JP2018011593A - Nucleic acid for inhibiting expression of mex3b gene, mex3b gene expression inhibitor, method for inhibiting mex3b gene expression, and prophylactic or therapeutic agent for disease caused by mex3b gene expression - Google Patents
Nucleic acid for inhibiting expression of mex3b gene, mex3b gene expression inhibitor, method for inhibiting mex3b gene expression, and prophylactic or therapeutic agent for disease caused by mex3b gene expression Download PDFInfo
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- JP2018011593A JP2018011593A JP2017133718A JP2017133718A JP2018011593A JP 2018011593 A JP2018011593 A JP 2018011593A JP 2017133718 A JP2017133718 A JP 2017133718A JP 2017133718 A JP2017133718 A JP 2017133718A JP 2018011593 A JP2018011593 A JP 2018011593A
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Abstract
Description
本発明は、ＭＥＸ３Ｂ遺伝子の発現を抑制する核酸、上記核酸を含むＭＥＸ３Ｂ遺伝子発現抑制剤、ＭＥＸ３Ｂ遺伝子発現を抑制する方法及びＭＥＸ３Ｂ遺伝子発現に起因する疾病の予防又は治療剤に関する。 The present invention relates to a nucleic acid that suppresses MEX3B gene expression, a MEX3B gene expression inhibitor that contains the nucleic acid, a method that suppresses MEX3B gene expression, and a preventive or therapeutic agent for a disease caused by MEX3B gene expression.
ＭＥＸ３Ｂ遺伝子は元来ＴＧＦ−βにより活性化される遺伝子として同定され、その後の解析から、ＭＥＸ３Ｂタンパク質は種々のｍＲＮＡに結合してそれらｍＲＮＡの機能（つまりタンパク質への翻訳）を制御する分子であることが知られている（例えば、非特許文献１）。
また、ＭＥＸ３Ｂタンパク質はアポトーシスを誘導するタンパク質であることが知られている（例えば、特許文献１）。
アポトーシスは、正常な生理学的過程以外にも、神経変性疾患等の重篤な疾患の発症にも関与していることが知られている。例えば、神経変性疾患（例えば、アルツハイマー病、パーキンソン病、筋萎縮性側索硬化症、ハンチントン舞踏病など）は、アポトーシスの異常な亢進が原因と考えられている（例えば、非特許文献２）。
The MEX3B gene was originally identified as a gene activated by TGF-β, and from subsequent analysis, the MEX3B protein is a molecule that binds to various mRNAs and regulates the function of those mRNAs (ie, translation into proteins). It is known (for example, Non-Patent Document 1).
Moreover, it is known that MEX3B protein is a protein which induces apoptosis (for example, patent document 1).
Apoptosis is known to be involved in the development of serious diseases such as neurodegenerative diseases in addition to normal physiological processes. For example, neurodegenerative diseases (for example, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea, etc.) are considered to be caused by abnormally increased apoptosis (for example, Non-Patent Document 2).
このような背景から、ＭＥＸ３Ｂ遺伝子の発現を抑制できる核酸医薬品の開発が求められている。 From such a background, development of a nucleic acid drug capable of suppressing the expression of the MEX3B gene has been demanded.
本発明は、上記事情に鑑みてなされたものであり、細胞内において、ＭＥＸ３Ｂ遺伝子の発現を抑制することができる核酸、上記核酸を含むＭＥＸ３Ｂ遺伝子発現抑制剤、ＭＥＸ３Ｂ遺伝子発現を抑制する方法及びＭＥＸ３Ｂ遺伝子発現に起因する疾病の予防又は治療剤の提供を目的とする。 This invention is made | formed in view of the said situation, The nucleic acid which can suppress the expression of MEX3B gene in a cell, the MEX3B gene expression inhibitor containing the said nucleic acid, the method of suppressing MEX3B gene expression, and MEX3B The object is to provide a preventive or therapeutic agent for diseases caused by gene expression.
本発明者らは、ＭＥＸ３Ｂのゲノム遺伝子及びｍＲＮＡ前駆体中に存在し、スプライシングにより除去されてｍＲＮＡ中には存在しないイントロン中のオリゴヌクレオチドに相補的な配列を有する核酸をヒト肺がん細胞に供与することによりＭＥＸ３Ｂ遺伝子のｍＲＮＡ発現が抑制されることを見出し、本発明を完成するに至った。
具体的には、本発明は以下の通りである。
The present inventors donate to human lung cancer cells a nucleic acid having a sequence complementary to an oligonucleotide in an intron that is present in the genomic gene and mRNA precursor of MEX3B, removed by splicing, and not present in mRNA. As a result, it was found that mRNA expression of the MEX3B gene was suppressed, and the present invention was completed.
Specifically, the present invention is as follows.
本発明の第１の態様は、
ＭＥＸ３Ｂ遺伝子のイントロン領域中の連続する少なくとも１０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有し、ＭＥＸ３Ｂ遺伝子の発現を抑制する核酸である。
The first aspect of the present invention is:
A nucleic acid having a sequence complementary to an oligonucleotide containing at least 10 consecutive nucleotides in the intron region of the MEX3B gene and suppressing the expression of the MEX3B gene.
本発明の第２の態様は、
第１の態様に係る核酸を含むＭＥＸ３Ｂ遺伝子発現抑制剤である。
本発明の第３の態様は、
第２の態様に係る剤を対象（ヒトの個体を除く。）に接触させることを含む、ＭＥＸ３Ｂ遺伝子発現を抑制する方法である。
本発明の第４の態様は、
第２の態様に係る剤を含む、ＭＥＸ３Ｂ遺伝子発現に起因する疾病の予防又は治療剤である。
The second aspect of the present invention is:
It is a MEX3B gene expression inhibitor containing the nucleic acid which concerns on a 1st aspect.
The third aspect of the present invention is:
It is a method for suppressing MEX3B gene expression, which comprises contacting an agent according to the second aspect with a subject (excluding a human individual).
The fourth aspect of the present invention is:
A prophylactic or therapeutic agent for a disease caused by MEX3B gene expression, comprising the agent according to the second aspect.
細胞内において、ＭＥＸ３Ｂ遺伝子の発現を抑制することができる核酸、上記核酸を含むＭＥＸ３Ｂ遺伝子発現抑制剤、ＭＥＸ３Ｂ遺伝子発現を抑制する方法及びＭＥＸ３Ｂ遺伝子発現に起因する疾病の予防又は治療剤を提供することができる。
上記核酸、上記ＭＥＸ３Ｂ遺伝子発現抑制剤、上記予防又は治療剤は、副作用としての細胞毒性が少ない。
副作用として細胞毒性が少ない理由は、エクソン（特に、アミノ酸をコードする領域（ＣＤＳ））に比べ、イントロンの方が、ＭＥＸ３Ｂホモログとの相同性が低く、オフターゲット効果が生じ難いことに基づくものと推測される。
To provide a nucleic acid capable of suppressing the expression of MEX3B gene in a cell, a MEX3B gene expression inhibitor containing the nucleic acid, a method for suppressing MEX3B gene expression, and a preventive or therapeutic agent for diseases caused by MEX3B gene expression. Can do.
The nucleic acid, the MEX3B gene expression inhibitor, and the preventive or therapeutic agent have low cytotoxicity as a side effect.
The reason for the low cytotoxicity as a side effect is that introns are less homologous to MEX3B homologs than exons (particularly the amino acid coding region (CDS)), and are less likely to produce off-target effects. Guessed.
以下、本発明の実施態様について詳細に説明するが、本発明は、以下の実施態様に何ら限定されるものではなく、本発明の目的の範囲内において、適宜変更を加えて実施することができる。 Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention. .
（ＭＥＸ３Ｂ遺伝子）
ＭＥＸ３Ｂ遺伝子はエクソン１、イントロン及びエクソン２を含み、この構成はヒト、マウス、その他の哺乳類において高度に保存されている。
後記の配列番号１はヒトＭＥＸ３Ｂ遺伝子のイントロン領域の８３６塩基を示す。ヒトＭＥＸ３ＢのｍＲＮＡをコードするヒトＭＥＸ３Ｂ遺伝子は後記の配列番号２で表される配列を有する。ヒトＭＥＸ３Ｂ遺伝子において、上記イントロン領域は、配列番号２で表される配列における６９２番目の塩基と６９３番目の塩基との間に存在する。
配列番号１０は、スプライシング前のヒトＭＥＸ３ＢのプレｍＲＮＡをコードする塩基配列を示す。配列番号１０で表されるヒトＭＥＸ３ＢのプレｍＲＮＡをコードする配列における６９３〜１５２８番目の領域が、配列番号１で表されるヒトＭＥＸ３Ｂ遺伝子のイントロン領域に相当する。
マウスＭＥＸ３ＢのｍＲＮＡをコードするマウスＭＥＸ３Ｂ遺伝子は後記の配列番号３で表される配列を有する。
また、ＭＥＸ３Ｂタンパク質（例えば、後記の配列番号４又は５で表されるアミノ酸配列を有するタンパク質）をコードする遺伝子は全てＭＥＸ３Ｂ遺伝子に属する。
(MEX3B gene)
The MEX3B gene contains exon 1, intron, and exon 2, and this organization is highly conserved in humans, mice, and other mammals.
Sequence number 1 of a postscript shows 836 bases of the intron area | region of a human MEX3B gene. The human MEX3B gene encoding human MEX3B mRNA has a sequence represented by SEQ ID NO: 2 described later. In the human MEX3B gene, the intron region is present between the 692nd base and the 693rd base in the sequence represented by SEQ ID NO: 2.
SEQ ID NO: 10 shows the nucleotide sequence encoding human MEX3B pre-mRNA before splicing. The 693-1528th region in the sequence encoding the human MEX3B pre-mRNA represented by SEQ ID NO: 10 corresponds to the intron region of the human MEX3B gene represented by SEQ ID NO: 1.
The mouse MEX3B gene encoding mouse MEX3B mRNA has a sequence represented by SEQ ID NO: 3 described later.
In addition, all genes encoding MEX3B protein (for example, a protein having an amino acid sequence represented by SEQ ID NO: 4 or 5 described below) belong to the MEX3B gene.
（ＭＥＸ３Ｂ遺伝子の取得）
ＭＥＸ３Ｂ遺伝子の取得方法は特に限定されない。本明細書の配列表の配列番号２〜５及び１０に記載した塩基配列およびアミノ酸配列の情報に基づいて適当なブローブやプライマーを調製し、それらを用いて、ヒトｃＤＮＡライブラリー（ＭＥＸ３Ｂ遺伝子が発現される適当な細胞より常法に従い調製したもの）から所望クローンを選択することにより、ＭＥＸ３Ｂ遺伝子を単離することができる。
(Acquisition of MEX3B gene)
The method for obtaining the MEX3B gene is not particularly limited. Appropriate probes and primers are prepared based on the nucleotide sequence and amino acid sequence information shown in SEQ ID NOs: 2 to 5 and 10 in the sequence listing of the present specification, and a human cDNA library (MEX3B gene is expressed using them) MEX3B gene can be isolated by selecting a desired clone from a suitable cell prepared according to a conventional method.
＜ＭＥＸ３Ｂ遺伝子の発現を抑制する核酸＞
第１の態様に係るＭＥＸ３Ｂ遺伝子の発現を抑制する核酸（以下、単に「第１の態様に係る核酸」ともいう。）は、ＭＥＸ３Ｂ遺伝子のイントロン領域中の連続する少なくとも１０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有する。
上記イントロンは、スプライシングにより除去されるので、ＭＥＸ３Ｂのゲノム遺伝子及びｍＲＮＡ前駆体中に存在し、かつｍＲＮＡ中には存在しない。
第１の態様に係る核酸は、ＭＥＸ３Ｂ遺伝子を発現する細胞の細胞質内のみならず、核内まで到達することができ、上記イントロンに作用してＭＥＸ３Ｂのゲノム遺伝子又はｍＲＮＡ前駆体を分解することにより、ＭＥＸ３ＢのｍＲＮＡ発現を抑制することができる。
例えば、上記イントロン中に含まれるオリゴヌクレオチドと、それと相補的な第１の態様に係る核酸とがハイブリッド形成することにより、生じたハイブリッド二本鎖に特異的なヌクレアーゼ（例えば、ＲＮアーゼＨ）によりヌクレオチド鎖が分解されることが好ましい。
第１の態様に係る核酸は、ＤＮＡであってもＲＮＡであってもよいが、ｍＲＮＡ前駆体中のイントロンに含まれるオリゴヌクレオチドとハイブリッド形成する観点から、ＤＮＡであることが好ましい。
<Nucleic acid that suppresses expression of MEX3B gene>
The nucleic acid that suppresses the expression of the MEX3B gene according to the first aspect (hereinafter simply referred to as “the nucleic acid according to the first aspect”) is an oligonucleotide containing at least 10 consecutive nucleotides in the intron region of the MEX3B gene. It has a complementary sequence.
Since the intron is removed by splicing, it is present in the genomic gene and mRNA precursor of MEX3B, but not in mRNA.
The nucleic acid according to the first aspect can reach not only the cytoplasm of the cell expressing the MEX3B gene but also the nucleus, and acts on the intron to degrade the genomic gene or mRNA precursor of MEX3B. , MEX3B mRNA expression can be suppressed.
For example, the oligonucleotide contained in the intron and the nucleic acid according to the first aspect complementary to the oligonucleotide are hybridized, and thus the resulting nuclease specific to the hybrid duplex (for example, RNase H) It is preferred that the nucleotide chain is degraded.
The nucleic acid according to the first embodiment may be DNA or RNA, but is preferably DNA from the viewpoint of hybridization with an oligonucleotide contained in an intron in the mRNA precursor.
第１の態様に係る核酸としては、ＭＥＸ３Ｂ遺伝子のイントロン領域中の連続する少なくとも１０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが好ましく、少なくとも１１ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることがより好ましく、少なくとも１２ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが更に好ましく、少なくとも１３ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが特に好ましく、少なくとも１４ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが最も好ましい。 The nucleic acid according to the first aspect is preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 10 consecutive nucleotides in the intron region of the MEX3B gene, and an oligonucleotide containing at least 11 nucleotides More preferably, it is an antisense oligonucleotide having a sequence complementary to the oligonucleotide, more preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide comprising at least 12 nucleotides, and an oligonucleotide comprising at least 13 nucleotides It is particularly preferred that the antisense oligonucleotide has a sequence complementary to the antisense, and the antisense has a sequence complementary to an oligonucleotide comprising at least 14 nucleotides And most preferably Rigo nucleotides.
また、第１の態様に係る核酸の塩基長の上限値としては、ＭＥＸ３Ｂ遺伝子のイントロン領域中の連続する４０ヌクレオチド以下のオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが好ましく、連続する３０ヌクレオチド以下のオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることがより好ましく、連続する２５ヌクレオチド以下のオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが更に好ましく、連続する２０ヌクレオチド以下のオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが特に好ましく、連続する１７ヌクレオチド以下のオリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドであることが最も好ましい。
第１の態様に係る核酸としては、例えば、ＭＥＸ３Ｂ遺伝子のイントロン領域中の連続する１２〜２０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有する核酸が挙げられ、上記イントロン領域を表す配列番号１における２８０〜８２２番目の配列中の連続する１２〜２０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有する核酸又はヒトＭＥＸ３ＢのプレｍＲＮＡを表す配列番号１０における８４９〜１２５８番目の配列中の連続する１２〜２０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有する核酸であることが好ましく、上記イントロン領域を表す配列番号１における２８０〜２９５番目のオリゴヌクレオチド、７０８〜７２３番目のオリゴヌクレオチド、７２１〜７３６番目のオリゴヌクレオチド、７３０〜７４５番目のオリゴヌクレオチド若しくは８０７〜８２２番目のオリゴヌクレオチド又はヒトＭＥＸ３ＢのプレｍＲＮＡを表す配列番号１０における８４９〜８６４番目のオリゴヌクレオチド、８８９〜９０４番目のオリゴヌクレオチド、９０８〜９２３番目のオリゴヌクレオチド、１０９３〜１１０８番目のオリゴヌクレオチド若しくは１２４３〜１２５８番目のオリゴヌクレオチドに相補的な配列を有する核酸であることがより好ましく、上記イントロン領域を表す配列番号１における７０８〜８２２番目の配列中の連続する１２〜２０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有する核酸又はヒトＭＥＸ３ＢのプレｍＲＮＡを表す配列番号１０における８８９〜１１０８番目の配列中の連続する１２〜２０ヌクレオチドを含むオリゴヌクレオチドに相補的な配列を有する核酸であることが更に好ましく、上記イントロン領域を表す配列番号１における７０８〜７２３番目のオリゴヌクレオチド、７２１〜７３６番目のオリゴヌクレオチド若しくは８０７〜８２２番目のオリゴヌクレオチド又はヒトＭＥＸ３ＢのプレｍＲＮＡを表す配列番号１０における９０８〜９２３番目のオリゴヌクレオチドに相補的な配列を有する核酸であることが特に好ましく、上記イントロン領域を表す配列番号１における７０８〜７２３番目のオリゴヌクレオチド若しくは７２１〜７３６番目のオリゴヌクレオチド又はヒトＭＥＸ３ＢのプレｍＲＮＡを表す配列番号１０における９０８〜９２３番目のオリゴヌクレオチドに相補的な配列を有する核酸であることが最も好ましい。
なお、配列番号１０における８４９〜８６４番目のオリゴヌクレオチド、８８９〜９０４番目のオリゴヌクレオチド、９０８〜９２３番目のオリゴヌクレオチド、１０９３〜１１０８番目のオリゴヌクレオチド及び１２４３〜１２５８番目のオリゴヌクレオチドは、それぞれ、上記イントロン領域を表す配列番号１における１５７〜１７２番目のオリゴヌクレオチド、１９７〜２１２番目のオリゴヌクレオチド、２１６〜２３１番目のオリゴヌクレオチド、４０１〜４１６番目のオリゴヌクレオチド及び５５１〜５６６番目のオリゴヌクレオチドに相当する。
The upper limit of the base length of the nucleic acid according to the first aspect is preferably an antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 40 nucleotides or less in the intron region of the MEX3B gene, More preferably, it is an antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 30 nucleotides or less, and further it is an antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 25 nucleotides or less. An antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 20 nucleotides or less is particularly preferred, and an antisense oligonucleotide having a sequence complementary to a continuous oligonucleotide of 17 nucleotides or less is preferred. And most preferably in the range of scan oligonucleotide.
Examples of the nucleic acid according to the first aspect include a nucleic acid having a sequence complementary to an oligonucleotide containing 12 to 20 consecutive nucleotides in the intron region of the MEX3B gene, and in SEQ ID NO: 1 representing the intron region. A nucleic acid having a sequence complementary to an oligonucleotide containing 12 to 20 nucleotides in the sequence of 280 to 822 or a premRNA of human MEX3B, and a sequence of 12 to 12 in the sequence of 849 to 1258 in SEQ ID NO: 10 It is preferably a nucleic acid having a sequence complementary to an oligonucleotide containing 20 nucleotides, the 280-295th oligonucleotide, the 708-723th oligonucleotide, the 721-736th nucleotide in SEQ ID NO: 1 representing the intron region. Oligonucleotide The 730-745th oligonucleotide, the 807-822th oligonucleotide, or the 849-864th oligonucleotide, the 889-904th oligonucleotide, the 908-923rd oligonucleotide in SEQ ID NO: 10 representing the pre-mRNA of human MEX3B More preferably, the nucleic acid has a sequence complementary to the oligonucleotides 1093 to 1108 or the oligonucleotides 1243 to 1258, and is continuous in the sequences 708 to 822 in SEQ ID NO: 1 representing the intron region. A nucleic acid having a sequence complementary to an oligonucleotide containing 12 to 20 nucleotides or a sequence of 12 to 20 nucleotides in the 889th to 1108th sequence in SEQ ID NO: 10 representing the pre-mRNA of human MEX3B More preferably, it is a nucleic acid having a sequence complementary to an oligonucleotide containing a leotide, the 708th to 723rd oligonucleotide, the 721th to 736th oligonucleotide or the 807th to 822th oligonucleotide in SEQ ID NO: 1 representing the intron region. Particularly preferred is a nucleic acid having a sequence complementary to the 908th to 923rd oligonucleotides in SEQ ID NO: 10 representing the oligonucleotide or human MEX3B pre-mRNA, and the 708th to 723rd positions in SEQ ID NO: 1 representing the intron region. Most preferred is a nucleic acid having a sequence complementary to the oligonucleotides 908 to 923 in SEQ ID NO: 10 representing the oligonucleotide or the oligonucleotides 721 to 736 or human MEX3B pre-mRNA. That's right.
The 849-864th oligonucleotide, the 889-904th oligonucleotide, the 908-923rd oligonucleotide, the 1093-1108th oligonucleotide and the 1243-1258th oligonucleotide in SEQ ID NO: 10 are respectively It corresponds to the 157th to 172nd oligonucleotides in SEQ ID NO: 1 representing the intron region, the 197th to 212th oligonucleotides, the 216th to 231st oligonucleotides, the 401st to 416th oligonucleotides and the 551st to 566th oligonucleotides .
第１の態様に係る核酸としては、ホスホロチオエート構造、架橋構造及びアルコキシ構造よりなる群から選択される少なくとも１つの構造を有するヌクレオチドを少なくとも１つ含むアンチセンスオリゴヌクレオチドが好ましい。
例えば、ヌクレオチド同士をつなぐリン酸ジエステル結合部がホスホロチオエート構造を有することにより、ヌクレアーゼ耐性を獲得することができ、また、疎水性が向上することから細胞内又は核内への取り込みも向上することができる。
また、ヌクレオチドの糖部が、２’，４’−ＢＮＡ（２’，４’−Ｂｒｉｄｇｅｄ Ｎｕｃｌｅｉｃ Ａｃｉｄ；別名ＬＮＡ（Ｌｏｃｋｅｄ Ｎｕｃｌｅｉｃ Ａｃｉｄ））、ＥＮＡ（２’−Ｏ，４’−Ｃ−Ｅｔｈｙｌｅｎｅ−ｂｒｉｄｇｅｄ Ｎｕｃｌｅｉｃ Ａｃｉｄ）等の架橋構造、２’−Ｏ−メチル化、２’−Ｏ−メトキシエチル化（２’−ＭＯＥ）等のアルコキシ構造を有することにより、ヌクレアーゼ耐性獲得及びｍＲＮＡの結合能を向上することができる。
The nucleic acid according to the first aspect is preferably an antisense oligonucleotide comprising at least one nucleotide having at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure, and an alkoxy structure.
For example, a phosphodiester bond that connects nucleotides to each other has a phosphorothioate structure, so that nuclease resistance can be obtained, and since hydrophobicity is improved, incorporation into cells or nuclei can be improved. it can.
In addition, the sugar part of the nucleotide is 2 ′, 4′-BNA (2 ′, 4′-Bridged Nucleic Acid; also known as LNA (Locked Nucleic Acid)), ENA (2′-O, 4′-C-Ethylene-bridged Nucleic Acid) etc. have a cross-linked structure, 2′-O-methylation, 2′-O-methoxyethylation (2′-MOE) and other alkoxy structures, thereby improving nuclease resistance acquisition and mRNA binding ability be able to.
第１の態様に係る核酸において、ヌクレオチド同士をつなぐ少なくとも１つのリン酸ジエステル結合部がホスホロチオエート構造を有することが好ましく、第１の態様に係る核酸中のリン酸ジエステル結合のうちの５０％以上がホスホロチオエート構造を有することがより好ましく、第１の態様に係る核酸中のリン酸ジエステル結合のうちの７０％以上がホスホロチオエート構造を有することが更に好ましく、第１の態様に係る核酸中のリン酸ジエステル結合のうちの９０％以上がホスホロチオエート構造を有することが特に好ましく、第１の態様に係る核酸の全てのリン酸ジエステル結合がホスホロチオエート構造を有することが最も好ましい。
第１の態様に係る核酸において、少なくともいずれか一方の末端のヌクレオチドが架橋構造又はアルコキシ構造を有することが好ましく、第１の態様に係る核酸の両末端のヌクレオチドが架橋構造又はアルコキシ構造を有することがより好ましく（いわゆるギャップマー（Ｇａｐｍｅｒ）型アンチセンスオリゴヌクレオチド）、第１の態様に係る核酸の両末端において、独立して、末端から４塩基までが架橋構造又はアルコキシ構造を有することが更に好ましく、末端から２又は３塩基が架橋構造又はアルコキシ構造を有することが特に好ましい。
第１の態様に係る核酸は、ＤＮＡ合成機及び公知の有機合成技術を用いて常法により製造することができる。
In the nucleic acid according to the first aspect, it is preferable that at least one phosphodiester bond connecting nucleotides has a phosphorothioate structure, and 50% or more of the phosphodiester bonds in the nucleic acid according to the first aspect More preferably, it has a phosphorothioate structure, and 70% or more of the phosphodiester bonds in the nucleic acid according to the first aspect further have a phosphorothioate structure, and the phosphodiester in the nucleic acid according to the first aspect. It is particularly preferred that 90% or more of the bonds have a phosphorothioate structure, and most preferred that all phosphodiester bonds of the nucleic acid according to the first aspect have a phosphorothioate structure.
In the nucleic acid according to the first aspect, at least one of the terminal nucleotides preferably has a crosslinked structure or an alkoxy structure, and the nucleotides at both ends of the nucleic acid according to the first aspect have a crosslinked structure or an alkoxy structure. Is more preferable (so-called gapmer type antisense oligonucleotide), and at both ends of the nucleic acid according to the first aspect, it is more preferable that from the end to 4 bases have a crosslinked structure or an alkoxy structure. It is particularly preferable that 2 or 3 bases from the terminal have a crosslinked structure or an alkoxy structure.
The nucleic acid according to the first aspect can be produced by a conventional method using a DNA synthesizer and a known organic synthesis technique.
インビボ（ｉｎ ｖｉｖｏ）又はインビトロ（ｉｎ ｖｉｔｒｏ）において、細胞内、好ましくは核内への取り込みは、第１の態様に係る核酸を細胞に接触させること、例えば、任意の細胞を培養する培地中に、第１の態様に係る核酸を添加することにより達成することができるが、第１の態様に係る核酸が、ホスホロチオエート構造、架橋構造及びアルコキシ構造よりなる群から選択される少なくとも１つの構造を有することにより、細胞内、好ましくは核内への取り込みを更に向上させることができる。
第１の態様に係る核酸は、ホスホロチオエート構造、架橋構造及びアルコキシ構造よりなる群から選択される少なくとも１つの構造を有することと、後述するリポフェクション用担体とを組み合わせて用いることにより細胞内、好ましくは核内への取り込みを更に向上させることができる。
In vivo or in vitro, intracellular uptake, preferably uptake into the nucleus, can be achieved by contacting the nucleic acid according to the first aspect with the cell, eg, in the culture medium of any cell. The nucleic acid according to the first aspect can be achieved by adding the nucleic acid according to the first aspect, but the nucleic acid according to the first aspect has at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure, and an alkoxy structure. Thereby, the uptake into cells, preferably into the nucleus, can be further improved.
The nucleic acid according to the first aspect has an intracellular structure, preferably by using a combination of at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure, and an alkoxy structure and a carrier for lipofection described later. Incorporation into the nucleus can be further improved.
第１の態様に係る核酸の細胞への導入方法としては、適当なベクター中に挿入し、更に適当な宿主細胞に導入する方法であってもよい。
上記適当なベクターの種類は特に限定されず、例えば、自律的に複製するベクター（例えば、プラスミド等）でもよいが、宿主細胞に導入された際に宿主細胞のゲノムに組み込まれ、組み込まれた染色体と共に複製されるものであることが好ましい。
上記適当なベクターとしては、大腸菌由来のプラスミド（例、ｐＢＲ３２２、ｐＵＣ１１８その他）、枯草菌由来のプラスミド（例、ｐＵＢ１１０、ｐＳＨ１９その他）、さらにバクテリオファージやレトロウイルスやワクシニアウイルス等の動物ウイルス等が利用できる。組み換えに際しては、適当な合成ＤＮＡアダプターを用いて翻訳開始コドンや翻訳終止コドンを付加することも可能である。
The method for introducing the nucleic acid into the cell according to the first aspect may be a method in which the nucleic acid is inserted into a suitable vector and further introduced into a suitable host cell.
The type of the appropriate vector is not particularly limited, and may be, for example, an autonomously replicating vector (for example, a plasmid). However, when it is introduced into the host cell, it is integrated into the host cell genome and integrated into the chromosome. It is preferable to be duplicated together.
Examples of suitable vectors include plasmids derived from E. coli (eg, pBR322, pUC118, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pSH19, etc.), and animal viruses such as bacteriophages, retroviruses and vaccinia viruses. it can. In recombination, a translation initiation codon and a translation termination codon can be added using an appropriate synthetic DNA adapter.
また、第１の態様に係る核酸は、必要に応じて、例えばヒト成長ホルモンターミネーター又は真菌宿主についてはＴＰＩ１ターミネーター若しくはＡＤＨ３ターミネーターのような適切なターミネーターに機能的に結合されていてもよい。組み換えベクターは更に、ポリアデニレーションシグナル（例えばＳＶ４０またはアデノウイルス５Ｅ１ｂ領域由来のもの）、転写エンハンサー配列（例えばＳＶ４０エンハンサー）及び翻訳エンハンサー配列（例えばアデノウイルスＶＡＲＮＡをコードするもの）のような要素を有していてもよい。
組み換えベクターは更に、該ベクターが宿主細胞内で複製することを可能にするＤＮＡ配列を具備してもよく、その一例としてはＳＶ４０複製起点（宿主細胞が哺乳類細胞のとき）が挙げられる。
組み換えベクターはさらに選択マーカーを含有してもよい。選択マーカーとしては、例えば、ジヒドロ葉酸レダクターゼ（ＤＨＦＲ）またはシゾサッカロマイセス・ポンベＴＰＩ遺伝子等のようなその補体が宿主細胞に欠けている遺伝子、又は例えばアンピシリン、カナマイシン、テトラサイクリン、クロラムフェニコール、ネオマイシン若しくはヒグロマイシンのような薬剤耐性遺伝子を挙げることができる。
Moreover, the nucleic acid according to the first aspect may be operably linked to an appropriate terminator such as a human growth hormone terminator or a TPI1 terminator or an ADH3 terminator for a fungal host, if necessary. The recombinant vector further has elements such as polyadenylation signals (eg, from SV40 or adenovirus 5E1b region), transcription enhancer sequences (eg, SV40 enhancer) and translation enhancer sequences (eg, those encoding adenovirus VARNA). You may do it.
The recombinant vector may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell).
The recombinant vector may further contain a selection marker. Selectable markers include, for example, genes that lack their complement in host cells, such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or such as ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of drug resistance genes such as neomycin or hygromycin.
第１の態様に係る核酸又はそれを含むベクターを導入される宿主細胞は、高等真核細胞、細菌、酵母、真菌等が挙げられるが、哺乳類細胞であることが好ましい。
哺乳類細胞の例としては、ＨＥＫ２９３細胞、ＨｅＬａ細胞、ＣＯＳ細胞（例えば、ＣＯＳ−７細胞など）、ＢＨＫ細胞、ＣＨＬ細胞またはＣＨＯ細胞、ＢＡＬＢ／ｃマウス細胞（例えば、ＢＡＬＢ／ｃマウス胎児繊維芽細胞）等が挙げられる。哺乳類細胞を形質転換し、該細胞に導入された遺伝子を発現させる方法も公知であり、例えば、リポフェクション法、エレクトロポレーション法、リン酸カルシウム法等を用いることができる。
Examples of the host cell into which the nucleic acid according to the first aspect or the vector containing the nucleic acid is introduced include higher eukaryotic cells, bacteria, yeasts, fungi and the like, and mammalian cells are preferable.
Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells (eg, COS-7 cells), BHK cells, CHL cells or CHO cells, BALB / c mouse cells (eg, BALB / c mouse fetal fibroblasts) ) And the like. Methods for transforming mammalian cells and expressing genes introduced into the cells are also known, and for example, lipofection method, electroporation method, calcium phosphate method and the like can be used.
第１の態様に係る核酸によるＭＥＸ３Ｂ遺伝子発現の抑制は、ＭＥＸ３Ｂ遺伝子の塩基配列情報を基にすれば、インビボ、インビトロでも、例えば該遺伝子の一部又は全部の塩基配列を有するプローブまたはプライマーを利用することにより検出することができる。
特に、ＭＥＸ３Ｂ遺伝子のｍＲＮＡレベルでの発現量の測定は、ＲＴ−ＰＣＲ、ノザンブロット等の常法により行うことができる。
Inhibition of MEX3B gene expression by the nucleic acid according to the first aspect uses, for example, a probe or primer having a part or all of the base sequence of the gene in vivo and in vitro based on the base sequence information of the MEX3B gene. This can be detected.
In particular, the expression level of the MEX3B gene at the mRNA level can be measured by a conventional method such as RT-PCR or Northern blot.
ＰＣＲを行なう場合、プライマーは、ＭＥＸ３Ｂ遺伝子のみを特異的に増幅できるものであれば特に限定されず、ＭＥＸ３Ｂ遺伝子の配列情報に基づき適宜設定することができる。例えば、ＭＥＸ３Ｂ遺伝子中の連続する少なくとも１０ヌクレオチドを含むオリゴヌクレオチド、並びに該オリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドをプローブまたはプライマーとして使用することができる。より具体的には、ＭＥＸ３Ｂ遺伝子中の連続した１０〜６０残基、好ましくは１０〜４０残基の塩基配列を有するオリゴヌクレオチド、並びに該オリゴヌクレオチドに相補的な配列を有するアンチセンスオリゴヌクレオチドを使用することができる。 When PCR is performed, the primer is not particularly limited as long as it can specifically amplify only the MEX3B gene, and can be appropriately set based on the sequence information of the MEX3B gene. For example, an oligonucleotide comprising at least 10 consecutive nucleotides in the MEX3B gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide can be used as a probe or primer. More specifically, an oligonucleotide having a base sequence of 10 to 60 residues, preferably 10 to 40 residues in the MEX3B gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide are used. can do.
＜ＭＥＸ３Ｂ遺伝子発現抑制剤＞
第２の態様に係るＭＥＸ３Ｂ遺伝子発現抑制剤（以下、単に「第２の態様に係る剤」ともいう。）は、第１の態様に係る核酸を含む。
第２の態様に係る剤は、細胞内、好ましくは核内への取り込みを向上させる観点から、リポフェクション用担体を更に含むことが好ましい。
リポフェクション用担体としては、細胞膜、好ましくは核膜との親和性の高い担体（例えばリポソームやコレステロール等）が挙げられ、リポフェクトアミン又はリポフェクチンが好ましく、リポフェクトアミンがより好ましい。
第１の態様に係る核酸がホスホロチオエート構造、架橋構造及びアルコキシ構造よりなる群から選択される少なくとも１つの構造を有することと、リポフェクション用担体とを組み合わせて用いることにより細胞内、好ましくは核内への取り込みを更に向上させることができる。
例えば、ホスホロチオエート構造、架橋構造及びアルコキシ構造よりなる群から選択される少なくとも１つの構造を有する第１の態様に係る核酸を、リポフェクション用担体共存下において、後述の対象と接触させることが好ましい。
<MEX3B gene expression inhibitor>
The MEX3B gene expression inhibitor (hereinafter also simply referred to as “agent according to the second aspect”) according to the second aspect includes the nucleic acid according to the first aspect.
The agent according to the second embodiment preferably further contains a lipofection carrier from the viewpoint of improving the uptake into cells, preferably into the nucleus.
Examples of the carrier for lipofection include carriers having high affinity with cell membranes, preferably nuclear membranes (for example, liposome and cholesterol), and lipofectamine or lipofectin is preferable, and lipofectamine is more preferable.
The nucleic acid according to the first aspect has at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure, and an alkoxy structure, and is used in combination with a lipofection carrier to enter the cell, preferably the nucleus. Can be further improved.
For example, the nucleic acid according to the first embodiment having at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure, and an alkoxy structure is preferably brought into contact with a target described later in the presence of a lipofection carrier.
第２の態様に係る剤は、第１の態様に係る核酸、上記リポフェクション用担体及びその他任意成分（例えば、水、緩衝液等）を混合して含む形態であってもよいが、第１の態様に係る核酸及びその他任意成分と、上記リポフェクション用担体及びその他任意成分とが個別の容器内に包装されたキットの形態であってもよい。
第２の態様に係る剤の形態としては、特に限定されるものではないが、液体、顆粒、錠剤、カプセル剤、貼り薬等の形態で使用することができる。インビボの場合には、第２の態様に係る剤を組織に直接暴露してもよい。より好ましくは生体に暴露（液体等）又は経口投与、あるいは、静脈又は動脈等の血管内、経口内、舌下、直腸内、腹膣内、皮膚、皮下、皮内、膀胱内、気管（気管支）、眼、鼻、耳内等への注入、噴霧、又は塗布等の手段により生体内に投与すればよい。
The agent according to the second aspect may be in a form containing a mixture of the nucleic acid according to the first aspect, the carrier for lipofection and other optional components (for example, water, buffer solution, etc.) It may be in the form of a kit in which the nucleic acid according to the embodiment and other optional components, and the lipofection carrier and other optional components are packaged in separate containers.
Although it does not specifically limit as a form of the agent which concerns on a 2nd aspect, It can use in forms, such as a liquid, a granule, a tablet, a capsule, a patch. In vivo, the agent according to the second aspect may be directly exposed to the tissue. More preferably, it is exposed to a living body (liquid etc.) or orally administered, or intravascularly or intravenously such as veins or arteries, sublingual, rectal, intravaginal, skin, subcutaneous, intradermal, intravesical, trachea (bronchi) ), Injection into the eye, nose, ear, etc., spraying, application, or the like.
＜ＭＥＸ３Ｂ遺伝子発現を抑制する方法＞
第３の態様に係るＭＥＸ３Ｂ遺伝子発現を抑制する方法は、第２の態様に係る剤を対象に接触させることを含む。
対象としては、生物個体、微生物、原虫、生物組織、生物組織切片、ヒト細胞、動物細胞等が挙げられる。
接触させる形態としては特に制限はないが、例えば、第２の態様に係る剤を、対象を含む媒体（例えば、培地、溶液、緩衝液等）に添加するか又は第２の態様に係る剤を予め含有する前記媒体を準備してもよい。接触させる際の温度、時間等は、特に制限はないが、対象の種類等に応じて適宜設定される。
<Method for suppressing MEX3B gene expression>
The method for suppressing MEX3B gene expression according to the third aspect includes bringing the agent according to the second aspect into contact with the subject.
Examples of the target include individual organisms, microorganisms, protozoa, biological tissues, biological tissue sections, human cells, animal cells, and the like.
Although there is no restriction | limiting in particular as a form made to contact, For example, the agent which concerns on a 2nd aspect is added to the medium (for example, culture medium, a solution, a buffer solution, etc.) containing object, or the agent which concerns on a 2nd aspect is used. The medium contained in advance may be prepared. There are no particular limitations on the temperature, time, and the like when contacting, but they are appropriately set according to the type of the target.
＜ＭＥＸ３Ｂ遺伝子発現に起因する疾病の予防又は治療剤＞
第４の態様に係るＭＥＸ３Ｂ遺伝子発現に起因する疾病の予防又は治療剤は、第２の態様に係るＭＥＸ３Ｂ遺伝子発現抑制剤を含む。
インターロイキン６（ＩＬ−６）は、炎症、造血、骨代謝、腫瘍増悪などに関与する重要なサイトカインであり、その活性は主に急性炎症から獲得免疫反応への移行や慢性炎症性疾患の発症に寄与することが知られている（例えば、Ｊ Ａｓｔｈｍａ．２００８；４５ Ｓｕｐｐｌ １：４１−４．）。
インターロイキン１３（ＩＬ−１３）は、炎症性サイトカインとして末梢組織でのアレルギー性炎症をより増強する役割を担うことが知られており、アレルギー性喘息の主な要因であるアレルギー反応を促進するという側面のみならず、ステロイド剤が効かなくなるという喘息の難治化に関与することが知られている。またＩＬ−１３は、喘息のみならず、炎症性腸疾患、アトピー性皮膚炎においてもその病態形成に関与している（例えば、Ｊ Ａｌｌｅｒｇｙ （Ｃａｉｒｏ）．２０１２；２０１２：３１６０４９、Ｎ Ｅｎｇｌ Ｊ Ｍｅｄ ２０１１；３６５：１０８８−１０９８）。
ＴＮＦ（腫瘍壊死因子：Ｔｕｍｏｒ Ｎｅｃｒｏｓｉｓ Ｆａｃｔｏｒ）、中でも、ＴＮＦ−αは、炎症反応を誘発するシグナル因子であり、感染防御の観点では重要な因子であるが、一方で炎症が亢進することによる障害にも同時に関与することが知られている。すなわち様々な疾患でＴＮＦは病態の悪化に関与しており、主に関節疾患（関節リウマチ、乾癬性関節炎、脊椎関節症、強直性脊椎炎）、炎症性腸疾患（潰瘍性大腸炎、クローン病）、がん（卵巣がん、乳がん）、精神疾患（うつ、双極性障害、てんかん、アルツハイマー病、パーキンソン病、多発性硬化症）、心血管疾患（心不全、動脈硬化）、呼吸器疾患（気管支喘息、慢性気管支炎、慢性閉塞性肺疾患、急性肺障害）、二型糖尿病、腎疾患（虚血性腎障害、移植後拒絶反応、糸球体腎炎）などに関与することが知られている（例えば、Ｊ Ａｌｌｅｒｇｙ Ｃｌｉｎ Ｉｍｍｕｎｏｌ．２００８ Ｊａｎ；１２１（１）：５−１０、Ｊ Ｐａｔｈｏｌ．２００８ Ｊａｎ；２１４（２）：１４９−６０．）。
また、Ｇ−ＣＳＦ（顆粒球コロニー刺激因子：Ｇｒａｎｕｌｏｃｙｔｅ−Ｃｏｌｏｎｙ Ｓｔｉｍｕｌａｔｉｎｇ Ｆａｃｔｏｒ）は顆粒球産出の促進、好中球の機能を高める作用があることが知られている。
また、ＩＬ−１３、ＴＮＦ、Ｇ−ＣＳＦも、喘息の重症化に関与することが知られている（例えば、Ｃｕｒｒ Ｏｐｉｎ Ｉｍｍｕｎｏｌ．２０１３ Ｄｅｃ；２５（６）：７５５−６０．）。
<Preventive or therapeutic agent for diseases caused by MEX3B gene expression>
The preventive or therapeutic agent for a disease caused by MEX3B gene expression according to the fourth aspect includes the MEX3B gene expression inhibitor according to the second aspect.
Interleukin 6 (IL-6) is an important cytokine involved in inflammation, hematopoiesis, bone metabolism, tumor exacerbation, etc., and its activity mainly shifts from acute inflammation to acquired immune response and development of chronic inflammatory diseases (For example, J Asthma. 2008; 45 Suppl 1: 41-4.).
Interleukin 13 (IL-13) is known to play a role of further enhancing allergic inflammation in peripheral tissues as an inflammatory cytokine, and promotes an allergic reaction that is a major factor in allergic asthma. It is known to be involved not only in the aspect but also in the intractable asthma that steroids become ineffective. IL-13 is also involved in the pathogenesis of not only asthma but also inflammatory bowel disease and atopic dermatitis (for example, J Allergy (Cairo). 2012; 2012: 316049, N Engl J Med 2011). 365: 1088-1098).
TNF (Tumor Necrosis Factor), in particular, TNF-α is a signal factor that induces an inflammatory response and is an important factor in terms of defense against infection. Is also known to be involved at the same time. In other words, TNF is involved in the deterioration of pathological conditions in various diseases, mainly joint diseases (rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, ankylosing spondylitis), inflammatory bowel diseases (ulcerative colitis, Crohn's disease) ), Cancer (ovarian cancer, breast cancer), mental illness (depression, bipolar disorder, epilepsy, Alzheimer's disease, Parkinson's disease, multiple sclerosis), cardiovascular disease (heart failure, arteriosclerosis), respiratory disease (bronchi) It is known to be involved in asthma, chronic bronchitis, chronic obstructive pulmonary disease, acute lung injury), type 2 diabetes, renal disease (ischemic kidney injury, post-transplant rejection, glomerulonephritis), etc. J Allergy Clin Immunol. 2008 Jan; 121 (1): 5-10, J Pathol.2008 Jan; 214 (2): 149-60.).
G-CSF (Granulocyte-Colony Stimulating Factor) is known to have the effect of promoting the production of granulocytes and enhancing the function of neutrophils.
IL-13, TNF, and G-CSF are also known to be involved in the severity of asthma (for example, Curr Opin Immunol. 2013 Dec; 25 (6): 755-60.).
また、ＣＸＣＬ１、ＣＸＣＬ２、ＣＸＣＬ５は炎症性ケモカインＣＸＣサブファミリーに属する。
気道粘膜における過剰な炎症の亢進により肺組織内でＣＸＣＬ１、ＣＸＣＬ２、ＣＸＣＬ５が分泌された場合には、ＣＸＣＬ１、ＣＸＣＬ２、ＣＸＣＬ５の受容体であるＣＸＣＲ２を高発現する好中球の浸潤が促される。結果的にはステロイド耐性の好中球浸潤が重症喘息を引き起こすことにより気道の不可逆的なリモデリングを誘導する慢性炎症を誘発する。
本発明者らは、ＭＥＸ３Ｂ遺伝子がＩＬ−６、ＩＬ−１３、ＴＮＦ、Ｇ−ＣＳＦ、ＣＸＣＬ１、ＣＸＣＬ２、又はＣＸＣＬ５に起因する疾病の発症に関わることを見出している。
したがって、第４の態様に係る予防又は治療剤は、ＩＬ−６、ＩＬ−１３、ＴＮＦ、Ｇ−ＣＳＦ、ＣＸＣＬ１、ＣＸＣＬ２、又はＣＸＣＬ５発現増加に起因する疾病（例えば、重症喘息、慢性閉塞性肺疾患、関節リウマチ、大腸炎、クローン病、アトピー皮膚炎、全身性エリテマトーデス、がん等の中でもＩＬ−６、ＩＬ−１３、ＴＮＦ、Ｇ−ＣＳＦ、ＣＸＣＬ１、ＣＸＣＬ２、又はＣＸＣＬ５に起因する重症喘息、慢性閉塞性肺疾患、関節リウマチ、大腸炎、クローン病、アトピー皮膚炎、全身性エリテマトーデス、がん等（Ｉｎｔ Ｉｍｍｕｎｏｌ．２０１５ Ｊａｎ；２７（１）：２１−９、Ｃａｎｃｅｒ Ｄｉｓｃｏｖ． ２０１６ Ｊａｎ；６（１）：８０−９５．））の予防又は治療剤として有効であると考えられる。
CXCL1, CXCL2, and CXCL5 belong to the inflammatory chemokine CXC subfamily.
When CXCL1, CXCL2, and CXCL5 are secreted in lung tissues due to excessive inflammation in the airway mucosa, infiltration of neutrophils that highly express CXCR2, which is a receptor for CXCL1, CXCL2, and CXCL5, is promoted. Consequently, steroid-resistant neutrophil infiltration induces chronic inflammation that induces irreversible remodeling of the airways by causing severe asthma.
The present inventors have found that the MEX3B gene is involved in the onset of diseases caused by IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5.
Therefore, the prophylactic or therapeutic agent according to the fourth aspect is a disease caused by increased expression of IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 (for example, severe asthma, chronic obstructive pulmonary lung) Severe asthma caused by IL-6, IL-13, TNF, G-CSF, CXCL1, CXCL2, or CXCL5 among diseases, rheumatoid arthritis, colitis, Crohn's disease, atopic dermatitis, systemic lupus erythematosus, cancer, etc. Chronic obstructive pulmonary disease, rheumatoid arthritis, colitis, Crohn's disease, atopic dermatitis, systemic lupus erythematosus, cancer, etc. (Int Immunol. 2015 Jan; 27 (1): 21-9, Cancer Discov. 2016 Jan; 6 ( 1): It is considered to be effective as a preventive or therapeutic agent for 80-95.)).
また、ＭＥＸ３Ｂタンパク質はアポトーシスを誘導するタンパク質であることが知られている（例えば、特許第４４２９２６９号公報）。
アポトーシスは、正常な生理学的過程以外にも、神経変性疾患等の重篤な疾患の発症にも関与していることが知られている。例えば、神経変性疾患（例えば、アルツハイマー病、パーキンソン病、筋萎縮性側索硬化症、ハンチントン舞踏病など）は、アポトーシスの異常な亢進が原因と考えられている（Ｗｏｌｏｚｉｎ，Ｂ．，ｅｔ ａｌ．，（１９９６）Ｓｃｉｅｎｃｅ，２７４，１７１０−１７１３）。
したがって、第４の態様に係る予防又は治療剤は、神経変性疾患の予防又は治療剤として有効であると考えられる。
MEX3B protein is known to be a protein that induces apoptosis (for example, Japanese Patent No. 4429269).
Apoptosis is known to be involved in the development of serious diseases such as neurodegenerative diseases in addition to normal physiological processes. For example, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea, etc.) are thought to be caused by abnormally elevated apoptosis (Wolozin, B., et al. (1996) Science 274, 1710-1713).
Therefore, the prophylactic or therapeutic agent according to the fourth aspect is considered to be effective as a prophylactic or therapeutic agent for neurodegenerative diseases.
第４の態様に係る予防又は治療剤は、経口または非経口的に全身又は局所的に投与することができる。非経口的な投与方法としては、点滴などの静脈内注射、筋肉内注射、腹腔内注射、皮下注射などを挙げることができる。患者の年齢、症状により適宜投与方法を選択することができる。その投与量は、年齢、投与経路、投与回数により異なり、当業者であれば適宜選択できる。
非経口投与に適した製剤形態として、例えば安定剤、緩衝剤、保存剤、等張化剤等の添加剤を含有したものは挙げられ、さらに薬学的に許容される担体や添加物を含むものでもよい。このような担体及び添加物の例として、水、有機溶剤、高分子化合物（コラーゲン、ポリビニルアルコールなど）、ステアリン酸、ヒト血清アルブミン（ＨＳＡ）、マンニトール、ツルビトール、ラクトース、界面活性剤などが挙げられるが、これらに限定されるものではない。
The prophylactic or therapeutic agent according to the fourth aspect can be administered systemically or locally orally or parenterally. Examples of parenteral administration methods include intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage varies depending on the age, administration route, and number of administrations, and can be appropriately selected by those skilled in the art.
Examples of the dosage form suitable for parenteral administration include those containing additives such as stabilizers, buffers, preservatives, tonicity agents, and further containing pharmaceutically acceptable carriers and additives. But you can. Examples of such carriers and additives include water, organic solvents, polymer compounds (collagen, polyvinyl alcohol, etc.), stearic acid, human serum albumin (HSA), mannitol, thurbitol, lactose, surfactants and the like. However, it is not limited to these.
有効成分である第１の態様に係る核酸の投与量としては、一般的には一回につき体重１ｋｇあたり０．１μｇ〜１００ｍｇ程度の範囲である。 The dosage of the nucleic acid according to the first embodiment, which is an active ingredient, is generally in the range of about 0.1 μg to 100 mg per kg of body weight per time.
以下、実施例を示して本発明を更に具体的に説明するが、本発明の範囲は、これらの実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further more concretely, the scope of the present invention is not limited to these Examples.
≪実施例１≫
＜ギャップマー（Ｇａｐｍｅｒ）型核酸の調製＞
イントロン領域を表す配列番号１における２８０〜２９５番目のオリゴヌクレオチドに相補的なアンチセンスオリゴヌクレオチドであるギャップマー型核酸、７０８〜７２３番目のオリゴヌクレオチドに相補的なアンチセンスオリゴヌクレオチドであるギャップマー型核酸、７２１〜７３６番目のオリゴヌクレオチドに相補的なアンチセンスオリゴヌクレオチドであるギャップマー型核酸、７３０〜７４５番目のオリゴヌクレオチドに相補的なアンチセンスオリゴヌクレオチドであるギャップマー型核酸、８０７〜８２２番目のオリゴヌクレオチドに相補的なアンチセンスオリゴヌクレオチドであるギャップマー型核酸を調製した。
各ギャップマー型核酸の両末端には２又は３塩基のＬＮＡ（２’，４’−ＢＮＡ）を配置し、それ以外の間を埋める塩基は通常のＤＮＡとし、各ヌクレオチドを結ぶリン酸ジエステル結合はホスホロチオエート化し、全長は１６塩基とした。
Example 1
<Preparation of Gapmer type nucleic acid>
Gapmer nucleic acid that is an antisense oligonucleotide complementary to the 280th to 295th oligonucleotides in SEQ ID NO: 1 representing an intron region, and a gapmer type that is an antisense oligonucleotide complementary to the 708th to 723rd oligonucleotides Gapmer nucleic acid that is an antisense oligonucleotide complementary to the nucleic acids 721 to 736, Gapmer nucleic acid that is an antisense oligonucleotide complementary to the 730 to 745th oligonucleotides, 807 to 822 A gapmer type nucleic acid which is an antisense oligonucleotide complementary to the above oligonucleotide was prepared.
Two or three bases of LNA (2 ', 4'-BNA) are placed at both ends of each gapmer type nucleic acid, and the base that fills the rest is normal DNA, and phosphodiester bonds linking each nucleotide Was phosphorothioated to a total length of 16 bases.
＜ギャップマー型核酸のトランスフェクション＞
上記調製した各ギャップマー型核酸を用いてトランスフェクション（細胞導入）した。
ギャップマー型核酸のトランスフェクションに用いた細胞はヒト肺がん細胞株Ａ５４９細胞であり、リポフェクトアミンＲＮＡｉＭａｘ（Ｉｎｖｉｔｒｏｇｅｎ社製）とその推奨プロトコルを採用して最終濃度２０ｎＭにて細胞に導入した。
＜ＲＴ定量的ＰＣＲ試験＞
トランスフェクション４８時間後に顕微鏡下にて細胞の状態を観察した後に細胞を回収して溶解バッファーＴＲＩｓｕｒｅ（ＢＩＯＬＩＮＥ社製）を用いて全ＲＮＡを回収した。Ｐｒｉｍｅｓｃｒｉｐｔ（ＴＡＫＡＲＡ社製）により逆転写反応を行いｃＤＮＡを得た。
その後、Ｌｉｇｈｔ Ｃｙｃｌｅｒ４８０（ＲＯＣＨＥ社製）を用いて定量的ＲＴ−ＰＣＲを行った。
<Gapmer nucleic acid transfection>
Transfection (cell introduction) was performed using each of the gapmer nucleic acids prepared above.
The cells used for transfection of the gapmer type nucleic acid were human lung cancer cell line A549 cells, which were introduced into the cells at a final concentration of 20 nM using Lipofectamine RNAiMax (manufactured by Invitrogen) and its recommended protocol.
<RT quantitative PCR test>
48 hours after transfection, the state of the cells was observed under a microscope, and then the cells were collected and total RNA was collected using a lysis buffer TRIsure (manufactured by BIOLINE). A reverse transcription reaction was performed using Primescript (manufactured by TAKARA) to obtain cDNA.
Then, quantitative RT-PCR was performed using Light Cycler 480 (manufactured by ROCHE).
定量的ＲＴ−ＰＣＲ試験に用いたプライマー配列は以下である。
ヒトＭＥＸ３Ｂプライマー１Ｆｏｒｗａｒｄ：５’−ＣＣＣＡＧＴＴＣＴＧＡＧＣＡＴＧＴＣＧ−３’（配列番号６）
ヒトＭＥＸ３Ｂプライマー１Ｒｅｖｅｒｓｅ：５’−ＴＣＡＧＧＡＡＡＡＡＡＴＧＣＧＧＡＴＧＧＣＣＴＧＡＧＴＧＡＣ−３’（配列番号７）
ヒトＧＡＰＤＨプライマー１Ｆｏｒｗａｒｄ：５’−ＡＧＡＧＡＣＡＧＣＣＧＣＡＴＣＴＴＣＴＴ−３’（配列番号８）
ヒトＧＡＰＤＨプライマー１Ｒｅｖｅｒｓｅ：５’−ＧＡＣＡＡＧＣＴＴＣＣＣＡＴＴＣＴＣＧＧ−３’（配列番号９）
The primer sequences used for the quantitative RT-PCR test are as follows.
Human MEX3B Primer 1 Forward: 5′-CCCAGTTCTGAGCATGTCG-3 ′ (SEQ ID NO: 6)
Human MEX3B Primer 1 Reverse: 5′-TCAGGAAAAAATGCGGATGGCCTGAGTGAC-3 ′ (SEQ ID NO: 7)
Human GAPDH Primer 1 Forward: 5′-AGAGACAGCCCGCATCTTCTT-3 ′ (SEQ ID NO: 8)
Human GAPDH Primer 1 Reverse: 5′-GACAAGCTTCCCATTCTCGG-3 ′ (SEQ ID NO: 9)
図１に示した定量的ＲＴ−ＰＣＲの結果から明らかなように、ギャップマー型核酸によるトランスフェクションを行っていないコントロールに対し、各ギャップマー型核酸を用いてトランスフェクションを行った細胞ではヒトＭＥＸ３ＢのｍＲＮＡ発現を抑制する効果が検出された。
特に、イントロン領域を表す配列番号１における７２１〜７３６番目のオリゴヌクレオチドに相補的な配列を有するギャップマー型核酸、及び７０８〜７２３番目のオリゴヌクレオチドに相補的な配列を有するギャップマー型核酸に、ヒトＭＥＸ３ＢのｍＲＮＡ発現を抑制する効果が検出された。
As is clear from the results of quantitative RT-PCR shown in FIG. 1, human MEX3B was not used in the cells transfected with each gapmer type nucleic acid compared to the control not transfected with the gapmer type nucleic acid. The effect of suppressing mRNA expression of was detected.
In particular, a gapmer type nucleic acid having a sequence complementary to the 721 to 736th oligonucleotide in SEQ ID NO: 1 representing the intron region, and a gapmer type nucleic acid having a sequence complementary to the 708 to 723rd oligonucleotide, The effect of suppressing human MEX3B mRNA expression was detected.
≪実施例２≫
＜ギャップマー型核酸の調製＞
イントロン領域の表１に示した各標的配列に相補的なアンチセンスオリゴヌクレオチドであるギャップマー型核酸ｈｍｒＧＤ−３３（配列番号１１）、ｈｍｒＧＤ−３４（配列番号１２）、ｈｍｒＧＤ−３５（配列番号１３）、ｈｍｒＧＤ−３６（配列番号１４）又はｈｍｒＧＤ−３７（配列番号１５）、ネガティブコントロールとしてのギャップマー型核酸（配列番号１６）、及び比較例としてＣＤＳ領域の表１に示した標的配列に相補的なアンチセンスオリゴヌクレオチドであるギャップマー型核酸ｈｍｒＧＤ−６８（配列番号１７）を調製した。
なお、各ギャップマー型核酸の両末端には２塩基のＬＮＡ（２’，４’−ＢＮＡ）を配置し、それ以外の間を埋める塩基は通常のＤＮＡとし、各ヌクレオチドを結ぶリン酸ジエステル結合はホスホロチオエート化し、ネガティブコントロールのギャップマー型核酸の全長は１５塩基とし、それ以外のギャップマー型核酸の全長は１６塩基とした。
<< Example 2 >>
<Preparation of gapmer type nucleic acid>
Gapmer nucleic acids hmrGD-33 (SEQ ID NO: 11), hmrGD-34 (SEQ ID NO: 12), and hmrGD-35 (SEQ ID NO: 13), which are antisense oligonucleotides complementary to each target sequence shown in Table 1 of the intron region. ), HmrGD-36 (SEQ ID NO: 14) or hmrGD-37 (SEQ ID NO: 15), gapmer-type nucleic acid (SEQ ID NO: 16) as a negative control, and complementary to the target sequence shown in Table 1 of the CDS region as a comparative example Gapmer nucleic acid hmrGD-68 (SEQ ID NO: 17), which is a typical antisense oligonucleotide, was prepared.
In addition, two bases of LNA (2 ', 4'-BNA) are arranged at both ends of each gapmer type nucleic acid, and the base that fills the other is normal DNA, and a phosphodiester bond linking each nucleotide Was phosphorothioated, and the total length of the negative control gapmer nucleic acid was 15 bases, and the total length of the other gapmer nucleic acids was 16 bases.
＜ギャップマー型核酸のトランスフェクション＞
上記調製した各ギャップマー型核酸を用いること以外は実施例１と同様にしてトランスフェクションを行った。
＜ＲＴ定量的ＰＣＲ試験＞
ＰＣＲプライマーとして以下のプライマーを用いること以外は実施例１と同様にして定量的ＲＴ−ＰＣＲ試験を行った。
ヒトＭＥＸ３Ｂプライマー２Ｆｏｒｗａｒｄ：５’−ＡＣＣＣＡＧＴＴＣＴＧＡＧＣＡＴＧＴＣＧ−３’（配列番号１８）
ヒトＭＥＸ３Ｂプライマー２Ｒｅｖｅｒｓｅ：５’−ＣＧＡＡＣＴＧＧＧＧＴＣＴＴＧＡＴＧＴＡＡ−３’（配列番号１９）
ヒトＧＡＰＤＨプライマー２Ｆｏｒｗａｒｄ：５’−ＧＣＡＣＣＧＴＣＡＡＧＧＣＴＧＡＧＡＡＣ−３’（配列番号２０）
ヒトＧＡＰＤＨプライマー２Ｒｅｖｅｒｓｅ：５’−ＴＧＧＴＧＡＡＧＡＣＧＣＣＡＧＴＧＧＡ−３’（配列番号２１）
<Gapmer nucleic acid transfection>
Transfection was performed in the same manner as in Example 1 except that each gapmer type nucleic acid prepared above was used.
<RT quantitative PCR test>
A quantitative RT-PCR test was performed in the same manner as in Example 1 except that the following primers were used as PCR primers.
Human MEX3B Primer 2 Forward: 5′-ACCCAGTTCTGAGCATGTCG-3 ′ (SEQ ID NO: 18)
Human MEX3B Primer 2 Reverse: 5'-CGAACTGGGGTCTTGATGTAA-3 '(SEQ ID NO: 19)
Human GAPDH Primer 2 Forward: 5′-GCACCGTCAAGGCTGAGAAC-3 ′ (SEQ ID NO: 20)
Human GAPDH Primer 2 Reverse: 5′-TGGTGAAGACGCCAGTGGA-3 ′ (SEQ ID NO: 21)
図２に示した定量的ＲＴ−ＰＣＲの結果から明らかなように、コントロールのギャップマー型核酸をトランスフェクションした細胞に対し、イントロン領域を標的領域とする各ギャップマー型核酸ｈｍｒＧＤ−３３、ｈｍｒＧＤ−３４、ｈｍｒＧＤ−３５、ｈｍｒＧＤ−３６又はｈｍｒＧＤ−３７を用いてトランスフェクションを行った細胞ではヒトＭＥＸ３ＢのｍＲＮＡ発現を抑制する効果が検出された。
ヒトＭＥＸ３ＢのプレｍＲＮＡを表す配列番号１０における９０８〜９２３番目のオリゴヌクレオチドに相補的な配列を有するギャップマー型核酸ｈｍｒＧＤ−３５は、ヒトＭＥＸ３ＢのｍＲＮＡ発現を抑制する効果が特に強く検出された。
As is clear from the results of quantitative RT-PCR shown in FIG. 2, the gapmer type nucleic acids hmrGD-33, hmrGD- having the intron region as the target region were compared with the cells transfected with the control gapmer type nucleic acid. No. 34, hmrGD-35, hmrGD-36 or hmrGD-37 was transfected with cells, and the effect of suppressing human MEX3B mRNA expression was detected.
The gapmer type nucleic acid hmrGD-35 having a sequence complementary to the 908th to 923rd oligonucleotides in SEQ ID NO: 10 representing the pre-mRNA of human MEX3B was detected particularly strongly in the effect of suppressing human MEX3B mRNA expression.
図３に示したように、ＣＤＳ領域の配列に相補的なギャップマー型核酸ｈｍｒＧＤ−６８を用いてトランスフェクションを行った細胞では、細胞毒性が著しく高く、多くの細胞が培養皿の底から剥がれて丸くなって浮いており、多くの細胞が死滅することが確認された。
一方、図３に示したように、イントロン領域の配列に相補的なギャップマー型核酸ｈｍｒＧＤ−３３、ｈｍｒＧＤ−３４、ｈｍｒＧＤ−３５、ｈｍｒＧＤ−３６又はｈｍｒＧＤ−３７を用いてトランスフェクションを行った細胞では、ネガティブコントロールと同様に、細胞毒性などの副作用は見られなかった。
As shown in FIG. 3, the cells transfected with the gapmer type nucleic acid hmrGD-68 complementary to the sequence of the CDS region are extremely high in cytotoxicity, and many cells are detached from the bottom of the culture dish. It was confirmed that many cells were killed.
On the other hand, as shown in FIG. 3, cells transfected with the gapmer type nucleic acid hmrGD-33, hmrGD-34, hmrGD-35, hmrGD-36 or hmrGD-37 complementary to the sequence of the intron region. Then, like the negative control, no side effects such as cytotoxicity were observed.
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| WO2021065686A1 (en) * | 2019-09-30 | 2021-04-08 | 国立大学法人 東京大学 | Nucleic acid that inhibits expression of mex3b gene, mex3b gene expression inhibiting agent, method for inhibiting mex3b gene expression, and prophylactic or therapeutic agent for disease caused by mex3b gene expression |
| WO2021221116A1 (en) * | 2020-05-01 | 2021-11-04 | 国立大学法人東京大学 | Prophylactic or therapeutic agent for at least one type of cancer selected from group consisting of pancreatic cancer, lung cancer, colorectal cancer, biliary tract cancer and liver cancer, prophylactic or therapeutic agent for said cancer which is used in combination drug in combination with said agent, combination drug comprising said agents, and method for screening for prophylactic or therapeutic agent for cancer |
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| JPWO2021065686A1 (en) * | 2019-09-30 | 2021-10-28 | 国立大学法人 東京大学 | Nucleic acid that suppresses MEX3B gene expression, MEX3B gene expression suppressant, method that suppresses MEX3B gene expression, and preventive or therapeutic agent for diseases caused by MEX3B gene expression |
| JP7033344B2 (en) | 2019-09-30 | 2022-03-10 | 国立大学法人 東京大学 | Nucleic acid that suppresses MEX3B gene expression, MEX3B gene expression inhibitor, method that suppresses MEX3B gene expression, and preventive or therapeutic agent for diseases caused by MEX3B gene expression. |
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