" means any action that suppresses or delays the onset of the target disease; "treatment" means any action by which the administration of the pharmaceutical composition according to the present invention improves or favorably alters the target disease and its associated metabolic abnormalities; and "improvement" means any action by which the administration of the composition according to the present invention reduces parameters related to the desired disease, such as the severity of symptoms.

[0051] The following examples are provided to aid in understanding the present invention. However, these examples are provided solely to facilitate understanding of the present invention and do not limit the scope of the present invention. [Modes for carrying out the invention]

[0052] [Examples] Example 1. Preparation of recombinant AAV (Adeno-associated virus) Recombinant AAV (Adeno-associated virus) is produced by triple transfection of HEK293 cells with a vector. Specifically, the first vector (pHelper vector) is included in all cases, the subtype is determined by the second vector (pRC vector), and the single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) are determined by the third vector. The gene of interest (GOI) is incorporated into the third vector. pAAV-DJ was used as the pRC vector, and GFP (Green fluorescent protein), IRES (internal ribosome entry site)-GFP, yeast NDi1 (GenBank: X61590.1), and hydra NDi1 (NCBI Reference Sequence: XM_002156815.3) were used as the gene of interest. The vector maps used in the experiment are shown in Figures 1 and 2.

[0053] All AAV preparation vectors were purchased from Cell Biolabs (San Diego, CA), and AAV-293 (Agilent, Santa Clara, CA) was used for 293 cells. 293 cells were cultured in 150mm dishes until approximately 90% confluence was reached. Then, the first, second, and third vectors were thoroughly mixed in proportions of 20 μg, 10 μg, and 10 μg, respectively. This mixture was then mixed with CaCl2 solution and uniformly sprayed onto the 293 cells. After 3 days, the 293 cells were collected using a cell scraper, purified using the AAVpro Purification Kit (Takara, San Jose, CA), and then the titer was measured using the company's AAVpro Titration Kit. The cells were then stored at -80°C until use.

[0054] Example 2. Confirmation of gene expression intensity and stability of scAAV To confirm the intensity and stability of gene expression induced by scAAV and ssAAV, fibroblasts were infected with scAAV-GFP and ssAAV-GFP, and the degree of green fluorescence expression was observed. The recombinant AAV shown in Figure 3a was used in the experiment. Specifically, skin fibroblasts derived from LHON patients were cultured in a 12-well plate until approximately 70% confluence was reached, and then approximately 10 ssAAV-2 / DJ-GFP or scAAV-2 / DJ-GFP were added per well. 9 Cells were infected by adding viral genomes (vg) one by one. Three days later, the expression level of GFP induced by each recombinant AAV in infected cells was observed under a FITC filter using an inverted fluorescence microscope. The results are shown in Figure 3b.

[0055] As shown in Figure 3b, it was observed that scAAV-GFP expressed green fluorescence more strongly in cells than ssAAV-GFP. This means that scAAV provides stable gene expression and high gene expression intensity.

[0056] Example 3. Confirmation of the cellular respiration restorative effect of scAAV-hydra NDi1. In a 24-well plate specifically for cellular respiration measurement, dermal fibroblasts derived from LHON patients were cultured until approximately 50% confluence was reached. Then, ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1 were each added to approximately 10 wells. 8Cells were infected with vg titer. In the culture medium, the oxygen consumption rate (OCR) rapidly decreases upon addition of rotenone, a complex I-specific inhibitor. Therefore, rotenone was added to cells 6 days after AAV infection to confirm the inhibitory effect of yeast NDi1 and hydra NDi1 on the reduction of cellular respiration. Cellular respiration was evaluated by measuring the oxygen consumption rate (OCR) using Agilent's Seahorse XF-24. The results are shown in Figure 4.

[0057] As shown in Figure 4, scAAV-hydra NDi1 more efficiently suppressed the decrease in rotenone-mediated cellular respiration compared to ssAAV-yeast NDi1.

[0058] Example 4. Confirmation of the cell death inhibitory effect of scAAV-hydra Ndi1 After culturing dermal fibroblasts from LHON patients in a 24-well plate until approximately 70% confluence was reached, ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1 were added to each well in approximately 10 units. 9 The cells were infected with VG titer. Rotenone treatment was performed 6 days later, and observation of cell death began 1 day after rotenone treatment. The results are shown in Figures 5a-5d. The presence or absence of cell death was determined by comprehensively considering the shape and morphology of individual cells, the degree of adhesion to the substrate, and the strength of contact with adjacent cells.

[0059] As shown in Figure 5a, one day after rotenone treatment, some cells in the control group (untransduced cells or ssAAV-IRES-GFP infected cells) began to die at 5 nM rotenone, while cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 showed slight cell death even at 20 nM rotenone. On the other hand, under the same conditions, almost no cell death was observed in the group infected with scAAV-hydra NDi1.

[0060] As shown in Figure 5b, two days after rotenone treatment, almost all cells in the untransduced cell population or the cell population infected with ssAAV-IRES-GFP died even with 5 nM rotenone, while cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 showed slight cell death with 20 nM rotenone. On the other hand, under the same conditions, almost no cell death was observed in the cell population infected with scAAV-hydra NDi1.

[0061] As shown in Figure 5c, three days after rotenone treatment, untransduced cells or cells infected with ssAAV-IRES-GFP were completely killed by 5 nM rotenone, while cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 began to die in large numbers even at 10 nM rotenone. On the other hand, under the same conditions, cells infected with scAAV-hydra NDi1 showed almost no death even at 20 nM rotenone.

[0062] As shown in Figure 5d, four days after rotenone treatment, some cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 were partially killed at 5 nM rotenone, and almost completely killed at 10 nM. On the other hand, cells infected with scAAV-hydra NDi1 showed almost no death even at 20 nM.

[0063] This means that scAAV-hydra NDi1 is significantly more effective than ssAAV-hydra NDi1 and ssAAV-yeast NDi1 in suppressing cell death in LHON patients caused by mitochondrial complex 1 abnormalities.

[0064] Based on these results, it has been confirmed that the scAAV-hydra NDi1 of the present invention has the effect of restoring the decrease in cellular respiration caused by rotenone and the effect of suppressing the death of LHON patient-derived cells caused by rotenone. Therefore, it is expected that this can be used not only for LHON but also for the prevention, improvement, or treatment of diseases caused by mitochondrial complex 1 abnormalities.

[0065] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that it can be readily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the embodiments described above should be understood to be illustrative and not restrictive in any way.

Claims

1. A pharmaceutical composition for the prevention or treatment of mitochondrial disorders, comprising a recombinant viral vector carrying the hydraNDi1 gene represented by Sequence ID No.

1.

2. The recombinant virus is an adeno-associated virus, wherein the pharmaceutically acceptable

3. The pharmaceutical composition for the prevention or treatment of mitochondrial disorders according to claim 2, wherein the adeno-associated virus is a self-complementary AAV.

4. The recombinant viral vector suppresses the decrease in cellular respiration, and is a pharmaceutical composition for the prevention or treatment of mitochondrial abnormalities according to claim 1.

5. The recombinant viral vector suppresses cell death, and is a pharmaceutical composition for the prevention or treatment of mitochondrial abnormalities according to claim 1.

6. The mitochondrial disorder is characterized by a mutation in one or more genes selected from the group consisting of ND1, ND4, and ND6, as described in claim 1, and is a pharmaceutical composition for the prevention or treatment of a mitochondrial disorder.

7. The mitochondrial disorder is selected from the group consisting of Leber's hereditary optic neuropathy, MELAS syndrome, neurogenic muscular atrophy, MERRF syndrome, mitochondrial encephalomyopathy, Leigh syndrome, and Kearns-Sayre syndrome, and is a pharmaceutical composition for the prevention or treatment of a mitochondrial disorder according to claim 1.

8. A method for preventing or treating mitochondrial disorders, comprising the step of administering to an individual a pharmaceutical composition for the prevention or treatment of mitochondrial disorders, comprising administering to an individual a pharmaceutical composition for the prevention or treatment of mitochondrial disorders, comprising administering to an individual a pharmaceutical composition for the prevention or treatment of mitochondrial disorders, comprising the step of

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