JP2019162111A - Antisense nucleic acid for the treatment of Fukuyama muscular dystrophy - Google Patents

Abstract

To provide antisense nucleic acids capable of treating Fukuyama muscular dystrophy by normalizing the abnormal splicing of the fukutin gene having the insertion mutation of the SVA type retrotransposon.SOLUTION: Provided is an antisense nucleic acid that suppresses abnormal splicing using a splicing donor site 3' of guanine at position 111177 and a splicing accepting site 5' of thymine at position 115943 of the specific sequence, in the transcription stage of fukutin genes having an insertion mutation of SVA type retrotransposon, the antisense nucleic acid targeting a range of positions 115937 to 115981 of a specific base sequence and consisting of 9 to 24 bases.SELECTED DRAWING: None

Description

  The present invention relates to an antisense nucleic acid for treating Fukuyama muscular dystrophy.

  Fukuyama type congenital muscular dystrophy (hereinafter sometimes abbreviated as “FCMD”) is a disease unique to Japan and is an autosomal recessive disease that presents three symptoms: congenital muscular dystrophy, type II spondylosis, and ocular malformation. It is a genetic disease. This is the second most common muscular disease in children in Japan. It is a serious disease that causes death in teens, but there is no cure yet. The Fctin gene, which is a disease responsible gene of FCMD, was identified by the positional cloning method in 1998 (see Non-Patent Documents 1 and 2). Most FCMD patients have an insertion mutation of an about 3 kb SVA retrotransposon (SVA: Sine-VNTR-Alu), which is a moving gene, in the 3'-untranslated region of the fuctin gene (see Non-Patent Document 2). This mutation occurred in one of Japanese ancestors about 100 generations ago. One in 88 Japanese was a carrier and reported to occur in about 1 in 30,000 births. (See Non-Patent Document 3). In the skeletal muscle of FCMD patients, the glycoprotein α-dystroglycan that connects the cell membrane and the basement membrane is deficient in the O-mannose-type sugar chain modification, and the binding between the cell membrane and the basement membrane via this sugar chain is broken, which is severe. It is known that muscular dystrophy develops (see Non-Patent Document 4).

  In addition to FCMD, it is known that various pathologies are caused by mutations in the fuctin gene (see Non-Patent Document 5). Among these, for example, adult-onset dilated cardiomyopathy (see Non-Patent Document 6) and Walker-Warburg syndrome-like severe congenital muscular dystrophy (see Non-Patent Document 7) are SVA type retrotransposon insertion mutations in the fuctin gene. Has been reported to be the cause.

  The present inventors have proved that FCMD is a splicing abnormality induced by insertion of an SVA type retrotransposon (see Non-Patent Document 8). More specifically, the strong splicing receptor site present in the sequence of the SVA type retrotransposon inserted in the 3 ′ untranslated region of the final exon (exon 10) of the fuctin gene which is a disease-responsible gene is a translated region of exon 10 It was revealed that this was caused by newly activating a potential splicing donor site (exon trapping).

Toda, T., Segawa, M., Nomura, Y. et al .: Localization of a gene for Fukuyama type congenital muscular dystrophy to chromosome 9q31-33. Nat. Genet., 5, 283-286 (1993) Kobayashi, K., Nakahori, Y., Miyake, M. et al .: An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy.Nature, 394, 388-392 (1998) Watanabe, M., Kobayashi, K., Jin, F. et al .: Founder SVA retrotransposal insertion in Fukuyama-type congenital muscular dystrophy and its origin in Japanese and Northeast Asian populations. Am. J. Med. Genet. A, 138 , 344-348 (2005) Michele, D. E., Barresi, R., Kanagawa, M. et al .: Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies. Nature, 418, 417-422 (2002) OMIM No.607440, http://omim.org/entry/607440 Murakami, T., Hayashi, Y. K., Noguchi, S., et al .: Fukutin gene mutations cause dilated cardiomyopathy with minimal muscle weakness. Ann. Neurol., 60, 597-602 (2006) Kondo-Iida, E., Kobayashi, K., Watanabe, M., et al .: Novel mutations and genotype-phenotype relationships in 107 families with Fukuyama-type congenital muscular dystrophy (FCMD). Hum. Mol. Genet., 8 , 2303-2309 (1999) Taniguchi-Ikeda, M., et al .: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy. Nature, 478, 127-131 (2011)

  An object of the present invention is to provide an antisense nucleic acid capable of treating FCMD and the like by normalizing abnormal splicing of a fuctin gene having an insertion mutation of SVA type retrotransposon. It is another object of the present invention to provide an antisense nucleic acid having excellent solubility in physiological saline and high safety.

The present invention includes the following inventions in order to solve the above problems.
[1] An antisense nucleic acid comprising 9 to 24 bases with a target sequence ranging from the 115th to 115981th positions of the base sequence represented by SEQ ID NO: 1.
[2] The antisense nucleic acid according to [1] above, comprising a base sequence selected from SEQ ID NOs: 6 to 70.
[3] The antisense nucleic acid according to [1] or [2] above, which is dissolved in physiological saline at 50 mg / mL or more.
[4] Selected from SEQ ID NOs: 18, 19, 26, 27, 32, 33, 37, 43, 53, 55, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69 and 70 The antisense nucleic acid according to any one of [1] to [3], wherein the antisense nucleic acid is composed of a base sequence.
[5] The above [1], wherein no abnormal value is observed in any of the AST value, the ALT value, the BUN value, and the creatinine value when an antisense nucleic acid at a dose of 60 mg / kg is administered to mice. Or the antisense nucleic acid as described in [2].
[6] Any one of [1], [2] or [5] above, comprising a base sequence selected from SEQ ID NOs: 32, 50, 51, 60, 62, 64, 65, 66 and 67 An antisense nucleic acid according to 1.
[7] When an antisense nucleic acid of 50 mg / mL or more dissolved in physiological saline and administered at a dose of 60 mg / kg is administered to mice, all of the AST, ALT, BUN and creatinine values are abnormal The antisense nucleic acid according to the above [1] or [2], wherein the antisense nucleic acid is not observed.
[8] The antisense nucleic acid according to any one of [1], [2] or [7] above, comprising a base sequence selected from SEQ ID NOs: 32, 60, 64, 65, 66 and 67 .
[9] The antisense nucleic acid according to any one of [1] to [8] above, wherein the 3 ′ end and / or the 5 ′ end of the antisense nucleic acid is unmodified.
[10] The antisense nucleic acid according to any one of [1] to [8] above, wherein the 3 ′ end and / or the 5 ′ end of the antisense nucleic acid is modified.
[11] One or more selected from the group consisting of triethylene glycol (TEG) modification, hexaethylene glycol (HEG) modification and dodecaethylene glycol (DODEG) modification at the 3 ′ end and / or 5 ′ end of the antisense nucleic acid The antisense nucleic acid according to [10] above, wherein two types of modifications are made.
[12] The antisense nucleic acid according to any one of [1] to [11], which is an oligodeoxyribonucleotide, oligoribonucleotide, morpholino oligomer, or a chimeric oligonucleotide thereof.
[13] The antisense nucleic acid according to [12], which is a morpholino oligomer.
[14] The antisense nucleic acid according to [13], which is a phosphorodiamidate morpholino oligomer.
[15] A pharmaceutical composition for treating a disease caused by an insertion mutation of an SVA type retrotransposon in a fuctin gene, wherein one of the antisense nucleic acids according to any one of the above [1] to [14] A pharmaceutical composition comprising two or more active ingredients.
[16] In the above [15], the disease caused by the insertion mutation of the SVA type retrotransposon in the fuctin gene is Fukuyama muscular dystrophy, adult-onset dilated cardiomyopathy, or severe congenital muscular dystrophy like Walker-Warburg syndrome The pharmaceutical composition as described.

  ADVANTAGE OF THE INVENTION By this invention, the antisense nucleic acid effective in the treatment of the disease resulting from the insertion variation | mutation of the SVA type retrotransposon in a fuctin gene can be provided.

It is a figure which shows the position of the primer for detecting the fukutin genome in a FCMD patient, an abnormal splicing product (abnormal mRNA), and a normal splicing product (normal mRNA), and abnormal mRNA and normal mRNA. It is a figure which shows the result of having introduce | transduced the antisense nucleic acid into the cell derived from FCMD patient, and confirming the sugar chain recovery | restoration of (alpha) dystroglycan. FMO model mice (Hp / −) were treated with PMO No. 9 shows the results of confirming recovery of α-dystroglycan sugar chain and recovery of normal fuctin protein in quadriceps muscle by intravenous administration of 9 antisense nucleic acids. FMO model mice (Hp / −) were treated with PMO No. It is a figure which shows the result of having confirmed the recovery | restoration of the sugar chain of alpha dystroglycan in the quadriceps muscle, and the recovery | restoration of normal fuctin protein by administering 27 antisense nucleic acids intravenously. FMO model mice (Hp / −) were treated with PMO No. 39 antisense nucleic acid or PMO no. It is a figure which shows the result of having confirmed the recovery | restoration of the sugar chain of (alpha) dystroglycan in the quadriceps muscle, and recovery | restoration of normal fuctin protein by administering 45 antisense nucleic acids intravenously. (A) shows a synthetic scheme of 5′-terminal triethylene glycol (TEG) modifying resin, (B) shows a synthetic scheme of 3′-terminal binding triethylene glycol (TEG) derivative, (C ) Shows the structures of a hexaethylene glycol (HEG) derivative for 3′-end bonding (compound 6) and a dodecaethylene glycol (DODEG) derivative for 3′-end bonding (compound 7), and (D) is a spacer compound (E) is a figure which shows the structure of a spacer.

[Antisense nucleic acid]
The present invention relates to a splicing acceptor at the 3 ′ side of guanine at position 111177 of SEQ ID NO: 1 and a splicing acceptor at the 5 ′ side of thymine at position 115943 in the transcription step of a fuctin gene having an insertion mutation of SVA type retrotransposon. An antisense nucleic acid that suppresses abnormal splicing using a site is provided.

The nucleotide sequence represented by SEQ ID NO: 1 is the genome sequence of an insertion mutant type fuctin gene in which an SVA type retrotransposon sequence (GenBank ACCESSION: AB185332) is inserted into the genome sequence of the fuctin gene (GenBank ACCESSION: AB038490). Positions to 118730 are SVA type retrotransposon sequences. In the insertion mutant type fuctin gene, by exon trapping, the 3 ′ side of guanine at position 111177 of SEQ ID NO: 1, that is, between guanine at position 111177 and guanine at position 111178 becomes a new splicing donor site, and position 115943 5 'side of thymine, that is, between guanine at position 115942 and thymine at position 115943 is a new splicing accepting site. In other words, in the insertion mutant type fuctin gene, abnormal splicing of guanine at position 111178 to guanine at position 115942 in SEQ ID NO: 1 is recognized as an intron, and guanine at position 120191 from thymine at position 115943 is recognized as an exon. Occurs. Therefore, if abnormal splicing using the 3 ′ splicing donor site of guanine at position 111177 and the splicing accepting site at the 5 ′ side of thymine at position 115943 is suppressed, the expression level of abnormal fuctin mRNA decreases and at the same time normal Expression of normal fuctin mRNA based on splicing is restored, and production of normal fuctin protein is restored.
The nucleotide sequence of the abnormal fuctin mRNA is shown in SEQ ID NO: 2, the amino acid sequence of the abnormal fuctin protein is shown in SEQ ID NO: 3, the nucleotide sequence of the normal fuctin mRNA is shown in SEQ ID NO: 4, and the amino acid sequence of the normal fuctin protein is shown in SEQ ID NO: 5.

  The antisense nucleic acid of the present invention suppresses the above abnormal splicing of the pre-mRNA, which is the primary transcription product, in the transcription step of the insertion mutant type fuctin gene (SEQ ID NO: 1), and expresses the normal fuctin mRNA based on the normal splicing, What is necessary is just to be able to recover the production of normal fuctin protein. As an antisense nucleic acid having such an effect, a nucleic acid having a target sequence as a peripheral sequence of a splicing accepting site used for abnormal splicing is preferable, and the range from 115159th to 115981 of the base sequence represented by SEQ ID NO: 1 A target sequence is more preferable.

  The antisense nucleic acid of the present invention contains a sequence complementary to the target sequence (base sequence of the pre-mRNA of the insertion mutant type fuctin gene), but is not required to be completely complementary. As long as it can form, it may contain a mismatch. The complementary sequence portion is 50% or more of the length (number of bases) of the antisense nucleic acid, more preferably 60% or more, further preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, Preferably it is 95% or more, More preferably, it is 98% or more, More preferably, it is 99% or more, Most preferably, it is 100%.

  The length of the antisense nucleic acid of the present invention is not particularly limited, but is preferably 24 bases or less, more preferably 23 bases or less, and even more preferably 22 bases or less. The lower limit is preferably 9 bases or more, and more preferably 10 bases or more. The length of the antisense nucleic acid of the present invention is preferably 9 to 24 bases.

  The fact that the expression of abnormal fuctin mRNA is suppressed and the expression of normal fuctin mRNA is restored is, for example, that the antisense nucleic acid of the present invention is introduced into a cell derived from an FCMD patient, and the cell is used as a sample for quantification. This can be confirmed by using a known mRNA measurement method such as RT-PCR. Moreover, the production of normal fuctin protein has been recovered, for example, by introducing the antisense nucleic acid of the present invention into a cell derived from an FCMD patient, and using a known protein measurement method such as ELISA or Western blotting using the cell as a sample. It can be confirmed by use. In addition, since the expression level of normal fuctin protein correlates with the expression level of normal fuctin mRNA generated by normal splicing, the production of normal fuctin protein is indirectly recovered by measuring the expression level of normal fuctin mRNA. Can be evaluated.

  A heterozygote (carrier) of a normal fuctin gene and an insertion mutant fuctin gene is a normal fuctin protein in which 50% of the expression level of the total fuctin protein is normal, but there is no abnormality in muscle function or brain, and α dystroglycan The amount of sugar chains is the same as that of healthy people. Therefore, it is considered that an antisense nucleic acid capable of restoring normal mRNA by 50% can treat a disease caused by an insertion mutation of SVA type retrotransposon. As a result of screening for antisense nucleic acids using myoblasts derived from FCMD patients (SVA type retrotransposon insertion mutant homozygote), the present inventors have found that the anti-virus comprising 65 kinds of base sequences shown in Table 1 Sense nucleic acid recovered normal fuctin mRNA by 50% or more.

  The antisense nucleic acid of the present invention is preferably an antisense nucleic acid consisting of a base sequence selected from SEQ ID NOs: 6 to 70 shown in Table 1. In addition, an antisense nucleic acid consisting of a sequence in which several bases are deleted, substituted or added as long as it can form a hybrid with the target sequence in the base sequence shown in any of SEQ ID NOs: 6 to 70 shown in Table 1 There may be. The number of bases to be deleted, substituted or added is preferably 1 to 5, more preferably 1 to 4, further preferably 1 to 3, and still more preferably 1 or 2. The target sequence of each antisense nucleic acid shown in Table 1 is a complementary sequence of each antisense nucleic acid.

  The antisense nucleic acid of the present invention preferably further has high solubility in physiological saline. An antisense nucleic acid having low solubility in physiological saline has a risk of being deposited and cannot be used during storage of the preparation, or having a risk of being deposited and not usable when using an infusion solution containing a salt. It has also been reported that drugs that precipitate easily show toxicity upon administration (Bulletin of Osaka University of Pharmaceutical Sciences 1 (2007) 91-99). Therefore, an antisense nucleic acid having high solubility in physiological saline is very useful as an active ingredient of a medicine.

The solubility in physiological saline is preferably 20 mg / mL or more, more preferably 30 mg / mL or more, further preferably 40 mg / mL or more, and particularly preferably 50 mg / mL or more. . The solubility of the antisense nucleic acid in physiological saline can be evaluated by dissolving the antisense nucleic acid in physiological saline at a desired concentration and confirming the presence or absence of precipitation after a certain time.
The present inventors have found that among the antisense nucleic acids shown in Table 1, an antisense nucleic acid comprising 22 kinds of base sequences has a solubility in physiological saline of 50 mg / mL or more. Specifically, SEQ ID NOs: 18, 19, 26, 27, 32, 33, 37, 43, 53, 55, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69 and 70 It is. Therefore, as the antisense nucleic acid of the present invention, SEQ ID NOs: 18, 19, 26, 27, 32, 33, 37, 43, 53, 55, 58, 59, 60, 61, 63, 64, 65, 66, 67 , 68, 69 and 70 are particularly preferred.

It goes without saying that highly safe antisense nucleic acids are preferred as an active ingredient of a medicine. Safety can be evaluated using, for example, blood aspartate aminotransferase (AST) value, alanine aminotransferase (ALT) value, urea nitrogen (BUN) value, and creatinine value after administration of antisense nucleic acid as indices. Specifically, for example, after antisense nucleic acid is administered to healthy mice, the blood AST value, ALT value, BUN value, and creatinine value are measured and compared with the measured values of the control group (solvent administration or no treatment). If an increase exceeding 1.3 times is observed, it can be determined as an abnormal value. In the present invention, an antisense nucleic acid solution is prepared using a 5% glucose aqueous solution, and blood is collected on the next day after administration into the C57BL / 6J male 6-week-old mouse at a dose of 60 mg / kg via the tail vein. ALT value, BUN value and creatinine value were measured, and when an increase exceeding 1.3 times was observed compared with the measured value of the control group (solvent administration or no treatment), it was judged as an abnormal value. If no abnormal value is observed, it can be determined that the array is highly safe.
The present inventors have found that among the antisense nucleic acids shown in Table 1, an antisense nucleic acid consisting of 9 types of base sequences is a very safe sequence. Specifically, it is sequence number 32, 50, 51, 60, 62, 64, 65, 66 and 67. Therefore, the antisense nucleic acid of the present invention is particularly preferably an antisense nucleic acid comprising a base sequence selected from SEQ ID NOs: 32, 50, 51, 60, 62, 64, 65, 66 and 67.

  The antisense nucleic acid of the present invention may be composed of any of a DNA strand, an RNA strand, and a mixed strand of DNA and RNA. That is, the antisense nucleic acid of the present invention is preferably an oligodeoxyribonucleotide, oligoribonucleotide, or a chimeric oligonucleotide of deoxyribonucleotide and ribonucleotide. Moreover, the antisense nucleic acid of the present invention may contain a nucleotide analog or a spacer. Antisense nucleic acids are preferably modified to increase nuclease resistance and / or increase affinity for the target sequence. Preferred nucleotide analogs include, for example, morpholino skeleton, carbamate skeleton, siloxane skeleton, sulfide skeleton, sulfoxide skeleton, sulfone skeleton, formacetyl skeleton, thioformacetyl skeleton, methyleneformacetyl skeleton, riboacetyl skeleton, alkene-containing skeleton, sulfomate skeleton, It includes modified skeletons such as a sulfonate skeleton, a sulfonamide skeleton, a methyleneimino skeleton, a methylenehydrazino skeleton, and an amide skeleton. Morpholino oligomers are modified oligonucleotides having an uncharged backbone in which the deoxyribose sugar of DNA is replaced with a six-membered ring and the phosphodiester bond is replaced with a phosphorodiamidate bond. Morpholino oligomers are more preferred as the antisense nucleic acid of the present invention because of their excellent resistance to enzymatic degradation.

  Further preferred nucleotide analogues are morpholino nucleotide derivatives, phosphorodiamidate morpholino oligomers in which the anionic phosphodiester bond between adjacent morpholino rings is replaced by a nonionic phosphorodiamidate bond, (Hereinafter referred to as “PMO”).

  Furthermore, the nucleotide analog preferably comprises one substitution of non-bridging oxygen in the phosphodiester bond. This modification adds significant resistance to nuclease degradation. Preferred nucleotide analogs include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, H-phosphonates such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, ethyl containing phosphonates. And other alkyl phosphonates, phosphinates, such as phosphoramidates including 3′-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, Contains selenophosphate or boranophosphate.

  In addition, nucleotide analogs can be -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, optionally having one or more heteroatoms in between, Alkenyl, alkynyl, alkaryl, allyl or aralkyl; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; O-, S- or N-allyl; Mono- or di-substituted at the 2 ', 3' and / or 5 'positions, such as O-alkyl-O-alkyl, -methoxy, -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; -dimethylaminoethoxyethoxy Preferably comprising one or more sugar moieties. The sugar moiety may be pyranose or a derivative thereof, or deoxypyranose or a derivative thereof, preferably ribose or a derivative thereof, or deoxyribose or a derivative thereof. Preferred derivatized sugar moieties include locked nucleic acids (LNA), where the 2 'carbon atom is linked to the 3' or 4 'carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. Preferred LNAs include 2'-O, 4'-C-ethylene bridged nucleic acids (Morita et al., 2001, Nucleic Acid Res Supplement No. 1: 241-242). These substitutions confer RNaseH and nuclease resistance to the nucleotide analog or equivalent and increase the affinity for the target RNA.

  The spacer that may be included in the antisense nucleic acid of the present invention is a compound that provides a space between regular arrangements of nucleic acid sequences by being present between residues constituting the antisense nucleic acid. It is. The spacer is not complementary to the target sequence and does not bind to the target sequence.

  Furthermore, the antisense nucleic acid of the present invention may be modified at the 5 'end and / or the 3' end. Such modifications include triethylene glycol (TEG) modification, hexaethylene glycol (HEG) modification, dodecaethylene glycol (DODEG) modification.

  The antisense nucleic acid of the present invention can be produced using a known nucleic acid synthesis method. As a known method, for example, the method described in International Publication No. WO2009 / 064471 or International Publication No. WO2013 / 100190 can be used.

[Pharmaceutical composition]
The present invention provides a pharmaceutical composition for treating a disease caused by an insertion mutation of an SVA type retrotransposon in the fuctin gene. The pharmaceutical composition of the present invention contains one or more of the above-described antisense nucleic acids of the present invention as an active ingredient. The antisense nucleic acid of the present invention uses a splicing donor site on the 3 ′ side of guanine at position 111177 and a splicing accepting site on the 5 ′ side of thymine at position 115943 of the insertion mutant-type fuctin gene (SEQ ID NO: 1). By suppressing the above, restoring the expression of normal fuctin mRNA based on normal splicing, and restoring the production of normal fuctin protein, it is possible to treat a disease caused by the insertion mutation of the SVA type retrotransposon in the fuctin gene.

  Examples of the disease caused by the insertion mutation of the SVA type retrotransposon in the fuctin gene include FCMD, adult-onset dilated cardiomyopathy (see Non-Patent Document 6), and Walker-Warburg syndrome-like severe congenital muscular dystrophy (Non-Patent Document). 7), but is not limited thereto. Diseases to which the pharmaceutical composition of the present invention is applied include diseases that will be caused in the future by SVA retrotransposon insertion mutations in the fuctin gene. FCMD is preferable.

  The pharmaceutical composition of the present invention may contain one type of antisense nucleic acid as an active ingredient, or may contain two or more types of antisense nucleic acids as active ingredients. The combination of two or more kinds of antisense nucleic acids is not particularly limited, but it is preferable to appropriately select a combination that enhances the effect as compared with the use alone and use it as an active ingredient.

  The pharmaceutical composition of the present invention can be formulated by appropriately blending a pharmaceutically acceptable carrier or additive with the above-described antisense nucleic acid of the present invention as an active ingredient. Specifically, oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do. A parenteral agent is preferable. The injection may be a lyophilized preparation. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ | adopted in the pharmaceutical field | area. Carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, excipients, binders, pH adjusters, disintegrants, absorption Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be prepared according to normal pharmaceutical practice (for example, dissolving or suspending an active ingredient in a solvent such as water for injection or natural vegetable oil). As an aqueous solution for injection, for example, isotonic solution (eg, D-sorbitol, D-mannitol, sucrose, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, etc. are used, and an appropriate dissolution is used. You may use together with adjuvant, for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. Furthermore, it may be a lyophilized preparation.

  The pharmaceutical composition of the present invention can be administered to a human who has developed a disease caused by an insertion mutation of an SVA type retrotransposon in the fuctin gene. The administration route is not particularly limited, but parenteral administration is preferred. Parenteral administration may be systemic administration such as intravenous administration, local administration such as intramuscular administration, transdermal administration, and transmucosal administration. Moreover, the antisense nucleic acid which is an active ingredient of the pharmaceutical composition of the present invention can be administered in the form of a non-viral vector or a viral vector. Furthermore, a method of introducing an antisense nucleic acid using liposome (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, carrier with gene gun (Gene Gun) For example, a method of transferring antisense nucleic acid into cells together with metal particles), a method of administering in combination with an ultrasonic introduction method, or the like can be used.

  The dosage of the pharmaceutical composition of the present invention varies depending on the kind of antisense nucleic acid contained, dosage form, administration route, age and weight of the patient, but when administered in the form of an injection, about 0.01 mg to 60 g per day. About 0.1 mg to about 24 g, more preferably about 0.1 mg to about 6 g can be administered. The administration interval can be set from once to several times a day, or from 1 day to 2 weeks.

Further, the present invention includes the following inventions.
(I) A method for treating a disease caused by an insertion mutation of an SVA type retrotransposon in a fuctin gene, which comprises administering an effective amount of the antisense nucleic acid of the present invention to the diseased patient Method.
(Ii) Use of the antisense nucleic acid of the present invention for producing a therapeutic agent for a disease caused by an insertion mutation of an SVA type retrotransposon in the fuctin gene.
(Iii) The antisense nucleic acid of the present invention described above for use in the treatment of a disease caused by an insertion mutation of an SVA type retrotransposon in the fuctin gene.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

[Example 1: Synthesis of non-terminal-modified antisense nucleic acid]
According to the method described in International Publication No. WO2013 / 100190, the PMO No. Various phosphorodiamidate morpholino oligomers (PMO) shown in 1 to 58 and 60 to 66 were synthesized.

[Example 2: PMO No. 59)
Step 1 Synthesis of Spacer Compound 3- (Methylamino) propan-1-ol (2 g, 22.44 mmol) and triethylamine (33.65 mmol) were dissolved in dimethylformamide (20 mL), and triphenylmethyl chloride (26 .92 mmol) was added and stirred at room temperature for 3 hours. The mixture was ice-cooled again, water (40 ml) was added and the mixture was stirred for 10 minutes, and then extracted with ethyl acetate. The organic layer was washed with water in a cloudy state, concentrated with an evaporator, diluted again with ethyl acetate (50 ml), and insolubles were removed by filtration. The filtrate was concentrated and directly purified by silica gel column chromatography. The obtained 3- [methyl (trityl) amino] propan-1-ol (4.8 g, 14 mmol) was dissolved in tetrahydrofuran (40 mL), and 1-methylimidazole (36 mmol) and N-ethylmorpholine (14 mmol) were added under ice cooling. , N, N-dimethylphosphoramide dichloridate (29 mmol) was added and stirred at room temperature for 3 hours. Liquid separation operation was performed with ethyl acetate and 1M sodium dihydrogen phosphate aqueous solution, and the organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification by silica gel column chromatography gave spacer compound 8 (4.6 g) shown in FIG. 6 (D).

Step 2 PMO No. In accordance with the method described in the synthesis international publication WO2013 / 100190 of No. 59 , PMO No. 59 was synthesized.
Note that PMO No. 59 is an antisense in which PMO having a base sequence of CTCG is bound to the 5 ′ end of spacer 9 shown in FIG. 6 (E), and PMO having the base sequence of SEQ ID NO: 71 is bound to the 3 ′ end of the spacer. It is a nucleic acid.
PMO No. The spacer included in 59 was introduced using the spacer compound obtained in Step 1.
PMO No. 59: CTCG- (Spacer) -CATCAGAGGGAGACC
ESI-TOF-MS calculated value: 6466.28
Measurement: 6465.23

[Example 3: Modified 5′-end ethylene glycol PMO No. 70 synthesis]
Step 1 Synthesis of N- tritylpiperazine Piperazine (150 g, 1741 mmol) was dissolved in a toluene (650 mL) / methanol (130 mL) mixture, and [chloro (diphenyl) methyl] benzene (48.5 g, 174 mmol) in toluene under ice-cooling. (244 mL) solution was added dropwise and stirred at room temperature overnight. The reaction solution was washed with water, and the toluene layer was added to a succinic acid (22.8 g, 193 mmol) / water (300 mL) solution and stirred under ice cooling. The precipitated crystals were collected by filtration, washed with acetone, and dried under reduced pressure at 50 ° C. to obtain 71.7 g of N-tritylpiperazine / succinate. The obtained compound (50 g, 64.5 mmol) was dissolved in dichloromethane and washed with an aqueous solution of potassium carbonate (322.6 mmol). The dichloromethane layer was washed with water, dried over sodium sulfate, and then dried under reduced pressure to obtain the title compound 1 (40 g) shown in FIG. 6 (A).

Step 2 Synthesis of 4-oxo-4- [2- [2- [2- (4- tritylpiperazine -1-carbonyl) oxyethyl] ethoxy] ethoxy] butanoic acid Triethylene glycol (370.03 mmol) was added to pyridine (500 mL). Then, 1,1′-carbonyldiimidazole (20 g, 123.34 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 30 minutes and at 40 ° C. for 2 hours. Next, Compound 1 (40 g, 121.8 mmol) obtained in Step 1 was added, and the mixture was stirred at 60 ° C. for 4 hours. The reaction solution was concentrated under reduced pressure and subjected to liquid separation operation with ethyl acetate and 1M aqueous sodium dihydrogen phosphate solution, and the organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 2 (43.3 g) shown in FIG. The obtained compound 2 and 4-dimethylaminopyridine (127.8 mmol) were dissolved in dichloromethane (500 mL), succinic anhydride (127.8 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was partitioned with 1M aqueous sodium dihydrogen phosphate solution, and the organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure to give the title compound 3 (51.6 g) shown in FIG. 6 (A). It was.

Step 3 Synthesis of 5′-terminal triethylene glycol modifying resin Compound 3 (43.50 mmol) obtained in Step 2, aminomethylpolystyrene resin (21.75 mmol), 4-dimethylaminopyridine (54.38 mmol), 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (217.5 mmol) was placed in an eggplant flask equipped with a filter, pyridine (420 mL) and then triethylamine (268.6 mmol) were added, and the mixture was shaken at room temperature for 3 days. The reaction solution was filtered, washed successively with pyridine, methanol and dichloromethane, and then dried under reduced pressure for 15 minutes. Tetrahydrofuran (312 mL), 2,6-lutidine (77.5 mL) and acetic anhydride (65 mL) were added, and the mixture was shaken at room temperature for 3 hours. After filtration through a filter, the product was washed successively with pyridine, methanol and dichloromethane, and dried under reduced pressure to obtain the title resin (compound 4) shown in FIG. 6A (loading: 310 μmol / g).

Step 4 PMO No. Using the resin (compound 4) obtained in synthesis step 3 of No. 70, according to the method described in International Publication WO2013 / 100190, 5′-end ethylene glycol modified PMO No. 70 was synthesized.
ESI-TOF-MS calculated value: 6994.47
Measurement: 6994.79

[Example 4: Modified 5′-end ethylene glycol PMO No. 68)
In the same manner as in Example 3, 5′-terminal ethylene glycol modified PMO No. 68 was synthesized.
ESI-TOF-MS calculated value: 6034.14
Measurement value: 6034.74

[Example 5: 3′-end ethylene glycol modified PMO No. 69 synthesis)
Step 1 Triethylene glycol (TEG) derivative Synthesis of 2- [2- [2- [bis (4-methoxyphenyl) (phenyl) methoxy] ethoxy] ethoxy] ethyldimethylphosphoramidochloridate Triethylene glycol (50 g, 332) .96 mmol) was added to pyridine (100 mL, 1236 mmol), dimethoxytrityl chloride (38 g, 112.2 mmol) was added little by little under an argon atmosphere, and the mixture was stirred for several hours. After evaporating pyridine under reduced pressure using an evaporator, the concentrated residue was dissolved in dichloromethane, and water was added to carry out a liquid separation operation. The organic layer was collected and dried over magnesium sulfate. Dichloromethane was distilled off under reduced pressure using an evaporator, and the residue was purified by silica gel column chromatography. 2- [2- [2- [bis (4-methoxyphenyl) -phenyl-methoxy] ethoxy] ethoxy] ethanol (35 g) was added. Obtained. The obtained compound (6 g, 13.25 mmol) was dissolved in tetrahydrofuran (60 mL), and 1-methylimidazole (33.146 mmol), N-ethylmorpholine (13.25 mmol), N, N-dimethylphosphole were added under ice cooling. Amide dichloridate (26.51 mmol) was added in this order. The mixture was stirred at room temperature for 2 hours, quenched with 1M aqueous sodium dihydrogen phosphate solution, and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over magnesium sulfate, and concentrated with an evaporator. The residue was purified by silica gel column chromatography to obtain 6.1 g of the title derivative (Compound 5) shown in FIG. 6 (B).

Step 2 PMO No. 69 synthetic PMO no. 27 (38.38 μmol) was placed in a 15 mL falcon tube and dissolved in dimethyl sulfoxide (1.2 mL). To this was added a mixed solution of Compound 5 (55 mg, 95.1 μmol) obtained in Step 1 and triethylamine (55 μL, 394.6 μmol) in THF (220 μL), and reacted at 40 ° C. in a water bath for 2 hours. After ice cooling, the reaction solution was diluted with 20 mM triethylamine-acetate buffer / acetonitrile (4/1, 18 mL), concentrated by reverse phase column purification (LiChroprep_RP18). The obtained residue was dissolved in 20% acetonitrile aqueous solution, and 0.1 M hydrochloric acid aqueous solution was added. After confirming that the pH was 2 with a pH test paper, the sample was allowed to stand for 1 hour. The reaction solution was neutralized by adding a 1M sodium hydroxide aqueous solution, and it was confirmed that the pH was 7 with a pH test paper. The reaction solution was filtered through a membrane filter (PVDF, 0.22 μm). After desalting and purification using a reverse phase column (YMC_GEL_TMS_HG), lyophilization, 3′-end ethylene glycol modified PMO No. 69 (237.6 mg) was obtained.
ESI-TOF-MS calculated value: 6882.40
Measurement: 6882.10.

[Example 6: 3′-end ethylene glycol modified PMO No. 67)
In the same manner as in Example 5, 3′-end ethylene glycol modified PMO No. 67 was synthesized. However, as the PMO in step 2, PMO No. 39 was used.
ESI-TOF-MS calculated value: 5922.08
Measurement value: 5922.25

[Example 7: 3′-end ethylene glycol modified PMO No. 71)
Step 1 Synthesis of Hexaethylene Glycol (HEG) Derivative The title derivative (Compound 6) shown in FIG. 6C was synthesized in the same manner as in Step 1 of Example 5 using hexaethylene glycol as a raw material.

Step 2 PMO No. 71 3′-terminal ethylene glycol modified PMO No. 71 was synthesized. However, Compound 6 obtained in Step 1 was used in place of Compound 5 as the ethylene glycol derivative in Step 2.
ESI-TOF-MS calculated value: 7014.49
Measurement value: 7014.98

[Example 8: 3′-end ethylene glycol modified PMO No. 73)
In the same manner as in Example 5, 3′-end ethylene glycol modified PMO No. 73 was synthesized. However, as the PMO in step 2, PMO No. 66 was used.
ESI-TOF-MS calculated value: 6659.37
Measurement: 6659.85

[Example 9: 3′-end ethylene glycol modified product PMO No. 72)
Step 1 Synthesis of Dodecaethylene Glycol (DODEG) Derivative The title derivative (Compound 7) shown in FIG. 6C was synthesized in the same manner as in Step 1 of Example 5 using dodecaethylene glycol as a raw material.

Step 2 PMO No. The title compound was synthesized in the same manner as in 72 Synthesis Example 5. However, Compound 7 obtained in Step 1 was used in place of Compound 5 as the ethylene glycol derivative in Step 2.
ESI-TOF-MS calculated value: 7278.64
Measurement: 7279.00

Table 3 shows the modified PMO terminal synthesized in Examples 3 to 9.

[Example 10: Screening of antisense nucleic acid for FCMD treatment]
(1) Cells used FCMD patient myoblasts (SVA type retrotransposon insertion mutant homozygote) were used. The cells were used with the approval of the Medical Ethics Review Board of the Graduate School of Medicine, Kobe University.
(2) Antisense nucleic acid PMO No. 1 synthesized in Examples 1-9. 1 to 73 were subjected to screening as test substances.

(3) Experimental method Myoblasts from FCMD patients were grown in growth medium (20% Fetal bovine serum (FBS, Invitrogen), 2.5 ng / mL basic fibroblast growth factor (SIGMA), 0.5% Antibiotic Antimycotic Solution (100 ×), Stabilized (SIGMA ) Containing Dulbecco's Modified Eagle's Medium (DMEM) -high glucose (SIGMA)), seeded in a cell culture dish at 30,000 cells per cm ^{2 and} cultured for 1 day.
The next day, the medium was changed to a differentiation medium (2% FBS-containing DMEM-high glucose), and PMO was added to 3 μM. Negative controls used randomized sequences or PMO targeted to the dystrophin gene. Transfection was performed using 6 μL Endo-Porter (Gene-Tools) per mL of medium. Two days later, total RNA was collected by RNeasy Mini Kit (Qiagen). CDNA synthesis was performed with random primers from the total RNA using PrimeScript RT-PCR Kit (Takara Bio). Using the obtained cDNA as a template, abnormal fuctin mRNA by abnormal splicing and normal fuctin mRNA by normal splicing were detected by PCR. The positions of the primers for detecting each mRNA are shown in FIG. PCR conditions were (94 ° C. for 30 seconds, 65 ° C. for 30 seconds, 72 ° C. for 60 seconds) × 32 cycles, and cooled to 4 ° C. after the reaction was completed. Using 1 μL of the PCR reaction sample, the size and concentration of the PCR amplified fragment were measured by Agilent DNA 1000 kit (Agilent Technologies) and 2100 Bioanalyzer (Agilent Technologies). Normal splicing from the quantitative value [A] (nmol / L) of the band derived from normal splicing detected near 219 bp and the quantitative value [B] (nmol / L) of the band derived from abnormal splicing detected near 265 bp The rate was calculated by the following formula.
Normal splicing rate = [A] / ([A] + [B])

(4) Conditions for selection of antisense nucleic acid for FCMD treatment In normal heterozygous heterozygotes (carriers) of normal fuctin and SVA type retrotransposon, no abnormalities were observed in muscle function and brain, and α-dystroglycan sugar chain amount was also healthy. It is no different from people. That is, it is considered that FCMD can be treated if normal mRNA can be recovered by 50%. Thus, the condition for selection of antisense nucleic acid for FCMD treatment in this screening was that normal splicing could be restored to 50% or more in FCMD patient myoblasts transfected at a nucleic acid concentration of 3 μM.

(5) Results As a result of screening, the 73 types of antisense nucleic acids shown in Tables 2 and 3 recovered normal splicing to 50% or more at a nucleic acid concentration of 3 μM. These 73 kinds of antisense nucleic acids are all antisense nucleic acids whose target sequence is a peripheral sequence of a splicing accepting site used for abnormal splicing of the SVA-inserted fuctin gene. Specifically, the range from 115937th to 115981 of the base sequence (SEQ ID NO: 1) of the SVA-inserted fuctin gene is used as the target sequence.

[Example 11: Sugar chain recovery of α-dystroglycan in FCMD patient cells]
(1) Experimental material and method The same FCMD patient myoblast as in Example 1 (inserted mutant homozygote of SVA type retrotransposon) was used. For cells subcultured in a growth medium (see Example 10), PMO was introduced by electroporation using Nucleofector (Lonza) and cultured in a differentiation medium (see Example 10). As PMO, PMO No. Five types of PMOs 1, 9, 27, 39 and 45 were used. As a negative control, PMO targeting the dystrophin gene was used.

  Five days after the introduction of PMO, the cells were washed once with PBS (−), 10% of the cell volume of 1% Triton TBS was added, and the cells were recovered with a scraper. The cells were lysed by mixing at 4 ° C. for 1 hour, and the supernatant after centrifugation was used as a cell extract. 15 μL of the cell extract was subjected to Western blot analysis using anti-α dystroglycan rat monoclonal antibody, anti-sugar chain-modified α dystroglycan mouse monoclonal antibody IIH6, and anti-β dystroglycan mouse monoclonal antibody 8D5. Western blot analysis was performed as described in the literature (Kanagawa, M. et al. Residual laminin-binding acivity and enhanced dystroglycan glycosylated by LARGE in novel model mice to dystroglycanopathy. Hum. Mol. Genet. 18, 621-31 (2009)). Carried out.

(2) Experimental results The results are shown in FIG. In FIG. 2, α-DG represents α dystroglycan, β-DG represents β dystroglycan, HSMM represents human skeletal muscle myoblast, and RD represents human fetal rhabdomyosarcoma. The result of using the anti-α dystroglycan rat monoclonal antibody in the upper part, the result of using the anti-sugar chain-modified α dystroglycan mouse monoclonal antibody IIH6, and the result of using the anti-β dystroglycan mouse monoclonal antibody 8D5 in the lower part.
FCMD patient cells lacked α-dystroglycan sugar chains, and α-dystroglycan was detected at a low molecular weight position (see water in FIG. 2). On the other hand, in FCMD patient cells into which PMO was introduced, it was confirmed that the sugar chain of α-dystroglycan was recovered in a concentration-dependent manner regardless of which PMO was introduced. In the FCMD patient cells into which PMO targeting the dystrophin gene as a negative control was introduced, the sugar chain of α-dystroglycan did not recover.

[Example 12: Efficacy evaluation using FCMD model mouse]
(1) Animals used All animal experiments were approved by the Laboratory Animal Ethics Review Board of the Graduate School of Medicine, Kobe University. FCMD knock-in mice and their nomenclature (Kanagawa, M. et al. Residual laminin-binding acivity and enhanced dystroglycan glycosylated by LARGE in novel model mice to dystroglycanopathy. Hum. Mol. Genet. 18, 621-31 (2009)) It is described in. Transgenic alleles with normal human exon 10 and SVA-inserted human exon 10 of the fuctin gene were designated Hn and Hp, respectively. Hp / Hp is a homozygote of an SVA-inserted human exon 10 allele, Hn / Hn is a homozygote of a normal human exon 10 allele, and Hp / − is an SVA-inserted human exon 10 allele and a mouse fuctin gene knockout allele. It is a composite heterozygote. The mice used in the experiment are 8-14 weeks old.

(2) Antisense nucleic acid administration PMO No. Four types of PMOs 9, 27, 39 and 45 were used.
Cardiotoxin 10 μM (SIGMA) was percutaneously administered to Hp / − mice at three sites: anterior tibial muscle (0.4 nmol), gastrocnemius muscle (2 nmol) and quadriceps muscle (1 nmol). Two mice were used for each dose of PMO. The day of cardiotoxin administration was defined as day 0, and PMO (20, 60, 200 mg / kg) dissolved in 5% mannitol was administered into the tail vein on the third day. On day 23, the mice were euthanized and each tissue was collected for analysis of fuctin protein and α-dystroglycan sugar chain.

(3) Detection of Fuctin Protein Anti-fuctin rabbit polyclonal antibody RY213 recognizes peptide CLKIESKDPRLDGIDS (SEQ ID NO: 72), and anti-fuctin goat polyclonal antibody 106G2 recognizes full-length fuctin lacking the N-terminal hydrophobic domain . Fuctin was obtained from tissue lysate obtained from muscle tissue 15-30 mg using 10-fold lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 20 mM Tris-HCl and 150 mM NaCl). Detection by the immunoprecipitation method used was followed by Western blot analysis using affinity purified RY213.

(4) Detection of dystroglycan A 10-fold amount of lysis buffer (1% Triton TBS) was added to muscle tissue to obtain a tissue lysate. Western blot analysis using 9 μL of the lysate using the same three types of antibodies as in Example 2 (anti-α dystroglycan rat monoclonal antibody, anti-sugar chain-modified α dystroglycan mouse monoclonal antibody IIH6, anti-β dystroglycan mouse monoclonal antibody 8D5) It was used for.

(5) Experimental results PMO No. The results of the antisense nucleic acid 9 are shown in FIG. The results of 27 antisense nucleic acids are shown in FIG. Results for 39 antisense nucleic acids and PMO no. The results for 45 antisense nucleic acids are shown in FIG. Both are the result of dystroglycan and fuctin protein in quadriceps. As is clear from FIGS. 3, 4 and 5, when any of the four types of antisense nucleic acid is administered, the sugar chain of α-dystroglycan and normal fuctin protein are recovered depending on the concentration of the antisense nucleic acid. It was confirmed that

[Example 13: Solubility of antisense nucleic acid in physiological saline]
(1) Experimental method Add 80.0 μL of water for injection to a 2.2 mL sample bottle (transparent) containing 5.0 mg of PMO, dissolve it using ultrasonic waves and vortex, and then add 20.0 μL of 5 × physiological saline. In addition, the mixture was stirred using vortex to obtain a 50 mg / mL physiological saline solution. The sample was allowed to stand at room temperature for 24 hours, and a sequence in which no precipitation occurred was evaluated as a highly soluble sequence.

(2) Evaluation results PMO No. 13, 14, 21, 22, 27, 28, 32, 38, 48, 50, 53-56, 58-66, 69-73 showed a solubility of 50 mg / mL or more in physiological saline. These 28 types of PMOs having high solubility in physiological saline were considered to be antisense nucleic acids having high utility value as pharmaceuticals.

[Example 14: Safety evaluation of antisense nucleic acid]
(1) Experimental method PMO was dissolved in a 5% aqueous glucose solution and administered into the tail vein of C57BL / 6J male 6-week-old mice. On the next day, serum was collected from the mice, and blood aspartate aminotransferase (AST) value, alanine aminotransferase (ALT) value, urea nitrogen amount (BUN) value, and creatinine value were measured. A measured value of a mouse administered with only a 5% glucose aqueous solution used as a vehicle or an untreated mouse was regarded as a normal value, and when an increase exceeding 1.3 times the normal value was observed, it was determined as an abnormal value. When no abnormal value was observed in any of the AST value, the ALT value, the BUN value, and the creatinine value at a dose of 60 mg / kg or less, it was determined that the antisense nucleic acid was highly safe.

(2) Evaluation results PMO No. For 27, 45, 46, 71, 55, 57, 60, 61, 62, 66, 73, abnormal values were observed in any of AST, ALT, BUN, and creatinine at doses of 60 mg / kg or less. It was confirmed that the nucleic acid was a highly safe antisense nucleic acid.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (15)

  An antisense nucleic acid comprising 9 to 24 bases, with the target sequence in the range from the 115th position to the 115981th position of the base sequence represented by SEQ ID NO: 1.   The antisense nucleic acid according to claim 1, comprising a base sequence selected from SEQ ID NOs: 6 to 70 (excluding SEQ ID NOs: 32, 60, 64, 65, 66 and 67).   The antisense nucleic acid according to claim 1 or 2, which is dissolved in physiological saline at 50 mg / mL or more.   It consists of a base sequence selected from SEQ ID NOs: 18, 19, 26, 27, 33, 37, 43, 53, 55, 58, 59, 61, 63, 68, 69 and 70. 4. The antisense nucleic acid according to any one of 3.   3. An abnormal value is not observed in any of AST value, ALT value, BUN value, and creatinine value when an antisense nucleic acid at a dose of 60 mg / kg is administered to mice. Antisense nucleic acids.   The antisense nucleic acid according to any one of claims 1, 2, and 5, which comprises a base sequence selected from SEQ ID NOs: 50, 51 and 62.   When an antisense nucleic acid of 50 mg / mL or more dissolved in physiological saline and an administration dose of 60 mg / kg was administered to mice, abnormal values were observed in any of the AST value, ALT value, BUN value, and creatinine amount. The antisense nucleic acid according to claim 1 or 2, wherein the antisense nucleic acid is not present.   The antisense nucleic acid according to any one of claims 1 to 7, wherein the 3 'end and / or the 5' end of the antisense nucleic acid is unmodified.   The antisense nucleic acid according to any one of claims 1 to 7, wherein the 3 'end and / or the 5' end of the antisense nucleic acid is modified.   One or two selected from the group consisting of triethylene glycol (TEG) modification, hexaethylene glycol (HEG) modification and dodecaethylene glycol (DODEG) modification at the 3 ′ end and / or 5 ′ end of the antisense nucleic acid The antisense nucleic acid according to claim 9, wherein the antisense nucleic acid is modified.   The antisense nucleic acid according to any one of claims 1 to 10, which is an oligodeoxyribonucleotide, an oligoribonucleotide, a morpholino oligomer, or a chimeric oligonucleotide thereof.   The antisense nucleic acid according to claim 11, which is a morpholino oligomer.   The antisense nucleic acid according to claim 12, which is a phosphorodiamidate morpholino oligomer.   A pharmaceutical composition for treating a disease caused by an insertion mutation of an SVA type retrotransposon in a fuctin gene, wherein one or more of the antisense nucleic acids according to any one of claims 1 to 13 are used as an active ingredient A pharmaceutical composition comprising:   The pharmaceutical composition according to claim 14, wherein the disease caused by the insertion mutation of the SVA type retrotransposon in the fuctin gene is Fukuyama muscular dystrophy, adult-onset dilated cardiomyopathy, or severe congenital muscular dystrophy like Walker-Warburg syndrome. object.