WO2024063570A1 - Antisense oligonucleotide fused with cell-penetrating peptide that targets myotonic dystrophy protein kinase and use thereof - Google Patents

Antisense oligonucleotide fused with cell-penetrating peptide that targets myotonic dystrophy protein kinase and use thereof Download PDF

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WO2024063570A1
WO2024063570A1 PCT/KR2023/014418 KR2023014418W WO2024063570A1 WO 2024063570 A1 WO2024063570 A1 WO 2024063570A1 KR 2023014418 W KR2023014418 W KR 2023014418W WO 2024063570 A1 WO2024063570 A1 WO 2024063570A1
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antisense oligonucleotide
dmpk
myotonic dystrophy
aso
cell
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PCT/KR2023/014418
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French (fr)
Korean (ko)
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조대웅
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주식회사 셀리버리
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to an antisense oligonucleotide (ASO) targeting myotonic dystrophy protein kinase (DMPK) and its use. More specifically, the present invention provides an antisense oligonucleotide fused with aMTD (advanced macromolecule transduction domain) as a cell-penetrating peptide targeting myotonic dystrophy protein kinase, and a myotonic dystrophy type 1 (DM1) agent comprising the same as an active ingredient. ), and a pharmaceutical composition for the prevention or treatment of, and a method of alleviating or treating amyotrophic dystrophy.
  • ASO antisense oligonucleotide
  • DM1 myotonic dystrophy type 1
  • RNA interference (RNAi) nucleic acid therapeutics such as siRNA or ASO have a clear treatment mechanism and can inhibit protein production in advance by matching the base sequence to the target mRNA, so they have high selectivity for targets with genetic mutations. Therefore, there is an advantage in that new drug substances can be developed by discovering the mRNA base sequence of the disease-causing gene. It can be an appropriate treatment for rare genetic diseases that occur in one gene. However, a disadvantage is that cell and tissue permeability is low and distribution is poor. Meanwhile, the Therapeuticmolecule Systemic Delivery Technology (TSDT) combines aMTD (advanced Macromolecule Transduction Domain), a hydrophobic cell-penetrating peptide (CPP), with a pharmacological substance (Cargo) with therapeutic efficacy. It has the advantage of being able to be delivered directly to cells and tissues through fusion.
  • TSDT Therapeuticmolecule Systemic Delivery Technology
  • Amyotrophic dystrophy (DM1) is estimated to affect approximately 1 in 8,000 people worldwide, and is an autosomal dominant trait that occurs immediately after birth until adulthood. The cause is an abnormal increase in the repetition of three base sequences (CTG; Cytosine, Thymine, Guanine) in the untranslated region (UTR) of myotonic dystrophy protein kinase (DMPK).
  • CTG three base sequences
  • UTR untranslated region
  • DMPK myotonic dystrophy protein kinase
  • RNA-binding Protein 1 (CUG-Binding Protein 1: CUGBP1) takes over the role, the proteins of factors related to muscle formation are not synthesized properly and lose their role.
  • CUG-Binding Protein 1 (CUG-Binding Protein 1: CUGBP1) takes over the role, the proteins of factors related to muscle formation are not synthesized properly and lose their role.
  • CUG-Binding Protein 1 RNA-binding Protein 1: CUGBP1
  • CUGBP1 RNA-binding Protein 1
  • the present inventors produced an antisense oligonucleotide (ASO) targeting myotonic dystrophy protein kinase (DMPK) and developed a cell-penetrating peptide (Cell-Penetrating peptide). After confirming that the expression of DMPK can be significantly reduced when fused with aMTD (advanced macromolecule transduction domain) as a peptide (CPP), the present invention was completed.
  • ASO antisense oligonucleotide
  • DMPK myotonic dystrophy protein kinase
  • Cell-Penetrating peptide cell-penetrating peptide
  • the present invention provides an antisense oligonucleotide fused with an advanced macromolecule transduction domain (aMTD) targeting myotonic dystrophy protein kinase, and a drug for the prevention or treatment of myotonic dystrophy type 1 (DM1) containing the same as an active ingredient.
  • aMTD advanced macromolecule transduction domain
  • DM1 myotonic dystrophy type 1
  • the present invention According to a first embodiment, the present invention
  • the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
  • the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
  • the antisense oligonucleotide can reduce the expression of DMPK by at least 20%.
  • a pharmaceutical composition for the prevention or treatment of myotonic dystrophy type 1 containing as an active ingredient an antisense oligonucleotide (ASO) fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD),
  • ASO antisense oligonucleotide fused to a cell-penetrating peptide
  • aMTD advanced macromolecule transduction domain
  • the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
  • the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
  • the pharmaceutical composition can prevent or treat symptoms associated with the onset of amyotrophic dystrophy, and the symptoms associated with the onset of amyotrophic dystrophy include muscle stiffness, myotonia, peripheral weakness, asthenia, facial and jaw muscle weakness. , difficulty swallowing, dropping of the eyelids (ptosis), neck muscle weakness, arm and leg muscle weakness, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, shortness of breath, irregular heartbeat, myocardial damage, numbness, insulin resistance, or May include cataracts.
  • Myotonic dystrophy type 1 which includes administering to a subject a pharmaceutical composition containing an antisense oligonucleotide (ASO) fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) as an active ingredient.
  • ASO antisense oligonucleotide
  • aMTD cell-penetrating peptide
  • Disclosed is a method for alleviating or treating DM1), wherein the antisense oligonucleotide is selected from the group consisting of SEQ ID NOs: 1 to 5.
  • the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
  • the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
  • the pharmaceutical composition may be administered orally, subcutaneously, intravenously, intracerebroventricularly, intrathecally, intramuscularly, intraarterially, intraperitoneally, or intracranially.
  • the subject may be a mammal other than a human.
  • a cell-penetrating peptide (CPP) targeting myotonic dystrophy protein kinase is an antisense oligonucleotide (ASO) fused with aMTD (advanced macromolecule transduction domain). ) can significantly reduce the expression of DMPK.
  • Figure 1 shows the hDMPK sequence for designing ASO according to the present invention.
  • Figure 2 shows five types of ASO DMPK selected according to Example 1 of the present invention.
  • Figure 3 shows sequence information of five types of ASO DMPK selected according to Example 1 of the present invention.
  • Figure 4 shows five types of aMTD designed according to Example 2 of the present invention.
  • Figure 5 shows aMTD-ASO DMPK produced according to Example 3 of the present invention.
  • Figure 6 shows the efficacy of aMTD-ASO DMPK in suppressing DMPK expression according to Example 3 of the present invention.
  • Figure 7 shows the results of evaluating the efficacy of aMTD-ASO DMPK in a myotonic dystrophy mouse model according to Example 3 of the present invention.
  • the terms “prevent,” “preventing,” and “prevention” refer to reducing or eliminating the possibility of contracting a disease.
  • the terms “treat,” “treating,” and “treatment” refer to eliminating all or part of a disease and/or its accompanying symptoms.
  • the terms “alleviate,” “alleviating,” and “alleviation” refer to alleviating all or part of a disease and/or its accompanying symptoms.
  • the term "subject” refers to an animal, preferably a mammal (e.g., primates (e.g., humans), cattle, sheep, goats, horses, etc., that is the object of treatment, observation, or experiment. , dog, cat, rabbit, rat, mouse, etc.), most preferably a person.
  • a mammal e.g., primates (e.g., humans), cattle, sheep, goats, horses, etc., that is the object of treatment, observation, or experiment. , dog, cat, rabbit, rat, mouse, etc.
  • the term "administration” means introducing the composition of the present invention into an individual by any appropriate method, and the route of administration of the composition of the present invention can be various oral or parenteral routes as long as it can reach the target tissue. It can be administered through
  • the term “therapeutically effective amount” refers to a biological or medical treatment in a tissue system, animal or human that is sought by a researcher, veterinarian, physician or other clinician and that includes alleviating the symptoms of the disease or disorder being treated. It refers to the amount of active compound or pharmaceutical agent that induces a reaction.
  • composition includes a product comprising specified ingredients in specified amounts and any product that results directly or indirectly from a combination of specified amounts of specified ingredients.
  • DMPK myotonic dystrophy protein kinase
  • DMPK expression means the level of mRNA transcribed from the gene encoding DMPK or the level of protein translated from mRNA. DMPK expression can be measured by methods known in the art, such as Northern or Western blot.
  • DMPK nucleic acid means any nucleic acid encoding DMPK.
  • the DMPK nucleic acid is a DNA sequence encoding DMPK, an RNA sequence transcribed from DNA encoding DMPK (including genomic DNA including introns and exons), and an mRNA or preparatory sequence encoding DMPK. -Contains an mRNA sequence.
  • DMPK mRNA means mRNA encoding DMPK protein.
  • targeting refers to the process of designing and selecting antessense oligonucleotides that induce a desired effect by specifically hybridizing to a target nucleic acid.
  • nucleoside refers to a nucleobase linked to a sugar. In certain embodiments, the nucleoside is linked to a phosphate group.
  • nucleotide refers to a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
  • oligonucleotide refers to a polymer of linked nucleosides, and each nucleoside and internucleoside linkage may be independently modified or unmodified.
  • phosphorothioate refers to a linkage between nucleosides in which the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom.
  • a phosphorothioate linkage is a modified internucleoside linkage.
  • 2'-O-methoxyethyl (2'-MOE and 2'-O(CH 2 ) 2 -OCH 3 ) refers to O-methoxy-ethyl modification at the 2' position of the furosyl ring.
  • 2'-O-methoxyethyl modified sugar is a modified sugar.
  • ASO Antisense oligonucleotide
  • the object is to provide an antisense oligonucleotide (ASO) targeting myotonic dystrophy protein kinase selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5.
  • ASO according to the present invention is shown in Table 1.
  • the ASO targets myotonic dystrophy protein kinase nucleic acid.
  • antisense to it involves hybridization to the target nucleic acid via hydrogen bonds, for example Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonds.
  • the present invention seeks to provide an antisense oligonucleotide fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) targeting myotonic dystrophy protein kinase (DMPK).
  • aMTD advanced macromolecule transduction domain
  • DMPK myotonic dystrophy protein kinase
  • the antisense oligonucleotide may be selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5.
  • the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
  • the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
  • Antisense oligonucleotides according to the invention reduce the expression of DMPK by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%. %, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99 % can be reduced.
  • the present invention seeks to provide a pharmaceutical composition for the prevention or treatment of myotonic dystrophy type 1 (DM1), which contains as an active ingredient an antisense oligonucleotide fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD).
  • DM1 myotonic dystrophy type 1
  • aMTD advanced macromolecule transduction domain
  • the antisense oligonucleotide may be selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5.
  • the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
  • the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
  • the pharmaceutical composition can prevent or treat symptoms related to the onset of amyotrophic dystrophy.
  • symptoms associated with the development of the above-mentioned amyotrophic dystrophy include muscle stiffness, myotonia, peripheral weakness atony, facial and jaw muscle weakness, dysphagia, eyelid drooping (ptosis), neck muscle weakness, arm and leg muscle weakness, These may include, but are not limited to, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, shortness of breath, irregular heartbeat, myocardial damage, numbness, insulin resistance, or cataracts.
  • the appropriate dosage of the pharmaceutical composition is determined by the formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and response sensitivity. It varies depending on the same factors, and usually a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.
  • the pharmaceutical composition may be administered once or in multiple doses, for example, once to four times a day.
  • the pharmaceutical composition uses a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. It can be formulated into a unit dose form or can be manufactured by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer. Additionally, the pharmaceutical composition may be administered in the form of a suppository, spray, ointment, cream, gel, inhalant, or skin patch.
  • the pharmaceutical composition may be prepared for administration to mammals, more preferably for administration to humans.
  • Pharmaceutically acceptable carriers may be solid or liquid and include excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavoring agents, lubricants, release regulators, wetting agents, It may be one or more selected from stabilizers, suspending agents, and lubricants.
  • the pharmaceutically acceptable carrier may be selected from saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.
  • suitable fillers include sugars (e.g., dextrose, sucrose, maltose, and lactose), starches (e.g., corn starch), sugar-alcohols (e.g., mannitol, sorbitol, maltitol, erythritol, and xylitol). ), starch hydrolysates (e.g., dextrin and maltodextrin), cellulose or cellulose derivatives (e.g., microcrystalline cellulose), or mixtures thereof may be used, but are not limited thereto.
  • sugars e.g., dextrose, sucrose, maltose, and lactose
  • starches e.g., corn starch
  • sugar-alcohols e.g., mannitol, sorbitol, maltitol, erythritol, and xylitol
  • starch hydrolysates e.g., dextrin
  • Suitable binders include magnesium aluminum silicate, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gum, sucrose, starch paste or A mixture of these may be used, but is not limited thereto.
  • Suitable preservatives include benzoic acid, sodium benzoate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorbutol, gallate, hydroxybenzoate, EDTA, or mixtures thereof. It is not limited to this.
  • Suitable disintegrants include, but are not limited to, sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked carboxymethylcellulose, starch, microcrystalline cellulose, or mixtures thereof.
  • No. Suitable sweeteners include, but are not limited to, sucralose, saccharin, sodium or potassium or calcium saccharin, acesulfame potassium or sodium cyclamate, mannitol, fructose, sucrose, maltose or mixtures thereof.
  • Suitable lubricants include, but are not limited to, silica, colloidal silicon dioxide, and talc.
  • Suitable lubricants include, but are not limited to, long-chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glyceride wax, or mixtures thereof.
  • the present invention is a treatment for myotonic dystrophy type 1 (DM1), comprising administering to a subject a pharmaceutical composition containing an antisense oligonucleotide fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) as an active ingredient.
  • DM1 myotonic dystrophy type 1
  • the goal is to provide relief or treatment methods.
  • the antisense oligonucleotide may be selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5.
  • the putative cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
  • the ASO fused to aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
  • the administration may include oral administration or parenteral administration.
  • the parenteral administration may include subcutaneous administration, intravenous administration, intracerebroventricular administration, intrathecal administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration.
  • the administration may be chronic, continuous, short-term, or intermittent.
  • the pharmaceutical composition can prevent or treat symptoms related to the onset of amyotrophic dystrophy.
  • symptoms associated with the development of the above-mentioned amyotrophic dystrophy include muscle stiffness, myotonia, peripheral weakness atony, facial and jaw muscle weakness, dysphagia, eyelid drooping (ptosis), neck muscle weakness, arm and leg muscle weakness, These may include, but are not limited to, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, shortness of breath, irregular heartbeat, myocardial damage, numbness, insulin resistance, or cataracts.
  • ASOs targeting the hDMPK sequence shown in Figure 1 were designed and transfected into GM04033 and GM03132, respectively, among cells derived from patients with amyotrophic dystrophy, and the efficacy of suppressing DMPK expression was analyzed through real-time quantitative polymerase chain reaction (qPCR) analysis. It was verified through the average of the gene expression inhibition efficacy values in the two cell lines, and finally five types with good efficacy (ASO DMPK 32, 29, 25, 30, and 8) were selected (Figure 2), and the five selected types were The sequence information of ASO DMPK is shown in Figure 3.
  • aMTD-ASO DMPK was produced by fusing aMTD to the 5' side of the ASO designed in Example 1 using Chemical Linker (Succinimidyl-4-(N-Maleimidomethyl Cyclohexane)-1-Carboxylate: SMCC). did.
  • ASO DMPK 8 and cell-permeable aMTD321-ASO DMPK 8 were administered intravenously to a myotonic dystrophy mouse model in which the hDMPK gene was inserted.
  • aMTD321-ASO DMPK 8 compared to ASO DMPK 8 in brain, heart, and muscle tissue (FIG. 7).
  • a cell-penetrating peptide (CPP) targeting myotonic dystrophy protein kinase is an antisense oligonucleotide (ASO) fused with aMTD (advanced macromolecule transduction domain). ) can significantly reduce the expression of DMPK. Therefore, it is expected that ASO fused with aMTD can be effectively used as a nucleic acid therapeutic agent for preventing, treating or alleviating myotonic dystrophy type 1 (DM1).

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Abstract

The present invention relates to an antisense oligonucleotide (ASO) that targets myotonic dystrophy protein kinase (DMPK) and use thereof. More specifically, the present invention relates to: an antisense oligonucleotide fused with an advanced macromolecule transduction domain (aMTD) as a cell-penetrating peptide that targets myotonic dystrophy protein kinase; a pharmaceutical composition for the prevention or treatment of myotonic dystrophy type 1 (DM1), comprising same as an active ingredient; and a method for alleviating or treating myotonic dystrophy type 1.

Description

근긴장성 이영양증 단백질 키나아제를 표적화하는 세포투과성 펩티드 융합 안티센스 올리고뉴클레오티드 및 이의 용도Cell-penetrating peptide fusion antisense oligonucleotide targeting myotonic dystrophy protein kinase and uses thereof
본 발명은 근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)를 표적화하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO) 및 이의 용도에 관한 것이다. 보다 구체적으로, 본 발명은 근긴장성 이영양증 단백질 키나아제를 표적화하는 세포투과성 펩티드로서 aMTD(advanced macromolecule transduction domain)와 융합된 안티센스 올리고뉴클레오티드, 및 이를 유효성분으로 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1)의 예방 또는 치료를 위한 약제학적 조성물, 및 근위축성 이영양증의 경감 또는 치료 방법에 관한 것이다. The present invention relates to an antisense oligonucleotide (ASO) targeting myotonic dystrophy protein kinase (DMPK) and its use. More specifically, the present invention provides an antisense oligonucleotide fused with aMTD (advanced macromolecule transduction domain) as a cell-penetrating peptide targeting myotonic dystrophy protein kinase, and a myotonic dystrophy type 1 (DM1) agent comprising the same as an active ingredient. ), and a pharmaceutical composition for the prevention or treatment of, and a method of alleviating or treating amyotrophic dystrophy.
siRNA나 ASO등 RNA 간섭 (RNAi) 핵산치료제는 치료기전이 명확하고, 염기서열만 표적 mRNA에 맞추어 주면 단백질 생성을 사전에 억제할 수 있어 유전적 변이를 가진 타깃에 대한 높은 선택성을 갖는다. 따라서 질병 원인 유전자의 mRNA 염기 서열을 발굴하면 신약 물질을 개발할 수 있다는 장점이 있다. 한가지 유전자에 일어나는 희귀유전질환에 적합한 치료법이 될 수 있다. 그러나 세포 및 조직 투과도가 낮아 분포가 잘 이루어지지 않는 것이 단점이다. 한편, 약리물질 생체 내 전송기술(Therapeuticmolecule Systemic Delivery Technology, TSDT)은 소수성 세포막 투과 펩타이드(Hydrophobic Cell-Penetrating Peptide: CPP)인 aMTD(advanced Macromolecule Transduction Domain)와 치료효능이 있는 약리물질(Cargo)을 융합(Fusion) 하여 세포 및 조직에 직접 전달할 수 있는 장점이 있다. RNA interference (RNAi) nucleic acid therapeutics such as siRNA or ASO have a clear treatment mechanism and can inhibit protein production in advance by matching the base sequence to the target mRNA, so they have high selectivity for targets with genetic mutations. Therefore, there is an advantage in that new drug substances can be developed by discovering the mRNA base sequence of the disease-causing gene. It can be an appropriate treatment for rare genetic diseases that occur in one gene. However, a disadvantage is that cell and tissue permeability is low and distribution is poor. Meanwhile, the Therapeuticmolecule Systemic Delivery Technology (TSDT) combines aMTD (advanced Macromolecule Transduction Domain), a hydrophobic cell-penetrating peptide (CPP), with a pharmacological substance (Cargo) with therapeutic efficacy. It has the advantage of being able to be delivered directly to cells and tissues through fusion.
근위축성 이영양증(DM1)은 전세계적으로 약 8천 명중 1명의 환자가 있을 것으로 추정되며, 상염색체의 우성 형질로 출생직후부터 성인까지 발병하는 유전성 질환이다. 원인은 근긴장성 이영양증 단백질 키나아제(DMPK)의 비번역부위(Untranslated Region: UTR)에서 3개 염기서열(CTG; Cytosine, Thymine, Guanine)의 반복이 비정상적으로 증가되는 것이다. 증가된 CTG 염기서열이 헤어핀 구조를 형성하고, 그 부위에 MBNL1(Muscleblind Like Splicing Regulator 1) 단백질이 결합되어 제 역할을 못하게 된다. 대신 다른 RNA 결합 단백질(CUG-Binding Protein 1: CUGBP1)이 역할을 대신하게 되면, 근육형성에 관련된 인자들의 단백질이 제대로 합성되지 못해 역할을 잃게 된다. 안면 및 근육들이 약화됨에 따라 걷기 힘들어지고, 심근병증, 부정맥, 위장기능 장애, 인슐린 저항성, 백내장, 인지지능 장애 등의 다양한 증상들이 나타난다. 특히, 호흡근의 결함에 의해 폐기능에 영향을 미쳐 호흡곤란이 오게 되거나 심블록(Heart Block)으로 인해 사망에 이르는 질병이다. 현재까지 확실한 치료제는 없으며, 호흡 보조기를 착용하고, 심장 기능 이상증상을 심박 조율기 및 이식형 제세동기를 사용하는 등 나타나는 증상에 따른 조치를 취하는 것이 최선이다.Amyotrophic dystrophy (DM1) is estimated to affect approximately 1 in 8,000 people worldwide, and is an autosomal dominant trait that occurs immediately after birth until adulthood. The cause is an abnormal increase in the repetition of three base sequences (CTG; Cytosine, Thymine, Guanine) in the untranslated region (UTR) of myotonic dystrophy protein kinase (DMPK). The increased CTG base sequence forms a hairpin structure, and MBNL1 (Muscleblind Like Splicing Regulator 1) protein binds to that site, preventing it from performing its role. Instead, when another RNA-binding protein (CUG-Binding Protein 1: CUGBP1) takes over the role, the proteins of factors related to muscle formation are not synthesized properly and lose their role. As the face and muscles weaken, it becomes difficult to walk, and various symptoms such as cardiomyopathy, arrhythmia, gastrointestinal dysfunction, insulin resistance, cataracts, and cognitive impairment appear. In particular, it is a disease that affects lung function due to defects in the respiratory muscles, leading to breathing difficulties or death due to heart block. There is currently no definite treatment, and it is best to take action according to the symptoms, such as wearing a breathing apparatus and using a pacemaker or implantable cardioverter-defibrillator to treat symptoms of cardiac dysfunction.
본 발명자들은 근위축성 이영양증 치료제를 개발하기 위하여 예의 노력한 결과, 근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)를 표적화하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)를 제작하고 세포투과성 펩티드(Cell-Penetrating Peptide: CPP)로서 aMTD(advanced Macromolecule Transduction Domain)와 융합하는 경우, DMPK의 발현을 현저히 감소시킬 수 있음을 확인한 후, 본 발명을 완성하기에 이르렀다. As a result of our diligent efforts to develop a treatment for amyotrophic dystrophy, the present inventors produced an antisense oligonucleotide (ASO) targeting myotonic dystrophy protein kinase (DMPK) and developed a cell-penetrating peptide (Cell-Penetrating peptide). After confirming that the expression of DMPK can be significantly reduced when fused with aMTD (advanced macromolecule transduction domain) as a peptide (CPP), the present invention was completed.
그러나, 본원이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the problem to be solved by the present application is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
본 발명은 근긴장성 이영양증 단백질 키나아제를 표적화하는 aMTD(advanced macromolecule transduction domain)와 융합된 안티센스 올리고뉴클레오티드, 및 이를 유효성분으로 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1)의 예방 또는 치료를 위한 약제학적 조성물, 및 근위축성 이영양증의 경감 또는 치료 방법을 개시한다. The present invention provides an antisense oligonucleotide fused with an advanced macromolecule transduction domain (aMTD) targeting myotonic dystrophy protein kinase, and a drug for the prevention or treatment of myotonic dystrophy type 1 (DM1) containing the same as an active ingredient. Disclosed is a medical composition and a method for alleviating or treating amyotrophic dystrophy.
제1구현예에 따르면, 본 발명은 According to a first embodiment, the present invention
근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)를 표적화하는 세포투과성 펩티드(advanced macromolecule transduction domain, aMTD)에 융합된 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)로서, 상기 안티센스 올리고뉴클레오티드는 서열번호 1 내지 5로 이루어진 군으로부터 선택되는 안티센스 올리고뉴클레오티드를 개시한다. An antisense oligonucleotide (ASO) fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) targeting myotonic dystrophy protein kinase (DMPK), wherein the antisense oligonucleotide is SEQ ID NO: 1 to Disclosed is an antisense oligonucleotide selected from the group consisting of 5.
본 발명에 있어서, 상기 세포투과성 펩티드는 서열번호 6 내지 10의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the present invention, the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
본 발명에 있어서, 상기 aMTD-에 융합된 ASO는 서열번호 11 내지 35의 염기서열로 이루어진 군으로부터 선택될 수 있다.In the present invention, the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
본 발명에 있어서, 상기 안티센스 올리고뉴클레오티드는 DMPK의 발현을 적어도 20% 감소시킬 수 있다. In the present invention, the antisense oligonucleotide can reduce the expression of DMPK by at least 20%.
제2구현예에 따르면, 본 발명은 According to a second embodiment, the present invention
세포투과성 펩티드(advanced macromolecule transduction domain, aMTD)에 융합된 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)를 유효성분으로 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1) 예방 또는 치료를 위한 약제학적 조성물로서, 상기 안티센스 올리고뉴클레오티드는 서열번호 1 내지 5로 이루어진 군으로부터 선택되는 것인, 약제학적 조성물을 개시한다. A pharmaceutical composition for the prevention or treatment of myotonic dystrophy type 1 (DM1) containing as an active ingredient an antisense oligonucleotide (ASO) fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD), Disclosed is a pharmaceutical composition in which the antisense oligonucleotide is selected from the group consisting of SEQ ID NOs: 1 to 5.
본 발명에 있어서, 상기 세포투과성 펩티드는 서열번호 6 내지 10의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the present invention, the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
본 발명에 있어서, 상기 aMTD-에 융합된 ASO는 서열번호 11 내지 35의 염기서열로 이루어진 군으로부터 선택될 수 있다.In the present invention, the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
본 발명에 있어서, 상기 약제학적 조성물은 근위축성 이영양증의 발병과 관련된 증상을 예방 또는 치료할 수 있고, 상기 근위축성 이영양증의 발병과 관련된 증상은 근육 경직, 근긴장증, 말초 약화 무력증, 안면 및 턱 근육 약화, 연하곤란, 눈꺼풀 수하(안검하수), 목근육 약화, 팔 및 다리 근육 약화, 계속된 근육 통증, 수면과잉, 근육 소모, 연하곤란증, 호흡 부족, 불규칙한 심장박동, 심근 손상, 무감각, 인슐린 저항 또는 백내장을 포함할 수 있다. In the present invention, the pharmaceutical composition can prevent or treat symptoms associated with the onset of amyotrophic dystrophy, and the symptoms associated with the onset of amyotrophic dystrophy include muscle stiffness, myotonia, peripheral weakness, asthenia, facial and jaw muscle weakness. , difficulty swallowing, dropping of the eyelids (ptosis), neck muscle weakness, arm and leg muscle weakness, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, shortness of breath, irregular heartbeat, myocardial damage, numbness, insulin resistance, or May include cataracts.
제3구현예에 따르면, According to the third implementation example,
세포투과성 펩티드(advanced macromolecule transduction domain, aMTD)에 융합된 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)를 유효성분으로 포함하는 약제학적 조성물을 대상체에 투여하는 단계를 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1)의 경감 또는 치료 방법으로서, 상기 안티센스 올리고뉴클레오티드는 서열번호 1 내지 5로 이루어진 군으로부터 선택되는 것인, 방법을 개시한다. Myotonic dystrophy type 1, which includes administering to a subject a pharmaceutical composition containing an antisense oligonucleotide (ASO) fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) as an active ingredient. Disclosed is a method for alleviating or treating DM1), wherein the antisense oligonucleotide is selected from the group consisting of SEQ ID NOs: 1 to 5.
본 발명에 있어서, 상기 세포투과성 펩티드는 서열번호 6 내지 10의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the present invention, the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
본 발명에 있어서, 상기 aMTD-에 융합된 ASO는 서열번호 11 내지 35의 염기서열로 이루어진 군으로부터 선택될 수 있다.In the present invention, the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
본 발명에 있어서, 상기 약제학적 조성물은 경구 투여, 피하 투여, 정맥내 투여, 뇌실내 투여, 척추강내 투여, 근육내 투여, 동맥내 투여, 복강내 투여 또는 두개내 투여될 수 있다. In the present invention, the pharmaceutical composition may be administered orally, subcutaneously, intravenously, intracerebroventricularly, intrathecally, intramuscularly, intraarterially, intraperitoneally, or intracranially.
본 발명에 있어서, 상기 대상체는 인간을 제외한 포유동물일 수 있다. In the present invention, the subject may be a mammal other than a human.
본 발명에 따른 근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)를 표적화하는 세포투과성 펩티드(Cell-Penetrating Peptide: CPP)로서 aMTD(advanced Macromolecule Transduction Domain)와 융합된 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)는 DMPK의 발현을 현저히 감소시킬 수 있다. According to the present invention, a cell-penetrating peptide (CPP) targeting myotonic dystrophy protein kinase (DMPK) is an antisense oligonucleotide (ASO) fused with aMTD (advanced macromolecule transduction domain). ) can significantly reduce the expression of DMPK.
한편, 전술한 바와 같은 효과들에 의해 본 발명의 범위가 제한되는 것은 아니다.Meanwhile, the scope of the present invention is not limited by the effects described above.
도 1은 본 발명에 따른 ASO를 설계하기 위한 hDMPK 서열을 나타낸다. Figure 1 shows the hDMPK sequence for designing ASO according to the present invention.
도 2는 본 발명의 실시예 1에 따라 선별된 5종의 ASODMPK를 나타낸다. Figure 2 shows five types of ASO DMPK selected according to Example 1 of the present invention.
도 3은 본 발명의 실시예 1에 따라 선별된 5종의 ASODMPK 서열정보를 나타낸다. Figure 3 shows sequence information of five types of ASO DMPK selected according to Example 1 of the present invention.
도 4는 본 발명의 실시예 2에 따라 설계된 5종의 aMTD를 나타낸다. Figure 4 shows five types of aMTD designed according to Example 2 of the present invention.
도 5는 본 발명의 실시예 3에 따라 제작된 aMTD-ASODMPK를 나타낸다. Figure 5 shows aMTD-ASO DMPK produced according to Example 3 of the present invention.
도 6은 본 발명의 실시예 3에 따른 aMTD-ASODMPK의 DMPK 발현 억제 효능을 나타낸다. Figure 6 shows the efficacy of aMTD-ASO DMPK in suppressing DMPK expression according to Example 3 of the present invention.
도 7은 본 발명의 실시예 3에 따른 aMTD-ASODMPK의 근긴장성 이영양증 마우스 모델에서의 효능 평가 결과를 나타낸다. Figure 7 shows the results of evaluating the efficacy of aMTD-ASO DMPK in a myotonic dystrophy mouse model according to Example 3 of the present invention.
이하, 발명의 구체적인 구현예에 따른 근긴장성 이영양증 단백질 키나아제를 표적화하는 세포투과성 펩티드 융합 안티센스 올리고뉴클레오티드 및 이의 용도에 대하여 상세하게 설명하기로 한다. 다만, 이는 발명의 하나의 예시로서 제시되는 것으로, 이에 의해 발명의 권리범위가 한정되는 것은 아니며, 발명의 권리범위 내에서 구현예에 대한 다양한 변형이 가능함은 당업자에게 자명하다. 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.Hereinafter, a cell-penetrating peptide fusion antisense oligonucleotide targeting myotonic dystrophy protein kinase according to a specific embodiment of the invention and its use will be described in detail. However, this is presented as an example of the invention, and the scope of the invention is not limited thereby, and it is obvious to those skilled in the art that various modifications to the embodiment are possible within the scope of the invention. Throughout this specification, unless otherwise specified, “include” or “contains” refers to the inclusion of any component (or component) without any particular limitation, excluding the addition of other components (or components). cannot be interpreted as
본 명세서에서 사용된 용어 “예방하다(prevent)”“예방하는(preventing)”및 “예방(prevention)”은 질병에 걸릴 가능성을 감소시키거나 가능성을 제거하는 것이다.As used herein, the terms “prevent,” “preventing,” and “prevention” refer to reducing or eliminating the possibility of contracting a disease.
본 명세서에서 사용된 용어 "치료하다(treat)", "치료하는(treating)" 및 "치료(treatment)"는 질병 및/또는 이의 수반되는 증상을 모두 또는 일부 제거하는 것이다.As used herein, the terms “treat,” “treating,” and “treatment” refer to eliminating all or part of a disease and/or its accompanying symptoms.
본 명세서에서 사용된 용어 "경감시키다(alleviate)", "경감시키는(alleviating)" 및 "경감(alleviation)"은 질병 및/또는 이의 수반되는 증상을 모두 또는 일부 완화시키는 것이다.As used herein, the terms “alleviate,” “alleviating,” and “alleviation” refer to alleviating all or part of a disease and/or its accompanying symptoms.
본 명세서에서 사용된 용어 "대상"은 치료, 관찰 또는 실험의 객체가 되는 동물, 바람직하게는 포유동물(예를 들면, 영장류(primates)(예를 들어, 사람), 소, 양, 염소, 말, 개, 고양이, 토끼, 쥐, 마우스 등), 가장 바람직하게는 사람을 의미한다.As used herein, the term "subject" refers to an animal, preferably a mammal (e.g., primates (e.g., humans), cattle, sheep, goats, horses, etc., that is the object of treatment, observation, or experiment. , dog, cat, rabbit, rat, mouse, etc.), most preferably a person.
본 명세서에서 사용된 용어 "투여"는 어떠한 적절한 방법으로 개체에게 본 발명의 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다.As used herein, the term "administration" means introducing the composition of the present invention into an individual by any appropriate method, and the route of administration of the composition of the present invention can be various oral or parenteral routes as long as it can reach the target tissue. It can be administered through
본 명세서에서 사용된 용어 "치료학적 유효량"은 연구원, 수의사, 의사 또는 다른 임상의에 의해 모색되며, 치료될 질환 또는 장애의 징후를 경감시킴을 포함하는, 조직계, 동물 또는 사람에서의 생물학적 또는 의학적 반응을 유도하는 활성 화합물 또는 약제학적 제제의 양을 의미한다.As used herein, the term “therapeutically effective amount” refers to a biological or medical treatment in a tissue system, animal or human that is sought by a researcher, veterinarian, physician or other clinician and that includes alleviating the symptoms of the disease or disorder being treated. It refers to the amount of active compound or pharmaceutical agent that induces a reaction.
본 명세서에서 사용된 용어 "조성물"은 특정 성분을 특정량으로 포함하는 생성물 및 특정량의 특정 성분의 배합물로부터 직접 또는 간접적으로 생성되는 임의의 생성물을 포함한다.As used herein, the term “composition” includes a product comprising specified ingredients in specified amounts and any product that results directly or indirectly from a combination of specified amounts of specified ingredients.
본 명세서에서 사용된 용어 "근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)"는 근긴장성 이영양증 단백질 키나아제의 임의의 핵산 또는 단백질을 의미한다. "DMPK 발현"은 DMPK을 암호화한 유전자로부터 전사된 mRNA의 수준 또는 mRNA로부터 번역된 단백질의 수준을 의미한다. DMPK 발현은 노던 또는 웨스턴 블럿과 같은 본 기술분야에서 공지된 방법에 의해서 측정될 수 있다. "DMPK 핵산"은 DMPK를 암호화하는 임의의 핵산을 의미한다. 예를 들어, 특정 구현예에서, DMPK 핵산은 DMPK를 암호화한 DNA 서열, DMPK를 암호화한 DNA (인트론 및 엑손을 포함하는 게놈 DNA를 포함)로부터 전사된 RNA 서열, 및 DMPK를 암호화한 mRNA 또는 예비-mRNA 서열을 포함한다. "DMPK mRNA"는 DMPK 단백질을 암호화한 mRNA를 의미한다.As used herein, the term “myotonic dystrophy protein kinase (DMPK)” refers to any nucleic acid or protein of myotonic dystrophy protein kinase. “DMPK expression” means the level of mRNA transcribed from the gene encoding DMPK or the level of protein translated from mRNA. DMPK expression can be measured by methods known in the art, such as Northern or Western blot. “DMPK nucleic acid” means any nucleic acid encoding DMPK. For example, in certain embodiments, the DMPK nucleic acid is a DNA sequence encoding DMPK, an RNA sequence transcribed from DNA encoding DMPK (including genomic DNA including introns and exons), and an mRNA or preparatory sequence encoding DMPK. -Contains an mRNA sequence. “DMPK mRNA” means mRNA encoding DMPK protein.
본 명세서에서 사용된 용어 “표적화” 또는 “표적화된”은 표적 핵산에 특이적으로 혼성화하여 원하는 효과를 유도하는 안테센스 올리고뉴클레오티드의 설계 및 선택 과정을 의미한다. As used herein, the term “targeting” or “targeted” refers to the process of designing and selecting antessense oligonucleotides that induce a desired effect by specifically hybridizing to a target nucleic acid.
본 명세서에서 사용된 용어 "뉴클레오사이드"는 당에 연결된 핵염기를 의미한다. 특정 구현예에서, 뉴클레오사이드는 인산기와 연결된다.As used herein, the term “nucleoside” refers to a nucleobase linked to a sugar. In certain embodiments, the nucleoside is linked to a phosphate group.
본 명세서에서 사용된 용어 "뉴클레오티드"는 뉴클레오사이드의 당 부분에 공유적으로 연결된 인산기를 갖는 뉴클레오사이드를 의미한다As used herein, the term “nucleotide” refers to a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
본 명세서에서 사용된 용어 "올리고뉴클레오티드"는 연결된 뉴클레오사이드의 중합체를 의미하고, 각각의 뉴클레오사이드 및 뉴클레오사이드간 연결은 서로 독립적으로 변형되거나 비변형될 수 있다.As used herein, the term “oligonucleotide” refers to a polymer of linked nucleosides, and each nucleoside and internucleoside linkage may be independently modified or unmodified.
본 명세서에서 사용된 용어 "포스포로티오에이트"는 포스포디에스테르 결합이 비-가교화 산소 원자들 중의 하나를 황 원자로 대체시킴으로써 변형된 뉴클레오사이드들 사이의 연결을 의미한다. 포스포로티오에이트 연결은 변형된 뉴클레오사이드간 연결이다.As used herein, the term “phosphorothioate” refers to a linkage between nucleosides in which the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
본 명세서에서 사용된 용어 “2'-O-메톡시에틸"(2'-MOE 및 2'-O(CH2) 2-OCH3)은 푸로실 환의 2' 위치의 O-메톡시-에틸 변형을 나타낸다. 2'-O-메톡시에틸 변형된 당은 변형된 당이다.As used herein, the term "2'-O-methoxyethyl"(2'-MOE and 2'-O(CH 2 ) 2 -OCH 3 ) refers to O-methoxy-ethyl modification at the 2' position of the furosyl ring. 2'-O-methoxyethyl modified sugar is a modified sugar.
1. 근긴장성 이영양증 단백질 키나아제를 표적화하는 안티센스 올리고뉴클레오티드(ASO)1. Antisense oligonucleotide (ASO) targeting myotonic dystrophy protein kinase
본 발명은 This invention
서열번호 1 내지 5의 염기서열로 이루어진 군으로부터 선택되는 근긴장성 이영양증 단백질 키나아제를 표적화하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)를 제공하고자 한다. 본 발명에 따른 ASO를 표 1에 나타내었다. The object is to provide an antisense oligonucleotide (ASO) targeting myotonic dystrophy protein kinase selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5. ASO according to the present invention is shown in Table 1.
Sequence( Sequence( <MOE><MOE> *PS**PS* <MOE><MOE> )) MerMer SEQ ID NO.SEQ ID NO.
88 <C*T*G*C*A>*<C*T*G*C*A>* T*G*T*C*T*G*C*C*T*G*T*G*T*C*T*G*C*C*T*G* <T*C*C*C*T><T*C*C*C*T> 2020 SEQ ID. 1SEQID. One
2525 <C*C*C*G*G><C*C*C*G*G> *C*T*A*C*A*A*G*G*A*C**C*T*A*C*A*A*G*G*A*C* <C*C*T*T*C><C*C*T*T*C> 2020 SEQ ID. 2SEQID. 2
2929 <C*A*G*C*A><C*A*G*C*A> *G*C*A*G*C*A*G*C*A*T**G*C*A*G*C*A*G*C*A*T* <T*C*C*C*G><T*C*C*C*G> 2020 SEQ ID. 3SEQID. 3
3030 <A*G*C*A*G><A*G*C*A*G> *C*A*G*C*A*G*C*A*G*C**C*A*G*C*A*G*C*A*G*C* <A*T*T*C*C><A*T*T*C*C> 2020 SEQ ID. 4SEQID. 4
3232 <G*C*G*C*A><G*C*G*C*A> *C*C*T*T*C*C*C*G*A*A**C*C*T*T*C*C*C*G*A*A* <T*G*T*C*C><T*G*T*C*C> 2020 SEQ ID. 5SEQID. 5
(상기 표 1의 안티센스 올리고뉴클레오티드에서 뉴클레오티드 사이의 * 표시는 포스포로티오에이트를 나타내고, 밑줄 표시된 뉴클레오티드는 2'-O-메톡시에틸변형된 뉴클레오티드를 나타낸다)상기 ASO는 표적 근긴장성 이영양증 단백질 키나아제 핵산에 대한 안티센스로서 수소 결합, 예를 들면 왓슨-크릭(Watson-Crick), 후그스텐(Hoogsteen) 또는 역전된 후그스텐(Hoogsteen) 수소 결합을 통해 표적 핵산에 대한 혼성화 수반한다. (* between nucleotides in the antisense oligonucleotides in Table 1 above indicates phosphorothioate, and underlined nucleotides indicate 2'-O-methoxyethyl modified nucleotides) The ASO targets myotonic dystrophy protein kinase nucleic acid. As antisense to, it involves hybridization to the target nucleic acid via hydrogen bonds, for example Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonds.
2. 세포투과성 펩티드(aMTD)-에 융합된 ASO 2. ASO fused to cell-penetrating peptide (aMTD)
본 발명은 근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)를 표적화하는 세포투과성 펩티드(advanced macromolecule transduction domain, aMTD)에 융합된 안티센스 올리고뉴클레오티드를 제공하고자 한다. The present invention seeks to provide an antisense oligonucleotide fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) targeting myotonic dystrophy protein kinase (DMPK).
본 발명에 따른 안티센스 올리고뉴클레오티드에 있어서, 상기 안티센스 올리고뉴클레오티드는 서열번호 1 내지 5의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the antisense oligonucleotide according to the present invention, the antisense oligonucleotide may be selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5.
본 발명에 따른 안티센스 올리고뉴클레오티드에 있어서, 상기 세포투과성 펩티드는 서열번호 6 내지 10의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the antisense oligonucleotide according to the present invention, the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
본 발명에 따른 안티센스 올리고뉴클레오티드에 있어서, 상기 aMTD-에 융합된 ASO는 서열번호 11 내지 35의 염기서열로 이루어진 군으로부터 선택될 수 있다.In the antisense oligonucleotide according to the present invention, the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
본 발명에 따른 안티센스 올리고뉴클레오티드는 DMPK의 발현을 적어도 약 15%, 적어도 약 20%, 적어도 약 25%, 적어도 약 30%, 적어도 약 35%, 적어도 약 40%, 적어도 약 45%, 적어도 약 50%, 적어도 약 55%, 적어도 약 60%, 적어도 약 65%, 적어도 약 70%, 적어도 약 75%, 적어도 약 80%, 적어도 약 85%, 적어도 약 90%, 적어도 약 95% 또는 적어도 약 99% 감소시킬 수 있다. Antisense oligonucleotides according to the invention reduce the expression of DMPK by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%. %, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99 % can be reduced.
3. 근위축성 이영양증 예방 또는 치료용 약제학적 조성물3. Pharmaceutical composition for preventing or treating muscular dystrophy
본 발명은 세포투과성 펩티드(advanced macromolecule transduction domain, aMTD)에 융합된 안티센스 올리고뉴클레오티드를 유효성분으로 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1) 예방 또는 치료를 위한 약제학적 조성물을 제공하고자 한다. The present invention seeks to provide a pharmaceutical composition for the prevention or treatment of myotonic dystrophy type 1 (DM1), which contains as an active ingredient an antisense oligonucleotide fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD).
본 발명에 따른 약제학적 조성물에 있어서, 상기 안티센스 올리고뉴클레오티드는 서열번호 1 내지 5의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the pharmaceutical composition according to the present invention, the antisense oligonucleotide may be selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5.
본 발명에 따른 약제학적 조성물에 있어서, 상기 세포투과성 펩티드는 서열번호 6 내지 10의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the pharmaceutical composition according to the present invention, the cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
본 발명에 따른 약제학적 조성물에 있어서, 상기 aMTD-에 융합된 ASO는 서열번호 11 내지 35의 염기서열로 이루어진 군으로부터 선택될 수 있다.In the pharmaceutical composition according to the present invention, the ASO fused to the aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
본 발명에 따른 약제학적 조성물에 있어서, 상기 약제학적 조성물은 근위축성 이영양증의 발병과 관련된 증상을 예방 또는 치료할 수 있다. 예를 들면, 상기 근위축성 이영양증의 발병과 관련된 증상은 근육 경직, 근긴장증, 말초 약화 무력증, 안면 및 턱 근육 약화, 연하곤란, 눈꺼풀 수하(안검하수), 목근육 약화, 팔 및 다리 근육 약화, 계속된 근육 통증, 수면과잉, 근육 소모, 연하곤란증, 호흡 부족, 불규칙한 심장박동, 심근 손상, 무감각, 인슐린 저항 또는 백내장을 포함할 수 있으나, 이에 한정되는 것은 아니다. In the pharmaceutical composition according to the present invention, the pharmaceutical composition can prevent or treat symptoms related to the onset of amyotrophic dystrophy. For example, symptoms associated with the development of the above-mentioned amyotrophic dystrophy include muscle stiffness, myotonia, peripheral weakness atony, facial and jaw muscle weakness, dysphagia, eyelid drooping (ptosis), neck muscle weakness, arm and leg muscle weakness, These may include, but are not limited to, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, shortness of breath, irregular heartbeat, myocardial damage, numbness, insulin resistance, or cataracts.
본 발명에 따른 약제학적 조성물에 있어서, 상기 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 상기 약제학적 조성물은 한 번 또는 여러 번의 투여량으로 투여될 수 있으며, 예를 들어, 하루 1회 내지 4회로 나누어 투여될 수 있다. In the pharmaceutical composition according to the present invention, the appropriate dosage of the pharmaceutical composition is determined by the formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and response sensitivity. It varies depending on the same factors, and usually a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. The pharmaceutical composition may be administered once or in multiple doses, for example, once to four times a day.
본 발명에 따른 약제학적 조성물에 있어서, 상기 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때, 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. 또한, 상기 약제학적 조성물은 좌약, 스프레이, 연고, 크림, 젤, 흡입제 또는 피부 패치의 형태로 투여될 수 있다. 또한 상기 약제학적 조성물은 포유동물 투여용, 더 바람직하게는 인간 투여용으로 제조될 수 있다. 약제학적으로 허용되는 담체는 고체이거나 액체일 수 있으며, 부형제, 항산화제, 완충액, 정균제, 분산제, 흡착제, 계면활성제, 결합제, 방부제, 붕해제, 감미제, 향미제, 활택제, 방출조절제, 습윤제, 안정화제, 현탁화제 및 윤활제에서 선택되는 1종 이상일 수 있다. 또한, 약제학적으로 허용되는 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들의 혼합물로부터 선택될 수 있다. 예를 들면, 적절한 부형제(filler)로는, 당(예컨대, 덱스트로스, 수크로스, 말토스 및 락토스), 전분(예컨대, 옥수수 전분), 당-알코올(예컨대, 만니톨, 솔비톨, 말티톨, 에리스리톨 및 자일리톨), 전분 가수분해물(starch hydrolysates)(예컨대, 덱스트린 및 말토덱스트린), 셀롤로스 또는 셀룰로스 유도체(예컨대, 미세결정질 셀룰로스) 또는 이들의 혼합물을 사용할 수 있으나, 이에 한정되지 않는다. 적절한 결합제(binder)로는, 마그네슘 알루미늄 실리케이트, 포비돈, 코포비돈, 메틸셀룰로오스, 히드록시메틸셀룰로오스, 히드록시프로필메틸셀룰로오스, 히드록시프로필셀룰로오스, 히드록시에틸셀룰로오스, 젤라틴, 검류, 수크로스, 전분 페이스트 또는 이들의 혼합물을 사용할 수 있으나, 이에 한정되지 않는다. 적절한 방부제(preservative)로는, 벤조산, 소듐 벤조에이트, 벤질 알콜, 부틸화 하이드록시아니솔, 부틸화 하이드록시톨루엔, 클로르부톨, 갈레이트, 하이드록시벤조에이트, EDTA 또는 이들의 혼합물을 사용할 수 있으나, 이에 한정되지 않는다. 적절한 붕해제(disintegrant)로는, 전분 글리콜레이트 소듐염(sodium starch glycolate), 가교된 폴리비닐 피롤리돈, 가교된 카르복시메틸셀룰로스, 전분, 미세결정질 셀룰로스 또는 이들의 혼합물을 사용할 수 있으나, 이에 한정되지 않는다. 적절한 감미제로는 수크랄로스, 사카린, 소듐 또는 포타슘 또는 칼슘 사카린, 아세설팜 포타슘 또는 소듐 시클라메이트, 만니톨, 프럭토스, 수크로스, 말토스 또는 이들의 혼합물을 사용할 수 있으나, 이에한정되지 않는다. 적절한 활택제(glidant)로는 실리카, 콜로이드성 실리콘 디옥사이드, 탈크 등을 사용할 수 있으나, 이에 한정되지 않는다. 적절한 윤활제(lubricant)로는, 장쇄 지방산 및 그 염, 예컨대, 마그네슘 스테아레이트 및 스테아르산, 탈크, 글리세라이드 왁스 또는 이들의 혼합물을 사용할 수 있으나, 이에 한정되지 않는다.In the pharmaceutical composition according to the present invention, the pharmaceutical composition uses a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. It can be formulated into a unit dose form or can be manufactured by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer. Additionally, the pharmaceutical composition may be administered in the form of a suppository, spray, ointment, cream, gel, inhalant, or skin patch. Additionally, the pharmaceutical composition may be prepared for administration to mammals, more preferably for administration to humans. Pharmaceutically acceptable carriers may be solid or liquid and include excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavoring agents, lubricants, release regulators, wetting agents, It may be one or more selected from stabilizers, suspending agents, and lubricants. Additionally, the pharmaceutically acceptable carrier may be selected from saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof. For example, suitable fillers include sugars (e.g., dextrose, sucrose, maltose, and lactose), starches (e.g., corn starch), sugar-alcohols (e.g., mannitol, sorbitol, maltitol, erythritol, and xylitol). ), starch hydrolysates (e.g., dextrin and maltodextrin), cellulose or cellulose derivatives (e.g., microcrystalline cellulose), or mixtures thereof may be used, but are not limited thereto. Suitable binders include magnesium aluminum silicate, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gum, sucrose, starch paste or A mixture of these may be used, but is not limited thereto. Suitable preservatives include benzoic acid, sodium benzoate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorbutol, gallate, hydroxybenzoate, EDTA, or mixtures thereof. It is not limited to this. Suitable disintegrants include, but are not limited to, sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked carboxymethylcellulose, starch, microcrystalline cellulose, or mixtures thereof. No. Suitable sweeteners include, but are not limited to, sucralose, saccharin, sodium or potassium or calcium saccharin, acesulfame potassium or sodium cyclamate, mannitol, fructose, sucrose, maltose or mixtures thereof. Suitable lubricants include, but are not limited to, silica, colloidal silicon dioxide, and talc. Suitable lubricants include, but are not limited to, long-chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glyceride wax, or mixtures thereof.
4. 근위축성 이영양증의 경감 또는 치료 방법4. Methods for alleviating or treating amyotrophic dystrophy
본 발명은 세포투과성 펩티드(advanced macromolecule transduction domain, aMTD)에 융합된 안티센스 올리고뉴클레오티드를 유효성분으로 포함하는 약제학적 조성물을 대상체에 투여하는 단계를 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1)의 경감 또는 치료 방법을 제공하고자 한다. The present invention is a treatment for myotonic dystrophy type 1 (DM1), comprising administering to a subject a pharmaceutical composition containing an antisense oligonucleotide fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) as an active ingredient. The goal is to provide relief or treatment methods.
본 발명에 따른 근위축성 이영양증의 경감 또는 치료 방법에 있어서, 상기 안티센스 올리고뉴클레오티드는 서열번호 1 내지 5의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the method for alleviating or treating muscular dystrophy according to the present invention, the antisense oligonucleotide may be selected from the group consisting of base sequences of SEQ ID NOs: 1 to 5.
본 발명에 따른 근위축성 이영양증의 경감 또는 치료 방법에 있어서, 상상기 세포투과성 펩티드는 서열번호 6 내지 10의 염기서열로 이루어진 군으로부터 선택될 수 있다. In the method for alleviating or treating amyotrophic dystrophy according to the present invention, the putative cell-penetrating peptide may be selected from the group consisting of base sequences of SEQ ID NOs: 6 to 10.
본 발명에 따른 근위축성 이영양증의 경감 또는 치료 방법에 있어서, 상기 aMTD-에 융합된 ASO는 서열번호 11 내지 35의 염기서열로 이루어진 군으로부터 선택될 수 있다.In the method for alleviating or treating muscular dystrophy according to the present invention, the ASO fused to aMTD- may be selected from the group consisting of base sequences of SEQ ID NOs: 11 to 35.
본 발명에 따른 근위축성 이영양증의 경감 또는 치료 방법에 있어서, 상기 투여는 경구 투여 또는 비경구 투여를 포함할 수 있다. 예를 들면, 상기 비경구 투여는 피하 투여, 정맥내 투여, 뇌실내 투여, 척추강내 투여, 근육내 투여, 동맥내 투여, 복강내 투여 또는 두개내 투여를 포함할 수 있다. 또한, 상기 투여는 만성적이거나, 연속적이거나, 단기간이거나 또는 간헐적일 수 있다. In the method for alleviating or treating amyotrophic dystrophy according to the present invention, the administration may include oral administration or parenteral administration. For example, the parenteral administration may include subcutaneous administration, intravenous administration, intracerebroventricular administration, intrathecal administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration. Additionally, the administration may be chronic, continuous, short-term, or intermittent.
본 발명에 따른 근위축성 이영양증의 경감 또는 치료 방법에 있어서, 상기 약제학적 조성물은 근위축성 이영양증의 발병과 관련된 증상을 예방 또는 치료할 수 있다. 예를 들면, 상기 근위축성 이영양증의 발병과 관련된 증상은 근육 경직, 근긴장증, 말초 약화 무력증, 안면 및 턱 근육 약화, 연하곤란, 눈꺼풀 수하(안검하수), 목근육 약화, 팔 및 다리 근육 약화, 계속된 근육 통증, 수면과잉, 근육 소모, 연하곤란증, 호흡 부족, 불규칙한 심장박동, 심근 손상, 무감각, 인슐린 저항 또는 백내장을 포함할 수 있으나, 이에 한정되는 것은 아니다. In the method for alleviating or treating amyotrophic dystrophy according to the present invention, the pharmaceutical composition can prevent or treat symptoms related to the onset of amyotrophic dystrophy. For example, symptoms associated with the development of the above-mentioned amyotrophic dystrophy include muscle stiffness, myotonia, peripheral weakness atony, facial and jaw muscle weakness, dysphagia, eyelid drooping (ptosis), neck muscle weakness, arm and leg muscle weakness, These may include, but are not limited to, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, shortness of breath, irregular heartbeat, myocardial damage, numbness, insulin resistance, or cataracts.
이하, 발명의 이해를 돕기 위해 다양한 실시예를 제시한다. 하기 실시예는 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 발명의 보호범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, various embodiments are presented to aid understanding of the invention. The following examples are provided to make the invention easier to understand, and the scope of protection of the invention is not limited to the following examples.
<실시예><Example>
실시예 1. hDMPK를 표적화하는 ASO의 설계 및 이의 효능 평가Example 1. Design of ASO targeting hDMPK and evaluation of its efficacy
도 1에 나타낸 hDMPK 서열을 표적화하는 ASO 36개를 설계하여 근위축성 이영양증 환자 유래 세포 중 GM04033 및 GM03132에 각각 Transfection 하여 DMPK의 발현 억제 효능을 실시간 중합효소연쇄반응 (quantitative Polymerase Chain Reaction: qPCR) 분석을 통해 검증하였으며, 두 세포주에서의 유전자발현 억제 효능 수치를 평균으로 하여 최종적으로 효능이 좋은 5종(ASODMPK 32, 29, 25, 30 및 8)을 선별하고(도 2), 선별된 5종의 ASODMPK의 서열정보를 도 3에 나타내었다. 36 ASOs targeting the hDMPK sequence shown in Figure 1 were designed and transfected into GM04033 and GM03132, respectively, among cells derived from patients with amyotrophic dystrophy, and the efficacy of suppressing DMPK expression was analyzed through real-time quantitative polymerase chain reaction (qPCR) analysis. It was verified through the average of the gene expression inhibition efficacy values in the two cell lines, and finally five types with good efficacy ( ASO DMPK 32, 29, 25, 30, and 8) were selected (Figure 2), and the five selected types were The sequence information of ASO DMPK is shown in Figure 3.
실시예 2. 세포투과성 펩티드의 설계Example 2. Design of cell-penetrating peptide
도 4에 나타낸 바와 같이 6가지 critical factors인 amino acid length (12개), rigidity/flexibility, hydropathy, bending potential, amino acid composition, structural feature를 기준으로 하여 세포투과능이 뛰어난 5종의 aMTD를 제작하였다.As shown in Figure 4, five types of aMTDs with excellent cell penetration ability were produced based on six critical factors: amino acid length (12), rigidity/flexibility, hydropathy, bending potential, amino acid composition, and structural feature.
실시예 3. aMTD-ASODMPK의 제작 및 효능평가Example 3. Production and efficacy evaluation of aMTD-ASO DMPK
도 5에 나타낸 바와 같이 상기 실시예 1에서 설계된 ASO의 5'에 Chemical Linker (Succinimidyl-4-(N-Maleimidomethyl Cyclohexane)-1-Carboxylate: SMCC)를 이용하여 aMTD을 융합시켜 aMTD-ASODMPK를 제작하였다. As shown in Figure 5, aMTD-ASO DMPK was produced by fusing aMTD to the 5' side of the ASO designed in Example 1 using Chemical Linker (Succinimidyl-4-(N-Maleimidomethyl Cyclohexane)-1-Carboxylate: SMCC). did.
근위축성 이영양증 환자 유래 세포에 단순 ASO의 주요 세포내 전송 메커니즘인 식포작용을 못하게 하는 식포작용 저해제(Endocytosis Inhibitor)를 처리하여 단순 ASO가 전송되지 않는 조건에서 5종의 ASODMPK 및 18종의 aMTD-ASODMPK를 처리하여 질병원인 유전자 DMPK의 발현 억제 효능을 qPCR 분석을 통해 검증하였다. 그 결과, aMTD-ASODMPK는 단순 ASO 보다 질병원인 유전자의 넉다운 효능을 통계적 유의성 99% 신뢰수준으로 향상시키며 이는 농도의존적으로 억제시킬 수 있음이 확인되었다. 특히, aMTD321-ASODMPK8, aMTD341-ASODMPK8, aMTD342-ASODMPK8, aMTD341-ASODMPK29, aMTD342-ASODMPK30 및 aMTD462-ASODMPK30의 경우 DMPK 발현 50% 저해농도 (IC50)를 최대 13배까지 향상시키는 것으로 확인되었다(도 6). By treating cells derived from patients with amyotrophic dystrophy with an endocytosis inhibitor that prevents phagocytosis, the main intracellular transport mechanism of simple ASOs, 5 types of ASO DMPK and 18 types of aMTD- The efficacy of ASO DMPK treatment to inhibit the expression of the disease-causing gene DMPK was verified through qPCR analysis. As a result, it was confirmed that aMTD-ASO DMPK improves the knockdown efficacy of disease-causing genes to a 99% confidence level of statistical significance compared to simple ASO, and that it can be suppressed in a concentration-dependent manner. In particular, for aMTD321-ASO DMPK 8, aMTD341-ASO DMPK 8, aMTD342-ASO DMPK 8, aMTD341-ASO DMPK 29, aMTD342-ASO DMPK 30 and aMTD462-ASO DMPK 30, the 50% inhibitory concentration (IC50) of DMPK expression was maximal. It was confirmed that it improved by up to 13 times (Figure 6).
또한, hDMPK 유전자가 삽입된 근긴장성 이영양증 마우스 모델에ASODMPK8와 세포투과성이 있는 aMTD321-ASODMPK8을 정맥 투여하여 효능을 분석한 결과, 뇌, 심장 및 근육조직에서 ASODMPK8 보다 aMTD321-ASODMPK8에 의해 DMPK 유전자 넉다운 효능이 유의성 있게 증가하는 것으로 확인되었다(도 7).In addition, ASO DMPK 8 and cell-permeable aMTD321-ASO DMPK 8 were administered intravenously to a myotonic dystrophy mouse model in which the hDMPK gene was inserted. As a result of analyzing the efficacy, it was confirmed that the DMPK gene knockdown efficacy was significantly increased by aMTD321-ASO DMPK 8 compared to ASO DMPK 8 in brain, heart, and muscle tissue (FIG. 7).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the practical scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)를 표적화하는 세포투과성 펩티드(Cell-Penetrating Peptide: CPP)로서 aMTD(advanced Macromolecule Transduction Domain)와 융합된 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)는 DMPK의 발현을 현저히 감소시킬 수 있다. 따라서, aMTD와 융합된 ASO는 근위축성 이영양증(myotonic dystrophy type 1, DM1)의 예방, 치료 또는 경감을 위한 핵산 치료제로서 효과적으로 사용될 수 있을 것으로 기대된다. According to the present invention, a cell-penetrating peptide (CPP) targeting myotonic dystrophy protein kinase (DMPK) is an antisense oligonucleotide (ASO) fused with aMTD (advanced macromolecule transduction domain). ) can significantly reduce the expression of DMPK. Therefore, it is expected that ASO fused with aMTD can be effectively used as a nucleic acid therapeutic agent for preventing, treating or alleviating myotonic dystrophy type 1 (DM1).

Claims (9)

  1. 근긴장성 이영양증 단백질 키나아제(myotonic dystrophy protein kinase, DMPK)를 표적화하는 세포투과성 펩티드(advanced macromolecule transduction domain, aMTD)에 융합된 안티센스 올리고뉴클레오티드(antisense oligonucleotide, ASO)로서, As an antisense oligonucleotide (ASO) fused to a cell-penetrating peptide (advanced macromolecule transduction domain, aMTD) targeting myotonic dystrophy protein kinase (DMPK),
    상기 안티센스 올리고뉴클레오티드는 서열번호 1 내지 5로 이루어진 군으로부터 선택되는 것인, 안티센스 올리고뉴클레오티드.The antisense oligonucleotide is an antisense oligonucleotide selected from the group consisting of SEQ ID NOs: 1 to 5.
  2. 제1항에 있어서,According to paragraph 1,
    상기 세포투과성 펩티드는 서열번호 6 내지 10으로 이루어진 군으로부터 선택되는 것인, 안티센스 올리고뉴클레오티드.The cell-penetrating peptide is an antisense oligonucleotide selected from the group consisting of SEQ ID NOs: 6 to 10.
  3. 제1항에 있어서,According to paragraph 1,
    상기 aMTD에 융합된 ASO는 서열번호 11 내지 35로 이루어진 군으로부터 선택되는 것인, 안티센스 올리고뉴클레오티드.The ASO fused to the aMTD is an antisense oligonucleotide selected from the group consisting of SEQ ID NOs: 11 to 35.
  4. 제1항에 있어서,According to paragraph 1,
    상기 안티센스 올리고뉴클레오티드는 DMPK의 발현을 적어도 20% 감소시키는 것을 특징으로 하는 것인, 안티센스 올리고뉴클레오티드. The antisense oligonucleotide is characterized in that it reduces the expression of DMPK by at least 20%.
  5. 제1항 내지 제4항 중 어느 한 항에 따른 안티센스 올리고뉴클레오티드를 유효성분으로 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1) 예방 또는 치료를 위한 약제학적 조성물. A pharmaceutical composition for preventing or treating myotonic dystrophy (myotonic dystrophy type 1, DM1), comprising the antisense oligonucleotide according to any one of claims 1 to 4 as an active ingredient.
  6. 제5항에 있어서,According to clause 5,
    상기 약제학적 조성물은 근위축성 이영양증의 발병과 관련된 증상을 예방 또는 치료할 수 있고, The pharmaceutical composition can prevent or treat symptoms related to the onset of amyotrophic dystrophy,
    상기 근위축성 이영양증의 발병과 관련된 증상은 근육 경직, 근긴장증, 말초 약화 무력증, 안면 및 턱 근육 약화, 연하곤란, 눈꺼풀 수하(안검하수), 목근육 약화, 팔 및 다리 근육 약화, 계속된 근육 통증, 수면과잉, 근육 소모, 연하곤란증, 호흡 부족, 불규칙한 심장박동, 심근 손상, 무감각, 인슐린 저항 또는 백내장을 포함하는 것을 특징으로 하는 것인, 약제학적 조성물. Symptoms associated with the onset of the above-mentioned amyotrophic dystrophy include muscle stiffness, myotonia, peripheral weakness, asthenia, facial and jaw muscle weakness, dysphagia, eyelid drooping (ptosis), neck muscle weakness, arm and leg muscle weakness, and persistent muscle pain. , hypersomnia, muscle wasting, dysphagia, shortness of breath, irregular heartbeat, myocardial damage, numbness, insulin resistance, or cataracts.
  7. 제1항 내지 제4항 중 어느 한 항에 따른 안티센스 올리고뉴클레오티드를 포함하는 약제학적 조성물을 대상체에 투여하는 단계를 포함하는 근위축성 이영양증(myotonic dystrophy type 1, DM1)의 경감 또는 치료 방법. A method for alleviating or treating muscular dystrophy (myotonic dystrophy type 1, DM1) comprising administering to a subject a pharmaceutical composition comprising the antisense oligonucleotide according to any one of claims 1 to 4.
  8. 제7항에 있어서, In clause 7,
    상기 약제학적 조성물은 경구 투여, 피하 투여, 정맥내 투여, 뇌실내 투여, 척추강내 투여, 근육내 투여, 동맥내 투여, 복강내 투여 또는 두개내 투여되는 것을 특징으로 하는 것인, 방법. The pharmaceutical composition is administered orally, subcutaneously, intravenously, intracerebroventricularly, intrathecally, intramuscularly, intraarterially, intraperitoneally, or intracranially.
  9. 제7항에 있어서,In clause 7,
    상기 대상체는 인간을 제외한 포유동물인 것을 특징으로 하는 것인, 방법. A method, wherein the subject is a mammal other than a human.
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