WO2021117122A1 - Prophylactic or therapeutic agent for lysosomal acid lipase deficiency syndrome - Google Patents

Abstract

[Problem] The purpose of the present invention is to provide a novel prophylactic or therapeutic agent for lysosomal acid lipase deficiency syndrome. [Solution] This prophylactic or therapeutic agent includes, as an active ingredient, 13-40 oligomers each: comprising a sequence having 80% or higher homology with the antisense strand of the 8th intron of human lysosomal acid type lipase gene; and including a sequence in which there are at least 13 bases identical to 5'-CCCAAAXGCACXCCXGGA-3' (X is T or U).

Description

Prophylactic or therapeutic agent for lysosomal acid lipase deficiency

The present invention relates to a prophylactic or therapeutic agent for lysosomal acid lipase deficiency.

Lysosomal acid lipase deficiency (LAL-D) 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. In 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.

It is known that the responsible gene for LAL-D is the lysosomal acid type lipase gene (LIPA) gene on chromosome 10. By mutating the base corresponding to the 894nd position of the cDNA of this gene from G (wild type) to A (mutant type) (hereinafter, this mutation is referred to as c.894G> A mutation), from the 8th exon. Splicing will occur to produce abnormal mRNAs lacking the transcriptional region of. As a result, LAL-D develops due to a marked decrease in the amount of lysosomal acidic lipase produced.

Conventionally, treatment of 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. It contains 17 or more sequences that are the same base with respect to 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U), and may be 17 or more and 40 or less. It contains a sequence of 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U), and may be 18 or more and 40 or less. In addition, 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. In addition, 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. To any one of 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.

In addition, 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. It may be selected from the group consisting of (GuNA), 2'-O, 4'-C-Spirocyclone bridged Nucleic Acid (scpBNA). 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. In addition, the nucleotide having the modified sugar moiety is 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'. It may be a 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.

Further, 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).

Further, the oligomer may contain a peptide nucleic acid.

Further, 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. ..

It is a figure which shows the detection method of the normal LIPA gene and the mutated LIPA gene and the result in the Example of this invention. (A) Each mutation in the GM03111 patient genome [c. 894G> A and 967_968delAG (p.S323Lfs * 44)] positions are shown. (B) The position of the primer / probe set for FL LIPA detection is shown. (C) The result of quantitative PCR (qPCR) using the primer / probe set for FL LIPA detection is shown. (D) The position of the primer / probe set for Delta8 LIPA detection is shown. (E) The result of qPCR using the primer / probe set for Delta8 LIPA detection is shown. In the examples of the present invention, c. 894G> A mutation and c. A patient (GM03111) with a 967_968 del AG (p. S323Lfs * 44) mutation in a complex heterozygotes and c. 894G> A No mutation but c. When 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. It is a figure which shows the detection principle (B) detection result of (A) LIPA cDNA. It is a figure which shows that the antisense oligomer for intron 8 of the LIPA gene has a splicing regulation function in LIPA gene expression in the Example of this invention. The results of evaluating (A) dose dependence and (B) time dependence of antisense oligomer L9-26 in the examples of the present invention are shown. It is a figure which shows the result of the experiment which evaluates the accuracy of the splicing adjustment by L9-26 in the Example of this invention. In the examples of the present invention, the results of examining the effect of the antisense oligomer that regulates LIPA splicing on the LAL enzyme activity are shown. In the examples of the present 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.

The object, feature, advantage, and idea thereof of the present invention will be apparent to those skilled in the art by the description of the present specification, and a person skilled in the art can easily reproduce the present invention from the description of the present specification. Embodiments and specific examples of the invention described below illustrate preferred embodiments of the invention and are shown for illustration or purposes only, to the present invention. It is not limited. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein within the intent and scope of the invention disclosed herein.

Unless otherwise specified in the embodiments and examples, M. R. Green & J. Sambrook (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor , New York (2012); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, JG Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in M Use the method described in a standard protocol collection such as Biology, John Wiley & Sons Ltd., or a modified or modified method. When using a commercially available reagent kit or measuring device, the protocol attached to them is used unless otherwise specified.

[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).

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. Here, 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.

Specifically, 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. In the case of 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. In the case of 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. Here, 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. For example, 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. Alternatively, 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. In that case, 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.

Further, a part or all of the sugar portion may be replaced with morpholine. As for the method for producing and using morpholin, a known method may be followed (for example, Biochem Biophys Res Communi. 2007 Jun 29; 358 (2): 521-7; WO2014189142; WO2016060135). In the present specification, the oligomer also includes a morpholino oligonucleotide in which a part or all of the sugar moiety is replaced with morpholine. Here, 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.

Further, 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.

By making these modifications to the oligomer, non-specific decomposition can be prevented, the stability and specificity of annealing can be enhanced, and therefore the efficiency of splicing adjustment can be improved.

When 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. 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.). For example, 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.

Further, 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. Suitable examples of 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. Preferable examples of sugar acids include ascorbic acid. Preferable examples of the sulfonic acid include 2-naphthalene sulfonic acid, 10-camphor sulfonic acid and methane sulfonic acid. Preferable examples of amides include nicotinamide, benzamide, lactamide, glycolamide and saccharin. Preferable examples of 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.

[Pharmaceutical]
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. For example, an oligomer in which a protecting group removed by an intracellular esterase is introduced into a phosphate diester site.

This pharmaceutical composition contains 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 ™, 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. Furthermore, the identity of the co-solvent component can be changed. For example, other surfactants may be used in place of Polysorbate 80 ™, other biocompatible polymers replace polyethylene glycol, such as polyvinylpyrrolidone, and other 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. For example, one or more pharmaceutically acceptable excipients, disintegrants, diluents, lubricants, flavoring agents, colorants, sweeteners, acidulants, flavoring agents, suspending agents, wetting agents, etc. Examples include emulsifiers, dispersants, auxiliaries, preservatives, buffers, binders, stabilizers, coatings, local anesthetics, isotonic agents and the like. Specifically, 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. as disintegrants, dried starch, sodium alginate, canten powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, etc. Salt, borosand, polyethylene glycol, etc., sodium citrate, sodium acetate, sodium phosphate, etc. as buffers, tragant, gum arabic, gelatin, sodium pyrosulfate, ethylenediamine tetraacetic acid (EDTA), etc. as stabilizers, Examples thereof 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. In either case, either the oral route or the parenteral route may be used. Examples of parenteral routes include intravenous administration, intraarterial administration, transdermal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and transmucosal administration. In the case of local 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 (particularly intracerebral 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. For example, for oral administration, 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. In general, suitable doses range, for example, from about 0.01 μg to about 100 mg, preferably about 0.1 μg 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. , Kamfer sulfonate, ethane sulfonate, nitate, fumarate, gentisate, gluconate, glucronate, glutamate, hydrobromide, hydrochloride, hydride dichloride, hydrogen iodide Latex, isonicotate, lactate, maleate, methanesulfonate, naphthalenesulfonate, nitrate, oxalate, pamonate, pantothenate, phosphate, propionate, glycosate , Salicylate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, trifluoroacetate, aluminum salt, ammonium salt, calcium salt, lithium salt, magnesium salt, potassium salt, sodium salt, zinc Examples thereof include, but are not limited to, salts and diethanolamine salts.

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).

In addition, in this specification, a normal mRNA means an mRNA generated by having exon 8 by splicing at a normal position, and 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 In this example, the following 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.

<Method>
The synthetic oligonucleotide used has the following objectives (principles are shown in FIGS. 1A, 1B, 1D).

05: The exon 8 sequence is deleted.

06: c. It has an 894G> A mutation but has an exon 8 sequence. The 894st nucleotide is the nucleotide on the exon 8 side of the boundary where exon 8 and exon 9 are bound.

07: Wild-type sequence.

08: C. Deletion of sequence AG at positions 967 to 968 of cDNA. It has 967_968 del AG. Since this deletion is on Primer 03, it is believed that Primer 03 does not work for this synthetic oligonucleotide.

And each oligonucleotide has the following sequence.

05-Standard delta8_E7_9_10 (5'-GACTTATTTGGAGACAAAGAATTTCTTCCCCAGAGTGCGTTTTTGAAGTGGCTGGGTACCCACGTTTGCACTCATGTCATACTGAAGGAGCTCTGTGGAAATCTCTGTTTTCTTCTGTGTGGATTTAATGAGAGAAATTTAAATATGGCTGTTAAATTCCAAAAGTTTCAAGCCTTTGACTGGGGAAGCAGTGCCAAGAATTATTTTCATTACAACCAGAGTTATCCTCCCACATACAATGTGAAGGACATGCTTGTGCCGACTGCAGTCTGGAGCGGG-3 ') (SEQ ID NO: 4);
06-Standard FL_mutant_894 G to A_E7_8_9_10 (5'-GACTTATTTGGAGACAAAGAATTTCTTCCCCAGAGTGCGTTTTTGAAGTGGCTGGGTACCCACGTTTGCACTCATGTCATACTGAAGGAGCTCTGTGGAAATCTCTGTTTTCTTCTGTGTGGATTTAATGAGAGAAATTTAAATATGTCTAGAGTGGATGTATATACAACACATTCTCCTGCTGGAACTTCTGTGCAAAACATGTTACACTGGAGCCAAGCTGTTAAATTCCAAAAGTTTCAAGCCTTTGACTGGGGAAGCAGTGCCAAGAATTATTTTCATTACAACCAGAGTTATCCTCCCACATACAATGTGAAGGACATGCTTGTGCCGACTGCAGTCTGGAGCGGG-3 ') (SEQ ID NO: 5);
07-Standard FL WT_894G to G_E7_8_9_10 (5'-GACTTATTTGGAGACAAAGAATTTCTTCCCCAGAGTGCGTTTTTGAAGTGGCTGGGTACCCACGTTTGCACTCATGTCATACTGAAGGAGCTCTGTGGAAATCTCTGTTTTCTTCTGTGTGGATTTAATGAGAGAAATTTAAATATGTCTAGAGTGGATGTATATACAACACATTCTCCTGCTGGAACTTCTGTGCAAAACATGTTACACTGGAGCCAGGCTGTTAAATTCCAAAAGTTTCAAGCCTTTGACTGGGGAAGCAGTGCCAAGAATTATTTTCATTACAACCAGAGTTATCCTCCCACATACAATGTGAAGGACATGCTTGTGCCGACTGCAGTCTGGAGCGGG-3 ') (SEQ ID NO: 6);
08-Standard FL c967_968delAG_894G to G_E7_8_9_10 (5'-GACTTATTTGGAGACAAAGAATTTCTTCCCCAGAGTGCGTTTTTGAAGTGGCTGGGTACCCACGTTTGCACTCATGTCATACTGAAGGAGCTCTGTGGAAATCTCTGTTTTCTTCTGTGTGGATTTAATGAGAGAAATTTAAATATGTCTAGAGTGGATGTATATACAACACATTCTCCTGCTGGAACTTCTGTGCAAAACATGTTACACTGGAGCCAGGCTGTTAAATTCCAAAAGTTTCAAGCCTTTGACTGGGGAAGCAGTGCCAAGAATTATTTTCATTACAACCAGTTATCCTCCCACATACAATGTGAAGGACATGCTTGTGCCGACTGCAGTCTGGAGCGGG-3 ') (SEQ ID NO: 7).

Specifically, a PCR reaction solution containing a qPCR probe (0.225 μM), a primer (0.6 μM), and a PCR enzyme was prepared using THUNDERBIRD® Probe qPCR Mix (Toyobo life science). 5 μL of synthetic oligonucleotide solution (05, 06, 07, 08) and 10 μL 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.

<For detection of Delta8 LIPA (detection of cDNA lacking exon 8)>
01-Fw-LIPA (Exon7, 9) (5'-ATGAGAGAAATTTAAATATGGCTGT-3') (SEQ ID NO: 8);
03-Rv-LIPA (5'-TTGTATGTGGGAGATAACTCTGG-3') (SEQ ID NO: 9);
02-Probe-LIPA (5'-/ 56-FAM / CCAAAAAGTT / ZEN / TCAAGCCTTTGACTGGGG / 3lABkFQ / -3') (SEQ ID NO: 10).

<For FL LIPA detection (detects cDNA with exon 8)>
04-Fw-LIPA (Exon8) (5'-AACATGTTACACTGGACCA) (SEQ ID NO: 11);
03-Rv-LIPA (5'-TTGTATGTGGGAGATAACTCTGG-3') (SEQ ID NO: 9);
02-Probe-LIPA (5'-/ 56-HEX / CCAAAAAGTT / ZEN / TCAAGCCTTTGACTGGGG / 3lABkFQ / -3') (SEQ ID NO: 12), or different fluorescent dyes detected (5'-/ 56-FAM / CCAAAAGT / ZEN / TCAAGCCTTTGACTGGGG / 3lABkFQ / -3') (SEQ ID NO: 10).

Ct was calculated from the obtained results, and the absolute copy number was calculated from the standard curve. Then, the obtained results were plotted on a graph of copy number (x-axis) and Ct (y-axis), and used as FIGS. 1C and 1E.

<Result>
From FIG. 1 (B), when the primer / probe set for FL LIPA detection is used, the cDNA in which exon 8 is present is detected, so 05 cannot be detected. From FIG. 1 (D), Delphi 8 LIPA is detected. When the primer / probe set of No. 1 is used, cDNA in which exon 8 is absent is detected, so it is predicted that 06 to 07 cannot be detected. In either case, it is expected that 08 cannot be detected because the primer 03 does not function.

When qPCR was actually performed, as shown in FIGS. 1C and 1E, 06 and 07 were detected and 05 and 08 were not detected in the FL LIPA detection primer / probe set, and the Delta 8 LIPA detection primer was not detected. In the / probe set, only 05 was detected and 06-08 were not detected. Therefore, the expected result was obtained.

Further, under this condition, it was clarified that the Ct value and the copy number of the template DNA showed a correlation, and the copy of the DNA contained in the reaction solution could be calculated from the Ct value. In (C), 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.

As described above, under the PCR conditions of Example 1, normal LIPA mRNA and abnormal LIPA mRNA could be discriminated, and the copy number could be calculated.

[Example 2] Measurement of endogenous LIPA mRNA content using RNA of fibroblasts of Wolman's disease patient In this example, fibroblasts of Wolman's disease patient were used and endogenous in the same manner as in Example 1. LIPA mRNA was measured.

<Method>
c. 849G> A and c. A patient with Wolman's disease (GM03111) having 967_968 del AG in a composite heterozygotes and c. Fibroblasts derived from carriers (GM03558) having 193C> T in heterozygotes (obtained from Coriell Cell Reports), DMEM (Life technologies) and High Glucose (Life technologies) containing 10% FBS. Cultivated using. 0.5x10 ⁴ cells were seeded in wells of a tissue culture treated 96-well plate (Corning), cultured for a predetermined time, and then washed with D-PBS (-) (wako). Next, using SuperPrep® Cell Lysis & RT Kit for qPCR (Toyobo life science), 25 μL / well of Lysis buffer was added to lyse the cells to decompose genomic DNA, and 5 μL of buffer for stopping the reaction. Was added to obtain an RNA-containing solution. 4 μL of RNA-containing solution was mixed with 16 μL 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.

As an internal standard, for measuring the number of mRNA copies of GAPDH, 20X gene expression PCR assay (Life Technologies, Inc., part number 4326317E) and standard-GAPDH (5'-AAATTGAGCCCGCAGCCTCCCGCTTCGCTCTCTGCTCCTCCTGTTCGACAGTCAGCCGCATCTTCTTTTGCGTCGCCAGCCGAGCCACATCGCTCAGACACCATGGGGAAGGTGAAGGTCGGAGTCAACGGATTTGGTCGTATTGGGCGCCTGGTCACCAGGGCTGCTTTTAACTCTGGTAAAGTGGATATTGTTGCCATCAATGACCCCTTCATTGACCTCAACTACATGGTTTACATGTTCCAATATGATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATCACCATCTTCCAGGAGCGAGATCCCTCCAAAATCAAGTGGGGCGATGCTGGCGCTGAGTACGTCGTGGAGTCCACTGGCGTCTTCACCACCATGGAGAAGGCTGGGGCTCATTTGCAGGGGGGAGCCAAAAGGGTCATCATCTCTGCCCCCTCTGCTGATGCCCCCATGTTCGTCAT-3 ') (SEQ ID NO: 13) was used. The quantitative value of the measurement result using cDNA was standardized by the quantitative value of GAPDH.

<Result>
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). In Wolman's disease patients, assuming that the total LIPA mRNA was 100%, 99.9% of abnormal mRNA Delta8 and 0.1% of normal mRNA FL LIPA were produced. On the other hand, in the carrier, Delta 8 was below the detection sensitivity.

As described above, 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 In this example, the presence or absence of exon 8 in LIPAC cDNA was detected using endpoint RT-PCR.

<Method>
By performing PCR on LIPAC cDNA using the following primers, as shown in FIG. 3A, a DNA fragment having a length of 320 bp was detected in the presence of exon 8, and 248 bp in the absence of exon 8. A DNA fragment of the length of is detected.

1113, [kpn] -hLIPA (E7) -F: 5'-TGTATACAAAGTTGACTTATTTGGAGACAAAGAATTTCTCCA-3'(SEQ ID NO: 14)
1098, hLIPA (E9)-(attB2) -R: 5'-GTACAAGAAAGCTGGGTGGTTGTAATGAAAATAATTTGGCAC-3'(SEQ ID NO: 15)
PCR was performed according to the package insert of TakaRa Ex Taq® Hot Start Version so that the final concentration of Primer was 1 μM. Then, 1 μL of XCdie (Nippon Gene) was mixed with 10 μL of the amplification product and electrophoresed on a 3% agarose gel. Visualization was performed with an ethidium bromide solution and BioDoc-It System (analytic jena US, An Endress + Hauser Company). The result is shown in FIG. 3B.

<Result>
As shown in FIG. 3B, patients with Wolman's disease (GM03111) obtained signals of both 320 bp and 248 bp, whereas carriers (GM03558) obtained signals of only 320 bp.

Considering the result of Example 2, 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.

[Example 4] Oligomers that regulate LIPA splicing In this example, the oligomers are c. It is shown that LIPA splicing is regulated to produce normal mRNA from alleles with the 894G> A mutation.

<Method>
In this example, 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. As the composition of the nucleotides, those in which LNA (Locked Nucleic Acid) nucleotides and deoxyribonucleotides were alternately arranged were used. The composition of the oligomer used is described below.

L4-21, 5'-a ^ T (L) ^ g ^ 5 (L) ^ a ^ 5 (L) ^ t ^ 5 (L) ^ ° C ^ T (L) ^ g ^ G (L) ^ a ^ A (L) ^ t ^ G (L) ^ c ^ c-3'(SEQ ID NO: 16)
L7-24, 5'-c ^ A (L) ^ a ^ A (L) ^ t ^ G (L) ^ c ^ A (L) ^ c ^ T (L) ^ c ^ 5 (L) ^ t ^ G (L) ^ g ^ A (L) ^ a ^ t-3'(SEQ ID NO: 17)
L8-25, 5'-c ^ 5 (L) ^ a ^ A (L) ^ a ^ T (L) ^ g ^ 5 (L) ^ a ^ 5 (L) ^ t ^ 5 (L) ^ c ^ T (L) ^ g ^ G (L) ^ a ^ a-3'(SEQ ID NO: 18)
L9-26, 5'-c ^ 5 (L) ^ c ^ A (L) ^ a ^ A (L) ^ t ^ G (L) ^ c ^ A (L) ^ c ^ T (L) ^ c ^ 5 (L) ^ t ^ G (L) ^ g ^ a-3'(SEQ ID NO: 19)
L10-27, 5'-c ^ 5 (L) ^ c ^ 5 (L) ^ a ^ A (L) ^ a ^ T (L) ^ g ^ 5 (L) ^ a ^ 5 (L) ^ t ^ 5 (L) ^ c ^ T (L) ^ g ^ g-3'(SEQ ID NO: 20)
L11-28, 5'-a ^ 5 (L) ^ c ^ 5 (L) ^ c ^ A (L) ^ a ^ A (L) ^ t ^ G (L) ^ c ^ A (L) ^ c ^ T (L) ^ c ^ 5 (L) ^ t ^ g-3'(SEQ ID NO: 21)
L12-29, 5'-a ^ A (L) ^ c ^ 5 (L) ^ c ^ 5 (L) ^ a ^ A (L) ^ a ^ T (L) ^ g ^ 5 (L) ^ a ^ 5 (L) ^ t ^ 5 (L) ^ c ^ t-3'(SEQ ID NO: 22)
L13-30, 5'-g ^ A (L) ^ a ^ 5 (L) ^ c ^ 5 (L) ^ c ^ A (L) ^ a ^ A (L) ^ t ^ G (L) ^ c ^ A (L) ^ c ^ T (L) ^ c ^ c-3'(SEQ ID NO: 23)
L14-31, 5'-t ^ G (L) ^ a ^ A (L) ^ c ^ 5 (L) ^ c ^ 5 (L) ^ a ^ A (L) ^ a ^ T (L) ^ g ^ 5 (L) ^ a ^ 5 (L) ^ t ^ c-3'(SEQ ID NO: 24)
Scramble ASO (negative control, NC), 5'-t ^ A (L) ^ a ^ 5 (L) ^ a ^ 5 (L) ^ g ^ T (L) ^ c ^ T (L) ^ a ^ T (L) ^ a ^ 5 (L) ^ g ^ 5 (L) ^ c ^ c-3'(SEQ ID NO: 25)
Scramble ASO (negate control-2, NC-2), 5'-a ^ T (L) ^ g ^ 5 (L) ^ a ^ T (L) ^ c ^ T (L) ^ c ^ A (L) ^ T ^ T (L) ^ g ^ T (L) ^ a ^ G (L) ^ t ^ c-3'(SEQ ID NO: 26)
In the above sequence, N (L) indicates an LNA having a ribose ring cross-linked and a modified nucleic acid having a nucleobase, and 5 (L) indicates an LNA having methylcytosine at the base. ^ Indicates phosphorothioate modification. Lowercase letters indicate deoxyribonucleotides.

These 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. FIG. 4 shows the average value of N = 2 and each value.

<Result>
As shown in FIG. 4, 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. In particular, 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 In this example, the dose dependence and time dependence of the splicing regulating action of oligomer L9-26 were evaluated.

<Method>
First, as in Example 4, 30 nM L9-26 was lipofected in fibroblasts derived from a Wolman's disease patient (GM03111), and the cells were collected after 1,6,24,48,72,96 hours to obtain cDNA. Synthesized. It was measured by quantitative PCR using qPCR (combination of 04, 02, 03) capable of specifically measuring FL LIPA or qPCR (combination of 01, 02, 03) capable of specifically measuring Delta8 LIPA. After standardizing with the measured values for GAPDH, the Δ value was calculated by subtracting the measured value of the negative control from the obtained value. The result of carrying out with N = 2 is shown in FIG. 5A.

On the other hand, L9-26 was introduced into fibroblasts derived from GM03111 at various concentrations, the cells were collected 48 hours later, and the results of quantitative PCR were calculated in the same manner. The result of carrying out with N = 2 is shown in FIG. 5B.

<Result>
As shown in FIG. 5A, the splicing-regulating action of oligomer L9-26 almost reaches a plateau 48 hours after lipofection.

Further, as shown in FIG. 5B, 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.

<Method>
Similar to Example 4, 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 μL EcoRII and 1 μL Buffer K (Takara) or 0.3 μL AluI and 1 μL Buffer L (Takara) were mixed with 8.7 μL of the PCR product and reacted at 37 ° C. for 1 hour. Next, a mixture of 0.5 μL of 200 mM EDTA solution (Wako) and 1 μL of XCdie (Nippon Gene) was electrophoresed on a 3% agarose gel, and the ethidium bromide solution and BioDoc-It System (analytic jena US, Inc.) were electrophoresed. Visualization was performed using An Endress + Hauser Company). The results are shown in FIG. 6 (B).

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 product obtained by amplifying the cDNA derived from fibroblasts derived from GM03558 (GM03558) was cleaved with each restriction enzyme and electrophoresed at the same time.

Here, in the expression of the wild-type allele, when splicing occurs accurately and a normal mRNA having exon 8 is obtained, an EcoRII recognition sequence is generated in the corresponding cDNA, and by cleaving with EcoRII, the length becomes 17 bases. A 98 base length fragment is produced. On the other hand, in the case of mutant alleles, c. Since it has the 894 G> A mutation, when splicing occurs accurately and a normal mRNA having exon 8 is obtained, an AluI recognition sequence is generated in the corresponding cDNA, and by cleaving with AluI, the length is 19 bases and 96. Base-length fragments are produced. This principle is shown in FIG. 6 (A).

<Result>
As shown in FIG. 6 (B), the by-product of oligonucleotide 06 is cleaved only by AluI, and the by-product of oligonucleotide 07 is cleaved only by EcoRII. In the case of fibroblast-derived cDNA derived from a carrier (GM03558) not treated with L9-26, since the exon 8 has a wild-type sequence, the amplified fragment by PCR is cleaved only with EcoRII, and at L9-26. In the case of untreated cDNA derived from fibroblasts derived from Wolman's disease patient (GM03111), since it does not have exons 8, no amplified fragment by PCR is present and no signal is detected.

However, in the case of a fibroblast-derived cDNA derived from a Wolman's disease patient (GM03111) that is not treated with L9-26, it is cleaved only by AluI. That is, c. Despite having the 894G> A mutation, splicing shows that it is occurring normally.

In this way, 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 In this example, in fibroblasts derived from Wolman's disease patients (GM03111), 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. After 48 hours, the cells were washed with PBS (−), cells were lysed with 50 μL of MilliQ solution containing 0.5% Triton X-100 (Wako Pure Chemical Industries, Ltd.), and the extract was recovered. Centrifugation at 13,200 xg for 5 minutes at 4 ° C. and the collected supernatant was used for measurement. For the measurement of enzyme activity, 5 μL of each supernatant was dispensed into 2 wells of a 384-well plate for fluorescence measurement (Corning Inc.). Add 1.25 μL of MilliQ solution to one well and 7.5 μM Lalistat2 (synthesized in house) to the other, incubate at 37 ° C. for 10 minutes, and then add 18.75 μL of substrate solution to each well [0. 4: 1: 14; 13.3 mM palmitol 4-Metyllum belieferone (CAYMAN): 0.5% Cardiolipin (SIGMA): 100 mM acetate buffer (pH 4.0)] was added, and the mixture was incubated at 37 ° C. for another 30 minutes. .. After the reaction, 5 uL of 1 M phosphate buffer (pH 12.0) was added, and the fluorescence intensity was measured using EnSpire (PerkinElmer) by setting the excitation and fluorescence wavelengths to 320 nm and 460 nm. For the standard curve, 4-Umbelliferone (synthesized in house) was used instead of the cell suspension. The enzyme activity was standardized by measuring the amount of protein in the same sample with the protein assay BCA kit (Wako Junyaku) and standardizing it to the substrate concentration (nmol / mg of protein / hour) that is degraded per hour with 1 mg protein amount. The t-test was performed using NC as a control (* P <0.05, ** p <0.01, *** p <0.001). The average value + SEM obtained at N = 4 is shown in FIG. As a negative control of the experiment itself, Intact shows the result of no treatment, and Vehicle shows the result of treatment with only the transfection reagent.

<Result>
As shown in FIG. 7, in GM03111, the oligomers of L7-24, L8-25, L9-26, and L10-27 showed a significant increase in LAL activity as compared with NC. L9-26 showed the strongest increase in activity, 15.1 times that of NC. On the other hand, no significant difference was observed between L4-21, L11-28, L12-29, and L13-30 as compared with NC. This result correlates with the enhancement of normal LIPA mRNA expression by oligomerization in Example 4.

On the other hand, c. In the case of GM06144 derived from a Welmann's disease patient without the 894G> A mutation, no significant difference was observed in any of the oligomer treatments. c. In the case of GM03558, which is a carrier having no 894G> A mutation, no significant difference was observed in any of the oligomer treatments.

Thus, 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.

<Result>
As shown in FIG. 8, 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.

It consists of a sequence having 80% or more complementary bases to the sense strand of the 8th intron of the human lysosomal acidic lipase gene, and 13 to 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U). Oligomers consisting of 13 or more and 40 or less nucleotides containing a sequence of one or more identical bases are described in c. Lysosome acidic lipase (LAL) deficiency, including Wolman's disease and cholesterol ester accumulation disease (CESD), as it can correct missplicing of LIPA gene transcripts with 894G> A mutations and enhance LAL enzyme activity to normal levels or higher. It can be used to treat illness.

Claims (31)

It consists of a sequence having 80% or more homology to the antisense strand of the 8th intron of the human lysosomal acidic lipase gene, and 13 or more to 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U). 13 or more and 40 or less oligomers containing sequences that are the same base. The oligomer according to claim 1, which comprises 15 or more sequences that are the same base with respect to 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U), and is 15 or more and 40 or less. The oligomer according to claim 1, which comprises 17 or more sequences that are the same base with respect to 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U), and is 17 or more and 40 or less. The oligomer according to claim 1, which comprises a sequence of 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U) and is 18 or more and 40 or less. It 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 contains a sequence consisting of 5'-CCCAAAAXGCACXCCXGGA-3'(X is T or U). The oligomer according to claim 1, wherein the number is 13 or more and 40 or less. 7th to 24th, 8th to 25th, 9th to 26th, and 5th from the 5'end in the nucleotide sequence of the sense strand of the 8th intron of the human lysosome acidic lipase gene shown in Sequence 1. From a base sequence complementary to any one of the base sequences selected from the group consisting of the 10th to 27th, the 11th to 28th, the 12th to 29th, the 13th to 30th, and the 14th to 31st. The oligomer according to claim 5. The oligomer according to any one of claims 1 to 6, which comprises an oligonucleotide or a modified oligonucleotide in which one or more sugar moieties and / or phosphate binding moieties are modified. The antisense oligomer according to claim 7, wherein the 2'position of the modified sugar moiety is 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).} The oligomer according to claim 8. The antisense oligomer according to any one of claims 7 to 9, wherein the -OH group at the 2'position of the modified sugar moiety _{is substituted with -OCH 3} or -OCH ₂ CH ₂ OCH _3. The oligomer according to claim 7 or 8, wherein the 2'position and the 4'position of the modified sugar moiety are 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. (GuNA). The oligomer according to claim 11, which is selected from the group consisting of 2'-O, 4'-C-Spilocyclone bridged Nucleic Acid (scpBNA). The oligomer according to any one of claims 7 to 9, wherein the nucleotide having the modified sugar moiety is a 2'-deoxy-ribonucleotide. The oligomer according to claim 13, wherein the 2'-deoxy-ribonucleotide is 2'-deoxy-adenosin or 2'-deoxy-guanosine. The nucleotide having the modified sugar moiety is 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino. The oligomer according to claim 7 or 8, wherein the nucleotide is selected from the group consisting of -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 The oligomer according to claim 7, which is a nucleotide selected from the group consisting of 6,6-diamino-purine, 4-thio-uridine, and 5-amino-allyl-uridine. Claim that the modified phosphate binding moiety is composed of a bond selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoromidate bond, and a borane phosphate bond. 7. The oligomer according to 7. The oligomer according to any one of claims 1 to 6, which contains a morpholino oligonucleotide. The oligomer according to claim 18, wherein the morpholino oligonucleotide contains a phosphorodiamidate-morpholino oligonucleotide. The oligomer according to claim 18 or 19, wherein the 5'end is a group according to any one of the following chemical formulas (1) to (3).

The oligomer according to any one of claims 1 to 6, which contains a peptide nucleic acid. The oligomer according to any one of claims 1 to 6, which comprises 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 oligomer according to claim 21, wherein the modified oligonucleotide is the modified oligonucleotide according to any one of claims 8 to 17. A splicing function modifier containing the oligomer according to any one of claims 1 to 23 or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient. A pharmaceutical composition containing the oligomer according to any one of claims 1 to 23 or a pharmaceutically acceptable salt or hydrate thereof as an active ingredient. A medicine containing the pharmaceutical composition according to claim 25. A therapeutic agent for lysosome acidic lipase deficiency, which comprises the pharmaceutical composition according to claim 25. In mammalian cells, a splicing mechanism that produces an abnormal mRNA lacking the transcription region from the 8th exon against the pre-mRNA of the lysosome acidic lipase gene, and a normal having a transcription region from the 8th exon. It is a method of regulating the splicing mechanism that makes it a splicing mechanism that produces mRNA.
A method for adjusting a splicing mechanism, which comprises a step of administering the oligomer according to any one of claims 1 to 23 or a pharmaceutically acceptable salt or hydrate thereof to the human cells. A method for preventing or treating lysosome acidic lipase deficiency in mammals.
A prophylactic or therapeutic method comprising administering to the mammal an effective amount of the oligomer according to any one of claims 1 to 23 or a pharmaceutically acceptable salt or hydrate thereof. The oligomer according to any one of claims 1 to 23 or a pharmaceutically acceptable salt or hydrate thereof for use in the prevention or treatment of lysosome acidic lipase deficiency. Use of the oligomer according to any one of claims 1 to 23 or a pharmaceutically acceptable salt or hydrate thereof for producing a prophylactic or therapeutic agent for lysosome acidic lipase deficiency.

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