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WO2021117122A1 - Agent prophylactique ou thérapeutique pour le syndrome de déficience en lipase acide lysosomale - Google Patents

Agent prophylactique ou thérapeutique pour le syndrome de déficience en lipase acide lysosomale Download PDF

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Publication number
WO2021117122A1
WO2021117122A1 PCT/JP2019/048232 JP2019048232W WO2021117122A1 WO 2021117122 A1 WO2021117122 A1 WO 2021117122A1 JP 2019048232 W JP2019048232 W JP 2019048232W WO 2021117122 A1 WO2021117122 A1 WO 2021117122A1
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oligomer according
modified
oligomer
group
sequence
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Japanese (ja)
Inventor
俊也 鈴木
信代 吉田
尚一 大久保
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Reborna Biosciences Inc
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Reborna Biosciences Inc
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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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular 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 a prophylactic or therapeutic agent for lysosomal acid lipase deficiency.
  • Lysosomal acid lipase deficiency is a progressive autosomal recessive disorder that clinically causes fatal Wolman disease in infancy and adults. Cholesteryl ester accumulation disease that develops in the early stages is known.
  • LAL-D patients the activity of lysosomal acid lipase (LAL) is genetically significantly reduced, resulting in chronic accumulation of lipids in organs such as the liver and spleen, resulting in serious liver disease. In some cases, death may occur early due to organ abnormalities such as, central diseases, cardiovascular diseases, etc.
  • LAL-D the responsible gene for LAL-D is the lysosomal acid type lipase gene (LIPA) gene on chromosome 10.
  • LIPA lysosomal acid type lipase gene
  • LAL-D has been performed by hematopoietic stem cell transplantation or administration of LAL enzyme (see, for example, WO2012 / 050695).
  • An object of the present invention is to provide a novel prophylactic or therapeutic agent for lysosomal acid lipase deficiency.
  • One embodiment of the present invention comprises a sequence having 80% or more homology to the antisense strand of the 8th intron of the human lysosomal acidic lipase gene, and 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or It is 13 or more and 40 or less oligomers containing a sequence of 13 or more identical bases with respect to U). It contains 15 or more sequences that are the same base with respect to 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U), and may be 15 or more and 40 or less.
  • the above oligomer consists of a sequence having a 100% complementary base to the sense strand of the 8th intron of the human lysosomal acidic lipase gene, and 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U). ) May be included.
  • the above-mentioned oligomer is the 7th to 24th, 8th to 25th, and 8th from the 5'end in the base sequence of the sense strand of the 8th intron of the human lysosome acidic lipase gene shown in Sequence 1.
  • the base sequences selected from the group consisting of 9th to 26th, 10th to 27th, 11th to 28th, 12th to 29th, 13th to 30th, and 14th to 31st. It may consist of a complementary base sequence.
  • the oligomer may contain an oligonucleotide or a modified oligonucleotide in which one or more sugar moieties and / or phosphate binding moieties are modified.
  • the 2'position of the modified sugar moiety may be modified.
  • the -OH group at the 2'position of the modified sugar moiety is -H, -OR, -R, -R'-OR, -SH, -SR, -NH 2 , -NHR, -NRR ",- Any group (-R, -R'' selected from the group consisting of ONH, -ONR, -N 3 , -CN, -F, -Cl, -Br and -I is independently C 1-.
  • the 2'position of the sugar moiety is modified by being selected from C 6 alkyl, alkenyl, alkynyl, C 1- C 6 alkyl carbonyl or aryl and substituted with (-R'representing alkylene). You may.
  • the -OH group at the 2'position of the modified sugar moiety may be substituted with -OCH 3 or -OCH 2 CH 2 OCH 3.
  • the 2'- and 4'-positions of the modified sugar moiety may be crosslinked.
  • Nucleotides having the modified sugar moiety are Locked Nucleic Acid (LNA), 2'-O, 4'-C-Ethylene-bridged Nucleic Acid (ENA), Amido-bridged Nucleic Acid (AmNA), and Guide.
  • the nucleotide having the modified sugar moiety may be a 2'-deoxy-ribonucleotide, and the 2'-deoxy-ribonucleotide is 2'-deoxy-adenosin or 2'-deoxy-guanosine. You may.
  • the nucleotide having the modified sugar moiety is 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'.
  • nucleotide selected from the group consisting of -amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, and 2'-amino-butyrylpyrene-uridine.
  • Nucleotides with the modified sugar moiety are 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribothymidine, 2-amino-purine, 5-fluoro-cytidine, 5-fluoro-uridine, 2 , 6-Diamino-purine, 4-thio-uridine, 5-amino-allyl-uridine may be a nucleotide selected from the group.
  • the modified phosphate binding moiety may be composed of a bond selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, and a borane phosphate bond.
  • the oligomer may be an oligomer containing a morpholino oligonucleotide.
  • the morpholino oligonucleotide may include a phosphorodiamidate-morpholino oligonucleotide.
  • the 5'end may be a group according to any of the following chemical formulas (1) to (3).
  • the oligomer may contain a peptide nucleic acid.
  • the oligomer may be an oligonucleotide containing both an oligonucleotide or a modified oligonucleotide in which one or more sugar moieties and / or phosphate binding moieties are modified, and a morpholino oligonucleotide.
  • the modified oligonucleotide may be the modified oligonucleotide according to any one of the above.
  • a further embodiment of the present invention is a splicing function modifier containing any of the above oligomers or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient.
  • a further embodiment of the present invention is a pharmaceutical composition containing any of the above oligomers or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient.
  • a further embodiment of the present invention is a drug containing the above-mentioned pharmaceutical composition or a therapeutic agent for lysosome acidic lipase deficiency.
  • a further embodiment of the present invention comprises a splicing mechanism in mammalian cells that produces an abnormal mRNA lacking the transcriptional region from the eighth exon against the pre-mRNA of the lysosome acidic lipase gene.
  • a method for regulating a splicing mechanism which comprises a splicing mechanism for producing a normal mRNA having a transcription region from an exon, wherein any of the above oligomers or a pharmaceutically acceptable salt or hydrate thereof is administered to the human cells. This is a method for adjusting the splicing mechanism, which includes a step of adjusting the splicing mechanism.
  • a further embodiment of the present invention is a method for preventing or treating lysosome acidic lipase deficiency in a mammal, wherein any of the above oligomers or a pharmaceutically acceptable salt thereof is used for the mammal.
  • a prophylactic or therapeutic method that includes the step of administering an effective amount of hydrate.
  • a further embodiment of the present invention is any of the above oligomers or pharmaceutically acceptable salts or hydrates thereof for use in the prevention or treatment of rhisosome acidic lipase deficiency.
  • a further embodiment of the present invention is the use of any of the above oligomers or pharmaceutically acceptable salts or hydrates thereof to produce a prophylactic or therapeutic agent for lysosome acidic lipase deficiency. ..
  • 894G> A mutation and c.
  • a carrier (GM03558) having a 193C> T mutation heterozygotically detects a normal cDNA containing (A) exon 8 and a mutant cDNA not containing (B) exon 8 by the qPCR method of Example 1. This is an example of the result of. It is a figure which showed the detection result of the patient-derived LIPACDNA by the endpoint RT-PCR in the Example of this invention.
  • the results of evaluating the concentration dependence of the antisense oligomer L9-26 on the enhancement of LAL enzyme activity are shown. It is a figure which showed the base sequence (SEQ ID NO: 1) of the human lysosomal acidic lipase gene (lipase A, lysosomal acid type [Homo sapiens], Gene ID: 3988). It is a figure which showed the base sequence of the sense strand of the 8th intron of a human lysosomal acidic lipase gene (lipase A, lysosomal acid type [Homo sapiens], Gene ID: 3988). The underline is the core sequence of the oligomer.
  • Oligomers according to an embodiment of the present invention include oligonucleotides, morpholino oligomers, or peptide nucleic acid (PNA) oligomers.
  • Oligonucleotides have nucleotides as a constituent unit.
  • the nucleotide may be either a ribonucleotide, a deoxyribonucleotide or a modified nucleotide.
  • a modified nucleotide means a ribonucleotide or a deoxyribonucleotide in which all or part of the nucleobase, sugar moiety, and phosphate-binding moiety that composes the ribonucleotide or deoxyribonucleotide is modified.
  • the oligomer of one embodiment of the invention consists of a sequence homologous to the antisense strand of the 8th intron of the human lysosome acidic lipase gene and is 5'-CCCAAAXGCACXCCXGGA-3'(X is T or U). It is 13 or more and 40 or less antisense oligomers containing 13 or more sequences that are the same base with respect to (SEQ ID NO: 3).
  • SEQ ID NO: 1 An example of the nucleotide sequence (SEQ ID NO: 1) of the human lysosomal acidic lipase gene (lipase A, lysosomal acid type [Homo sapiens], Gene ID: 3988) and the nucleotide sequence of the sense strand of the 8th intron (SEQ ID NO: 2). , 9 and 10.
  • the homology between this oligomer and the antisense strand of the 8th intron may be 80% or more, preferably 85% or more, more preferably 90% or more, and 95% or more. Is more preferable, and 100% is most preferable.
  • the measurement of homology can be determined by Pro.Natl.Acad.Sci.USA, 90 , 5873 (1993)). Each parameter shall be set to the default value.
  • the number of these oligomers is 13 or more, preferably 15 or more, more preferably 17 or more, still more preferably 18 or more, 40 or less, preferably 32 or less, more preferably 24 or less, still more preferably 16. Consists of up to 13 oligomers, preferably 13 or more, preferably 15 or more, more preferably 17 or more, still more preferably 17 or more, relative to 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U) (SEQ ID NO: 3). It has 18 identical bases.
  • this oligomer is contained in the nucleotide sequence of the sense strand of the 8th intron of the human lysosome acidic lipase gene shown in Sequence 1, from the 5'end to the 4th to 21st and 7th to 24th. Selected from the group consisting of th, 8th to 25th, 9th to 26th, 10th to 27th, 11th to 28th, 12th to 29th, 13th to 30th, and 14th to 31st. It may consist of a base sequence complementary to any one of the base sequences.
  • the nucleotide constituting this oligomer may be a natural nucleic acid or an artificial nucleic acid.
  • a natural nucleic acid it may be adenine, cytosine, guanine, thymine, uracil, ribonucleotide (RNA) or deoxyribonucleotide (DNA).
  • This oligomer may be RNA or DNA in all nucleotides, but may be a chimeric nucleic acid in which RNA and DNA are mixed.
  • an artificial nucleic acid for example, one or more sugar portions and / or phosphate binding portions may be modified, but the artificial nucleic acid is not particularly limited.
  • the sugar moiety refers to an atomic group constituting ribose or deoxybose of each nucleotide.
  • the phosphate bond portion refers to an atomic group constituting a phosphodiester bond between each nucleotide.
  • the sugar moiety may be modified at any atom, preferably at the 2'position.
  • the -OH group at the 2'position of the sugar moiety is -H, -OR, -R, -R'-OR, -SH, -SR, -NH 2 , -NHR, -NRR'', -ONH, Any group (-R, -R'' selected from the group consisting of -ONR, -N 3 , -CN, -F, -Cl, -Br and -I is independently C 1 -C 6 alkyl, alkenyl, alkynyl, selected from C 1 -C 6 alkylcarbonyl or aryl, -R 'is an alkylene may be substituted with a representative.), but especially in -OCH 3 or -OCH 2 CH 2 OCH 3 It is preferably substituted.
  • the nucleotide is 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-citidine, 2'-amino-uridine, 2'- It may be selected from the group consisting of amino-adenosine, -amino-guanosine, and 2'-amino-butyrylpyrrene-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-methyl-citidine, ribothymidine, 2- It may be selected from the group consisting of amino-purine, 5-fluoro-citidine, 5-fluoro-uridine, 2,6-diamino-purine, 4-thio-uridine, 5-amino-allyl-uridine.
  • the 2'and 4'positions of the sugar moiety may be directly or indirectly crosslinked with a linker or the like.
  • the nucleotides having a sugar moiety are Locked Nucleic Acid (LNA), 2'-O, 4'-C-Ethylene-bridged Nucleic Acid (ENA), Amido-bridged Nucleic Acid (AmNA), and Guide. GuNA)), 2'-O, 4'-C-Spilocyclone bridged Nucleic Acid (scpBNA) can be exemplified.
  • the sugar portion may be replaced with morpholine.
  • a known method may be followed (for example, Biochem Biophys Res Communi. 2007 Jun 29; 358 (2): 521-7; WO2014189142; WO2016060135).
  • the oligomer also includes a morpholino oligonucleotide in which a part or all of the sugar moiety is replaced with morpholine.
  • the morpholin may be a modified morpholin such as phosphorodiamidate morpholin.
  • the modified phosphate bond moiety is not particularly limited, but is composed of a bond selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond, and a boranephosphate bond. It is preferable to be done.
  • the 5'end of the oligomer may be a group according to any of the following chemical formulas (1) to (3).
  • the oligomer of one embodiment of the present invention may contain a plurality of these modifications.
  • the oligomer or its modified portion has an isomer such as an optical isomer, a stereoisomer, a positional isomer, or a rotational isomer, it may be either one of the isomers or a mixture thereof.
  • an isomer such as an optical isomer, a stereoisomer, a positional isomer, or a rotational isomer
  • it may be either one of the isomers or a mixture thereof.
  • Each of these isomers can be obtained as a single item by a known synthesis method or separation method (eg, concentration, solvent extraction, column chromatography, recrystallization, etc.).
  • the optical isomer may be an optical isomer separated from the racemate.
  • the oligomer may be a pharmaceutically acceptable crystal, and may have a single crystal form or a mixture of crystalline forms. Crystals can be produced by applying a known crystallization method.
  • the oligomer may be a pharmaceutically acceptable co-crystal or co-crystal salt.
  • the co-crystal or co-crystal salt can be produced according to a known co-crystallization method.
  • Examples of a co-crystal or co-crystal salt counter molecule in an oligomer include acids (eg, carboxylic acids, phosphoric acids, sugar acids, sulfonic acids), amides, ureas, bases, maltors, amino acids and the like.
  • carboxylic acids include fumaric acid, citric acid, glutaric acid, malonic acid, succinic acid, maleic acid, malic acid, tartaric acid, mandelic acid, lactic acid, gluconic acid, acetic acid, benzoic acid, gentic acid, salicylic acid, Succinic acid can be mentioned.
  • sugar acids include ascorbic acid.
  • sulfonic acid include 2-naphthalene sulfonic acid, 10-camphor sulfonic acid and methane sulfonic acid.
  • amides include nicotinamide, benzamide, lactamide, glycolamide and saccharin.
  • the base include tromethamine and meglumine.
  • Ethyl maltol is a good example of maltol.
  • Suitable examples of amino acids are tyrosine, alanine, serine, threonine, isoleucine, leucine, arginine, lysine, proline, tryptophan, valine, glutamic acid, aspartic acid, glycine, aspartic acid, methinenin, cysteine, phenylalanine, glutamine , Histidine is mentioned.
  • the oligomer may be a hydrate, a non-hydrate, a solvate, or a non-solvate.
  • Oligomers may be labeled with isotopes (eg, 2 H, 3 H, 11 C, 14 C, 35 S, 125 I).
  • the isotope-labeled or substituted compound (I) can be used, for example, as a tracer (PET tracer) used in positron emission tomography (PET), and is useful in fields such as medical diagnosis.
  • PET tracer positron emission tomography
  • the oligomer of one embodiment of the present invention or a pharmaceutically acceptable salt or hydrate thereof can be used as a splicing function modifier, a pharmaceutical composition, a pharmaceutical product, or a therapeutic agent for lysosome acidic lipase deficiency.
  • the administration target is not particularly limited, and any mammal may be used, but a human is more preferable.
  • the oligomer may be a prodrug that becomes a biologically active substance after being administered to animals including humans and metabolized.
  • a prodrug that becomes a biologically active substance after being administered to animals including humans and metabolized.
  • This pharmaceutical composition contains a co-solvent system.
  • a co-solvent system for example, benzyl alcohol, non-polar surfactant, water-miscible organic polymer, aqueous phase, VPD co-solvent system (3 w / v% benzyl alcohol, 8 w / v% non-polar surfactant Polysolvate 80 TM, and A solution of anhydrous ethanol containing 65 w / v% polyethylene glycol 300), but is not limited thereto.
  • the proportions of co-solvents can vary significantly without significantly altering their solubility and toxic properties.
  • the identity of the co-solvent component can be changed.
  • polyethylene glycol such as polyvinylpyrrolidone
  • sugars or polysaccharides are dextrose. Can replace, but is not limited to.
  • these may contain various ingredients for various purposes other than the active ingredient.
  • one or more pharmaceutically acceptable excipients include emulsifiers, dispersants, auxiliaries, preservatives, buffers, binders, stabilizers, coatings, local anesthetics, isotonic agents and the like.
  • the excipients include water, ethanol, polyethylene glycol, gelatin, lactose, sucrose, sodium chloride, glucose, starch, amylase, calcium carbonate, magnesium stearate, kaolin, hydroxymethyl cellulose, microcrystalline cellulose, and talc. , Silicic acid, viscous paraffin, polyvinylpyrrolidone, etc., as binders, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac. , Calcium phosphate, polyvinylpyrrolidone, etc.
  • disintegrants examples include thioglycolic acid and thiolactic acid, prokine hydrochloride and lidocaine hydrochloride as local anesthetics, and sodium chloride and glucose as isotonic agents.
  • the administration route of the above-mentioned drug can be selected from either systemic administration or topical administration.
  • either the oral route or the parenteral route may be used.
  • parenteral routes include intravenous administration, intraarterial administration, transdermal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and transmucosal administration.
  • parenteral routes include intravenous administration, intraarterial administration, transdermal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and transmucosal administration.
  • subarachnoid injection, intraventricular injection, intrathecal injection of the cerebral spinal cord (for example, intrathecal injection) and the like can be exemplified.
  • Systemic administration and topical administration may be used in combination to take into account the passage of the blood-brain barrier.
  • the dosage form is not particularly limited, and any dosage form suitable for the above administration route may be used.
  • tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, liquor, syrups, extracts and elixirs can be used.
  • Parenteral preparations include, for example, injections such as subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections; transdermal or patches, ointments or lotions; sublingual preparations for oral administration. , Oral patches; and aerosols for nasal administration; suppositories. These preparations can be produced by a known method usually used in the preparation process. Further, the agent according to the present invention may be in a long-acting or sustained-release dosage form.
  • the amount of the active ingredient contained in the above-mentioned medicine can be appropriately determined depending on the dose range of the active ingredient, the number of doses, and the like.
  • the dose range is not particularly limited, and the efficacy of the ingredients contained, the form of administration, the route of administration, the type of disease, the nature of the subject (weight, age, medical condition and the use of other drugs, etc.), and the doctor in charge It can be selected as appropriate according to the judgment.
  • suitable doses range, for example, from about 0.01 ⁇ g to about 100 mg, preferably about 0.1 ⁇ g to about 1 mg per kg body weight of the subject.
  • the administration may be once a day or may be divided into several doses.
  • Pharmaceutically acceptable salts may be salts of various categories such as organic salts, inorganic salts, acidic salts, basic salts, metal salts, non-metal salts, acid addition salts, base addition salts, and salts of various embodiments. Be done. Examples include acetates, acidic phosphates, ascorbates, benzoates, benzenesulfonates, bicarbonates, hydrogen tartrates, borates, butyrates, chlorides, citrates, succinates.
  • the splicing function regulator provides a splicing mechanism for producing a mutant mRNA lacking the transcription region from the 8th exon to the pre-mRNA of the lysosome acidic lipase gene in mammalian cells from the 8th exon. It has a function to normalize the splicing mechanism that produces normal mRNA having a transcription region of.
  • the cells may be cultured cells or cells in an individual (which may be human or non-human).
  • a normal mRNA means an mRNA generated by having exon 8 by splicing at a normal position
  • an abnormal mRNA means an mRNA generated by splicing at a normal position and exon 8 is not generated.
  • Means the mRNA produced by skipping. Examples of mutations include c. 894G> A can be mentioned. Therefore, c. Even if it has the 894G> A mutation, the mRNA produced with exon 8 due to splicing at a normal position is called normal mRNA.
  • Example 1 Detection and quantification of normal sequences and mutant sequences using synthetic oligonucleotides as templates
  • synthetic oligonucleotides having the sequences of exons 7 to 10 of the LIPA gene are used as templates, and primers and primers are used. It was confirmed that the probe works with qPCR.
  • the synthetic oligonucleotide used has the following objectives (principles are shown in FIGS. 1A, 1B, 1D).
  • the 894st nucleotide is the nucleotide on the exon 8 side of the boundary where exon 8 and exon 9 are bound.
  • each oligonucleotide has the following sequence.
  • a PCR reaction solution containing a qPCR probe (0.225 ⁇ M), a primer (0.6 ⁇ M), and a PCR enzyme was prepared using THUNDERBIRD® Probe qPCR Mix (Toyobo life science). 5 ⁇ L of synthetic oligonucleotide solution (05, 06, 07, 08) and 10 ⁇ L of PCR reaction solution were mixed. In order to accurately evaluate the amplification efficiency of PCR and quantify the copy number as an absolute value, synthetic oligocreothides (gBlocks, Integrated DNA Technologies, Inc./IDT) having known concentrations were serially diluted, and the obtained Ct value was obtained. Created a calibration line with. ViiA7 (Applied Biosystems) was used for qPCR. Initial denaturation: 95 ° C (1 minute); Cycle: The reaction was carried out at 95 ° C. (15 seconds) / 60 ° C. (1 minute) under the condition of 40 times or more. The following primers and probes were used.
  • the amplification efficiency of PCR is 92% for the oligonucleotide of 06 and can be quantified at 150 copies / well or more, and the amplification efficiency of PCR is 100% for the oligonucleotide of 05. It was possible to quantify at 15 copies / well or higher.
  • Example 2 Measurement of endogenous LIPA mRNA content using RNA of fibroblasts of Wolman's disease patient
  • fibroblasts of Wolman's disease patient were used and endogenous in the same manner as in Example 1. LIPA mRNA was measured.
  • RNA-containing solution was mixed with 16 ⁇ L of reverse transcriptase buffer containing dNTP, random and oligo dT primers and reverse transcriptase. The reaction was carried out at 37 ° C. (15 minutes), 50 ° C. (5 minutes), and 98 ° C. (5 minutes), and cDNA was obtained by reverse transcription reaction.
  • the cDNA was diluted 4-fold with Ultra-pure water (Invitrogen) to measure LIPA cDNA, or 44-fold to measure GAPDH. Using the cDNA thus obtained, quantitative PCR (qPCR) was performed in the same manner as in Example 1.
  • FIG. 2 shows the analysis results of LIPA mRNA using mRNA derived from (A) Wolman's disease patient (GM03111) and (B) mRNA derived from a carrier (GM03558).
  • GM03111 Wolman's disease patient
  • GM03558 mRNA derived from a carrier
  • Example 1 the detection method of Example 1 is based on c.I. Abnormal splicing due to 894G> A mutation can be specifically detected. Then, the Wolman's disease patient (GM03111) is described in c. It can be seen that nearly 100% of mRNA from mutant alleles having 894G> A occupy abnormal mRNA.
  • Example 3 Detection of endogenous LIPA mRNA by endpoint RT-PCR
  • the presence or absence of exon 8 in LIPAC cDNA was detected using endpoint RT-PCR.
  • the signal of 248 bp of GM03111 lacks 72 bp of exon 8 from 320 bp, and c. It can be seen that the cDNA is from an allele with the 894G> A mutation.
  • oligomers that regulate LIPA splicing are c. It is shown that LIPA splicing is regulated to produce normal mRNA from alleles with the 894G> A mutation.
  • an oligomer of intron 8 was administered to fibroblasts derived from a Wolman's disease patient (GM03111), and the mRNA of the LIPA gene expressed by the cells was examined.
  • the oligomer sequence corresponds to the p-th to q-th nucleotide of the intron 8 of the LIPA gene, and is named Lp-q.
  • LNA Locked Nucleic Acid
  • oligomers were introduced into fibroblasts derived from Wolman's disease patients using Lipofectamine 2000 (Life technologies). Specifically, 0.5x10 4 cells per well were seeded on a tissue culture treated 96-well plate (Corning) the day before introduction, and 24 hours after the introduction of the oligomer (30 or 100 nM), D- The cells were washed with PBS ( ⁇ ) (wako), and quantitative PCR (qPCR) was performed in the same manner as in Example 2.
  • the oligomers other than L14-31 are c. It had the effect of regulating LIPA splicing, such as producing normal mRNA from alleles with the 894G> A mutation.
  • L7-24, L8-25, L9-26, and L10-27 had excellent actions, and among them, L9-26 had the most excellent action.
  • Example 5 Evaluation of dose dependence and time dependence of oligomer L9-26
  • the dose dependence and time dependence of the splicing regulating action of oligomer L9-26 were evaluated.
  • the oligomer L9-26 exerts a splicing regulating action from the nM order.
  • Example 6 Evaluation of accuracy of splicing adjustment by L9-26 In this example, it is shown that splicing caused by L9-26 occurs at an accurate position similar to that of the wild type.
  • PCR was performed with primers 03 and 04 on the cDNA solution obtained from the cells collected 24 hours after introducing L9-26 into fibroblasts derived from a Wolman's disease patient (GM03111). .. 0.3 ⁇ L EcoRII and 1 ⁇ L Buffer K (Takara) or 0.3 ⁇ L AluI and 1 ⁇ L Buffer L (Takara) were mixed with 8.7 ⁇ L of the PCR product and reacted at 37 ° C. for 1 hour.
  • oligonucleotide 06 As a control, it has the oligonucleotide 06 (c.894G> A mutation) used in Example 1, but has the sequence of exon 8. Note that the 894th nucleotide is the boundary where exon 8 and exon 9 are bound.
  • Exon 8-side nucleotides) and oligonucleotide 07 wild-type sequence cleaved with each restriction enzyme, fibroblasts and carriers from Wolman's disease patients (GM03111) not treated with L9-26.
  • the oligomer L9-26 restores the original splicing mechanism and causes the same accurate splicing as the wild type.
  • Example 7 Effect of oligomers that regulate LIPA splicing on LAL enzyme activity
  • the oligomer that regulates LIPA splicing is lysosomal acidic lipase (LAL). It is shown to enhance the enzyme activity of.
  • ⁇ Method> 1.0x10 4 patients with Wolman's disease (GM03111) fibroblasts per well, c. Patients with Wolman's disease (GM06144) and carrier (GM03558) fibroblasts without the 894G> A mutation were seeded in tissue culture treated 96-well plates (Corning). After 16 hours, L4-21, L7-24, L8-25, L9-26, L10-27, L11-28, L12-29, L13-30 oligomers and 100 nM each of negative control oligomers (NC) for comparison. / Well-dose was lipofect.
  • the oligomers of L7-24, L8-25, L9-26, and L10-27 are the c.I. It has an action of specifically correcting missplicing due to the 894G> A mutation and enhancing the activity of the LAL enzyme, and among them, L9-26 has the strongest action.
  • Example 8 Evaluation of concentration dependence of oligomer L9-26 on enhancement of LAL enzyme activity Using oligomer L9-26, the effect of enhancing LAL enzyme activity was examined in the same manner as in Example 9 except for the concentration used. The concentration of the oligomer was measured at 10, 30, and 100 nM. The result is shown in FIG.
  • the LAL activity was significantly lower in the GM03111-derived fibroblasts than in the GM03558-derived fibroblasts, but in the GM03111-derived fibroblasts into which L9-26 was introduced, the LAL activity was LAL. Activity increased to the same level as untreated GM03558 at 10 nM, and a significant increase was observed at 30 nM compared to untreated GM03111.
  • LAL Lysosome acidic lipase
  • CED cholesterol ester accumulation disease

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Abstract

Le but de la présente invention est de fournir un nouvel agent prophylactique ou thérapeutique pour le syndrome de déficience en lipase acide lysosomale. La solution selon l'invention porte sur un agent prophylactique ou thérapeutique qui comprend, en tant que principe actif, 13 à 40 oligomères comprenant chacun : une séquence présentant une homologie de 80 % ou plus avec le brin antisens du 8ème intron du gène de lipase de type acide lysosomale humaine ; et une séquence dans laquelle il y a au moins 13 bases identiques à 5'-CCCAAAXGCACXCCXGGA-3' (X est T ou U).
PCT/JP2019/048232 2019-12-10 2019-12-10 Agent prophylactique ou thérapeutique pour le syndrome de déficience en lipase acide lysosomale Ceased WO2021117122A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013540733A (ja) * 2010-09-09 2013-11-07 シナジーバ バイオファーマ コープ 患者のリソソーム酸リパーゼ欠乏症を治療するためのリソソーム酸リパーゼの使用
WO2019213525A1 (fr) * 2018-05-04 2019-11-07 Stoke Therapeutics, Inc. Méthodes et compositions pour le traitement d'une maladie de stockage d'ester de cholestéryle
JP2020000097A (ja) * 2018-06-27 2020-01-09 株式会社リボルナバイオサイエンス ライソゾーム酸性リパーゼ欠損症の予防または治療剤

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013540733A (ja) * 2010-09-09 2013-11-07 シナジーバ バイオファーマ コープ 患者のリソソーム酸リパーゼ欠乏症を治療するためのリソソーム酸リパーゼの使用
WO2019213525A1 (fr) * 2018-05-04 2019-11-07 Stoke Therapeutics, Inc. Méthodes et compositions pour le traitement d'une maladie de stockage d'ester de cholestéryle
JP2020000097A (ja) * 2018-06-27 2020-01-09 株式会社リボルナバイオサイエンス ライソゾーム酸性リパーゼ欠損症の予防または治療剤

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