WO2022024833A1 - アンギオテンシン変換酵素2遺伝子のエクソンのスキッピングを誘導するアンチセンス核酸 - Google Patents
アンギオテンシン変換酵素2遺伝子のエクソンのスキッピングを誘導するアンチセンス核酸 Download PDFInfo
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- C12N15/1137—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 against enzymes
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- C12N2320/33—Alteration of splicing
Definitions
- the present invention relates to an antisense nucleic acid that induces exon skipping of the angiotensin converting enzyme 2 gene.
- Angiotensin converting enzyme 2 is a approximately 92 kDa type I transmembrane protein consisting of 805 amino acids, encoded by the 107 kb ACE2 gene located on the X chromosome Xp22. The size of the cDNA is about 3 kb and consists of 19 exons (NM_021804.3). ACE2 has a hydrolyzing effect that cuts off the amino acid at the C'terminal of the peptide, and converts angiotensin II to angiotensin (1-7) (Ang (1-7)) as carboxypeptidase.
- ACE2 is a receptor for the invasion of the coronavirus SARS-CoV into the body. It attracted attention as a key molecule for the establishment of infection. It was also clarified that the SARS-CoV-2 virus, which is currently spreading worldwide as COVID-19, also uses ACE2 as a receptor (Non-Patent Document 1).
- ACE2 is a transmembrane protein consisting of three domains: extracellular, transmembrane, and intracellular, and has a virus-binding domain in the extracellular domain. This virus-binding domain binds to the SARS-CoV-2 virus with high affinity and functions as a virus receptor.
- Non-Patent Document 2 Splicing is a reaction in which an intron is excised from a pre-mRNA transcribed from a gene to produce a mature mRNA composed only of exons.
- the splicing site is determined by the sequence of GT-AGs present at both ends of the intron, which is called the splicing consensus sequence.
- the splicing-promoting sequence acts as a cis factor, resulting in an accurate splicing reaction.
- Antisense nucleic acid (ASO) to this splicing-promoting sequence inhibits the splicing-promoting function and results in exon skipping.
- ASO Antisense nucleic acid
- the ACE2 gene is a gene consisting of exon 19, and the transmembrane domain is encoded by exon 18.
- Skipping exon 18 eliminates 195 bases from the mRNA and shortens the ACE2 protein by 65 amino acids.
- This protein is a novel lytic ACE2 lacking a transmembrane domain and is expected to be a decoy receptor for the virus. Therefore, skipping of exon 18 brings about an increase in Otori receptor as well as a decrease in SARS-CoV-2 receptor, and is highly expected as a powerful and epoch-making therapeutic method for preventing virus infection.
- An object of the present invention is to provide an antisense nucleic acid (ASO) that induces exon skipping of the ACE2 gene for the purpose of suppressing the expression of ACE2 protein and promoting the expression of lysed ACE2.
- ASO antisense nucleic acid
- the present inventors have identified an antisense nucleic acid (ASO) that induces skipping of exon 18 of the ACE2 gene.
- ASO antisense nucleic acid
- the gist of the present invention is as follows. (1) An antisense oligonucleotide having 15 to 30 bases having a base sequence complementary to the target site of exon 18 of the angiotensin converting enzyme 2 gene, and inducing exon skipping of the angiotensin converting enzyme 2 gene. The antisense oligonucleotide, a salt thereof or a solvent product thereof. (2) The base sequence of exon 18 of the angiotensin converting enzyme 2 gene is the base sequence of SEQ ID NO: 1, and the target site of exon 18 of the angiotensin converting enzyme 2 gene is the base sequence of the base sequence of SEQ ID NO: 1 to 195.
- the base sequence of the antisense oligonucleotide is continuous in any of the base sequences of SEQ ID NOs: 2 to 17 (where t in the sequence may be u and u may be t).
- the base sequence of the antisense oligonucleotide is any of the base sequences of SEQ ID NOs: 2 to 17 (however, t in the sequence may be u and u may be t) (. 4) The antisense oligonucleotide, a salt thereof or a solvent product thereof. (6) The antisense oligonucleotide according to any one of (1) to (5), wherein at least one nucleotide is modified, a salt thereof, or a solvate thereof.
- a pharmaceutical agent comprising the antisense oligonucleotide according to any one of (1) to (8), a pharmaceutically acceptable salt or solvate thereof.
- the drug according to (9) for suppressing the infectivity of SARS-CoV-2 virus (11) It has the effect of inhibiting the uptake of virus into cells by reducing the receptor-type angiotensin converting enzyme 2 and / or capturing the virus extracellularly by increasing the lytic angiotensin-converting enzyme 2 capable of binding to the virus (10). ) The listed drug. (12) The drug according to any one of (8) to (11) for preventing and / or treating SARS-CoV-2 infection.
- a polynucleotide comprising the nucleotide sequence encoding the lysed angiotensin converting enzyme 2 according to (14) and / or a sequence complementary thereto.
- SARS-CoV-2 comprising administering to the subject an effective amount of the antisense oligonucleotide according to any one of (1) to (8), a pharmaceutically acceptable salt or solvate thereof. How to control the infectivity of viruses.
- SARS-CoV-2 comprising administering to a subject an effective amount of the antisense oligonucleotide according to any one of (1) to (8), a pharmaceutically acceptable salt or solvate thereof. How to prevent and / or treat an infection.
- the angiotensin converting enzyme 2 comprising administering to the subject an effective amount of the antisense oligonucleotide according to any one of (1) to (8), a pharmaceutically acceptable salt or solvate thereof.
- a pharmaceutically acceptable salt or solvate is a pharmaceutically acceptable salt or solvate.
- the antisense oligonucleotide according to any one of (1) to (8) in the production of a drug for suppressing the expression of the protein of angiotensin converting enzyme 2 and / or promoting the expression of the dissolved angiotensin converting enzyme 2.
- Exon 18 skipping of the ACE2 gene produces an mRNA encoded by exon 18 that is 195 bases shorter.
- the result is the ACE2 protein, which is 65 amino acids shorter.
- these 65 amino acids contain all the amino acid sequences that make up the transmembrane domain, producing soluble ACE2 that lacks the transmembrane domain.
- suppression of SARS-CoV-2 infection receptor expression, decoy receptor expression, and peptidase expression are achieved. It is expected that a large preventive effect on SARS-CoV-2 infection will be obtained.
- This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2020-127142, and / or the drawings, which are the basis of the priority of the present application.
- Target site for ACE2 pre-mRNA and ASO The ACE2 pre-mRNA produced by transcription from the ACE2 gene is composed of 19 exons. ASO1, 2, and 3 complementary to the sequence in exon 18 of ACE2 were prepared for the purpose of skipping exon 18. Comparison of ASO effectiveness. ASO1, 2 and 3 are introduced into human hepatocellular carcinoma cells (HepG2), respectively, and the results of RT-PCR of their ACE2 mRNA are shown. a) The results of RT-PCR of ACE2 mRNA and GAPDH mRNA of human liver cancer cells (HepG2) are shown. w / o is ASO unprocessed.
- ASO9, In16Ex17, and Ex17In17 that are complementary to exon 18 of ACE2 and intron sequences on both sides. Comparison of ASO effectiveness. ASO9, In16Ex17, and Ex17In17 are introduced into human hepatocellular carcinoma cells (HepG2), respectively, and the results of RT-PCR of their ACE2 mRNA are shown. a) The results of RT-PCR of ACE2 mRNA and GAPDH mRNA of human liver cancer cells (HepG2) are shown. w / o is ASO unprocessed. The white arrowhead indicates ACE2, and the black arrowhead indicates ACE2 in which exon 18 is skipped.
- ASO5 + 5, 5 + 6, 5 + 7, 5 + 8, 5 + 10, 5 + 12 complementary to the sequence in exon 18 of ACE2 to search for an effective ASO to skip exon 18.
- a) The results of RT-PCR of ACE2 mRNA and GAPDH mRNA of human liver cancer cells (HepG2) are shown.
- w / o is ASO unprocessed.
- the white arrowhead indicates ACE2, and the black arrowhead indicates ACE2 in which exon 18 is skipped.
- ASO5 + 7 and 5c complementary to the sequence in exon 18 of ACE2.
- the target sequence of ASO5c is the same as that of ASO5, but the positions of ENA and 2'OMe in ASO are different from those of ASO5.
- ASO5 + 7, 5c are introduced into human liver cancer cells (HepG2), respectively, and the results of RT-PCR of their ACE2 mRNA are shown.
- the present invention is an antisense oligonucleotide having 15 to 30 bases having a base sequence complementary to the target site of exon 18 of the angiotensin converting enzyme 2 gene, and induces exon skipping of the angiotensin converting enzyme 2 gene.
- said antisense oligonucleotides capable of being, salts thereof or solvates thereof.
- the nucleotide sequence of exon 18 of the human angiotensin converting enzyme 2 gene is shown in SEQ ID NO: 1.
- the target site of exon 18 of the angiotensin converting enzyme 2 gene is the base number 1 to the base sequence of the base sequence of SEQ ID NO: 1. It should be in the area of 195.
- the base sequence of the antisense oligonucleotide may be any of the base sequences of SEQ ID NOs: 2 to 17 (provided that t in the sequence may be u and u may be t. ) May contain a sequence consisting of at least 15 consecutive bases.
- the base length of the antisense oligonucleotide may be 18, and the base sequence of the antisense oligonucleotide may be any of the base sequences of SEQ ID NOs: 2 to 17 (provided that t in the sequence may be u. u may be t).
- the nucleotide constituting the antisense oligonucleotide may be a natural DNA, a natural RNA, or a modified product thereof, but it is preferable that at least one of them is a modified nucleotide.
- Modified nucleotides include those modified with sugar (eg, 2'-O-alkylated D-ribofuranose, 2'-O, 4'-C-alkyleneated D-ribofuranose.
- sugar eg, 2'-O-alkylated D-ribofuranose, 2'-O, 4'-C-alkyleneated D-ribofuranose.
- the 2'-hydroxyl group of D-ribofuranose is modified (for example, thioated)
- the phosphate diester bond is modified (for example, thioated)
- the base is modified, or a combination thereof.
- at least one D-ribofuranose constituting an antisense oligonucleotide is 2'-O-alkylated or 2'-O, 4'-C-alkyleneated.
- oligonucleotide that is, oligo DNA, oligo RNA
- at least one constituent of the oligonucleotide at least one constituent of the oligonucleotide.
- Those having a thioated phosphate diester bond can also be expected to have a higher therapeutic effect than natural nucleotides (that is, oligo DNA and oligo RNA) because of their high resistance to nucleases. Since the oligonucleotide containing both of the modified phosphoric acid has higher resistance to the nuclease, even higher therapeutic effect can be expected.
- sugar modifications include 2'-O-alkylation of D-ribofuranose (eg, 2'-O-methylation, 2'-O-aminoethylation, 2'-O. -Propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, 2'-O-propargylization, etc.), D-ribo 2'-O, 4'-C-alkyleneation of furanose (eg 2'-O, 4'-C-ethylation, 2'-O, 4'-C-methyleneation, 2'-O, 4' -C-propyleneation, 2'-O, 4'-C-tetramethyleneation, 2'-O, 4'-C-pentamethyleneation, etc.), 3'-deoxy-3'-amino-2'-deoxy -D-ribofuranose,
- D-ribofuranose eg, 2'
- examples of modification of the phosphate diester bond include phosphorothioate bond, methylphosphonate bond, methylthiophosphonate bond, phosphorodithioate bond, phosphoramidate bond and the like.
- examples of base modifications include 5-methylation, 5-fluoromation, 5-bromolation, 5-iodolation, N4-methylation of cytosine, and 5-demethylation of thymidine (uracil).
- 5-Fluoration, 5-Bromolation, 5-Iodization, N6-methylation of adenine, 8-bromolation, N2-methylation of guanine, 8-bromolation, pseudouridineization of uridine, 1-methylpseudouridine Uracilization and the like can be mentioned.
- the antisense oligonucleotide of the present invention may be in the form of a salt.
- the salt may be a pharmaceutically acceptable salt, and such salts include alkali metal salts such as sodium salt, potassium salt and lithium salt.
- Alkaline earth metal salts such as calcium salt, magnesium salt, metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt; inorganic salt such as ammonium salt, t-octylamine salt , Dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt , Chloroprocine salt, prokine salt, diethanolamine salt, N-benzyl-phenetylamine salt, piperazine salt, tetramethylammonium salt, amine salts such as organic salts such as tris (hydroxymethyl) aminomethane salt; Inorganic acid salts such as hydrochlorides, hydrobromates, halogen atomized amine
- Lower alcanul sulfonates such as acid salts, ethane sulfonates, benzene sulfonates, aryl sulfonates such as p-toluene sulfonates, acetates, apple salts, fumarates, succinates.
- Organic acid salts such as citrate, tartrate, oxalate, maleate; amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, asparaginate and the like. These salts can be produced by known methods.
- the antisense oligonucleotide may also exist as a solvate (for example, a hydrate), and may be such a solvate.
- the antisense oligonucleotide may be administered in the form of a prodrug, and examples of the prodrug include amides, esters, carbamates, carbonates, ureidos, phosphates and the like. These prodrugs can be produced by known methods.
- the method for synthesizing the antisense oligonucleotide is not particularly limited, and a conventionally known method can be adopted.
- Examples of the synthesis method include a synthesis method by a genetic engineering method, a chemical synthesis method, and the like.
- Examples of the genetic engineering method include an in vitro transcriptional synthesis method, a method using a vector, and a method using a PCR cassette.
- the vector is not particularly limited, and examples thereof include non-viral vectors such as plasmids and viral vectors.
- the chemical synthesis method is not particularly limited, and examples thereof include a phosphoramidite method and an H-phosphonate method.
- As the chemical synthesis method for example, a commercially available automatic nucleic acid synthesizer can be used.
- amidite is generally used as the chemical synthesis method.
- the amidite is not particularly limited, and in the examples described later, the antisense oligonucleotide was synthesized by the phosphoramidite method using ENA-2CE phosphoramidite and 2'OMe-2CE phosphoramidite.
- Phosphoramidite reagents include natural nucleosides and 2'-O-methyl nucleosides (ie, 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytosine, 2'-O- For methyluridine), commercially available reagents can be used. The following is for 2'-O-alkylguanosine, adenosine, cytosine and uridine having 2 to 6 carbon atoms in the alkyl group.
- 2'-O-aminoethylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725.).
- 2'-O-propylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, E.A. et al. Biochemistry (1993), 32, 7832-7838.).
- reagents can be used for 2'-O-allyl guanosine, adenosine, cytosine, and uridine.
- 2'-O-Methoxyethylguanosine, adenosine, cytosine, and uridine can be synthesized according to the patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.
- 2'-O-butylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, E.A. et al. Biochemistry (1993), 32, 7832-7838.).
- 2'-O-pentylguanosine, adenosine, cytosine, and uridine can be synthesized according to the literature (Lesnik, E.A. et al. Biochemistry (1993), 32, 7832-7838.).
- antisense having a phosphorothioate bond is reacted by reacting with a reagent such as sulfur, tetraethylthium disulfide (TETD, Applied Biosystem), Beaucage reagent (Glen Research), or xanthan hydride.
- a reagent such as sulfur, tetraethylthium disulfide (TETD, Applied Biosystem), Beaucage reagent (Glen Research), or xanthan hydride.
- TETD tetraethylthium disulfide
- Beaucage reagent Glen Research
- xanthan hydride xanthan hydride
- CPG controlled pore glass
- a commercially available one with 2'-O-methylnucleoside bonded can be used.
- 2'-O, 4'-C-methyleneguanosine, adenosine, 5-methylcytosine and thymidine 2'-O having 2 to 5 carbon atoms in the alkylene group according to the method described in WO99 / 14226.
- nucleosides prepared according to the method described in WO00 / 47599 were prepared according to the literature (Oligonucleotide Synthesis, Edited by MJGait, Oxford University Press, 1984). Can be combined with CPG.
- CPG modified CPG (described in Example 12b of JP-A-778982)
- an oligonucleotide in which a 2-hydroxyethyl phosphate group is attached to the 3'end can be synthesized.
- the antisense oligonucleotide of the present invention can be used in medicine.
- the antisense oligonucleotide may be in the form of a pharmaceutically acceptable salt, solvate or prodrug thereof. Therefore, the present invention is an antisense oligonucleotide having 15 to 30 bases having a base sequence complementary to the target site of exon 18 of the angiotensin converting enzyme 2 gene, and skipping the exon of the angiotensin converting enzyme 2 gene.
- a pharmaceutical comprising said antisense oligonucleotide that can be induced, a pharmaceutically acceptable salt or admixture thereof.
- the drug may be intended to suppress the infectivity of the SARS-CoV-2 virus, but is not limited to it.
- the pharmaceutical agent of the present invention exhibits the effect of inhibiting the uptake of virus into cells by reducing the receptor-type angiotensin converting enzyme 2 and / or increasing the extracellular angiotensin-converting enzyme 2 capable of binding to the virus to capture the virus extracellularly. I can have it.
- the pharmaceuticals of the present invention may be for preventing and / or treating SARS-CoV-2 infection.
- the present invention also provides a method for suppressing the infectivity of SARS-CoV-2 virus, which comprises administering to a subject an effective amount of the above antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof. do.
- the present invention also comprises a method of preventing and / or treating SARS-CoV-2 infection, comprising administering to the subject an effective amount of the antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof.
- the subject can be a human or an animal. Examples of animals include mammals such as dogs, cats, minks, tigers, lions, mice, rats, rabbits, sheep, pigs, cows, and horses.
- prevention of infection includes prevention of viral infection, prevention of the onset of unfavorable symptoms due to viral infection (viral infection), prevention of aggravation of viral infection, and “prevention” includes This includes reducing the rate of viral infections, reducing the incidence of unwanted symptoms due to viral infections, reducing the rate of aggravation of viral infections, and reducing the degree of aggravation of viral infections.
- treatment of infection includes cure from viral infection, relief of unfavorable symptoms due to viral infection, prevention or delay of aggravation of viral infection.
- the antisense oligonucleotide of the present invention may be used alone or as a pharmacologically acceptable carrier or dilution.
- active ingredient a pharmaceutically acceptable salt, solvate or prodrug thereof
- it can be administered orally or parenterally to a mammal (eg, human, rabbit, dog, cat, rat, mouse) as a suitable formulation.
- a mammal eg, human, rabbit, dog, cat, rat, mouse
- the dose varies depending on the administration subject, symptoms, administration route, etc., but for example, the single dose of the active ingredient is usually about 0.1 to 50 mg / kg body weight, preferably about 0.5 mg / kg body weight 1-3 times a day.
- It may be administered intranasally or intravenously (preferably continuously or every other day) at a frequency of about 1 degree, preferably about once a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.
- Formulations for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. , Emulsion, suspending agent, etc.
- Such a formulation can be produced by a conventional method, and may contain a carrier, a diluent or an excipient commonly used in the formulation field.
- examples of carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate and the like.
- nasal drops examples include nasal drops, injections, suppositories, etc.
- the nasal drops may be a dosage form such as nasal drops, nasal drops, etc. Is preferably a dosage form such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection.
- the active ingredient may be made into moderately fine particles, and if necessary, it may be mixed with the additive to make it homogeneous. It may be suspended and filtered if necessary. An tonicity agent, a pH regulator, or the like may be used.
- the additive include a preservative such as benzalkonium chloride, a binder such as hydroxypropyl cellulose, and an excipient such as lactose.
- physiological saline is usually used, but alcohol (for example, ethanol, isopropyl alcohol, etc.), glycol (for example, propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether, glycerol, etc.), polyoxyethylene alcohol, etc.
- a solubilizing agent may be added.
- Injections are prepared by conventional methods, that is, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily solution normally used for injections.
- Aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, etc.). It may be used in combination with polyethylene glycol), a nonionic surfactant (for example, polysorbate 80, HCO-50 (polyoxythethylene (50 mol) added of hydrogenated castor oil)) and the like. Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
- the prepared injection solution is usually filled in a suitable ampoule. Suppositories used for rectal administration can be prepared by mixing the active ingredient with a conventional suppository base.
- the above-mentioned oral or parenteral pharmaceutical preparation may be prepared in a dosage form of a dosage unit suitable for the dose of the active ingredient.
- a dosage form of such a dosage unit include tablets, pills, capsules, nasal preparations filled in nasal drops, injections (ampoules), suppositories, etc., and each dosage unit dosage form It is usually preferable that the active ingredient is contained in an amount of 0.1 to 1.000 mg.
- the antisense oligonucleotide of the present invention enables suppression of protein expression of angiotensin converting enzyme 2 and / or promotion of expression of lysed angiotensin converting enzyme 2. Therefore, the present invention is an antisense oligonucleotide having 15 to 30 bases having a base sequence complementary to the target site of exon 18 of the angiotensin converting enzyme 2 gene, and skipping the exon of the angiotensin converting enzyme 2 gene.
- the agent of the present invention can be used as a pharmaceutical or as an experimental reagent.
- the present invention comprises the administration of the above antisense oligonucleotide, a pharmaceutically acceptable salt or solvate thereof to a subject in an effective amount, and suppresses the expression of the protein of angiotensin converting enzyme 2 and / or dissolves the angiotensin.
- a method for promoting the expression of converting enzyme 2 is also provided.
- the subject can be a human or an animal.
- animals include mammals such as dogs, cats, minks, tigers, lions, mice, rats, rabbits, sheep, pigs, cows, and horses.
- the antisense oligonucleotide may be in the form of a salt, solvate or prodrug.
- salts, solvates or prodrugs include pharmaceutically acceptable salts, solvates or prodrugs, which have been described above.
- the expression of the angiotensin converting enzyme 2 protein When used as an experimental reagent, the expression of the angiotensin converting enzyme 2 protein by treating the cells, tissues or organs expressing the angiotensin converting enzyme 2 with the antisense oligonucleotide of the present invention, a salt thereof or a solvate thereof. Can be suppressed and the expression of lysed angiotensin converting enzyme 2 can be promoted.
- the antisense oligonucleotide of the present invention, a salt thereof and a solvate thereof can be used in an amount effective for suppressing the expression of the protein of angiotensin converting enzyme 2 or promoting the expression of the dissolved angiotensin converting enzyme 2. good.
- Examples of cells expressing angiotensin converting enzyme 2 include nasal mucosal epithelial goblet cells, type II alveolar epithelial cells and resorbable intestinal epithelial cells, liver cancer cells, vascular endothelial cells, renal tubule epithelial cells and the like. In addition to naturally occurring cells, recombinant cells into which the angiotensin converting enzyme 2 gene has been introduced can also be exemplified. Examples of tissues and organs expressing angiotensin converting enzyme 2 include heart, kidney, testis, lung, testis, small intestine, kidney, and prostate.
- angiotensin converting enzyme 2 For the expression of angiotensin converting enzyme 2, the angiotensin converting enzyme 2 mRNA in the sample is analyzed by RT-PCR, the angiotensin converting enzyme 2 protein in the sample is detected by Western blotting, or by mass spectrometry. It can be analyzed by this.
- Exon skipping of the angiotensin converting enzyme 2 gene induced by the antisense oligonucleotide of the present invention, a salt thereof or a solvate thereof produces a lysed angiotensin converting enzyme 2 lacking a transmembrane domain.
- the present invention also provides this lysed angiotensin converting enzyme 2.
- the present invention also provides a polynucleotide containing a nucleotide sequence encoding lytic angiotensin converting enzyme 2 and / or a sequence complementary thereto.
- the polynucleotide containing the lysed angiotensin converting enzyme 2 of the present invention and the nucleotide sequence encoding the same can be used as an exon skipping of the angiotensin converting enzyme 2 gene by the antisense oligonucleotide of the present invention in a cell expressing the angiotensin converting enzyme 2. Is produced by inducing.
- the lytic angiotensin converting enzyme 2 of the present invention extracts RNA from cells in which exon skipping of the angiotensin converting enzyme 2 gene has been induced, synthesizes cDNA using a reverse transcription enzyme and a random primer, and amplifies it by PCR.
- sequence analysis was performed to determine the sequence, and then restriction enzyme recognition sequences were added to the 5'and 3'sides of the sequence for which the codon usage frequency of the open reading frame was optimized, and then incorporated into an appropriate vector. It can be produced by introducing it into a suitable host cell and producing it as a recombinant protein.
- Vectors include E. coli-derived viruses (eg, pBR322, pBR325, pUC12, pUC13, pUC19, pET-44, pBlueScriptII) and bacteriophage-derived plasmids (eg, YEp13, pYES2, YRp7, YIp5, pYAC2, pUB110, pTP5.
- E. coli-derived viruses eg, pBR322, pBR325, pUC12, pUC13, pUC19, pET-44, pBlueScriptII
- bacteriophage-derived plasmids eg, YEp13, pYES2, YRp7, YIp5, pYAC2, pUB110, pTP5.
- yeast-derived plasmids eg, pSH19, pSH15
- bacteriophages such as ⁇ phage, retroviruses, adenoviruses, lentiviruses, adeno-associated viruses, animal viruses such as vaccinia virus, insect-pathogenic viruses such as baculovirus, etc. Can be used.
- a addition signal, selectable marker, SV40 replication origin, or the like may be added to the expression vector.
- Hosts include bacterial cells (eg, Escherichia spp., Bacillus spp., Bacillus subtilis, etc.), fungal cells (eg, yeast, Aspergillus, etc.), insect cells (eg, S2 cells, Sf cells, etc.), animal cells (eg, S2 cells, Sf cells, etc.). , CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), plant cells and the like can be exemplified.
- bacterial cells eg, Escherichia spp., Bacillus spp., Bacillus subtilis, etc.
- fungal cells eg, yeast, Aspergillus, etc.
- insect cells eg, S2 cells, Sf cells, etc.
- animal cells eg, S2 cells, Sf cells, etc.
- plant cells and the like can be exemplified.
- Transformed cells can be cultured in a medium and lysed angiotensin converting enzyme 2 can be collected from the culture.
- the medium When the dissolved angiotensin converting enzyme 2 is secreted into the medium, the medium may be recovered, and the dissolved angiotensin converting enzyme 2 may be separated from the medium and purified.
- the lysed angiotensin converting enzyme 2 When the lysed angiotensin converting enzyme 2 is produced in the transformed cell, the cell may be lysed, and the lysed angiotensin converting enzyme 2 may be separated from the lysate and purified.
- Separation and purification of the dissolved angiotensin converting enzyme 2 can be performed by a known method.
- known separation and purification methods differences in molecular weight such as methods using solubility such as salting out and solvent precipitation, dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis can be used.
- ASO antisense oligonucleotide
- Table 1 The antisense oligonucleotide (ASO) shown in Table 1 was synthesized. The sequence locations of ASO complementary to ACE2 pre-mRNA are shown in FIGS. 1 and 3.
- ENA® (2'-O, 4'-C-Ethylene-bridged Nucleic Acids) nucleic acids modified to A (adenine), G (guanine), C (cytosine) and T (thymine) in the ASO sequence. was introduced to improve affinity and stability.
- gCTgTaTCCCCagaaaCT (ACE2ASO1) Synthesis (Example 1) This was performed on a 1 ⁇ mol scale using an automatic nucleic acid synthesizer (DNA / RNA synthesizer NTS H-6 manufactured by Nippon Techno Service Co., Ltd.).
- the concentrations of solvent, reagent, and phosphoramidite in each synthesis cycle are the same as in the case of natural oligonucleotide synthesis, and the reagent, phosphoramidite of 2'-O-methylnucleoside (adenosine product No. 10-3100-10, The guanosine product No. 10-3121-10) was manufactured by Glen Research.
- the solvent used was that of Wako Pure Chemical Industries.
- the oligomer is cut out from the support by heat-treating the protected oligonucleotide analog having the target sequence with concentrated aqueous ammonia (55 ° C, 8 hours), and the protecting group cyanoethyl group on the phosphorus atom and the protection on the nucleic acid base are protected. I removed the base.
- This ammonia solution was de-DMTed in a cartridge using Glen-Pak DNA Purification Cartridge (product No. 60-5100 manufactured by Glen Research) according to the Glen Research recommended protocol, the recovered solution was distilled off under reduced pressure, and the residue was reversed.
- Phase HPLC (LC-2a manufactured by Shimadzu Corporation, column (Triart C18 (10 x 150 mm) manufactured by YMC), solution A: 0.1 M aqueous solution of triethylamine acetate (TEAA), pH 7.0, solution B: acetonitrile, B%: 10% ⁇ Purification was performed at 25% (30 min, linear gradient); 50 ° C; 4.7 mL / min; 280 nm). After distilling off the solvent, it was dissolved in a 10 mM NaOH solution, replaced with pure water by ultrafiltration using a Microsep centrifugal filtration device (product No. MAP003C manufactured by Nippon Paul Co., Ltd.), and freeze-dried to obtain the target compound.
- a Microsep centrifugal filtration device product No. MAP003C manufactured by Nippon Paul Co., Ltd.
- This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6461, measured value: 6461).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36796-36813 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of gaTCCCagTgaagaTCag (ACE2ASO2) (Example 2)
- Example 2 The compound of Example 2 having the same target sequence as the compound of Example 1 was synthesized.
- This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6486, measured value: 6487).
- the nucleotide sequence of this compound is complementary to nucleotide number 36906-36923 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TCTTCCgaTCTCTgaTCC (ACE2ASO3) (Example 3) The compound of Example 3 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6456, measured value: 6456).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36919-36936 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TgaTaCggCTCCgggaCa (ACE2ASO4) (Example 4) The compound of Example 4 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6504, measured value: 6504).
- the nucleotide sequence of this compound is complementary to nucleotide number 36745-36762 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of ggCTgTTgTCaTTCagaC (ACE2ASO5) (Example 5) The compound of Example 5 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6495, measured value: 6495).
- the nucleotide sequence of this compound is complementary to nucleotide number 36775-36792 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TaggaggTCCaagTgTTg (ACE2ASO6) (Example 6) The compound of Example 6 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6520, measured value: 6521).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36814-36831 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of CCaTaTggaaaCaggggg (ACE2ASO7) (Example 7) The compound of Example 7 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6513, measured value: 6513).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36837-36854 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of CaCaaCTCCaaaaCaaT (ACE2ASO8) (Example 8) The compound of Example 8 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6407, measured value: 6407).
- the nucleotide sequence of this compound is complementary to nucleotide number 36858-36875 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of aaTgCCaaCCaCTaTCaC (ACE2ASO9) (Example 9) The compound of Example 9 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6428, measured value: 6429).
- the nucleotide sequence of this compound is complementary to nucleotide number 36882-36899 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of CgggaCaTCcTaTTTgCa (ACE2ASOIn16Ex17) (Example 10) The compound of Example 10 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6452, measured value: 6452).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36734-36751 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of gCCacTTacTTcTTCCga (ACE2ASOEx17In17) (Example 11) The compound of Example 11 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6375, measured value: 6377).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36929-36946 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of CggCTCCgggaCaTccTa (ACE2ASO4 + 5) (Example 12) The compound of Example 12 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6440, measured value: 6440).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36740-36757 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of CggaAAgcATCaTTgaTA (ACE2ASO4-13) (Example 13) The compound of Example 13 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 649, measured value: 6492).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36758-36775 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of CTCTaggcTgTTgTcaTT (ACE2ASO5-5) (Example 14)
- ACE2ASO5-5 The compound of Example 14 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6433, measured value: 6434).
- the nucleotide sequence of this compound is complementary to nucleotide number 36780-36797 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TCcaagTgTTggCTgTAT (ACE2ASO6 + 7) (Example 15)
- ACE2ASO6 + 7 The compound of Example 15 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6482, measured value: 6482).
- the nucleotide sequence of this compound is complementary to nucleotide number 36807-36824 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of AgggggcTGgTTAggagG (ACE2ASO6-11) (Example 16)
- ACE2ASO6-11 The compound of Example 16 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6482, measured value: 6482).
- the nucleotide sequence of this compound is complementary to nucleotide number 36825-36842 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TTgTCaTTCagaCggaaa (ACE2ASO5 + 5) (Example 17)
- ACE2ASO5 + 5 The compound of Example 17 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6474, measured value: 6474).
- the nucleotide sequence of this compound is complementary to nucleotide number 36770-36787 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TgTCaTTCagaCggaaag (ACE2ASO5 + 6) (Example 18) The compound of Example 18 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6487, measured value: 6489).
- the nucleotide sequence of this compound is complementary to nucleotide number 36769-36786 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of gTCaTTCagaCggaaagC (ACE2ASO5 + 7) (Example 19) The compound of Example 19 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6486, measured value: 6488).
- the nucleotide sequence of this compound is complementary to nucleotide number 36768-36785 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TCaTTCagaCggaaagCa (ACE2ASO5 + 8) (Example 20) The compound of Example 20 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6470, measured value: 6474).
- the nucleotide sequence of this compound is a sequence complementary to nucleotide number 36767-36784 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of aTTCagaCggaaagCaTC (ACE2ASO5 + 10) (Example 21) The compound of Example 21 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6470, measured value: 6472).
- the nucleotide sequence of this compound is complementary to nucleotide number 36765-36782 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of TCagaCggaaagCaTCaT (ACE2ASO5 + 12) (Example 22) The compound of Example 22 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6470, measured value: 6472).
- the nucleotide sequence of this compound is complementary to nucleotide number 36763-36780 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1). Synthesis of GgcTgtTgtCaTtCagaC (ACE2ASO5c) (Example 23) The compound of Example 23 having the same target sequence as the compound of Example 1 was synthesized. This compound was identified by the negative ion MALDI-TOFMS (calculated value: 6403, measured value: 6399).
- nucleotide sequence of this compound is complementary to nucleotide number 36775-36792 of Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1).
- ACE2 Homo sapiens angiotensin I converting enzyme 2
- RefSeqGene on chromosome X Gene Bank accession No. NG_012575.1
- Table 1 ASO sequence synthesized in this example.
- Uppercase is ENA nucleic acid
- lowercase is 2'OMe.
- RNA preparation 1 The cells transfected with each ASO were cultured for 24 hours, washed once with PBS, and 300 ⁇ l of RNA extraction reagent of High Pure RNA Isolation Kit (# 11828665001, Roche Life Science) was added to the cells. 2) After leaving at room temperature for 10 minutes, the RNA extraction reagent in the well was collected in a tube. 3) RNA was extracted according to the protocol of the High Pure RNA Isolation Kit, and finally 50 ⁇ l of RNA lysate was obtained.
- Random primers # 48190011, Thermo Fisher Scientific
- dNTP Mixture 2.5 mM each
- M-MLV Reverse Transcriptase # 28025013, Thermo Fisher Scientific
- RNaseOUT Recombinant Ribonuclease Inhibitor # 10777-019, Thermo Fisher Scientific
- DTT attached to M-MLV
- 5x First Strand Buffer (M) -Attached to MLV) was added and incubated at 37 ° C for 55 minutes and 70 ° C for 10 minutes to obtain cDNA.
- primer ACE2F2 (5'-ctgttccgatcatctgttgc-3': SEQ ID NO: 18) 1 ⁇ l
- primer ACE2R2 (5'-gagaccaaatacacactttccc-3': SEQ ID NO: 19) 1 ⁇ l
- Takara Ex Taq DNA polymerase (# RR001A, Takara) 0.1 ⁇ l
- dNTP Mixture 2.5 mM each
- 10x Ex Taq Buffer 2 ⁇ l 10x Ex Taq Buffer 2 ⁇ l
- Nuclease-Free Water 12.3 ⁇ l were added.
- ACE2 is required for virus infection as a virus receptor, inhibition of ACE2 receptor function is attracting attention as a prophylactic treatment method for viruses. In fact, it has been reported that viral infection is reduced when ACE2 antibody or peptide is allowed to act on cells to inhibit the function of ACE2. A method of inhibiting viral infection by using siRNA to reduce the expression level of ACE2 has also been verified. ACE2 is composed of 18 exons, and it was expected that by skipping exons 18, it would be possible to create ACE2 in which the transmembrane domain was eliminated.
- ACE2ASO3, 4 + 5, 4, 4-13, 5, 5-5, 5 + 5, 5 + 7, 5 + 8, 5 + 10, 5 + 12, 5c of the present invention are ACE2 in which exon 18 is skipped. It is expected to be effective in preventing virus infection because it has increased the proportion of ACE2 in all ACE2. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
- the present invention can be applied as a preventive treatment method by ASO. It was revealed that ASO induces exon skipping of the ACE2 gene. This result is expected to exert the virus neutralizing action and Ang (1-7) action by the virus decoy by the inhibition of the virus invasion by the decrease of ACE2 protein and the production of lysed ACE2. Therefore, intranasal administration as a preventive / therapeutic method for viral infection and intravenous administration as a therapeutic method for preventing the aggravation of the infection can be considered.
- the advantages of prevention and treatment using this ASO are 1. Easy access to the route of administration: Residual evaluation at the site of administration is required 2. Double effect 1) ACE2 decrease 2) Virus Otori increase 3) Ang (1-7) increase 3. Mass synthesis is possible.
- ⁇ SEQ ID NO: 1> The base sequence of exon 18 of the angiotensin converting enzyme 2 gene is shown.
- the base sequence of ASO synthesized in the example is shown.
- the nucleotide constituting the antisense oligonucleotide may be any of natural DNA, natural RNA, DNA / RNA chimera, and modified products thereof, and at least one of them may be a modified nucleotide.
- SEQ ID NOS: 18-21> The base sequences of the primers used in the test examples are shown.
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Abstract
Description
一方、2002-2003年の重症急性呼吸器症候群(severe acute respiratory syndrome(SARS))の流行時に、ACE2がコロナウィルスSARS-CoVの体内への侵入の際のレセプターである事が明らかにされ,ウイルス感染成立のキー分子として注目を浴びた。そして、COVID-19として現在全世界に蔓延中のSARS-CoV-2ウイルスもACE2をリセプターとして利用する事が明らかにされた(非特許文献1)。
ACE2は細胞外・膜貫通・細胞内の3ドメインからなる膜貫通タンパクで、細胞外ドメインにウィルス結合ドメインが存在する。このウイルス結合ドメインがSARS-CoV-2ウィルスと親和性高く結合し、ウィルスの受容体としての機能を担う。そのため、ACE2は、SARS-CoV-2治療標的の一つとなり、ACE2の発現を阻止する方法あるいは細胞外ドメインのみからなる溶解型ACE2をおとり受容体として活用する方法などが勢力的に研究されている(非特許文献2)。
スプライシングは、遺伝子から転写されたpre-mRNAからイントロンを切り取り、エクソンのみから構成される成熟mRNAを産生する反応である。スプライシング部位は、スプライシングコンセンサス配列と呼ばれるイントロンの両端に存在するGT-AGの配列により決定される。これに加え、スプライシング促進配列がシス因子として作用し、正確なスプライシング反応となる。このスプライシング促進配列に対するアンチセンス核酸(ASO)は、スプライシング促進機能を阻害しエクソンのスキッピングをもたらす。遺伝病などを治療するために、エクソンのスキッピングを誘導するASOの開発は盛んである(非特許文献3)。
本アンチセンス核酸開発研究を開始した後、Rehmanらはインシリコ研究でASOを用いたACE2遺伝子のエクソンスキッピングを例示した(Rehman S, and Tabish M. Alternative splicing of ACE2 possibly generates variants that may limit the entry of SARS-CoV-2: a potential therapeutic approach using SSOs. Clin Sci (Lond). 2020;134(10):1143-5)。
本発明は、ACE2のタンパクの発現の抑制と溶解型ACE2の発現促進を目的としてACE2遺伝子のエクソンスキッピングを誘導するアンチセンス核酸(ASO)を提供することを目的とする。
(1)アンギオテンシン変換酵素2遺伝子のエクソン18の標的部位に相補的な塩基配列を有する、塩基数15~30のアンチセンスオリゴヌクレオチドであって、アンギオテンシン変換酵素2遺伝子のエクソンのスキッピングを誘導することができる前記アンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(2)アンギオテンシン変換酵素2遺伝子のエクソン18の塩基配列が配列番号1の塩基配列であり、アンギオテンシン変換酵素2遺伝子のエクソン18の標的部位が、配列番号1の塩基配列の塩基番号1~195の領域内に存在する(1)記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(3)アンチセンスオリゴヌクレオチドの塩基配列が、配列番号2~17のいずれかの塩基配列(但し、配列中のtはuであってもよく、uはtであってもよい)中の連続する少なくとも15個の塩基からなる配列を含む(1)又は(2)に記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(4)アンチセンスオリゴヌクレオチドの塩基長が18である(1)~(3)のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(5)アンチセンスオリゴヌクレオチドの塩基配列が、配列番号2~17のいずれかの塩基配列(但し、配列中のtはuであってもよく、uはtであってもよい)である(4)記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(6)少なくとも1個のヌクレオチドが修飾されている(1)~(5)のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(7)修飾ヌクレオチドを構成する糖がD-リボフラノースであり、D-リボフラノースの2’位の水酸基が修飾されている(6)記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(8)D-リボフラノースが2’-O-アルキル化及び/又は2’-O, 4’-C-アルキレン化されている(7)記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
(9)(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を含む、医薬。
(10)SARS-CoV-2ウィルスの感染性を抑制するための(9)記載の医薬。
(11)受容体型アンギオテンシン変換酵素2の減少によるウイルスの細胞内への取り込み阻害及び/又はウイルスと結合可能な溶解型アンギオテンシン変換酵素2の増加による細胞外でのウイルスの捕捉の効果を持つ(10)記載の医薬。
(12)SARS-CoV-2感染を予防及び/又は治療するための(8)~(11)のいずれかに記載の医薬。
(13)(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物を含む、アンギオテンシン変換酵素2のタンパクの発現抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進のための薬剤。
(14)(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物により誘導された、アンギオテンシン変換酵素2遺伝子のエクソンのスキッピングにより産生される、膜貫通ドメインを欠いた溶解型アンギオテンシン変換酵素2。
(15)(14)記載の溶解型アンギオテンシン変換酵素2をコードするヌクレオチド配列及び/又はそれに相補的な配列を含むポリヌクレオチド。
(16)(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を有効な量で被験者に投与することを含む、SARS-CoV-2ウイルスの感染性を抑制する方法。
(17)(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を有効な量で被験者に投与することを含む、SARS-CoV-2感染を予防及び/又は治療する方法。
(18)(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を有効な量で被験者に投与することを含む、アンギオテンシン変換酵素2のタンパクの発現抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進方法。
(19)SARS-CoV-2ウイルスの感染性を抑制する方法に使用するための、(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物。
(20)SARS-CoV-2感染を予防及び/又は治療する方法に使用するための、(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物。
(21)アンギオテンシン変換酵素2のタンパクの発現抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進方法に使用するための、(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物。
(22)SARS-CoV-2ウイルスの感染性を抑制するための医薬の製造における、(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物の使用。
(23)SARS-CoV-2感染を予防及び/又は治療するための医薬の製造における、(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物の使用。
(24)アンギオテンシン変換酵素2のタンパクの発現抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進のための薬剤の製造における、(1)~(8)のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物の使用。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2020‐127142の明細書および/または図面に記載される内容を包含する。
本明細書において、「感染の予防」とは、ウイルス感染予防、ウイルス感染による好ましくない症状の発症(ウイルス感染症)の予防、ウイルス感染症の重症化の予防を含み、「予防」には、ウイルス感染率の低減、ウイルス感染による好ましくない症状の発症率の低減、ウイルス感染症の重症化率の低減、ウイルス感染症の重症化の程度の低減が含まれる。
また、本明細書において、「感染の治療」とは、ウイルス感染からの治癒、ウイルス感染による好ましくない症状の軽快、ウイルス感染症の重症化の防止や遅延を含む。
〔実施例1~8〕アンチセンスオリゴヌクレオチド(ASO)の合成
表1に示すアンチセンスオリゴヌクレオチド(ASO)を合成した。ACE2 pre-mRNAに相補的なASOの配列場所を図1、3に示す。ASOの配列中のA(アデニン)、G(グアニン)、C(シトシン)およびT(チミン)に修飾核酸のENA(登録商標)(2'-O,4'-C-Ethylene-bridged Nucleic Acids)を導入し、親和性と安定性を向上させた。
gCTgTaTCCCCagaaaCTの合成 (ACE2ASO1) の合成(実施例1)
核酸自動合成機(日本テクノサービス社製DNA/RNA合成装置 NTS H-6)を用い、1μmolスケールで行った。各合成サイクルにおける溶媒、試薬、ホスホロアミダイトの濃度は天然オリゴヌクレオチド合成の場合と同じであり、試薬、2'-O-メチルヌクレオシドのホスホロアミダイト(アデノシン体product No. 10-3100-10、グアノシン体product No. 10-3121-10)はグレンリサーチ社製のものを用いた。溶媒は和光純薬工業のものを用いた。非天然型のホスホロアミダイトは特開2000-297097の実施例22(5'-O-ジメトキシトリチル-2'-O,4'-C-エチレン-4-N-ベンゾイル-5-メチルシチジン-3'-O-(2-シアノエチルN,N-ジイソプロピル)ホスホロアミダイト)、実施例9(5'-O-ジメトキシトリチル-2'-O,4'-C-エチレン-5-メチルウリジン-3'-O-(2-シアノエチルN,N-ジイソプロピル)ホスホロアミダイト)の化合物を用いた。固相担体としてユニバーサルコントロールポアグラス(CPG)(グレンリサーチ社製product No. 25-5040)を用い、表記の化合物を合成した。但し、アミダイトの縮合に要する時間は、15分とした。
目的配列を有する保護されたオリゴヌクレオチド類縁体を濃アンモニア水で加熱処理(55℃、8時間)することによってオリゴマーを支持体から切り出すとともに、リン原子上の保護基シアノエチル基と核酸塩基上の保護基を外した。本アンモニア溶液をGlen-Pak DNA Purification Cartridge(グレンリサーチ社製product No. 60-5100)を用い、グレンリサーチ推奨プロトコルに従いカートリッジ内で脱DMTを行い、回収した溶液を減圧留去し、残渣を逆相HPLC(島津製作所製LC-2a、カラム(YMC製Triart C18 (10×150 mm))、A溶液:0.1M酢酸トリエチルアミン水溶液(TEAA), pH 7.0、B溶液:アセトニトリル、B%:10%→25%(30 min, linear gradient);50°C;4.7 mL/min;280 nm)にて精製した。溶媒留去後、10mM NaOH溶液に溶解し、マイクロセップ遠心濾過デバイス(日本ポール社製product No. MAP003C)を用いて限外濾過により純水置換し、凍結乾燥後目的化合物を得た。
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6461、測定値:6461)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36796-36813に相補的な配列である。
gaTCCCagTgaagaTCag (ACE2ASO2) の合成(実施例2)
実施例1の化合物と同様に目的配列を有する実施例2の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6486、測定値:6487)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36906-36923に相補的な配列である。
TCTTCCgaTCTCTgaTCC (ACE2ASO3) の合成(実施例3)
実施例1の化合物と同様に目的配列を有する実施例3の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6456、測定値:6456)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36919-36936に相補的な配列である。
TgaTaCggCTCCgggaCa (ACE2ASO4) の合成(実施例4)
実施例1の化合物と同様に目的配列を有する実施例4の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6504、測定値:6504)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36745-36762に相補的な配列である。
ggCTgTTgTCaTTCagaC (ACE2ASO5) の合成(実施例5)
実施例1の化合物と同様に目的配列を有する実施例5の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6495、測定値:6495)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36775-36792に相補的な配列である。
TaggaggTCCaagTgTTg (ACE2ASO6) の合成(実施例6)
実施例1の化合物と同様に目的配列を有する実施例6の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6520、測定値:6521)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36814-36831に相補的な配列である。
CCaTaTggaaaCaggggg (ACE2ASO7) の合成(実施例7)
実施例1の化合物と同様に目的配列を有する実施例7の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6513、測定値:6513)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36837-36854に相補的な配列である。
CaCaaCTCCaaaaaCaaT (ACE2ASO8) の合成(実施例8)
実施例1の化合物と同様に目的配列を有する実施例8の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6407、測定値:6407)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36858-36875に相補的な配列である。
aaTgCCaaCCaCTaTCaC (ACE2ASO9) の合成(実施例9)
実施例1の化合物と同様に目的配列を有する実施例9の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6428、測定値:6429)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36882-36899に相補的な配列である。
CgggaCaTCcTaTTTgCa (ACE2ASOIn16Ex17) の合成(実施例10)
実施例1の化合物と同様に目的配列を有する実施例10の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6452、測定値:6452)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36734-36751に相補的な配列である。
gCCacTTacTTcTTCCga (ACE2ASOEx17In17) の合成(実施例11)
実施例1の化合物と同様に目的配列を有する実施例11の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6375、測定値:6377)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36929-36946に相補的な配列である。
CggCTCCgggaCaTccTa (ACE2ASO4+5) の合成(実施例12)
実施例1の化合物と同様に目的配列を有する実施例12の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6440、測定値:6440)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36740-36757に相補的な配列である。
CggaAAgcATCaTTgaTA (ACE2ASO4-13) の合成(実施例13)
実施例1の化合物と同様に目的配列を有する実施例13の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6493、測定値:6492)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36758-36775に相補的な配列である。
CTCTaggcTgTTgTcaTT (ACE2ASO5-5) の合成(実施例14)
実施例1の化合物と同様に目的配列を有する実施例14の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6433、測定値:6434)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36780-36797に相補的な配列である。
TCcaagTgTTggCTgTAT (ACE2ASO6+7) の合成(実施例15)
実施例1の化合物と同様に目的配列を有する実施例15の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6482、測定値:6482)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36807-36824に相補的な配列である。
AgggggcTGgTTAggagG (ACE2ASO6-11) の合成(実施例16)
実施例1の化合物と同様に目的配列を有する実施例16の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6482、測定値:6482)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36825-36842に相補的な配列である。
TTgTCaTTCagaCggaaa (ACE2ASO5+5) の合成(実施例17)
実施例1の化合物と同様に目的配列を有する実施例17の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6474、測定値:6474)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36770-36787に相補的な配列である。
TgTCaTTCagaCggaaag (ACE2ASO5+6) の合成(実施例18)
実施例1の化合物と同様に目的配列を有する実施例18の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6487、測定値:6489)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36769-36786に相補的な配列である。
gTCaTTCagaCggaaagC (ACE2ASO5+7) の合成(実施例19)
実施例1の化合物と同様に目的配列を有する実施例19の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6486、測定値:6488)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36768-36785に相補的な配列である。
TCaTTCagaCggaaagCa (ACE2ASO5+8) の合成(実施例20)
実施例1の化合物と同様に目的配列を有する実施例20の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6470、測定値:6474)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36767-36784に相補的な配列である。
aTTCagaCggaaagCaTC (ACE2ASO5+10) の合成(実施例21)
実施例1の化合物と同様に目的配列を有する実施例21の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6470、測定値:6472)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36765-36782に相補的な配列である。
TCagaCggaaagCaTCaT (ACE2ASO5+12) の合成(実施例22)
実施例1の化合物と同様に目的配列を有する実施例22の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6470、測定値:6472)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36763-36780に相補的な配列である。
GgcTgtTgtCaTtCagaC (ACE2ASO5c) の合成(実施例23)
実施例1の化合物と同様に目的配列を有する実施例23の化合物を合成した
本化合物は負イオンMALDI-TOFMSにより、同定した(計算値:6403、測定値:6399)。
本化合物の塩基配列は、Homo sapiens angiotensin I converting enzyme 2 (ACE2), RefSeqGene on chromosome X (Gene Bank accession No. NG_012575.1のヌクレオチド番号36775-36792に相補的な配列である。
(表1)
本実施例で合成したASOの配列。大文字はENA核酸、小文字は2'OMe。
実験方法
ACE2 mRNAの評価
ASO導入後のACE2のスプライシングパターンの変化をヒト肝癌細胞 (HepG2, ATCC) でRT-PCRにより評価した。
細胞培養
ヒト肝癌細胞 (HepG2) は10%FBS (10270-106, gibco) を含むE-MEM培地 (051-07615, 富士フイルム和光純薬) で培養した。
ASOトランスフェクション
1) Opti-MEM培地 (31985070, Thermo Fisher Scientific) 100 μlにASO (Nuclease-Free Water (AM9932, Thermo Fisher Scientific)で50 pmol/μlとしたもの) を各々2 μl混合した。ASO無処理は、Nuclease-Free Waterを2 μl混合した。
2) 別のチューブでOpti-MEM培地 100 μlにLipofectamine 3000 Transfection Reagent (L3000015, Thermo Fisher Scientific) を4 μl混合した。
3) 1)液と2)液を混合し、15分間室温で放置した。
4) 12-ウェルプレートで培養したヒト肝癌細胞 (HepG2) をPBSで1回洗浄した後、ウェルにOpti-MEM培地を800 μl加えた。
5) 3)液を4)に添加し (ASO最終濃度100 nM)、37℃ 5%CO2下で3時間培養した後、培地を、10%FBSを含むE-MEM培地に交換し、さらに培養を継続した。
RNA調製
1) 各ASOをトランスフェクションした細胞を、24時間培養した後、PBSにて1回洗浄し、High Pure RNA Isolation Kit (#11828665001, Roche Life Science) のRNA抽出試薬300 μlを細胞に添加した。
2) 10分間室温に放置した後、ウェル内のRNA抽出試薬をチューブに回収した。
3) High Pure RNA Isolation Kitのプロトコルに従ってRNAを抽出し、最終的に50 μlのRNA溶解液を得た。
逆転写反応
1) RNA 500 ngにRandom primers (#48190011, Thermo Fisher Scientific)、dNTP Mixture (各2.5 mM) (#4030, Takara) を加え、65℃で5分、25℃で10分間インキュベートした。
2) 1)液にM-MLV Reverse Transcriptase (#28025013, Thermo Fisher Scientific)、RNaseOUT Recombinant Ribonuclease Inhibitor (#10777-019, Thermo Fisher Scientific)、DTT (M-MLVに添付)、5x First Strand Buffer (M-MLVに添付) を加え、37℃で55分、70℃で10分間インキュベートしてcDNAを得た。
PCR反応と反応産物の確認
1) 得られたcDNA 2 μlに対し、プライマーACE2F2 (5'-ctgttccgatcatctgttgc-3':配列番号18) 1 μl、プライマーACE2R2 (5'-gagaccaaatacacactttccc-3':配列番号19) 1 μl、Takara Ex Taq DNAポリメラーゼ (#RR001A, Takara) 0.1 μl、dNTP Mixture (各2.5 mM) 1.6 μl、10x Ex Taq Buffer 2 μl、Nuclease-Free Water 12.3 μlを添加した。
2) 94℃ 3分間加熱した。
3) 94℃ 0.5分・60℃ 0.5分・72℃ 1.5分の処理を30サイクル行った。
4) 72℃ 3分間加熱した。
5) PCR反応の反応産物は、Agilent2100バイオアナライザ電気泳動システム(アジレント・テクノロジー株式会社)を用いてPCR反応産物の泳動、定量を行った。
6) GAPDHに対して、プライマーGAPDH H_F (5'-cccttcattgacctcaac-3':配列番号20)、GAPDH H_R (5'-ttcacacccatgacgaac-3':配列番号21)を用いて上記の1)~5)を行った(3)は18サイクルで行った)。
実験結果
ACE2のエクソン18のスキップを目的に、ACE2 pre-mRNAのエクソン18に対して相補的配列を持つ18塩基のASOを23種類作製した (図1、3、5、7、9、11、13)。各ASOは、ACE2 pre-mRNAにおけるスプライシング因子の結合予測を基に作製した。ヒト肝癌細胞 (HepG2) をそれぞれのASOで24時間処理した後、ACE2 mRNAをRT-PCRで検証したところ、ACE2ASO3、4+5、4、4-13、5、5-5、5+5、5+7、5+8、5+10、5+12、5c処理で全ACE2に占めるエクソン18スキップACE2の割合の増加が有意に認められた (図2、4、8、10、12、14)。
考察
ACE2はウイルスの受容体としてウイルス感染に必要であることから、ACE2の受容体機能の阻害はウイルスに対する予防的治療方法として注目されている。実際、細胞においてACE2抗体やペプチドを作用させてACE2の機能を阻害するとウイルス感染が減少する報告がなされている。また、siRNAを使用してACE2の発現量を下げることでウイルス感染を阻害する方法も検証されてる。ACE2は18エクソンから構成され、エクソン18をスキップさせることにより膜貫通ドメインを消失させたACE2を作製することができると予想された。そこで本研究ではASOを利用してACE2のエクソン18をスキップさせ、膜貫通ドメイン消失による受容体型ACE2の減少ならびに遊離型ACE2の増加を引き起こすことを目的とした。これにより受容体型ACE2の減少によるウイルスの細胞内への取り込み阻害とウイルスと結合可能な遊離型ACE2の増加による細胞外でのウイルスの捕捉という二重の効果が期待できる。本発明のACE2ASO3、4+5、4、4-13、5、5-5、5+5、5+7、5+8、5+10、5+12、5cはエクソン18がスキップされたACE2の全ACE2に占める割合を増加させたことからウイルス感染予防に効果を発揮すると期待される。
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
1. 投与経路がアクセス容易:投与部位での残存評価が必要
2. 効果が2重作用 1)ACE2減 2)ウィルスオトリ増 3)Ang(1-7)増
3. 大量合成が可能
なことである。
gatgtcccggagccgtatcaatgatgctttccgtctgaatgacaacagcctagagtttctggggatacagccaacacttggacctcctaaccagccccctgtttccatatggctgattgtttttggagttgtgatgggagtgatagtggttggcattgtcatcctgatcttcactgggatcagagatcggaagaa
<配列番号2~17及び22~28>実施例で合成したASOの塩基配列を示す。アンチセンスオリゴヌクレオチドを構成するヌクレオチドは、天然型DNA、天然型RNA、DNA/RNAのキメラ、これらの修飾体のいずれであってもよく、また、少なくとも1つが修飾ヌクレオチドであるとよい。
<配列番号18~21>試験例で用いたプライマーの塩基配列を示す。
Claims (24)
- アンギオテンシン変換酵素2遺伝子のエクソン18の標的部位に相補的な塩基配列を有する、塩基数15~30のアンチセンスオリゴヌクレオチドであって、アンギオテンシン変換酵素2遺伝子のエクソンのスキッピングを誘導することができる前記アンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- アンギオテンシン変換酵素2遺伝子のエクソン18の塩基配列が配列番号1の塩基配列であり、アンギオテンシン変換酵素2遺伝子のエクソン18の標的部位が、配列番号1の塩基配列の塩基番号1~195の領域内に存在する請求項1記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- アンチセンスオリゴヌクレオチドの塩基配列が、配列番号2~17のいずれかの塩基配列(但し、配列中のtはuであってもよく、uはtであってもよい)中の連続する少なくとも15個の塩基からなる配列を含む請求項1又は2に記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- アンチセンスオリゴヌクレオチドの塩基長が18である請求項1~3のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- アンチセンスオリゴヌクレオチドの塩基配列が、配列番号2~17のいずれかの塩基配列(但し、配列中のtはuであってもよく、uはtであってもよい)である請求項4記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- 少なくとも1個のヌクレオチドが修飾されている請求項1~5のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- 修飾ヌクレオチドを構成する糖がD-リボフラノースであり、D-リボフラノースの2’位の水酸基が修飾されている請求項6記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- D-リボフラノースが2’-O-アルキル化及び/又は2’-O, 4’-C-アルキレン化されている請求項7記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物。
- 請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を含む、医薬。
- SARS-CoV-2ウィルスの感染性を抑制するための請求項9記載の医薬。
- 受容体型アンギオテンシン変換酵素2の減少によるウイルスの細胞内への取り込み阻害及び/又はウイルスと結合可能な溶解型アンギオテンシン変換酵素2の増加による細胞外でのウイルスの捕捉の効果を持つ請求項10記載の医薬。
- SARS-CoV-2感染を予防及び/又は治療するための請求項8~11のいずれかに記載の医薬。
- 請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物を含む、アンギオテンシン変換酵素2のタンパクの発現の抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進のための薬剤。
- 請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その塩又は溶媒和物により誘導された、アンギオテンシン変換酵素2遺伝子のエクソンのスキッピングにより産生される、膜貫通ドメインを欠いた溶解型アンギオテンシン変換酵素2。
- 請求項14記載の溶解型アンギオテンシン変換酵素2をコードするヌクレオチド配列及び/又はそれに相補的な配列を含むポリヌクレオチド。
- 請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を有効な量で被験者に投与することを含む、SARS-CoV-2ウイルスの感染性を抑制する方法。
- 請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を有効な量で被験者に投与することを含む、SARS-CoV-2感染を予防及び/又は治療する方法。
- 請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物を有効な量で被験者に投与することを含む、アンギオテンシン変換酵素2のタンパクの発現抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進方法。
- SARS-CoV-2ウイルスの感染性を抑制する方法に使用するための、請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物。
- SARS-CoV-2感染を予防及び/又は治療する方法に使用するための、請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物。
- アンギオテンシン変換酵素2のタンパクの発現抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進方法に使用するための、請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物。
- SARS-CoV-2ウイルスの感染性を抑制するための医薬の製造における、請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物の使用。
- SARS-CoV-2感染を予防及び/又は治療するための医薬の製造における、請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物の使用。
- アンギオテンシン変換酵素2のタンパクの発現抑制及び/又は溶解型アンギオテンシン変換酵素2の発現促進のための薬剤の製造における、請求項1~8のいずれかに記載のアンチセンスオリゴヌクレオチド、その医薬的に許容できる塩又は溶媒和物の使用。
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