JP6519842B2 - Antisense nucleic acid for treatment of Fukuyama muscular dystrophy - Google Patents
Antisense nucleic acid for treatment of Fukuyama muscular dystrophy Download PDFInfo
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- JP6519842B2 JP6519842B2 JP2014204750A JP2014204750A JP6519842B2 JP 6519842 B2 JP6519842 B2 JP 6519842B2 JP 2014204750 A JP2014204750 A JP 2014204750A JP 2014204750 A JP2014204750 A JP 2014204750A JP 6519842 B2 JP6519842 B2 JP 6519842B2
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- antisense nucleic
- nucleic acid
- fukutin
- pmo
- sva
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Description
本発明は、福山型筋ジストロフィー治療用アンチセンス核酸に関するものである。 The present invention relates to an antisense nucleic acid for the treatment of Fukuyama muscular dystrophy.
福山型筋ジストロフィー（Fukuyama type congenital muscular dystrophy、以下「ＦＣＭＤ」と略記する場合がある。）は日本に特有の疾患であり、先天性筋ジストロフィー、ＩＩ型滑脳症、眼奇形の３症状を示す常染色体劣性の遺伝疾患である。わが国で２番目に多い小児の筋疾患で、１０代のうちに死に至る重篤な疾患であるが、未だ治療法がない。ＦＣＭＤの疾患責任遺伝子であるフクチン遺伝子は、１９９８年にポジショナルクローニング法により同定された（非特許文献１、２参照）。ほとんどのＦＣＭＤ患者はフクチン遺伝子の３’側非翻訳領域に、動く遺伝子である約３ｋｂのＳＶＡ型レトロトランスポゾン（SVA：Sine-VNTR-Alu）の挿入変異を認める（非特許文献２参照）。この変異は約１００世代前に日本人祖先のひとりに生じたもので、日本人の８８人に１人が保因者であり、約３万の出生に１人の割合で発症することが報告されている（非特許文献３参照）。ＦＣＭＤ患者の骨格筋には細胞膜と基底膜をつなぐ糖タンパク質αジストログリカンのＯ−マンノース型糖鎖修飾に欠損があり、この糖鎖を介する細胞膜と基底膜とのあいだの結合が破綻するため重度の筋ジストロフィーが発症することが知られている（非特許文献４参照）。 Fukuyama-type muscular dystrophy (Fukuyama type congenital muscular dystrophy, hereinafter abbreviated as "FCMD") is a disease unique to Japan and is an autosomal recessive showing congenital muscular dystrophy, type II slip disorder and type III eye deformity. Is a hereditary disease of It is the second most common muscle disease of children in Japan, and it is a serious disease that will die in the teens, but there is still no cure. The Fukutin gene, which is a disease responsible gene of FCMD, was identified by positional cloning method in 1998 (see Non-Patent Documents 1 and 2). Most FCMD patients have an insertion mutation of the moving gene approximately 3 kb of SVA type retrotransposon (SVA: Sine-VNTR-Alu) in the 3 'untranslated region of the fukutin gene (see non-patent document 2). This mutation occurred in one of the Japanese ancestry about 100 generations ago, and it is reported that 1 out of 88 Japanese people are carriers and about 1 in 30,000 births. (See Non-Patent Document 3). Skeletal muscle of FCMD patient is deficient in O-mannose type glycosylation of glycoprotein α dystroglycan, which connects cell membrane and basement membrane, and this sugar chain mediates severe breakdown between cell membrane and basement membrane Muscle dystrophy is known to develop (see Non-Patent Document 4).
ＦＣＭＤ以外にも、フクチン遺伝子の変異に起因して様々な病態が引き起こされることが知られている（非特許文献５参照）。これらの中で、例えば、成人発症の拡張型心筋症（非特許文献６参照）およびＷａｌｋｅｒ−Ｗａｒｂｕｒｇ症候群様の重症型先天性筋ジストロフィー（非特許文献７参照）はフクチン遺伝子におけるＳＶＡ型レトロトランスポゾン挿入変異が原因であることが報告されている。 Besides FCMD, it is known that various pathological conditions are caused due to mutation of fukutin 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 retrotransposon insertion mutations in the fukutin gene. Is reported to be the cause.
本発明者らは、ＦＣＭＤがＳＶＡ型レトロトランスポゾンの挿入により誘導されるスプライシング異常症であることを証明した（非特許文献８参照）。より詳細には、疾患責任遺伝子であるフクチン遺伝子の最終エクソン（エクソン１０）の３’側非翻訳領域に挿入されたＳＶＡ型レトロトランスポゾンの配列に存在する強力なスプライシング受容部位がエクソン１０の翻訳領域にある潜在的なスプライシング供与部位を新たに活性化すること（エクソントラッピング）が原因であることを明らかにした。 The present inventors proved that FCMD is a splicing disorder induced by insertion of SVA type retrotransposon (see Non-patent Document 8). More specifically, the strong splicing acceptor site present in the sequence of SVA type retrotransposon inserted in the 3 'untranslated region of the last exon (exon 10) of the disease responsible gene fukutin gene is the translation region of exon 10 It turned out that the cause is new activation (exon trapping) of the potential splicing donor site.
本発明は、ＳＶＡ型レトロトランスポゾンの挿入変異を有するフクチン遺伝子の異常スプライシングを正常化することにより、ＦＣＭＤ等を治療し得るアンチセンス核酸を提供することを課題とする。さらに、生理食塩水に対する溶解性に優れ、安全性が高いアンチセンス核酸を提供することを課題とする。 An object of the present invention is to provide an antisense nucleic acid capable of treating FCMD or the like by normalizing aberrant splicing of a fukutin gene having an insertion mutation of SVA type retrotransposon. Furthermore, it is an object of the present invention to provide an antisense nucleic acid which is excellent in solubility in physiological saline and high in safety.
本発明は、上記課題を解決するために、以下の各発明を包含する。
［１］配列番号１で示される塩基配列の第１１５９３７位〜第１１５９８１位の範囲を標的配列とし９〜２４塩基からなることを特徴とするアンチセンス核酸。
［２］配列番号６〜７０から選択される塩基配列からなることを特徴とする前記［１］に記載のアンチセンス核酸。
［３］生理食塩水に５０ｍｇ／ｍＬ以上溶解することを特徴とする前記［１］または［２］に記載のアンチセンス核酸。
［４］配列番号１８、１９、２６、２７、３２、３３、３７、４３、５３、５５、５８、５９、６０、６１、６３、６４、６５、６６、６７、６８、６９および７０から選択される塩基配列からなることを特徴とする前記［１］〜［３］のいずれかに記載のアンチセンス核酸。
［５］投与用量６０ｍｇ／ｋｇのアンチセンス核酸をマウスに投与したときに、ＡＳＴ値、ＡＬＴ値、ＢＵＮ値およびクレアチニン値のいずれにも異常値がみられないことを特徴とする前記［１］または［２］に記載のアンチセンス核酸。
［６］配列番号３２、５０、５１、６０、６２、６４、６５、６６および６７から選択される塩基配列からなることを特徴とする前記［１］、［２］または［５］のいずれかに記載のアンチセンス核酸。
［７］生理食塩水に５０ｍｇ／ｍＬ以上溶解し、かつ、投与用量６０ｍｇ／ｋｇのアンチセンス核酸をマウスに投与したときに、ＡＳＴ値、ＡＬＴ値、ＢＵＮ値およびクレアチニン値のいずれにも異常値がみられないことを特徴とする前記［１］または［２］に記載のアンチセンス核酸。
［８］配列番号３２、６０、６４、６５、６６および６７から選択される塩基配列からなることを特徴とする前記［１］、［２］または［７］のいずれかに記載のアンチセンス核酸。
［９］アンチセンス核酸の３’末端および／または５’末端が非修飾であることを特徴とする前記［１］〜［８］のいずれかに記載のアンチセンス核酸。
［１０］アンチセンス核酸の３’末端および／または５’末端が修飾されていることを特徴とする前記［１］〜［８］のいずれかに記載のアンチセンス核酸。
［１１］アンチセンス核酸の３’末端および／または５’末端がトリエチレングリコール（ＴＥＧ）修飾、ヘキサエチレングリコール（ＨＥＧ）修飾およびドデカエチレングリコール（ＤＯＤＥＧ）修飾からなる群から選択される１種または２種の修飾がされていることを特徴とする前記［１０］に記載のアンチセンス核酸。
［１２］オリゴデオキシリボヌクレオチド、オリゴリボヌクレオチド、モルホリノオリゴマーまたはこれらのキメラオリゴヌクレオチドである前記［１］〜［１１］のいずれかに記載のアンチセンス核酸。
［１３］モルホリノオリゴマーである前記［１２］に記載のアンチセンス核酸。
［１４］ホスホロジアミデートモルホリノオリゴマーである前記［１３］に記載のアンチセンス核酸。
［１５］フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患を治療するための医薬組成物であって、前記［１］〜［１４］のいずれかに記載のアンチセンス核酸の１種または２種以上を有効成分として含有することを特徴とする医薬組成物。
［１６］フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患が、福山型筋ジストロフィー、成人発症の拡張型心筋症、またはＷａｌｋｅｒ−Ｗａｒｂｕｒｇ症候群様の重症型先天性筋ジストロフィーである前記［１５］に記載の医薬組成物。
The present invention includes the following inventions in order to solve the above-mentioned problems.
[1] An antisense nucleic acid comprising 9 to 24 bases as a target sequence, wherein the range of positions 115937 to 115981 of the base sequence shown in SEQ ID NO: 1 is a target sequence.
[2] The antisense nucleic acid according to the above [1], which comprises a base sequence selected from SEQ ID NOs: 6 to 70.
[3] The antisense nucleic acid according to [1] or [2], which is dissolved in physiological saline by 50 mg / mL or more.
[4] Selection 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 the above [1] to [3], which comprises the base sequence
[5] Dosing When a 60 mg / kg dose of antisense nucleic acid is administered to mice, no abnormal values are found in any of the AST value, ALT value, BUN value and creatinine value [1]. Or the antisense nucleic acid according to [2].
[6] Any one of the above-mentioned [1], [2] or [5], characterized in that it consists of a base sequence selected from SEQ ID NO: 32, 50, 51, 60, 62, 64, 65, 66 and 67 Antisense nucleic acid as described in.
[7] When dissolved in saline at least 50 mg / mL and an administration dose of 60 mg / kg of antisense nucleic acid 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], which is not found.
[8] The antisense nucleic acid according to any one of the above [1], [2] or [7], which comprises 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 the above [1] to [8], 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 the above [1] to [8], 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 the 5' end of the antisense nucleic acid The antisense nucleic acid according to the above-mentioned [10], characterized in that two kinds of modifications are made.
[12] The antisense nucleic acid according to any one of the above [1] to [11], which is an oligodeoxyribonucleotide, an oligoribonucleotide, a morpholino oligomer, or a chimeric oligonucleotide thereof.
[13] The antisense nucleic acid according to [12] above, which is a morpholino oligomer.
[14] The antisense nucleic acid according to the above [13], which is a phosphorodiamidate morpholino oligomer.
[15] A pharmaceutical composition for treating a disease caused by an insertion mutation of SVA type retrotransposon in fukutin gene, which is one of the antisense nucleic acids according to any one of the above [1] to [14] or Pharmaceutical composition characterized by containing 2 or more types as an active ingredient.
[16] The disease resulting from the insertion mutation of SVA type retrotransposon in the fukutin gene is Fukuyama muscular dystrophy, dilated cardiomyopathy of adult onset, or severe congenital muscular dystrophy described in Walker-Warburg syndrome like severe congenital muscular dystrophy Pharmaceutical composition as described.
本発明により、フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患の治療に有効なアンチセンス核酸を提供することができる。 According to the present invention, it is possible to provide an antisense nucleic acid effective for the treatment of a disease caused by an insertion mutation of SVA type retrotransposon in the fukutin gene.
〔アンチセンス核酸〕
本発明は、ＳＶＡ型レトロトランスポゾンの挿入変異を有するフクチン遺伝子の転写段階において、配列番号１の第１１１１７７位のグアニンの３’側のスプライシング供与部位および第１１５９４３位のチミンの５’側のスプライシング受容部位を用いる異常スプライシングを抑制するアンチセンス核酸を提供する。
[Antisense nucleic acid]
According to the present invention, at the transcription stage of the fukutin gene having the insertion mutation of SVA type retrotransposon, the splicing reception site 3 'of guanine at position 111177 and the 5' side of thymine at position 115943 of SEQ ID NO: 1 Provided are antisense nucleic acids that suppress aberrant splicing using a site.
配列番号１で示される塩基配列は、フクチン遺伝子のゲノム配列（GenBank ACCESSION: AB038490）にＳＶＡ型レトロトランスポゾン配列（GenBank ACCESSION: AB185332）が挿入された挿入変異型フクチン遺伝子のゲノム配列であり、第１１５６６９位〜第１１８７３０位がＳＶＡ型レトロトランスポゾン配列である。挿入変異型フクチン遺伝子では、エクソントラッピングにより、配列番号１の第１１１１７７位のグアニンの３’側、つまり第１１１１７７位のグアニンと第１１１１７８位のグアニンの間が新たなスプライシング供与部位となり、第１１５９４３位のチミンの５’側、つまり第１１５９４２位のグアニンと第１１５９４３位のチミンの間が新たなスプライシング受容部位となる。換言すれば、挿入変異型フクチン遺伝子では、配列番号１の第１１１１７８位のグアニンから第１１５９４２位のグアニンをイントロンと認識し、第１１５９４３位のチミンから第１２０１９１位のグアニンをエクソンと認識する異常スプライシングが生じる。それゆえ、第１１１１７７位のグアニンの３’側のスプライシング供与部位および第１１５９４３位のチミンの５’側のスプライシング受容部位を用いる異常スプライシングを抑制すれば異常フクチンｍＲＮＡの発現量が減少し、同時に正常スプライシングに基づく正常フクチンｍＲＮＡの発現が回復し、正常フクチンタンパク質の生成が回復する。
異常フクチンｍＲＮＡの塩基配列を配列番号２に、異常フクチンタンパク質のアミノ酸配列を配列番号３に、正常フクチンｍＲＮＡの塩基配列を配列番号４に、正常フクチンタンパク質のアミノ酸配列を配列番号５にそれぞれ示す。
The base sequence shown by SEQ ID NO: 1 is the genome sequence of the inserted mutant fukutin gene in which the SVA type retrotransposon sequence (GenBank ACCESSION: AB185332) is inserted into the genome sequence of fukutin gene (GenBank ACCESSION: AB038490), Positions 118730 are SVA retrotransposon sequences. In the insertion variant fukutin gene, due to exon trapping, a new splicing donor site is provided at the 3 'end of the guanine at position 111177 of SEQ ID NO: 1, that is, between the guanine at position 111177 and the guanine at position 111178; 5 'of thymine, that is, between guanine at position 115942 and thymine at position 115943, becomes a new splicing acceptor site. In other words, in the insertion mutant fukutin gene, abnormal splicing that recognizes guanine at position 111178 to guanine at position 115942 of SEQ ID NO: 1 as an intron, and recognizes thymine at position 115943 to guanine at position 120191 as exon Will occur. Therefore, suppressing abnormal splicing using the 3'-side splicing donor site of guanine at position 111177 and the 5'-side splicing acceptor site of thymine at position 115943 reduces the expression level of abnormal fukutin mRNA and simultaneously normal Splicing-based expression of normal fukutin mRNA is restored, and production of normal fukutin protein is restored.
The nucleotide sequence of abnormal fukutin mRNA is shown in SEQ ID NO: 2, the amino acid sequence of abnormal fukutin protein is shown in SEQ ID NO: 3, the nucleotide sequence of normal fukutin mRNA is shown in SEQ ID NO: 4, and the amino acid sequence of normal fukutin protein is shown in SEQ ID NO: 5.
本発明のアンチセンス核酸は、挿入変異型フクチン遺伝子（配列番号１）の転写段階において、一次転写産物であるプレｍＲＮＡの上記異常スプライシングを抑制し、正常スプライシングに基づいて正常フクチンｍＲＮＡを発現させ、正常フクチンタンパク質の生成を回復させることができるものであればよい。このような効果を奏するアンチセンス核酸としては、異常スプライシングに用いられるスプライシング受容部位の周辺配列を標的配列とするものが好ましく、配列番号１で示される塩基配列の第１１５９３７位〜第１１５９８１位の範囲を標的配列とするものがより好ましい。 The antisense nucleic acid of the present invention suppresses the above-mentioned aberrant splicing of the pre-mRNA, which is the primary transcript, at the transcription stage of the insertion mutant fukutin gene (SEQ ID NO: 1), and expresses normal fukutin mRNA based on normal splicing. Anything that can restore the production of normal fukutin protein may be used. As an antisense nucleic acid which exerts such an effect, one having as a target sequence a sequence around the splicing acceptor site used for abnormal splicing is preferable, and the range of position 115937 to 115981 of the base sequence shown in SEQ ID NO: 1 It is more preferable that the target sequence be
本発明のアンチセンス核酸は、標的配列（挿入変異型フクチン遺伝子のプレｍＲＮＡの塩基配列）に相補的な配列を含むものであるが、完全に相補的であることを要するものではなく、標的配列とハイブリッドを形成できる限りにおいてミスマッチを含むものであってもよい。相補的な配列部分はアンチセンス核酸の長さ（塩基数）の５０％以上、より好ましくは６０％以上、さらに好ましくは７０％以上、さらに好ましくは８０％以上、さらに好ましくは９０％以上、さらに好ましくは９５％以上、さらに好ましくは９８％以上、さらに好ましくは９９％以上、最も好ましくは１００％である。 The antisense nucleic acid of the present invention contains a sequence complementary to the target sequence (the base sequence of the pre-mRNA of the insertion mutant fukutin gene), but is not required to be completely complementary, and is hybrid with the target sequence As long as it can form, it may contain a mismatch. The complementary sequence portion is 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more of the length (number of bases) of the antisense nucleic acid. It is preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, and most preferably 100%.
本発明のアンチセンス核酸の長さは特に限定されないが、２４塩基以下であることが好ましく、２３塩基以下であることがより好ましく、２２塩基以下であることがさらに好ましい。下限は９塩基以上であることが好ましく、１０塩基以上であることがより好ましい。本発明のアンチセンス核酸の長さは、９〜２４塩基であることが好ましい。 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 still more preferably 22 bases or less. The lower limit is preferably 9 bases or more, more preferably 10 bases or more. The length of the antisense nucleic acid of the present invention is preferably 9 to 24 bases.
異常フクチンｍＲＮＡの発現が抑制されていること、および正常フクチンｍＲＮＡの発現が回復していることは、例えば、ＦＣＭＤ患者由来の細胞に本発明のアンチセンス核酸を導入し、当該細胞を試料として定量ＲＴ−ＰＣＲ等の公知のｍＲＮＡ測定方法を用いることにより確認することができる。また、正常フクチンタンパク質の生成が回復していることは、例えば、ＦＣＭＤ患者由来の細胞に本発明のアンチセンス核酸を導入し、当該細胞を試料としてＥＬＩＳＡやウエスタンブロッティング等の公知のタンパク質測定法を用いることにより確認することができる。なお、正常フクチンタンパク質の発現量は正常なスプライシングにより生成される正常フクチンｍＲＮＡの発現量に相関するので、正常フクチンｍＲＮＡの発現量を測定することにより、間接的に正常フクチンタンパク質の生成が回復していることを評価できる。 The suppression of abnormal fukutin mRNA expression and the restoration of normal fukutin mRNA expression can be determined, for example, by introducing the antisense nucleic acid of the present invention into cells derived from FCMD patients and quantifying the cells as a sample. It can confirm by using well-known mRNA measurement methods, such as RT-PCR. In addition, recovery of the production of normal fukutin protein means that, for example, the antisense nucleic acid of the present invention is introduced into cells derived from FCMD patients, and the cells are used as samples for known protein measurement methods such as ELISA and Western blotting. It can confirm by using. Since the expression level of the normal fukutin protein correlates with the expression level of the normal fukutin mRNA produced by normal splicing, the production of the normal fukutin protein is indirectly restored by measuring the expression level of the normal fukutin mRNA. Can be assessed.
正常フクチン遺伝子と挿入変異フクチン遺伝子とのヘテロ接合体（保因者）は、全フクチンタンパク質発現量の５０％が正常フクチンタンパク質であるが、筋機能および脳に異常は見られず、αジストログリカン糖鎖量も健常人と変わらない。したがって、正常ｍＲＮＡを５０％回復させることができるアンチセンス核酸は、ＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患を治療することができると考えられる。本発明者らは、ＦＣＭＤ患者由来の筋芽細胞（ＳＶＡ型レトロトランスポゾンの挿入変異ホモ接合体）を用いてアンチセンス核酸のスクリーニングを行った結果、表１に示す６５種類の塩基配列からなるアンチセンス核酸が正常フクチンｍＲＮＡを５０％以上回復させた。 In heterozygotes (carriers) of the normal fukutin gene and the insertion mutant fukutin gene (carrier), 50% of the total fukutin protein expression level is normal fukutin protein, but no abnormality is observed in muscle function and brain, and α dystroglycan The amount of sugar chains is also the same as that of healthy people. Therefore, it is considered that an antisense nucleic acid capable of restoring 50% of normal mRNA can treat a disease caused by insertion mutation of SVA type retrotransposon. The present inventors performed screening of antisense nucleic acid using myoblasts (insertion homozygote of SVA type retrotransposon) derived from FCMD patient, and as a result, it was found that antitype consisting of 65 kinds of nucleotide sequences shown in Table 1 The sense nucleic acid restored 50% or more of normal fukutin mRNA.
本発明のアンチセンス核酸は、表１に示された配列番号６〜７０から選択される塩基配列からなるアンチセンス核酸であることが好ましい。また、表１に示した配列番号６〜７０のいずれかで示される塩基配列において、標的配列とハイブリッドを形成できる限りいくつかの塩基が欠失、置換または付加された配列からなるアンチセンス核酸であってもよい。欠失、置換または付加される塩基数は、１〜５個が好ましく、より好ましくは１〜４個、さらに好ましくは１〜３個、さらに好ましくは１または２個である。なお、表１に示された各アンチセンス核酸の標的配列は、各アンチセンス核酸の相補配列である。 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. Furthermore, in the base sequence represented by any one of SEQ ID NOs: 6 to 70 shown in Table 1, an antisense nucleic acid consisting of a sequence in which several bases are deleted, substituted or added as long as they can form a hybrid with the target sequence. It may be. The number of bases to be deleted, substituted or added is preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, 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.
本発明のアンチセンス核酸は、さらに、生理食塩水に対する溶解性が高いことが好ましい。生理食塩水に対する溶解性が低いアンチセンス核酸は、製剤保存時に析出して使用できなくなる危険性や、塩を含む輸液を利用した際に析出し、使用できなくなる危険性を有する。また、析出しやすい薬剤は投与時に毒性を示すことも報告されている（Bulletin of Osaka University of Pharmaceutical Sciences 1 (2007)91-99）。したがって、生理食塩水に対する溶解性が高いアンチセンス核酸は、医薬の有効成分として非常に利用価値が高い。 The antisense nucleic acid of the present invention is further preferably highly soluble in saline. Antisense nucleic acids having low solubility in physiological saline have a risk of being deposited and becoming unusable during storage of the preparation, or being deposited when using an infusion containing a salt and becoming unusable. Also, it has been reported that drugs that tend to precipitate show toxicity upon administration (Bulletin of Osaka University of Pharmaceutical Sciences 1 (2007) 91-99). Therefore, antisense nucleic acid having high solubility in saline is very useful as an active ingredient of medicine.
生理食塩水に対する溶解性は、２０ｍｇ／ｍＬ以上であることが好ましく、３０ｍｇ／ｍＬ以上であることがより好ましく、４０ｍｇ／ｍＬ以上であることがさらに好ましく、５０ｍｇ／ｍＬ以上であることが特に好ましい。アンチセンス核酸の生理食塩水に対する溶解性は、アンチセンス核酸を目的の濃度で生理食塩水に溶解し、一定時間後に沈殿の有無を確認することにより、評価することができる。
本発明者らは、表１に示したアンチセンス核酸のうち、２２種類の塩基配列からなるアンチセンス核酸は生理食塩水に対する溶解性が５０ｍｇ／ｍＬ以上であることを見出した。具体的には、配列番号１８、１９、２６、２７、３２、３３、３７、４３、５３、５５、５８、５９、６０、６１、６３、６４、６５、６６、６７、６８、６９および７０である。したがって、本発明のアンチセンス核酸としては、配列番号１８、１９、２６、２７、３２、３３、３７、４３、５３、５５、５８、５９、６０、６１、６３、６４、６５、６６、６７、６８、６９および７０から選択される塩基配列からなるアンチセンス核酸が特に好ましい。
The solubility in physiological saline is preferably 20 mg / mL or more, more preferably 30 mg / mL or more, still more 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 target concentration and confirming the presence or absence of precipitation after a predetermined time.
The present inventors have found that among the antisense nucleic acids shown in Table 1, an antisense nucleic acid consisting of 22 types of base sequences has a solubility of 50 mg / mL or more in physiological saline. 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 NO: 18, 19, 26, 27, 32, 33, 37, 43, 53, 55, 58, 59, 60, 61, 63, 64, 65, 66, 67 Particularly preferred is an antisense nucleic acid consisting of a nucleotide sequence selected from 68, 69 and 70.
また、安全性の高いアンチセンス核酸が医薬の有効成分として好ましいことは言うまでもない。安全性は、例えば、アンチセンス核酸投与後の血中アスパラギン酸アミノトランスフェラーゼ（ＡＳＴ）値、アラニンアミノトランスフェラーゼ（ＡＬＴ）値、尿素窒素（ＢＵＮ）値およびクレアチニン値を指標にして評価することができる。具体的には、例えば、健常マウスにアンチセンス核酸を投与した後、血中ＡＳＴ値、ＡＬＴ値、ＢＵＮ値およびクレアチニン値を測定し、対照群（溶媒投与または無処置）の測定値と比較して１．３倍を超える上昇が見られた場合に異常値と判断することができる。本発明においては、５％グルコース水溶液を用いてアンチセンス核酸溶液を調製し、Ｃ５７ＢＬ／６Ｊ雄性６週齢マウスに６０ｍｇ／ｋｇの用量で尾静脈内投与した翌日に採血して血中ＡＳＴ値、ＡＬＴ値、ＢＵＮ値およびクレアチニン値を測定し、対照群（溶媒投与または無処置）の測定値と比較して１．３倍を超える上昇が見られた場合に異常値と判断し、いずれの値にも異常値がみられない場合に安全性が高い配列であると判定することができる。
本発明者らは、表１に示したアンチセンス核酸のうち、９種類の塩基配列からなるアンチセンス核酸は非常に安全性の高い配列であることを見出した。具体的には、配列番号３２、５０、５１、６０、６２、６４、６５、６６および６７である。したがって、本発明のアンチセンス核酸としては、配列番号３２、５０、５１、６０、６２、６４、６５、６６および６７から選択される塩基配列からなるアンチセンス核酸が特に好ましい。
Needless to say, highly safe antisense nucleic acids are preferred as the active ingredient of medicines. Safety can be evaluated, for example, using blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN) and creatinine levels after administration of antisense nucleic acid as an index. Specifically, for example, after administering antisense nucleic acid to healthy mice, blood AST value, ALT value, BUN value and creatinine value are measured, and compared with those of the control group (solvent administration or no treatment). If a rise of more than 1.3 times is observed, it can be judged as an outlier. In the present invention, an antisense nucleic acid solution is prepared using a 5% glucose aqueous solution and blood AST value is collected on the day after C57BL / 6J male 6-week-old mice were administered in a tail vein at a dose of 60 mg / kg. The ALT value, BUN value and creatinine value were measured, and it was judged as an outlier when there was a 1.3-fold increase compared to the measurement of the control group (solvent or no treatment), whichever value If no outliers are found, it can be determined that the array is highly safe.
The present inventors found that among the antisense nucleic acids shown in Table 1, the antisense nucleic acid consisting of 9 kinds of base sequences is a very safe sequence. Specifically, SEQ ID NOs: 32, 50, 51, 60, 62, 64, 65, 66 and 67. Therefore, as the antisense nucleic acid of the present invention, an antisense nucleic acid consisting of a nucleotide sequence selected from SEQ ID NOs: 32, 50, 51, 60, 62, 64, 65, 66 and 67 is particularly preferable.
本発明のアンチセンス核酸は、ＤＮＡ鎖、ＲＮＡ鎖、ＤＮＡとＲＮＡの混合鎖のいずれからなるものでもよい。すなわち、本発明のアンチセンス核酸は、オリゴデオキシリボヌクレオチド、オリゴリボヌクレオチドまたはデオキシリボヌクレオチドとリボヌクレオチドのキメラオリゴヌクレオチドであることが好ましい。また、本発明のアンチセンス核酸は、ヌクレオチド類似体またはスペーサーを含むものであってもよい。アンチセンス核酸は、ヌクレアーゼ耐性を増加させ、および／または標的配列に対する親和性を増加させるように修飾されることが好ましい。好ましいヌクレオチド類似体は、例えば、モルホリノ骨格、カルバメート骨格、シロキサン骨格、スルフィド骨格、スルホキシド骨格、スルホン骨格、ホルムアセチル骨格、チオホルムアセチル骨格、メチレンホルムアセチル骨格、リボアセチル骨格、アルケン含有骨格、スルホメート骨格、スルホネート骨格、スルホンアミド骨格、メチレンイミノ骨格、メチレンヒドラジノ骨格、アミド骨格などの修飾骨格を含む。モルホリノオリゴマーは、ＤＮＡのデオキシリボース糖が六員環に置き換えられ、ホスホジエステル結合がホスホロジアミデート結合に置き換えられた無電荷の骨格を有する修飾オリゴヌクレオチドである。モルホリノオリゴマーは酵素分解耐性に優れていることから、本発明のアンチセンス核酸としてより好ましい。 The antisense nucleic acid of the present invention may be 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, an oligoribonucleotide or a chimeric oligonucleotide of deoxyribonucleotide and ribonucleotide. Furthermore, the antisense nucleic acid of the present invention may contain nucleotide analogues or a spacer. Antisense nucleic acids are preferably modified to increase nuclease resistance and / or to increase the affinity for the target sequence. Preferred nucleotide analogues are, 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 sulfonate skeleton, sulfonamide skeleton, methyleneimino skeleton, methylenehydrazino skeleton and amide skeleton. Morpholino oligomers are modified oligonucleotides having an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six-membered ring and the phosphodiester bond is replaced by a phosphorodiamidate bond. Morpholino oligomers are more preferable as the antisense nucleic acid of the present invention because they are excellent in enzymatic degradation resistance.
さらに好ましいヌクレオチド類似体は、モルホリノヌクレオチド誘導体であり、隣接モルホリノ環の間の陰イオン性ホスホジエステル結合が、非イオン性ホスホロジアミデート結合に置き換えられたホスホロジアミデートモルホリノオリゴマー（phosphorodiamidate morpholino oligomer、以下「ＰＭＯ」と記す）である。 A further preferred nucleotide analogue is a morpholino nucleotide derivative, wherein a phosphorodiamidate morpholino oligomer, wherein the anionic phosphodiester bond between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate bond, Hereinafter, it is referred to as "PMO".
さらに、ヌクレオチド類似体は、ホスホジエステル結合中の非架橋酸素の１つの置換を含むことが好ましい。この修飾により、ヌクレアーゼ分解に対する重要な耐性が加えられる。好ましいヌクレオチド類似体は、ホスホロチオエート、キラルなホスホロチオエート、ホスホロジチオエート、ホスホトリエステル、アミノアルキルホスホトリエステル、Ｈ−ホスホネート、例えば３’−アルキレンホスホネート、５’−アルキレンホスホネート、キラルなホスホネートを含むエチルおよび他のアルキルホスホネート、ホスフィネート、例えば３’−アミノホスホルアミデートおよびアミノアルキルホスホルアミデートを含むホスホルアミデート、チオノホスホルアミデート、チオノアルキルホスホネート、チオノアルキルホスホトリエステル、セレノホスフェートまたはボラノホスフェートを含む。 Furthermore, it is preferred that the nucleotide analogues comprise one substitution of non-bridging oxygen in the phosphodiester bond. This modification adds important resistance to nuclease degradation. Preferred nucleotide analogues are phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, H-phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, ethyl containing chiral phosphonates And other alkyl phosphonates, phosphinates, eg phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, Contains selenophosphate or boranophosphate.
さらに、ヌクレオチド類似体は、−ＯＨ；−Ｆ；１つまたは複数のヘテロ原子が間に存在してもよい、置換または非置換の、直鎖状または分岐状の低級（Ｃ１〜Ｃ１０）アルキル、アルケニル、アルキニル、アルカリル、アリルまたはアラルキル；Ｏ−、Ｓ−またはＮ−アルキル；Ｏ−、Ｓ−またはＮ−アルケニル；Ｏ−、Ｓ−またはＮ−アルキニル；Ｏ−、Ｓ−またはＮ−アリル；Ｏ−アルキル−Ｏ−アルキル、−メトキシ、−アミノプロポキシ；−メトキシエトキシ；−ジメチルアミノオキシエトキシ；−ジメチルアミノエトキシエトキシなどの、２’、３’および／または５’位で一または二置換される１つまたは複数の糖部分を含むことが好ましい。糖部分は、ピラノースもしくはその誘導体、またはデオキシピラノースもしくはその誘導体、好ましくはリボースもしくはその誘導体、またはデオキシリボースもしくはその誘導体であってよい。好ましい誘導体化された糖部分は、ロックト核酸（ＬＮＡ）を含み、２’炭素原子が糖環の３’または４’炭素原子に連結し、それによって二環式糖部分を形成する。好ましいＬＮＡは、２’−Ｏ，４’−Ｃ−エチレン架橋核酸を含む（Morita et al., 2001, Nucleic Acid Res Supplement No.1:241−242）。これらの置換により、ヌクレオチド類似体または同等物に、ＲＮａｓｅＨおよびヌクレアーゼ耐性が与えられ、標的ＲＮＡに対する親和性が増加する。 Furthermore, the nucleotide analogues may be substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, wherein -OH; -F; one or more heteroatoms may be present 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; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; -methoxyethoxy; -dimethylaminooxyethoxy; -dimethylaminoethoxyethoxy, etc., mono or disubstituted at the 2 ', 3' and / or 5 'positions It is preferred to include 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), and the 2 'carbon atom is linked to the 3' or 4 'carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. A preferred LNA comprises a 2'-O, 4'-C-ethylene crosslinked nucleic acid (Morita et al., 2001, Nucleic Acid Res Supplement No. 1: 241-242). These substitutions confer RNase H and nuclease resistance to the nucleotide analogue or equivalent and increase the affinity for the target RNA.
本発明のアンチセンス核酸に含まれてもよいスペーサーは、アンチセンス核酸を構成する残基と残基の間に存在することで核酸配列の規則的な並びの間にスペース（空間）を与える化合物である。スペーサーは標的配列に対して相補的でなく、標的配列に結合しない。 The spacer which may be contained in the antisense nucleic acid of the present invention is a compound which provides a space between the regular arrangement of nucleic acid sequences by being present between the residues constituting the antisense nucleic acid and the residue. It is. The spacer is not complementary to the target sequence and does not bind to the target sequence.
さらにまた、本発明のアンチセンス核酸は、５’末端および／または３’末端が修飾されていてもよい。かかる修飾は、トリエチレングリコール（ＴＥＧ）修飾、ヘキサエチレングリコール（ＨＥＧ）修飾、ドデカエチレングリコール（ＤＯＤＥＧ）修飾を含む。 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.
本発明のアンチセンス核酸は、公知の核酸合成方法を用いて製造することができる。公知の方法としては、例えば、国際公開公報WO2009/064471または国際公開公報WO2013/100190に記載の方法を用いることができる。 The antisense nucleic acid of the present invention can be produced using known nucleic acid synthesis methods. As a known method, for example, the method described in International Publication WO2009 / 064471 or International Publication WO2013 / 100190 can be used.
〔医薬組成物〕
本発明は、フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患を治療するための医薬組成物を提供する。本発明の医薬組成物は、上記本発明のアンチセンス核酸の１種または２種以上を有効成分として含有する。本発明のアンチセンス核酸は、挿入変異型フクチン遺伝子（配列番号１）の第１１１１７７位のグアニンの３’側のスプライシング供与部位および第１１５９４３位のチミンの５’側のスプライシング受容部位を用いる異常スプライシングを抑制し、正常スプライシングに基づく正常フクチンｍＲＮＡの発現を回復させ、正常フクチンタンパク質の生成を回復させることにより、フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患を治療することができる。
[Pharmaceutical composition]
The present invention provides a pharmaceutical composition for treating a disease caused by an insertion mutation of SVA type retrotransposon in the fukutin 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 is aberrantly spliced using the splicing donor site 3 'of guanine at position 111177 and the 5' splice acceptor site of thymine at position 115943 of the insertion mutant fukutin gene (SEQ ID NO: 1) By suppressing the expression of normal fukutin mRNA based on normal splicing and restoring the production of normal fukutin protein, it is possible to treat a disease caused by the insertion mutation of SVA type retrotransposon in the fukutin gene.
フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患としては、例えば、ＦＣＭＤ、成人発症の拡張型心筋症（非特許文献６参照）Ｗａｌｋｅｒ−Ｗａｒｂｕｒｇ症候群様の重症型先天性筋ジストロフィー（非特許文献７参照）等が挙げられるが、これらに限定されない。本発明の医薬組成物の適用対象となる疾患には、将来、フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異が原因であることが明らかとなる疾患も含まれる。好ましくはＦＣＭＤである。 Examples of diseases caused by insertion mutation of SVA type retrotransposon in the fukutin gene include, for example, FCMD, adult onset dilated cardiomyopathy (see non-patent document 6) and severe congenital muscular dystrophy like Walker-Warburg syndrome (non-patent document) 7) and the like, but not limited thereto. Diseases to which the pharmaceutical composition of the present invention is to be applied also include diseases which are revealed to be due to insertion mutation of SVA type retrotransposon in the fukutin gene in the future. Preferably it is FCMD.
本発明の医薬組成物は、１種のアンチセンス核酸を有効成分とするものでもよく、２種以上のアンチセンス核酸を有効成分とするものでもよい。２種以上のアンチセンス核酸の組み合わせは特に限定されないが、単独で使用するより効果が増強される組み合わせを適宜選択して有効成分とすることが好ましい。 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 acid as an active ingredient. The combination of two or more kinds of antisense nucleic acids is not particularly limited, but it is preferable to appropriately select a combination whose effect is enhanced more than used alone as an active ingredient.
本発明の医薬組成物は、上記本発明のアンチセンス核酸を有効成分とし、薬学的に許容される担体または添加剤を適宜配合して製剤化することができる。具体的には錠剤、被覆錠剤、丸剤、散剤、顆粒剤、カプセル剤、液剤、懸濁剤、乳剤等の経口剤；注射剤、輸液、坐剤、軟膏、パッチ剤等の非経口剤とすることができる。好ましくは、非経口剤である。注射剤は凍結乾燥製剤としてもよい。担体または添加剤の配合割合については、医薬品分野において通常採用されている範囲に基づいて適宜設定すればよい。配合できる担体または添加剤は特に制限されないが、例えば、水、生理食塩水、その他の水性溶媒、水性または油性基剤等の各種担体、賦形剤、結合剤、ｐＨ調整剤、崩壊剤、吸収促進剤、滑沢剤、着色剤、矯味剤、香料等の各種添加剤が挙げられる。 The pharmaceutical composition of the present invention can be formulated by using the above-mentioned antisense nucleic acid of the present invention as an active ingredient and appropriately combining a pharmaceutically acceptable carrier or additive. Specifically, oral agents such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, and emulsions; and parenteral agents such as injections, infusions, suppositories, ointments, patches and the like can do. Preferably, it is a parenteral agent. The injection may be a freeze-dried preparation. The compounding ratio of the carrier or the additive may be appropriately set based on the range generally employed in the pharmaceutical field. Carriers or additives that can be incorporated are not particularly limited, but, for example, various carriers such as water, saline, other aqueous solvents, aqueous or oily bases, excipients, binders, pH adjusters, disintegrants, absorption Various additives such as accelerators, lubricants, coloring agents, flavoring agents, and perfumes may be mentioned.
錠剤、カプセル剤などに混和することができる添加剤としては、例えば、ゼラチン、コーンスターチ、トラガント、アラビアゴムのような結合剤、結晶性セルロースのような賦形剤、コーンスターチ、ゼラチン、アルギン酸などのような膨化剤、ステアリン酸マグネシウムのような潤滑剤、ショ糖、乳糖またはサッカリンのような甘味剤、ペパーミント、アカモノ油またはチェリーのような香味剤などが用いられる。調剤単位形態がカプセルである場合には、上記タイプの材料にさらに油脂のような液状担体を含有することができる。注射のための無菌組成物は通常の製剤業務（例えば有効成分を注射用水、天然植物油等の溶媒に溶解または懸濁させる等）に従って調製することができる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、Ｄ−ソルビトール、Ｄ−マンニトール、スクロース、塩化ナトリウムなど）などが用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン性界面活性剤（例、ポリソルベート８０ＴＭ、ＨＣＯ−５０）などと併用してもよい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、溶解補助剤である安息香酸ベンジル、ベンジルアルコールなどと併用してもよい。また、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤（例えば、ベンジルアルコール、フェノールなど）、酸化防止剤などと配合してもよい。さらに、凍結乾燥製剤としてもよい。 Examples of additives that can be incorporated into tablets, capsules, etc. include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid etc. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, akamono oil or cherry, and the like. When the dosage unit form is a capsule, it may further contain a liquid carrier such as fat and oil in the above-mentioned type of material. Sterile compositions for injection can be prepared according to the usual formulation practices (eg, dissolving or suspending the active ingredient in a solvent such as water for injection, natural vegetable oil, etc.). As the 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 appropriate dissolution You may use together with adjuvants, for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysorbate 80TM , HCO-50) and the like. As an oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with a solubilizer such as benzyl benzoate and benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation It may be blended with an agent (eg, benzyl alcohol, phenol etc.), an antioxidant and the like. Furthermore, it may be a freeze-dried preparation.
本発明の医薬組成物は、フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患を発症しているヒトに対して投与することができる。投与経路は特に限定されないが、非経口投与が好ましい。非経口投与は、静脈内投与等の全身性投与、筋肉内投与、経皮投与、経粘膜投与等の局所投与のいずれであってもよい。また、本発明の医薬組成物の有効成分であるアンチセンス核酸は、非ウイルスベクターまたはウイルスベクターの形態で投与することができる。さらに、リポソームを用いてアンチセンス核酸を導入する方法（リポソーム法、ＨＶＪ−リポソーム法、カチオニックリポソーム法、リポフェクション法、リポフェクトアミン法など）、マイクロインジェクション法、遺伝子銃（Ｇｅｎｅ Ｇｕｎ）でキャリア（金属粒子）とともにアンチセンス核酸を細胞に移入する方法、超音波導入法等を併用して投与する方法などを利用することができる。 The pharmaceutical composition of the present invention can be administered to a human who has developed a disease caused by an insertion mutation of SVA type retrotransposon in the fukutin gene. Although the administration route is not particularly limited, parenteral administration is preferred. Parenteral administration may be any of systemic administration such as intravenous administration, intramuscular administration, transdermal administration, and topical administration such as transmucosal administration. Furthermore, an antisense nucleic acid, which is an active ingredient of the pharmaceutical composition of the present invention, can be administered in the form of a nonviral vector or a viral vector. Furthermore, the method of introducing antisense nucleic acid using liposome (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method etc.), microinjection method, carrier by gene gun (Gene Gun) A method of transferring an antisense nucleic acid to cells together with metal particles, a method of administering in combination with an ultrasonic wave introduction method and the like, and the like can be used.
本発明の医薬組成物の用量は、含有するアンチセンス核酸の種類、剤形、投与経路、患者の年齢や体重によって異なるが、注射剤の形で投与する場合、１日当たり約０．０１ｍｇ〜６０ｇ程度、好ましくは約０．１ｍｇ〜２４ｇ程度、より好ましくは約０．１ｍｇ〜６ｇ程度を投与することができる。投与間隔は１日１回〜数回、または１日〜２週間の間隔を設定することができる。 The dose of the pharmaceutical composition of the present invention varies depending on the type of antisense nucleic acid contained, dosage form, administration route, age and body weight of the patient, but when administered in the form of injection, it is about 0.01 mg to 60 g per day A dose of about 0.1 mg to about 24 g, preferably about 0.1 mg to about 6 g, can be administered. The administration interval can be set once to several times a day, or an interval of one day to two weeks.
さらに本発明には以下の発明が含まれる。
（ｉ）フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患の治療方法であって、当該疾患患者に対して、上記本発明のアンチセンス核酸の有効量を投与することを特徴とする治療方法。
（ｉｉ）フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患の治療薬を製造するための、上記本発明のアンチセンス核酸の使用。
（ｉｉｉ）フクチン遺伝子におけるＳＶＡ型レトロトランスポゾンの挿入変異に起因する疾患の治療に使用するための、上記本発明のアンチセンス核酸。
Further, the present invention includes the following inventions.
(I) A method for treating a disease caused by insertion mutation of SVA type retrotransposon in a fukutin gene, which comprises administering an effective amount of the above-mentioned 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 insertion mutation of SVA type retrotransposon in fukutin gene.
(Iii) The above-mentioned antisense nucleic acid of the present invention for use in the treatment of a disease caused by an insertion mutation of SVA type retrotransposon in the fukutin gene.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.
〔実施例１：末端非修飾型アンチセンス核酸の合成〕
国際公開公報WO2013/100190に記載の方法に従い、表２のＰＭＯ Ｎｏ．１〜５８、６０〜６６に示す各種ホスホロジアミデートモルホリノオリゴマー（ＰＭＯ）を合成した。
[Example 1: Synthesis of non-terminally modified antisense nucleic acid]
According to the method described in International Publication WO 2013/100190, PMO No. 1 in Table 2 is obtained. Various phosphorodiamidate morpholino oligomers (PMO) shown in 1 to 58, 60 to 66 were synthesized.
〔実施例２：ＰＭＯ Ｎｏ．５９の合成〕
工程１ スペーサー化合物の合成
３−（メチルアミノ）プロパン−１−オール（２ｇ，２２．４４ｍｍｏｌ）、トリエチルアミン（３３．６５ｍｍｏｌ）をジメチルホルムアミド（２０ｍＬ）に溶解し、氷冷下トリフェニルメチルクロリド（２６．９２ｍｍｏｌ）を加え室温で３時間撹拌した。再度氷冷し、水（４０ｍｌ）を加えて１０分撹拌した後、酢酸エチルで抽出した。有機層を白濁のまま水洗し、エバポレーターにて濃縮後、再度酢酸エチル（５０ｍｌ）希釈して不溶物をろ過して除いた。ろ液を濃縮してそのままシリカゲルカラムクロマトグラフィーにて精製した。得られた３−［メチル（トリチル）アミノ］プロパン−１−オール（４．８ｇ，１４ｍｍｏｌ）をテトラヒドロフラン（４０ｍＬ）に溶解し氷冷下１−メチルイミダゾール（３６ｍｍｏｌ）、Ｎ−エチルモルホリン（１４ｍｍｏｌ）、Ｎ，Ｎ−ジメチルホスホルアミドジクロリデート（２９ｍｍｏｌ）を加え室温で３時間撹拌した。酢酸エチルと１Ｍりん酸二水素ナトリウム水溶液にて分液操作を行い、有機層を飽和食塩水にて洗浄、硫酸ナトリウムで乾燥後、減圧濃縮した。シリカゲルカラムクロマトグラフィーにて精製し、図６（Ｄ）に示すスペーサー化合物８（４．６ｇ）を得た。
Example 2: PMO No. Synthesis of 59]
Step 1 Synthesis of spacer compound Dissolve 3- (methylamino) propan-1-ol (2 g, 22.44 mmol) and triethylamine (33.65 mmol) in dimethylformamide (20 mL), and under ice-cooling, 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 extracted with ethyl acetate. The organic layer was washed with white turbidity, concentrated by an evaporator, diluted again with ethyl acetate (50 ml) and filtered to remove insolubles. 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) is dissolved in tetrahydrofuran (40 mL), 1-methylimidazole (36 mmol), N-ethyl morpholine (14 mmol) under ice-cooling And N, N-dimethylphosphoramide dichloridate (29 mmol) were added and stirred at room temperature for 3 hours. Liquid separation operation was performed with ethyl acetate and 1 M aqueous sodium dihydrogen phosphate solution, and the organic layer was washed with saturated brine, dried over sodium sulfate and concentrated under reduced pressure. It refine | purified by silica gel column chromatography, and obtained spacer compound 8 (4.6 g) shown to FIG. 6 (D).
工程２ ＰＭＯ Ｎｏ．５９の合成
国際公開公報WO2013/100190に記載の方法に従い、ＰＭＯ Ｎｏ．５９を合成した。
なお、ＰＭＯ Ｎｏ．５９は、図６（Ｅ）に示すスペーサー９の５’末端側に塩基配列がＣＴＣＧからなるＰＭＯが結合し、配列番号７１の塩基配列からなるＰＭＯがスペーサーの３’末端側に結合したアンチセンス核酸である。
ＰＭＯ Ｎｏ．５９に含まれるスペーサーは、工程１で得られたスペーサー化合物を用いて導入した。
ＰＭＯ Ｎｏ．５９： ＣＴＣＧ−（スペーサー）−ＣＡＴＣＡＧＡＧＧＧＡＧＡＣＣ
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：６４６６．２８
測定値：６４６５．２３
Process 2 PMO No. According to the method described in US Patent Application Publication No. WO 2013/100190, 59 PMO No. 59 was synthesized.
In addition, PMO No. 59 is an antisense wherein a PMO consisting of CTCG is attached to the 5 'end of the spacer 9 shown in FIG. 6E and a PMO consisting of the nucleotide sequence of SEQ ID NO: 71 is joined to the 3' end of the spacer It is a nucleic acid.
PMO No. The spacer contained in 59 was introduced using the spacer compound obtained in step 1.
PMO No. 59: CTCG-(spacer)-CATCAGAGGGA G ACC
ESI-TOF-MS calculated value: 6466.28
Measurement value: 6465.23
〔実施例３：５’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．７０の合成〕
工程１ Ｎ−トリチルピペラジンの合成
ピペラジン（１５０ｇ，１７４１ｍｍｏｌ）をトルエン（６５０ｍＬ）／メタノール（１３０ｍＬ）混合液に溶解し、氷冷下［クロロ（ジフェニル）メチル］ベンゼン（４８．５ｇ，１７４ｍｍｏｌ）のトルエン（２４４ｍＬ）溶液を滴下して加え、室温で終夜撹拌した。反応液を水洗し、トルエン層をコハク酸（２２．８ｇ，１９３ｍｍｏｌ）／水（３００ｍＬ）溶液に添加し氷冷下撹拌した。析出した結晶をろ取し、アセトンで洗浄後、５０℃にて減圧乾燥し、Ｎ−トリチルピペラジン／コハク酸塩７１．７ｇを得た。得られた化合物（５０ｇ，６４．５ｍｍｏｌ）をジクロロメタンに溶解し、炭酸カリウム（３２２．６ｍｍｏｌ）水溶液で洗浄した。ジクロロメタン層を水洗、硫酸ナトリウムで乾燥後、減圧乾固し、図６（Ａ）に示す標記化合物１（４０ｇ）を得た。
Example 3 5'-Terminal Ethylene Glycol Modified Product PMO No. Synthesis of 70]
Step 1 Synthesis of N- tritylpiperazine Dissolve piperazine (150 g, 1741 mmol) in a mixed solution of toluene (650 mL) / methanol (130 mL), and add toluene under ice-cooling of [chloro (diphenyl) methyl] benzene (48.5 g, 174 mmol) The solution (244 mL) was added dropwise and stirred at room temperature overnight. The reaction solution was washed with water, and the toluene layer was added to a solution of succinic acid (22.8 g, 193 mmol) / water (300 mL) 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 resulting compound (50 g, 64.5 mmol) was dissolved in dichloromethane and washed with aqueous potassium carbonate (322.6 mmol) solution. The dichloromethane layer was washed with water, dried over sodium sulfate and evaporated to dryness under reduced pressure to obtain the title compound 1 (40 g) shown in FIG. 6 (A).
工程２ ４−オキソ−４−［２−［２−［２−（４−トリチルピペラジン−１−カルボニル）オキシエチル］エトキシ］エトキシ］ブタン酸の合成
トリエチレングリコール（３７０．０３ｍｍｏｌ）をピリジン（５００ｍＬ）に溶解し、氷冷下１，１’−カルボニルジイミダゾール（２０ｇ，１２３．３４ｍｍｏｌ）を加え、室温で３０分、４０℃で２時間撹拌した。次いで、工程１で得た化合物１（４０ｇ，１２１．８ｍｍｏｌ）を加え、６０℃で４時間撹拌した。反応液を減圧濃縮して酢酸エチルと１Ｍリン酸二水素ナトリウム水溶液で分液操作を行い、有機層を飽和食塩水で洗浄、硫酸ナトリウムで乾燥後、減圧濃縮した。残渣をシリカゲルカラムクロマトグラフィーで精製し、図６（Ａ）に示す化合物２（４３．３ｇ）を得た。得られた化合物２と４−ジメチルアミノピリジン（１２７．８ｍｍｏｌ）をジクロロメタン（５００ｍＬ）に溶解し無水コハク酸（１２７．８ｍｍｏｌ）を加えて室温で１時間撹拌した。反応液を１Ｍリン酸二水素ナトリウム水溶液で分液し、有機層を飽和食塩水で洗浄、硫酸ナトリウムで乾燥後、減圧濃縮し図６（Ａ）に示す標記化合物３（５１．６ｇ）を得た。
Step 2 Synthesis of 4-oxo-4- [2- [2- [2- (4- tritylpiperazine -1-carbonyl) oxyethyl] ethoxy] ethoxy] butanoic acid Triethylene glycol (370.03 mmol) in pyridine (500 mL) The solution was dissolved in water, and 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. Then, Compound 1 (40 g, 121.8 mmol) obtained in Step 1 was added, and stirred at 60 ° C. for 4 hours. The reaction solution is concentrated under reduced pressure, liquid separation operation is performed with ethyl acetate and 1 M aqueous sodium dihydrogen phosphate solution, the organic layer is 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. 6 (A). 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 solution is separated with 1 M aqueous sodium dihydrogen phosphate solution, and the organic layer is 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. The
工程３ ５’−末端トリエチレングリコール修飾用樹脂の合成
工程２で得た化合物３（４３．５０ｍｍｏｌ）、アミノメチルポリスチレン樹脂（２１．７５ｍｍｏｌ）、４−ジメチルアミノピリジン（５４．３８ｍｍｏｌ）、１−（３−ジメチルアミノプロピル）−３−エチルカルボジイミドヒドロクロリド（２１７．５ｍｍｏｌ）をフィルター付きナスフラスコに入れ、ピリジン（４２０ｍＬ）、次いでトリエチルアミン（２６８．６ｍｍｏｌ）を加え室温で３日間振とうした。反応液をろ過し、ピリジン、メタノール、ジクロロメタンで順次洗浄後、１５分減圧乾燥をした。テトラヒドロフラン（３１２ｍＬ）、２，６−ルチジン（７７．５ｍＬ）、無水酢酸（６５ｍＬ）を加えて室温で３時間振とうした。フィルターろ過後、ピリジン、メタノール、ジクロロメタンで順次洗浄し、減圧乾燥後に図６（Ａ）に示す標記樹脂（化合物４）を得た（ローディング：３１０μｍｏｌ／ｇ）。
Step 3 Synthesis of resin for 5'-end triethylene glycol modification Compound 3 (43.50 mmol) obtained in Step 2 of amino methyl polystyrene resin (21.75 mmol), 4-dimethylaminopyridine (54.38 mmol), 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (217.5 mmol) was placed in a filtered eggplant flask, pyridine (420 mL) and then triethylamine (268.6 mmol) were added and shaken at room temperature for 3 days. The reaction solution was filtered, washed successively with pyridine, methanol and dichloromethane and dried under reduced pressure for 15 minutes. Tetrahydrofuran (312 mL), 2,6-lutidine (77.5 mL) and acetic anhydride (65 mL) were added and shaken at room temperature for 3 hours. After filtration through a filter, the residue was washed successively with pyridine, methanol and dichloromethane, and dried under reduced pressure to obtain the title resin (compound 4) shown in FIG. 6 (A) (loading: 310 μmol / g).
工程４ ＰＭＯ Ｎｏ．７０の合成
工程３で得た樹脂（化合物４）を用い、国際公開公報WO2013/100190に記載の方法に従い５’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．７０を合成した。
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：６９９４．４７
測定値：６９９４．７９
Process 4 PMO No. Using the resin (compound 4) obtained in Synthesis Step 3 of 70, according to the method described in International Publication WO 2013/100190, 5'-terminal ethylene glycol modified product PMO No. 70 was synthesized.
ESI-TOF-MS calculated value: 6994.47
Measurement value: 6994.79
〔実施例４：５’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．６８の合成〕
実施例３と同様の方法にて５’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．６８を合成した。
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：６０３４．１４
測定値：６０３４．７４
Example 4 5'-Terminal Ethylene Glycol Modified Product PMO No. Synthesis of 68]
In the same manner as in Example 3, 5'-terminal ethylene glycol modified product PMO No. 68 was synthesized.
ESI-TOF-MS calculated value: 6034.14
Measurement value: 6034.74
〔実施例５：３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．６９の合成〕
工程１ トリエチレングリコール（ＴＥＧ）誘導体 ２−［２−［２−［ビス（４−メトキシフェニル）（フェニル）メトキシ］エトキシ］エトキシ］エチルジメチルホスホルアミドクロリデートの合成
トリエチレングリコール（５０ｇ，３３２．９６ｍｍｏｌ）をピリジン（１００ｍＬ，１２３６ｍｍｏｌ）に加え、アルゴン雰囲気下ジメトキシトリチルクロリド（３８ｇ，１１２．２ｍｍｏｌ）を少しずつ加えて数時間攪拌した。エバポレーターにてピリジンを減圧留去後、濃縮残渣をジクロロメタンに溶解し、水を加え分液操作を行った。有機層を回収し硫酸マグネシウムにて乾燥した。エバポレーターにてジクロロメタンを減圧留去し、残渣をシリカゲルカラムクロマトグラフィーで精製し、２−［２−［２−［ビス（４−メトキシフェニル）−フェニル−メトキシ］エトキシ］エトキシ］エタノール（３５ｇ）を得た。得られた化合物（６ｇ，１３．２５ｍｍｏｌ）をテトラヒドロフラン（６０ｍＬ）に溶解し、氷冷下１−メチルイミダゾール（３３．１４６ｍｍｌ）、Ｎ−エチルモルホリン（１３．２５ｍｍｏｌ）、Ｎ，Ｎ−ジメチルホスホルアミドジクロリデート（２６．５１ｍｍｏｌ）をこの順で加えた。室温で２時間撹拌し、１Ｍリン酸二水素ナトリウム水溶液でクエンチ後、酢酸エチルにて抽出した。有機層を飽和食塩水で洗浄、硫酸マグネシウムにて乾燥後、エバポレーターで濃縮した。残渣をシリカゲルカラムクロマトグラフィーで精製し、図６（Ｂ）に示す標記誘導体（化合物５）６．１ｇを得た。
Example 5 3'-Terminal Ethylene Glycol Modified Product PMO No. 5 Synthesis of 69]
Step 1 Triethylene Glycol (TEG) Derivative Synthesis of 2- [2- [2- [bis (4-methoxyphenyl) (phenyl) methoxy] ethoxy] ethoxy] ethyl dimethyl phosphoramidochloridate 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 with an evaporator, the concentrated residue was dissolved in dichloromethane, water was added, and a liquid separation operation was performed. The organic layer was collected and dried over magnesium sulfate. Dichloromethane is distilled off under reduced pressure with an evaporator, and the residue is purified by silica gel column chromatography to give 2- [2- [2- [bis (4-methoxyphenyl) -phenyl-methoxy] ethoxy] ethoxy] ethanol (35 g) Obtained. The obtained compound (6 g, 13.25 mmol) is dissolved in tetrahydrofuran (60 mL), 1-methylimidazole (33.146 mml), N-ethyl morpholine (13.25 mmol), N, N-dimethylphosphor 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 1 M aqueous sodium dihydrogenphosphate 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 titled derivative (Compound 5) shown in FIG. 6 (B).
工程２ ＰＭＯ Ｎｏ．６９の合成
ＰＭＯ Ｎｏ．２７（３８．３８μｍｏｌ）を１５ｍＬファルコンチューブに入れ、ジメチルスルホキシド（１．２ｍＬ）に溶解させた。これに工程１で得た化合物５（５５ｍｇ，９５．１μｍｏｌ）、トリエチルアミン（５５μＬ，３９４．６μｍｏｌ）のＴＨＦ（２２０μＬ）混合溶液を加え、水浴４０℃で２時間反応させた。氷冷後、反応液を２０ｍＭトリエチルアミン−酢酸緩衝液／アセトニトリル（４／１、１８ｍＬ）で希釈し、逆相カラム精製（ＬｉＣｈｒｏｐｒｅｐ＿ＲＰ１８）を行い濃縮した。得られた残渣を２０％アセトニトリル水溶液に溶解し、０．１Ｍ塩酸水溶液を加えた。ｐＨ試験紙でｐＨが２になったことを確認後１時間静置した。反応液に１Ｍ水酸化ナトリウム水溶液を加えて中和し、ｐＨ試験紙でｐＨが７になったことを確認した。反応液をメンブレンフィルター（ＰＶＤＦ，０．２２μｍ）でろ過した。逆相カラム（ＹＭＣ＿ＧＥＬ＿ＴＭＳ＿ＨＧ）で脱塩および精製を行い、凍結乾燥後、３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．６９（２３７．６ｍｇ）を得た。
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：６８８２．４０
測定値：６８８２．１０
Process 2 PMO No. 69 synthetic PMO No. 27 (38.38 μmol) was placed in a 15 mL Falcon tube and dissolved in dimethylsulfoxide (1.2 mL). 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) was added to this, and reacted at 40 ° C. for 2 hours. After ice-cooling, the reaction solution was diluted with 20 mM triethylamine-acetic acid buffer / acetonitrile (4/1, 18 mL), reverse phase column purification (LiChroprep_RP18) was performed, and the solution was concentrated. The resulting residue was dissolved in 20% aqueous acetonitrile and 0.1 M aqueous hydrochloric acid was added. After confirming that the pH had reached 2 with pH paper, it was allowed to stand for 1 hour. The reaction solution was neutralized by adding 1 M aqueous sodium hydroxide solution, and it was confirmed by pH paper that pH reached 7. The reaction solution was filtered through a membrane filter (PVDF, 0.22 μm). After desalting and purification on a reverse phase column (YMC_GEL_TMS_HG) and lyophilization, the 3'-terminal ethylene glycol modified product PMO No. 69 (237.6 mg) were obtained.
ESI-TOF-MS calculated value: 6882.40
Measurement value: 6882.10
〔実施例６：３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．６７の合成〕
実施例５と同様の方法にて３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．６７を合成した。ただし、工程２のＰＭＯとしてＰＭＯ Ｎｏ．３９を使用した。
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：５９２２．０８
測定値：５９２２．２５
Example 6 3'-Terminal Ethylene Glycol Modified Product PMO No. Synthesis of 67]
In the same manner as in Example 5, 3'-terminal ethylene glycol modified product PMO No. 5 67 was synthesized. However, as PMO of process 2, PMO no. I used 39.
ESI-TOF-MS calculated value: 5922.08
Measurement value: 5922.25
〔実施例７：３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．７１の合成〕
工程１ ヘキサエチレングリコール（ＨＥＧ）誘導体の合成
ヘキサエチレングリコールを原料として、実施例５の工程１と同様の方法で図６（Ｃ）に示す標記誘導体（化合物６）を合成した。
Example 7 3'-Terminal Ethylene Glycol Modified Product PMO No. Synthesis of 71]
Step 1 Synthesis of Hexaethylene Glycol (HEG) Derivative The title derivative (Compound 6) shown in FIG. 6 (C) was synthesized in the same manner as in Step 1 of Example 5 using hexaethylene glycol as a raw material.
工程２ ＰＭＯ Ｎｏ．７１の合成
実施例５と同様の方法にて３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．７１を合成した。ただし、工程２のエチレングリコール誘導体として、化合物５に代えて工程１で得た化合物６を用いた。
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：７０１４．４９
測定値：７０１４．９８
Process 2 PMO No. Synthesis of 71 3 'end ethylene glycol modified product PMO No. 71 was synthesized. However, Compound 6 obtained in Step 1 was used instead of Compound 5 as the ethylene glycol derivative of Step 2.
ESI-TOF-MS calculated value: 7014.49
Measurement value: 7014.98
〔実施例８：３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．７３の合成〕
実施例５と同様の方法にて３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．７３を合成した。ただし、工程２のＰＭＯとしてＰＭＯ Ｎｏ．６６を使用した。
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：６６５９．３７
測定値：６６５９．８５
Example 8 3'-Terminal Ethylene Glycol Modified Product PMO No. Synthesis of 73]
In the same manner as in Example 5, 3'-terminal ethylene glycol modified product PMO No. 5 73 was synthesized. However, as PMO of process 2, PMO no. 66 was used.
ESI-TOF-MS calculated value: 6659.37
Measurement value: 659.85
〔実施例９：３’末端エチレングリコール修飾体ＰＭＯ Ｎｏ．７２の合成〕
工程１ ドデカエチレングリコール（ＤＯＤＥＧ）誘導体の合成
ドデカエチレングリコールを原料として、実施例５の工程１と同様の方法で図６（Ｃ）に示す標記誘導体（化合物７）を合成した。
Example 9 3'-Terminal Ethylene Glycol Modified Product PMO No. Synthesis of 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.
工程２ ＰＭＯ Ｎｏ．７２の合成
実施例５と同様の方法にて標記化合物を合成した。ただし、工程２のエチレングリコール誘導体として、化合物５に換えて工程１で得た化合物７を用いた。
ＥＳＩ−ＴＯＦ−ＭＳ 計算値：７２７８．６４
測定値：７２７９．００
Process 2 PMO No. Synthesis of 72 The title compound was synthesized in the same manner as in Example 5. However, Compound 7 obtained in Step 1 was used instead of Compound 5 as the ethylene glycol derivative of Step 2.
ESI-TOF-MS calculated value: 7278.64
Measurement value: 7279.00
実施例３〜９において合成したＰＭＯ末端修飾体を表３に示す。
〔実施例１０：ＦＣＭＤ治療用アンチセンス核酸のスクリーニング〕
（１）使用細胞
ＦＣＭＤ患者筋芽細胞（ＳＶＡ型レトロトランスポゾンの挿入変異ホモ接合体）を用いた。この細胞は、神戸大学大学院医学研究科の医学倫理審査委員会の承認を受けて使用した。
（２）アンチセンス核酸
実施例１〜９で合成したＰＭＯ Ｎｏ．１〜７３を試験物質としてスクリーニングに供した。
Example 10 Screening of Antisense Nucleic Acid for Treatment of FCMD
(1) Cells used Myoblasts from FCMD patients (SVA type retrotransposon insertion mutation 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）実験方法
ＦＣＭＤ患者筋芽細胞を増殖培地（20% Fetal bovine serum (FBS, Invitrogen)、2.5 ng/mL basic fibroblast growth factor（SIGMA）、0.5% Antibiotic Antimycotic Solution (100×), Stabilized (SIGMA)含有Dulbecco's Modified Eagle's Medium (DMEM)- high glucose(SIGMA)）で継代培養し、１ｃｍ２当たり３０，０００細胞になるように細胞培養ディッシュに播種して１日培養した。
翌日、分化培地（2% FBS含有DMEM-high glucose）に交換し、３μＭとなるようＰＭＯを添加した。陰性対照にはランダム化配列またはジストロフィン遺伝子を標的としたＰＭＯを用いた。培地１ｍＬ当たり６μＬのＥｎｄｏ−Ｐｏｒｔｅｒ（Gene-Tools）を用いてトランスフェクションを行った。２日後、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（Qiagen）によりトータルＲＮＡを回収した。トータルＲＮＡからＰｒｉｍｅＳｃｒｉｐｔ ＲＴ−ＰＣＲ Ｋｉｔ（タカラバイオ）を用いて、ランダムプライマーによりｃＤＮＡ合成を行った。得られたｃＤＮＡを鋳型として異常スプライシングによる異常フクチンｍＲＮＡおよび正常スプライシングによる正常フクチンｍＲＮＡをＰＣＲにより検出した。各ｍＲＮＡを検出するためのプライマーの位置を図１に示した。ＰＣＲの条件は（９４℃３０秒、６５℃３０秒、７２℃６０秒）×３２サイクルとし、反応終了後４℃に冷却した。ＰＣＲ反応サンプル１μＬを用いて、Ａｇｉｌｅｎｔ ＤＮＡ １０００ ｋｉｔ（Agilent Technologies）および２１００ Ｂｉｏａｎａｌｙｚｅｒ（Agilent Technologies）によりＰＣＲ増幅断片のサイズと濃度の測定を行った。２１９ｂｐ付近に検出される正常スプライシングに由来するバンドの定量値［Ａ］（nmol/L）および２６５ｂｐ付近に検出される異常スプライシングに由来するバンドの定量値［Ｂ］（nmol/L）から正常スプライシング率を以下の計算式で算出した。
正常スプライシング率＝［Ａ］／（［Ａ］＋［Ｂ］）
(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) The cells were subcultured with Dulbecco's Modified Eagle's Medium (DMEM) -high glucose (SIGMA)), seeded in a cell culture dish at 30,000 cells / cm 2 and cultured for 1 day.
The next day, the medium was replaced with differentiation medium (DMEM-high glucose containing 2% FBS), and PMO was added to a concentration of 3 μM. PMO targeting random sequence or dystrophin gene was used as negative control. Transfection was performed using 6 μL Endo-Porter (Gene-Tools) per mL of culture medium. Two days later, total RNA was recovered by RNeasy Mini Kit (Qiagen). CDNA synthesis was performed from total RNA using a PrimeScript RT-PCR Kit (Takara Bio) with random primers. Using the obtained cDNA as a template, abnormal fukutin mRNA by abnormal splicing and normal fukutin mRNA by normal splicing were detected by PCR. The positions of primers for detecting each mRNA are shown in FIG. The conditions for PCR were (94.degree. C. for 30 seconds, 65.degree. C. for 30 seconds, 72.degree. C. for 60 seconds) .times.32 cycles, and the reaction was cooled to 4.degree. Using 1 μL of the PCR reaction sample, measurement of size and concentration of PCR amplified fragment was performed with Agilent DNA 1000 kit (Agilent Technologies) and 2100 Bioanalyzer (Agilent Technologies). The quantitative value [A] (nmol / L) of the band derived from normal splicing detected around 219 bp and the quantified value [B] (nmol / L) of the band derived from aberrant splicing detected around 265 bp The rate was calculated by the following formula.
Normal splicing rate = [A] / ([A] + [B])
（４）ＦＣＭＤ治療用アンチセンス核酸の選択条件
正常フクチンとＳＶＡ型レトロトランスポゾンの挿入変異ヘテロ接合体（保因者）は筋機能および脳に異常は見られず、αジストログリカン糖鎖量も健常人と変わらない。すなわち、正常ｍＲＮＡを５０％回復させることができればＦＣＭＤが治療可能であると考えられる。そこで、核酸濃度３μＭでトランスフェクションしたＦＣＭＤ患者筋芽細胞において５０％以上に正常スプライシングが回復できることを本スクリーニングにおけるＦＣＭＤ治療用アンチセンス核酸の選択条件とした。
(4) Conditions for selection of antisense nucleic acid for FCMD treatment Insertion mutation heterozygote (carrier) of normal fukutin and SVA type retrotransposon shows no abnormality in muscle function and brain, and the amount of α dystroglycan sugar chain is also healthy It is not different from people. That is, if normal mRNA can be recovered by 50%, FCMD is considered to be treatable. Therefore, the condition that normal splicing can be restored to 50% or more in FCMD patient myoblasts transfected at a nucleic acid concentration of 3 μM was set as the selection condition for the antisense nucleic acid for FCMD treatment in this screening.
（５）結果
スクリーニングの結果、表２および表３に示した７３種のアンチセンス核酸は、核酸濃度３μＭにおいて５０％以上に正常スプライシングを回復した。これら７３種のアンチセンス核酸は、いずれもＳＶＡ挿入フクチン遺伝子の異常スプライシングに用いられるスプライシング受容部位の周辺配列を標的配列とするアンチセンス核酸である。具体的には、ＳＶＡ挿入フクチン遺伝子の塩基配列（配列番号１）の第１１５９３７位〜第１１５９８１位の範囲を標的配列とするものである。
(5) Results As a result of screening, 73 types of antisense nucleic acids shown in Tables 2 and 3 restored normal splicing to 50% or more at a nucleic acid concentration of 3 μM. These 73 types of antisense nucleic acids are all antisense nucleic acids whose target sequence is a sequence around a splicing acceptor site used for abnormal splicing of the SVA insertion fukutin gene. Specifically, the range of positions 115937 to 115981 of the base sequence (SEQ ID NO: 1) of the SVA insertion fukutin gene is used as a target sequence.
〔実施例１１：ＦＣＭＤ患者細胞におけるαジストログリカンの糖鎖回復〕
（１）実験材料および方法
実施例１と同じＦＣＭＤ患者筋芽細胞（ＳＶＡ型レトロトランスポゾンの挿入変異ホモ接合体）を用いた。増殖培地（実施例１０参照）で継代培養した細胞に対して、Ｎｕｃｌｅｏｆｅｃｔｏｒ（Lonza）を用いて電気穿孔法によりＰＭＯを導入し、分化培地（実施例１０参照）で培養した。ＰＭＯとしてＰＭＯ Ｎｏ．１、９、２７、３９および４５の５種類のＰＭＯを用いた。陰性対照にはジストロフィン遺伝子を標的としたＰＭＯを用いた。
[Example 11: Sugar chain restoration of α dystroglycan in FCMD patient cells]
(1) Experimental Materials and Methods The same FCMD myoblasts (SVA type retrotransposon insertion mutant homozygote) as in Example 1 were used. PMO was introduced into cells subcultured in growth medium (see Example 10) by electroporation using Nucleofector (Lonza), and cultured in differentiation medium (see Example 10). PMO as PMO No. Five PMOs were used: 1, 9, 27, 39 and 45. PMO targeted to the dystrophin gene was used as a negative control.
ＰＭＯ導入から５日後に、ＰＢＳ（−）で細胞を１回洗浄し、細胞容量の１０倍量の１％ Ｔｒｉｔｏｎ ＴＢＳを加えてスクレーパーで回収した。４℃で１時間混和して細胞を溶解し、遠心後の上清を細胞抽出液とした。細胞抽出液１５μＬを抗αジストログリカンラットモノクローナル抗体、抗糖鎖修飾αジストログリカンマウスモノクローナル抗体ＩＩＨ６、抗βジストログリカンマウスモノクローナル抗体８Ｄ５を用いたウエスタンブロット分析に供した。ウエスタンブロット分析は、文献(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))の記載に従って実施した。 Five days after PMO introduction, cells were washed once with PBS (-), and 10 volumes of 1% Triton TBS was added to the cell volume and collected 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 an anti-α dystroglycan rat monoclonal antibody, an anti-carbohydrate modified α dystroglycan mouse monoclonal antibody IIH6 and an anti-β dystroglycan mouse monoclonal antibody 8D5. Western blot analysis is carried out according to the description in the literature (Kanagawa, M. et al. Residual laminin-binding acivity and enhanced dysglycosylation by LARGE in novel model mice to dystroglycanopathy. Hum. Mol. Genet. 18, 621-31 (2009)) Carried out.
（２）実験結果
結果を図２に示した。図２中、α−ＤＧはαジストログリカン、β−ＤＧはβジストログリカン、ＨＳＭＭはヒト骨格筋筋芽細胞、ＲＤはヒト胎児横紋筋肉腫を表す。上段が抗αジストログリカンラットモノクローナル抗体を用いた結果、中段が抗糖鎖修飾αジストログリカンマウスモノクローナル抗体ＩＩＨ６を用いた結果、下段が抗βジストログリカンマウスモノクローナル抗体８Ｄ５を用いた結果である。
ＦＣＭＤ患者細胞はαジストログリカンの糖鎖が欠損しており、αジストログリカンは低分子量の位置に検出された（図２のｗａｔｅｒ参照）。一方、ＰＭＯを導入したＦＣＭＤ患者細胞では、いずれのＰＭＯを導入した場合でも濃度依存的にαジストログリカンの糖鎖が回復していることが確認できた。陰性対照のジストロフィン遺伝子を標的としたＰＭＯを導入したＦＣＭＤ患者細胞では、αジストログリカンの糖鎖が回復しなかった。
(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 myoblasts, and RD represents human fetal rhabdomyosarcoma. The upper row shows the results of using the anti-α dystroglycan rat monoclonal antibody, the middle row shows the results of using the anti-carbohydrate modified α dystroglycan mouse monoclonal antibody IIH6, and the lower row shows the results of using the anti-β dystroglycan mouse monoclonal antibody 8D5.
FCMD patient cells were deficient in α-dystroglycan sugar chains, and α-dystroglycan was detected at the position of low molecular weight (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 restored in a concentration dependent manner regardless of which PMO was introduced. In FCMD patient cells transfected with PMO targeting the negative control dystrophin gene, α-dystroglycan sugar chain was not restored.
〔実施例１２：ＦＣＭＤモデルマウスを用いた薬効評価〕
（１）使用動物
全ての動物実験は神戸大学大学院医学研究科の実験動物倫理審査委員会の承認を受けた。ＦＣＭＤノックインマウスおよびその命名については文献（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)）に記載されている。フクチン遺伝子の正常ヒトエクソン１０およびＳＶＡ挿入ヒトエクソン１０を有するトランスジェニックアレルをそれぞれＨｎおよびＨｐと命名した。Ｈｐ／ＨｐはＳＶＡ挿入ヒトエクソン１０アレルのホモ接合体であり、Ｈｎ／Ｈｎは正常ヒトエクソン１０アレルのホモ接合体であり、Ｈｐ／−はＳＶＡ挿入ヒトエクソン１０アレルとマウスフクチン遺伝子ノックアウトアレルの複合ヘテロ接合体である。実験に用いたマウスは８〜１４週齢である。
[Example 12: Efficacy evaluation using FCMD model mouse]
(1) Use animals All animal experiments were approved by the Experimental Animal Ethics Review Board of the Graduate School of Medicine, Kobe University. For the FCMD knock-in mouse and its nomenclature, see (Kanagawa, M. et al., Residual laminin-binding activity and enhanced dysglycosylation by LARGE in novel model mice to dystropathy. Hum. Mol. Genet. 18, 621-31 (2009)) It is described in. Transgenic alleles having normal human exon 10 and SVA inserted human exon 10 of the fukutin gene were designated as Hn and Hp, respectively. Hp / Hp is homozygous for the SVA inserted human exon 10 allele, Hn / Hn is homozygous for the normal human exon 10 allele, Hp /-is the SVA inserted human exon 10 allele and the mouse fukutin gene knockout allele It is a complex heterozygote. The mice used for the experiment are 8 to 14 weeks old.
（２）アンチセンス核酸投与
ＰＭＯとしてＰＭＯ Ｎｏ．９、２７、３９および４５の４種類のＰＭＯを用いた。
Ｈｐ／−マウスにカルジオトキシン１０μＭ（SIGMA）を経皮的に前脛骨筋肉内（0.4nmol）、腓腹筋（2nmol）および大腿四頭筋（1nmol）の３か所に投与した。ＰＭＯの投与用量毎に２匹のマウスを用いた。カルジオトキシン投与日を０日目とし、３日目に５％マンニトールに溶解したＰＭＯ（20, 60, 200mg/kg）を尾静脈内投与した。２３日目にマウスを安楽死させ、フクチンタンパク質、αジストログリカン糖鎖を解析するために各組織を回収した。
(2) Antisense nucleic acid administration PMO no. Four types of PMO, 9, 27, 39 and 45 were used.
Hp / − mice were orally administered cardiotoxin 10 μM (SIGMA) in three places, intratibial muscle (0.4 nmol), gastrocnemius muscle (2 nmol) and quadriceps femoris muscle (1 nmol). Two mice were used for each dose of PMO. The day of cardiotoxin administration was day 0, and on day 3, PMO (20, 60, 200 mg / kg) dissolved in 5% mannitol was administered via the tail vein. The mice were euthanized on the 23rd day, and each tissue was recovered for analysis of fukutin protein and α-dystroglycan sugar chain.
（３）フクチンタンパク質の検出
抗フクチンウサギポリクローナル抗体ＲＹ２１３は、ペプチドCLKIESKDPRLDGIDS（配列番号７２）を認識し、抗フクチンヤギポリクローナル抗体１０６Ｇ２はＮ末端の疎水性ドメインを欠失している全長フクチンを認識する。フクチンは、筋組織１５〜３０ｍｇから１０倍量の溶解バッファー（1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 20mM Tris-HCl および150mM NaCl）を用いて得た組織溶解液から１０６Ｇ２を用いた免疫沈降法により検出し、続いてアフィニティー精製したＲＹ２１３を用いるウエスタンブロット分析に供した。
(3) Detection of fukutin protein The anti-fuctin rabbit polyclonal antibody RY213 recognizes the peptide CLKIESKDPRLDGIDS (SEQ ID NO: 72), and the anti-fuctin goat polyclonal antibody 106G2 recognizes full-length fukutin lacking the hydrophobic domain at the N-terminus . Fukutin is a 106G2 solution from tissue lysates obtained using 15-30 mg of muscle tissue and 10 volumes of lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 20 mM Tris-HCl and 150 mM NaCl) Detection by immunoprecipitation used was followed by Western blot analysis using affinity purified RY213.
（４）ジストログリカンの検出
筋組織に１０倍量の溶解バッファー（1% Triton TBS）を加えて組織溶解液を得た。溶解液９μＬを、実施例２と同じ３種類の抗体（抗αジストログリカンラットモノクローナル抗体、抗糖鎖修飾αジストログリカンマウスモノクローナル抗体ＩＩＨ６、抗βジストログリカンマウスモノクローナル抗体８Ｄ５）を用いたウエスタンブロット分析に供した。
(4) Detection of dystroglycan A tissue lysate was obtained by adding 10 volumes of a lysis buffer (1% Triton TBS) to muscle tissue. Western blot analysis using 9 μL of the lysate as the same three antibodies as in Example 2 (anti-α dystroglycan rat monoclonal antibody, anti-carbohydrate modified α dystroglycan mouse monoclonal antibody IIH6, anti-β dystroglycan mouse monoclonal antibody 8D5) Provided for.
（５）実験結果
ＰＭＯ Ｎｏ．９のアンチセンス核酸の結果を図３に、ＰＭＯ Ｎｏ．２７のアンチセンス核酸の結果を図４に、ＰＭＯ Ｎｏ．３９のアンチセンス核酸の結果およびＰＭＯ Ｎｏ．４５のアンチセンス核酸の結果を図５にそれぞれ示した。いずれも大腿四頭筋におけるジストログリカンおよびフクチンタンパク質の結果である。図３、図４および図５から明らかなように、４種類のアンチセンス核酸のいずれを投与した場合においても、アンチセンス核酸の濃度依存的にαジストログリカンの糖鎖および正常フクチンタンパク質が回復していることが確認できた。
(5) Experimental results PMO No. The results of the antisense nucleic acid of 9 are shown in FIG. The results of 27 antisense nucleic acids are shown in FIG. Results of 39 antisense nucleic acids and PMO no. The results of 45 antisense nucleic acids are shown in FIG. 5 respectively. Both are the result of dystroglycan and fukutin protein in quadriceps. As is clear from FIGS. 3, 4 and 5, even when any of the four types of antisense nucleic acid is administered, the sugar chain and alpha fulctoglycan of alpha dystroglycan are recovered in a concentration dependent manner of the antisense nucleic acid. Was confirmed.
〔実施例１３：アンチセンス核酸の生理食塩水に対する溶解性〕
（１）実験方法
ＰＭＯ５．０ｍｇの入った２．２ｍＬサンプル瓶（透明）に注射用水８０．０μＬを加え、超音波とボルテックスを使って溶解させた後、５倍濃度生理食塩水２０．０μＬを加え、ボルテックスを使って撹拌し５０ｍｇ／ｍＬの生理食塩水溶液とした。室温で２４時間放置し、沈殿が生じないものを溶解性が高い配列と評価した。
Example 13 Solubility of Antisense Nucleic Acid in Saline
(1) Experimental method 80.0 μL of water for injection is added to a 2.2 mL sample bottle (transparent) containing 5.0 mg of PMO, dissolved using ultrasound and vortex, and then 20.0 μL of 5 times concentration saline In addition, it was stirred using a vortex into a 50 mg / mL saline solution. It was left at room temperature for 24 hours, and those which did not form a precipitate were evaluated as highly soluble sequences.
（２）評価結果
ＰＭＯ Ｎｏ．１３、１４、２１、２２、２７、２８、３２、３８、４８、５０、５３〜５６、５８〜６６、６９〜７３は、生理食塩水に対して５０ｍｇ／ｍＬ以上の溶解性を示した。生理食塩水に対する溶解性の高いこれら２８種類のＰＭＯは、医薬として利用価値が高いアンチセンス核酸であると考えられた。
(2) Evaluation results PMO No. 13, 14, 21, 22, 27, 28, 32, 38, 48, 50, 53 to 56, 58 to 66, and 69 to 73 exhibited a solubility of 50 mg / mL or more in saline. These 28 types of PMO highly soluble in saline were considered to be antisense nucleic acids having high utility as pharmaceuticals.
〔実施例１４：アンチセンス核酸の安全性評価〕
（１）実験方法
ＰＭＯを５％グルコース水溶液に溶解し、Ｃ５７ＢＬ／６Ｊ雄性６週齢マウスの尾静脈内へ投与した。翌日、マウスより血清を回収し、血中アスパラギン酸アミノトランスフェラーゼ（ＡＳＴ）値、アラニンアミノトランスフェラーゼ（ＡＬＴ）値、尿素窒素量（ＢＵＮ）値およびクレアチニン値を測定した。媒体として使用した５％グルコース水溶液のみを投与したマウスもしくは無処置マウスの測定値を正常値とし、正常値の１．３倍を超える上昇が見られた場合、異常値と判定した。６０ｍｇ／ｋｇ以下の用量においてＡＳＴ値、ＡＬＴ値、ＢＵＮ値およびクレアチニン値のいずれにも異常値がみられない場合、安全性が高いアンチセンス核酸であると判定した。
[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 a C57BL / 6J male 6-week-old mouse. The next day, serum was collected from the mice and blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN) and creatinine values were measured. The measured value of a mouse that received only the 5% glucose aqueous solution used as a vehicle or an untreated mouse was regarded as a normal value, and when an increase of more than 1.3 times the normal value was observed, it was judged as an abnormal value. When no abnormal value was found in any of AST value, ALT value, BUN value and creatinine value at a dose of 60 mg / kg or less, it was determined to be a highly safe antisense nucleic acid.
（２）評価結果
ＰＭＯ Ｎｏ．２７、４５、４６、７１、５５、５７、６０、６１、６２、６６、７３について、６０ｍｇ／ｋｇ以下の用量においてＡＳＴ値、ＡＬＴ値、ＢＵＮ値およびクレアチニン値のいずれにも異常値がみられず、安全性が高いアンチセンス核酸であることを確認した。
(2) Evaluation results PMO No. Abnormal values were observed for all of AST, ALT, BUN and creatinine at doses of 60 mg / kg or less for 27, 45, 46, 71, 55, 57, 60, 61, 62, 66, 73 It was confirmed that the nucleic acid was a highly safe antisense nucleic acid.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 The present invention is not limited to the above-described embodiments and examples, but various modifications are possible within the scope of the claims, and the technical means disclosed in each of the different embodiments may be combined as appropriate. The resulting embodiments are also included in the technical scope of the present invention. Also, all of the academic literature and patent literature described in the present specification are incorporated herein by reference.
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