Composition for preventing, alleviating, or treating mitochondrial complex 1 disorders, comprising recombinant adeno-associated virus vector loaded with hydra ndi1

Abstract

The present invention relates to a composition for the prevention, alleviation, or treatment of mitochondrial complex 1 disorders, including a recombinant adeno-associated virus (AAV) vector loaded with Hydra NDi1. The inventors have confirmed that the scAAV-Hydra NDi1 of the present invention has an effect of restoring cellular respiration reduction caused by rotenone and an effect of inhibiting the apoptosis of LHON patient-derived cells by rotenone. Thus, the ScAAV-Hydra NDi1 is expected to be useful not only for LHON but also for preventing, alleviating, or treating diseases caused by abnormalities in mitochondrial complex 1.

Description

Composition for preventing, improving or treating mitochondrial complex 1 abnormality disease, comprising a recombinant adeno-associated virus vector loaded with HYDRA NDI1

[0001] The present invention relates to a composition for preventing, improving or treating mitochondrial complex 1 abnormality disease, including a recombinant adeno-associated virus vector loaded with Hydra NDi1.

[0002] Mitochondrial genetic diseases caused by mutations in the mitochondrial genome were first reported approximately 30 years ago. Since then, there has been a remarkable leap forward in understanding the numerous diseases caused by mutations in the mitochondrial genome and the mitochondrial genetics involved in the transmission of mitochondrial genomes. Approximately thousands of mitochondrial genomes exist within a cell, and most mutated mitochondrial genomes exist intermixed with normal genomes, a phenomenon known as heteroplasmy. The severity of disease symptoms varies depending on the proportion of mutated genomes. Higher proportions of mutated genomes, or heteroplasmy, correlate with more severe symptoms. The heteroplasmy produced by the same mitochondrial genome mutations varies greatly across tissues and individuals. Much research is still needed to elucidate the mechanisms that regulate heteroplasmy.

[0003] Leber's hereditary optic neuropathy (LHON) is a hereditary mitochondrial disease characterized by sudden, painless, bilateral vision loss at a young age due to characteristic optic nerve damage. It was first described in 1871 by the German ophthalmologist Theodore Leber. It affects men more often than women (approximately 80%), with women experiencing a later onset and being more severely affected. The disease typically presents between the ages of 18 and 30, but can occur at any age, with sudden or subacute vision loss in both eyes, either simultaneously or sequentially. The exact incidence is not yet known. LHON was the first genetic disorder identified, caused by mutations in the mitochondrial DNA (mtDNA), and is inherited exclusively through the maternal line. While most patients have a maternal family history, 40% of cases are spontaneous.

[0004] Mitochondria are vital intracellular structures that produce energy (ATP) used for biological activities. Unlike nuclear genes, they have independent genes. Most genetic diseases are caused by defects in nuclear genes, but some can also be caused by abnormalities in mitochondrial genes. While a pair of genes in the cell nucleus are inherited from each parent, mitochondrial genes are inherited only from the mother because the paternal genes, including the mitochondrial genes, are separated from the sperm's tail during fertilization and cannot enter the egg. LHON accounts for 30-50% of cases of idiopathic optic neuropathy that occurs in both eyes and 2% of legal blindness. Approximately 90% of LHON cases carry one of three point mutations: G11778A (approximately 70% worldwide), G3460A, and T14484C (most common in people of mixed French and Canadian descent). Many other point mutations have been identified. Most cases have a single mutated gene (homoplasic), but rare cases have two or more mutated genes (heteroplasic). While the exact pathogenesis of the disease is unknown, it is believed to involve a defect in the mitochondrial gene, which disrupts normal cell division and growth processes, causing problems with energy production in optic nerve cells and ultimately leading to cell destruction.

[0005] Vitamin C and vitamin B as drug treatment 12 , or administration of idebenone may alleviate damage to retinal ganglion cells in patients with the disease, but the effect is minimal, so it is not widely used in clinical practice, and it has the limitation of not being able to fundamentally prevent it. Therefore, there is currently no proper treatment, and gene therapy is the only fundamental treatment.

[0006] Accordingly, the present inventors constructed a recombinant viral vector containing hydra NDi1 to effectively treat mitochondrial abnormalities, and experimentally demonstrated that the vector can be used to prevent, improve, and treat mitochondrial abnormalities, thereby completing the present invention.

[0007] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating mitochondrial abnormalities, comprising a recombinant viral vector loaded with the hydra NDi1 gene represented by sequence number 1.

[0008] In addition, an object of the present invention is to provide a method for preventing or treating a mitochondrial abnormality, comprising a step of administering to a subject a pharmaceutical composition for preventing or treating a mitochondrial abnormality, the pharmaceutical composition comprising a recombinant viral vector loaded with a hydra NDi1 gene represented by sequence number 1.

[0009] However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the art from the description below.

[0010] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating mitochondrial abnormality, comprising a recombinant viral vector loaded with the hydra NDi1 gene represented by sequence number 1.

[0011] In addition, the present invention provides a method for preventing or treating a mitochondrial disorder, comprising a step of administering a pharmaceutical composition comprising the vector of the present invention as an active ingredient to a subject in need thereof.

[0012] In addition, the present invention provides a use of a pharmaceutical composition comprising the vector of the present invention as an active ingredient for preventing or treating mitochondrial abnormalities.

[0013] The present invention also provides a use of the vector of the present invention for producing a drug for preventing or treating mitochondrial abnormalities.

[0014] The present 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 inhibiting the death of cells derived from LHON patients caused by rotenone, and therefore, it is expected that it can be used for the prevention, improvement, or treatment of diseases caused by abnormalities in mitochondrial complex 1 as well as LHON.

[0015] Figure 1 is a schematic diagram showing the production of ssAAV-2 / DJ-GFP, ssAAV-2 / DJ-IRES-GFP, or scAAV-2 / DJ-GFP recombinant adeno-associated virus.

[0016] Figure 2 is a schematic diagram showing the production of ssAAV-2 / DJ-yeast NDi1, ssAAV-2 / DJ-hydra NDi1, and scAAV-2 / DJ-hydra NDi1 recombinant adeno-associated viruses.

[0017] Figure 3a shows a cleavage map of a recombinant adeno-associated virus vector used in an experiment to confirm the gene expression intensity and stability of ssAAV and scAAV.

[0018] Figure 3b shows the results of confirming the level of green fluorescence expression of ssAAV-GFP and scAAV-GFP.

[0019] Figure 4 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.

[0020] Figures 5a to 5d show 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.

[0021] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0022] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0023]

[0024] Hereinafter, the present invention will be described in more detail.

[0025] The present invention provides a pharmaceutical composition for preventing or treating mitochondrial abnormalities, comprising a recombinant viral vector loaded with a hydra NDi1 gene represented by sequence number 1.

[0026] In the present invention, "NDi1 (NADH dehydrogenase or NADH-ubiquinone oxidoreductase)" is an NADH dehydrogenase present in the mitochondria of organisms that do not have a large enzyme complex such as mammalian mitochondrial complex 1, and performs 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 was used, and the NDi1 derived from Hydra vulgaris has a smaller molecular weight than the NDi1 derived from Saccharomyces cerevisiae. In one embodiment of the present invention, the hydra NDi1 gene represented by the following SEQ ID NO: 1 was used.

[0027]

[0028] Sequence number 1:

[0029] agttttagaa tttataccag cagctagtta taagcgcaaa atgccattct ctttatcatc 60

[0030] tgttctgtat cggaaaaagac ttgttttatt aggaacagga tggggatgtt atagtgtgtt 120

[0031] aaaaaatatt aacaagaagc tttatgatgt gattgttgtt agtccacgaa atcattttct 180

[0032] ttttacaccc ttgttaaata gcacaactgt tgggacttta gaatttcgaa gcataattga 240

[0033] accgattcga aacactaagt ttcgggatga tcatcatttt caattggctg aagcaattca 300

[0034] cttaaatcca catgataaat tagttgtttg taaagcagtc tcatctgata aagaatatac 360

[0035] tttatcatac gataaagttg taattggtgt tggagctgtt agtaatactt ttggaattcc 420

[0036] tggtgtccca aagtatgcat attttttgaa agaaatagca gatgctagaa aaatacgtaa 480

[0037] tcaaatcata tctaattttg agcaaagttt gtttccatt gtaaatgaag aggaaaggtt 540

[0038] atcactgttg cattttgtca ttgttggtgg tggtccaact ggtattgaat ttggagctga 600

[0039] gttgtatgac tttataacac atgatgttgc aagacttttt ccaggagaga agaatgatgt 660

[0040] tcatgtaaca ttagttgaag gagattcaat attaccttca tttgaccagc gattacgtaa 720

[0041] atttgctgag agaaaaaatta cacaaagaga aaatttccac ttaatcaaag attttgttgt 780

[0042] ggaagttggt gaaaactatg tgaagttgaa atcaggaaaa gttcttttaa ctaagcttgt 840

[0043] agtgtggtca accggtcttg gtcccagaaa gtttatagaa agtttagaacc tacctaaagg 900

[0044] taaatcaaaa caactcaagg tagatgatca tcttcgtgta gttggttatg acagtatatt 960

[0045] tgcaattggt gattgttcat acatagatgg atctccttta ccaagcacgg ctcaagtggc 1020

[0046] tgaaagacaa ggtcgatatg ttgcacagta cttatcctta ttagaaacaa attctaaaac 1080

[0047] agacagtaaa ccatttatgt ggtccaatgc tggtatgctt gcatatattg gtggatataa 1140

[0048] agctgttgca gatcttccaa caaaagctgg gaagataact ggatttaaaa gttggttaat 1200

[0049] atggcgatcg gtttatctta ctcgcttagg gagctggaga aatcgaatgc aagttccatt 1260

[0050] tgactgggct aggacttttt ttttggcag ggatatatct aggttttaaa ttacaaactt 1320

[0051] tatttaatat ttagtatttt ttgtatctta ttaatttgtt tttaa

[0052]

[0053] In the present invention, a "recombinant vector" is a genetic construct containing essential regulatory elements operably linked to allow a genetic insert to be expressed, and may be a plasmid vector, a cosmid vector, a bacteriophage vector, or a 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.

[0054] In the present invention, the recombinant viral vector comprises the hydra NDi1 gene represented by SEQ ID NO: 1. The hydra NDi1 gene sequence is preferably present in a suitable expression construct. In the expression construct, the hydra NDi1 gene sequence is preferably operatively linked to a promoter.

[0055] In the present invention, "operably linked" means a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the control sequence controls transcription and / or translation of the other nucleic acid sequence.

[0056] In the present invention, the promoter linked to the hydra NDi1 gene sequence may be any promoter capable of initiating expression of hydra NDi1, but it is preferable to select a promoter capable of operating in an animal cell, more specifically, a mammalian cell, to regulate transcription of the hydra NDi1 gene sequence. The promoter includes a promoter derived from a mammalian virus and a promoter derived from the genome of a mammalian cell, and includes, but is not limited to, a U6 promoter, a T7 promoter, an H1 promoter, a CMV (cytomegalovirus) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, an RSV promoter, an EF1 alpha promoter, a metallothionine promoter, a beta-actin promoter, a promoter of a human IL-2 gene, a promoter of a human IFN gene, a promoter of a human IL-4 gene, a promoter of a human lymphotoxin gene, a promoter of a human GM-CSF gene, an inducible promoter, or a tissue-specific promoter (e.g., an albumin promoter). The above vector can be produced using a genetic recombination technique well known in the art, and site-specific DNA cutting and ligation can be performed using enzymes generally known in the art.

[0057] In the present invention, the adeno-associated virus (AAV) is a single-stranded DNA virus belonging to the parvovirus. It is a defective virus that cannot replicate on its own, but can replicate and proliferate when infected with a helper virus 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 expression intensity, and low immunogenicity.

[0058] In the present invention, the recombinant viral vector can suppress, but is not limited to, a decrease in cellular respiration or apoptosis caused by mitochondrial complex 1 or more. The mitochondrial complex 1 or more can be caused by, but is not limited to, a mutation in the ND1, ND4, or ND6 genes.

[0059] In the present invention, "mitochondrial abnormality" refers to a disease caused by a mutation in mitochondrial DNA (mtDNA, mitochondrial gene), and various symptoms are expressed due to changes in mitochondrial morphology or biochemical properties in various tissues. The mitochondrial abnormality may be caused by an abnormality in mitochondrial complex 1, but is not limited thereto. In addition, the mitochondrial abnormality may be caused by a mutation 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. In addition, the above mitochondrial abnormality disease is 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, Kearns-Sayre syndrome, multiple sclerosis, encephalomyelitis, cranial neuropathy, peripheral neuropathy, Friedreich's ataxia, Alper disease, stroke, migraine, psychosis, depression, seizure, dementia, apoplexy, optic atrophy, optic neuropathy, glaucoma, retinitis pigmentosa, cataract, hyperaldosteronism, hypoparathyroidism, myopathy, muscular atrophy, Myoglobinuria, muscle tone impairment, myalgia, decreased exercise tolerance, renal tubulopathy, renal dysfunction, hepatic failure, hepatomegaly, sideroblastic anemia, neutropenia, thrombocytopenia, diarrhea, villous atrophy, polyvomiting, dysphagia, constipation, sensorineural hearing loss,It may be selected from the group consisting of mental retardation, epilepsy, Alzheimer's disease, Parkinson's disease, and Huntington's disease, and preferably selected from the group consisting of 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, and in one embodiment of the present invention, it was Leber's hereditary optic neuropathy (LHON), but is not limited thereto.

[0060] In the present invention, the Leber's hereditary optic neuropathy (LHON) may be called Leber's optic atrophy, etc., and refers to a disease in which complex 1 in mitochondria does not function properly due to mitochondrial ND1, ND4, or ND6 mtDNA (mitochondria-DNA) mutations, causing abnormalities in the optic nerve system and resulting in blindness.

[0061] The content of the vector of the present invention in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt% or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0062] 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 excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0063] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0064] 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0065] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0066] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen 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, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate,

[0067] Binders such as carboxymethylcellulose, carboxymethylcellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinyl pyrrolidone can be used, and hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, Disintegrants such as anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao 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 ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0068] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0069] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0070] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0071] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0072] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, 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 cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0073] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), and Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0074] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0075] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0076] 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. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0077] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0078] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0079] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0080] In the present invention, the term "subject" means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0081] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0082] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0083]

[0084] Hereinafter, the following examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0085] [Example]

[0086] Example 1. Production of recombinant AAV (Adeno-associated virus)

[0087] Recombinant adeno-associated virus (AAV) is produced by triple transfection of HEK293 cells with vectors. Specifically, the first vector (pHelper vector) is included in common, the subtype is determined by the second vector (pRC vector), and single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) are determined by the third vector. In addition, the gene of interest (GOI) was included in the third vector. The pRC vector used was pAAV-DJ, and the genes of interest used were GFP (Green fluorescent protein), IRES (internal ribosome entry site)-GFP, yeast NDi1 (GenBank: X61590.1), and hydra NDi1 (NCBI Reference Sequence: XM_002156815.3). Individual maps of the vectors used in the experiment are shown in Figures 1 and 2.

[0088] All of the above AAV production vectors were purchased from Cell Biolabs (San Diego, CA), and AAV-293 (Agilent, Santa Clara, CA) was used for 293 cells. After culturing 293 cells in 150 mm dishes to approximately 90% confluence, the first vector, the second vector, and the third vector were mixed well at a ratio of 20 µg, 10 µg, and 10 µg, respectively, and mixed with CaCl2 solution, and then evenly distributed over the 293 cells. After 3 days, the 293 cells were scraped with a cell scraper, purified with the AAVpro Purificatin Kit (Takara, San Jose CA), and the titer was measured with the company's AAVpro Titration Kit. The cells were then stored at -80°C until use.

[0089]

[0090] Example 2. Confirmation of gene expression intensity and stability of scAAV

[0091] To confirm the intensity and stability of gene expression by scAAV and ssAAV, scAAV-GFP and ssAAV-GFP were infected into fibroblasts and the degree of green fluorescence expression was confirmed. The experiment was conducted using the recombinant AAV shown in Fig. 3a. Specifically, skin fibroblast cells derived from LHON patients were cultured in a 12-well plate to about 70% confluence, and then ssAAV-2 / DJ-GFP or scAAV-2 / DJ-GFP was added at about 10 per well, respectively. 9 Cells were infected with each viral genome (vg). After 3 days, the level of GFP expression by each recombinant AAV in the infected cells was observed using an inverted fluorescence microscope under an FITC filter. The results are shown in Figure 3b.

[0092] As shown in Figure 3b, scAAV-GFP can be observed to express green fluorescence more strongly within cells than ssAAV-GFP. This indicates that scAAV stably expresses genes and has high gene expression intensity.

[0093]

[0094] Example 3. Confirmation of the cellular respiration restoration effect of scAAV-hydra NDi1.

[0095] Dermal fibroblasts derived from LHON patients were cultured to approximately 50% confluence in a 24-well plate dedicated to measuring cell respiration, and then ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1 were added at approximately 10 per well, respectively. 8 vg was infected with the titer. In the culture medium, the oxygen consumption rate (OCR) rapidly decreased with the addition of rotenone, a complex I-specific inhibitor. Therefore, 6 days after AAV infection, the addition of rotenone to the cells was confirmed to have an inhibitory effect on the reduction of cellular respiration by yeast NDi1 and hydra NDi1. Cellular respiration was confirmed by measuring the oxygen consumption rate (OCR) using Agilent's Seahorse XF-24. The results are shown in Fig. 4.

[0096] As shown in Fig. 4, scAAV-hydra NDi1 inhibited the reduction of cellular respiration induced by rotenone more efficiently than ssAAV-yeast NDi1.

[0097]

[0098] Example 4. Confirmation of the cell death inhibition effect of scAAV-hydra Ndi1.

[0099] After culturing LHON patient dermal fibroblasts to approximately 70% confluence in a 24-well plate, ssAAV-IRES-GFP, ssAAV-yeast NDi1, ssAAV-hydra NDi1, and scAAV-hydra NDi1 were added at approximately 10 per well, respectively. 9 The cells were infected with the titer of vg. Six days later, rotenone was treated, and cell death was observed starting one day after rotenone treatment. The results are shown in Figures 5a to 5d. Cell death was determined by comprehensively considering the shape and morphology of individual cells, the degree of attachment to the substrate, and the strength of contact with adjacent cells.

[0100] As shown in Fig. 5a, 1 day after rotenone treatment, some cells in the untransduced control group or the ssAAV-IRES-GFP-infected group began to die at 5 nM rotenone, whereas cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 showed some death at 20 nM rotenone. In contrast, cells infected with scAAV-hydra NDi1 under the same conditions showed almost no death.

[0101] As shown in Fig. 5b, 2 days after rotenone treatment, almost all cells in the untransduced cell group or the cell group infected with ssAAV-IRES-GFP were killed even at 5 nM rotenone, whereas cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 showed some cell death at 20 nM rotenone. In contrast, cells infected with scAAV-hydra NDi1 showed almost no cell death under the same conditions.

[0102] As shown in Fig. 5c, 3 days after rotenone treatment, untransduced cells or cells infected with ssAAV-IRES-GFP were completely killed at 5 nM rotenone, and cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 began to die significantly even at 10 nM rotenone. In contrast, cells infected with scAAV-hydra NDi1 under the same conditions were barely killed even at 20 nM rotenone.

[0103] As shown in Fig. 5d, 4 days after rotenone treatment, cells infected with ssAAV-yeast NDi1 or ssAAV-hydra NDi1 also showed some death at 5 nM rotenone and almost no death at 10 nM. In contrast, cells infected with scAAV-hydra NDi1 showed almost no death even at 20 nM.

[0104] This means that scAAV-hydra NDi1 has a significantly superior inhibitory effect on apoptosis of LHON patients-derived cells mediated by mitochondrial complex 1 or higher compared to ssAAV-hydra NDi1 and ssAAV-yeast NDi1.

[0105]

[0106] Through the above results, it was confirmed that the scAAV-hydra NDi1 of the present invention has an effect of restoring the decrease in cellular respiration caused by rotenone and an effect of inhibiting the death of cells derived from LHON patients caused by rotenone. Therefore, it is expected that this can be used for the prevention, improvement, or treatment of diseases caused by abnormalities in mitochondrial complex 1 as well as LHON.

[0107]

[0108] The above description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating mitochondrial abnormalities, comprising a recombinant viral vector loaded with the hydra NDi1 gene represented by sequence number 1.

2. In paragraph 1, A pharmaceutical composition for preventing or treating mitochondrial abnormality, wherein the recombinant virus is an adeno-associated virus.

3. In paragraph 2, A pharmaceutical composition for preventing or treating mitochondrial abnormality, wherein the above adeno-associated virus is a self-complementary AAV (scAAV).

4. In paragraph 1, The above recombinant viral vector is a pharmaceutical composition for preventing or treating mitochondrial abnormalities, which inhibits a decrease in cellular respiration.

5. In paragraph 1, The above recombinant viral vector is a pharmaceutical composition for preventing or treating mitochondrial abnormalities that inhibits apoptosis.

6. In paragraph 1, A pharmaceutical composition for preventing or treating a mitochondrial abnormality, wherein the mitochondrial abnormality is a mutation 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).

7. In paragraph 1, A pharmaceutical composition for preventing or treating a mitochondrial abnormality, wherein the mitochondrial abnormality is selected from the group consisting of Leber's hereditary optic neuropathy (LHON), MELAS (Mitochondrial Encephalopathy with Lactic acidosis and stroke-like episodes) syndrome, neurogenic muscle weakness, MERRF (Myoclonic Epilepsy with Ragged Red Fiber) syndrome, mitochondrial encephalomyopathy, Leigh syndrome, and Kearns-Sayre syndrome.

8. A method for preventing or treating a mitochondrial disorder, comprising administering to a subject a pharmaceutical composition for preventing or treating a mitochondrial disorder, the pharmaceutical composition comprising a recombinant viral vector having a hydra NDi1 gene represented by sequence number 1.

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