JP7386507B2 - Muscle differentiation promoting agent, method for promoting muscle differentiation, and oligo DNA for promoting muscle differentiation - Google Patents
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Description
本開示は、筋萎縮症の予防・治療、横紋筋肉腫の増殖・転移の抑制、ＥＳ・ｉＰＳ細胞もしくは体性幹細胞の筋分化誘導、筋疾患の再生医療、創薬のスクリーニングに供する骨格筋（幹）細胞の作成、食肉用家畜・家禽の飼料の添加物、食肉用家畜・家禽の骨格筋の形成・生育の促進に利用可能な筋分化促進剤および筋分化促進剤方法および筋分化促進オリゴＤＮＡに関する。 The present disclosure relates to prevention and treatment of muscular atrophy, suppression of rhabdomyosarcoma growth and metastasis, induction of muscle differentiation of ES/iPS cells or somatic stem cells, regenerative medicine for muscle diseases, and skeletal muscle used for drug discovery screening. Muscle differentiation promoters and muscle differentiation promoters that can be used to create (stem) cells, feed additives for meat-producing livestock and poultry, and promoting the formation and growth of skeletal muscle in meat-producing livestock and poultry.Methods and muscle differentiation promotion Regarding oligo DNA.
超高齢社会では、加齢性の骨格筋萎縮（サルコペニア）を主徴とするロコモティブ症候群（運動器症候群）が急増しており、日本における要支援・要介護となった原因の第一位を占めている。また、日本人の死因の第一位である癌の患者の数十％が癌悪液質（カヘキシー）を合併するが、癌悪液質による骨格筋量の減少は生命予後と強く相関する。また、死因の第２、３位を占める心血管疾患の最終病像である心不全においては、骨格筋の萎縮は予後の独立した危険因子である。さらに、糖尿病患者の一部でも糖尿病性の筋萎縮が認められる。このように、骨格筋の萎縮は、老化や多様な疾患と連動している。したがって、日本を含む超高齢社会において、筋萎縮の予防および治療法の開発は、人々の運動機能を維持し、生活の質（ＱＯＬ）を向上させ、健康寿命を延伸するために不可欠である。 In a super-aging society, locomotive syndrome (locomotor syndrome), whose main symptom is age-related skeletal muscle atrophy (sarcopenia), is rapidly increasing, and it is the number one cause of people requiring support and nursing care in Japan. ing. Additionally, several tens of percent of patients with cancer, which is the number one cause of death in Japan, develop cancer cachexia, and the decrease in skeletal muscle mass due to cancer cachexia is strongly correlated with life prognosis. Furthermore, in heart failure, which is the final manifestation of cardiovascular disease, which is the second or third leading cause of death, skeletal muscle atrophy is an independent risk factor for prognosis. Furthermore, diabetic muscle atrophy is also observed in some diabetic patients. In this way, skeletal muscle atrophy is linked to aging and various diseases. Therefore, in super-aging societies including Japan, the development of methods for preventing and treating muscle atrophy is essential for maintaining people's motor functions, improving quality of life (QOL), and extending healthy lifespans.
骨格筋は、衛星細胞と呼ばれる骨格筋幹細胞の増殖と分化によって、組織としての恒常性が保たれる。筋再生時には、衛星細胞は筋芽細胞と呼ばれる筋前駆細胞へと活性化される。筋芽細胞は数回の細胞分裂によって増殖した後、筋細胞へと分化する。筋細胞は互いに融合して多核の筋管を形成し、骨格筋組織を再生する。ところが老化が進行すると、筋組織中の衛星細胞の数が減少し、また、個々の衛星細胞の再生能力も低下する。衛星細胞の老化を抑制する、あるいは再生能力を活性化する機能性分子を探索・同定することは、筋萎縮症に対する新しい予防戦略の提唱につながると期待されている。 Skeletal muscle tissue homeostasis is maintained through the proliferation and differentiation of skeletal muscle stem cells called satellite cells. During muscle regeneration, satellite cells are activated into muscle precursor cells called myoblasts. Myoblasts proliferate through several rounds of cell division and then differentiate into muscle cells. Muscle cells fuse with each other to form multinucleated myotubes and regenerate skeletal muscle tissue. However, as aging progresses, the number of satellite cells in muscle tissue decreases, and the regenerative ability of individual satellite cells also decreases. Searching for and identifying functional molecules that suppress aging or activate the regenerative ability of satellite cells is expected to lead to the proposal of new preventive strategies for muscular atrophy.
老化防止という観点から、テロメア相同オリゴＤＮＡ（配列番号２、３）を細胞に曝露することにより、平均テロメア長を増加させる技術が開示されている（特許文献１）。また、最近では、乳酸菌ゲノム配列由来のテロメア配列を有する塩基長１８のオリゴＤＮＡ（配列番号１）を筋芽細胞に曝露することにより、骨格筋への分化を促進する技術が提案されている（特許文献２）。 From the viewpoint of preventing aging, a technique has been disclosed in which the average telomere length is increased by exposing cells to telomere homologous oligo DNA (SEQ ID NO: 2, 3) (Patent Document 1). Furthermore, recently, a technology has been proposed that promotes differentiation into skeletal muscle by exposing myoblasts to an 18 base long oligo DNA (SEQ ID NO: 1) having a telomere sequence derived from the lactic acid bacteria genome sequence ( Patent Document 2).
しかし、特許文献１の方法は現状維持に関する方法、すなわちテロメア長を維持することによる細胞の修復やＤＮＡ欠損に由来するガン化の抑制を目的としたものであり、筋分化を促進しようとするものではない。また、特許文献２のオリゴＤＮＡは、乳酸菌ゲノム配列由来ではあるが実際に商品として生産するには化学合成的な手法を用いざるを得ず、コスト的な問題があった。 However, the method of Patent Document 1 is a method for maintaining the status quo, that is, a method aimed at repairing cells by maintaining telomere length and suppressing canceration resulting from DNA defects, and is aimed at promoting muscle differentiation. isn't it. Further, although the oligo DNA of Patent Document 2 is derived from the lactic acid bacterium genome sequence, chemical synthesis methods must be used to actually produce it as a commercial product, which poses a cost problem.
そこで本発明者らは、上記課題を解決するために鋭意検討を行い、新たな知見を見出すに至った。 Therefore, the present inventors conducted intensive studies to solve the above problems, and came to discover new knowledge.
本開示の一態様に係る筋分化促進剤は、３連グアニン配列と４連グアニン配列がアデニンかチミンのいずれか又は両方を含む１乃至３個の塩基を介して結合した塩基配列（配列番号２０または配列番号２１または配列番号２２）を有し、前記３連グアニン配列と前記４連グアニン配列がそれぞれ対向する立体構造を有したオリゴＤＮＡを含む。 The muscle differentiation promoting agent according to one aspect of the present disclosure has a base sequence (SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22), and includes an oligo DNA having a three-dimensional structure in which the triple guanine sequence and the quadruple guanine sequence face each other.
前記オリゴＤＮＡの塩基長は８以上１６以下であってもよい。 The oligo DNA may have a base length of 8 or more and 16 or less.
前記オリゴＤＮＡの塩基長は１４以下であってもよい。 The oligo DNA may have a base length of 14 or less.
前記オリゴＤＮＡは、５’ＧＧＧＴＧＧＧＧ３’（配列番号４）または５’ＧＧＧＴＧＡＧＧＧＧ３’（配列番号５）のいずれかで表されてもよい。 The oligo DNA may be represented by either 5'GGGTGGGG3' (SEQ ID NO: 4) or 5'GGGTGAGGGG3' (SEQ ID NO: 5).
前記オリゴＤＮＡは、５’ＴＴＧＧＧＴＧＧＧＧＡＡ３’（配列番号６）または５’ＴＴＧＧＧＴＧＡＧＧＧＧＡＴ３’（配列番号７）で表されてもよい。 The oligo DNA may be represented by 5'TTGGGTGGGGAA3' (SEQ ID NO: 6) or 5'TTGGGTGAGGGGAT3' (SEQ ID NO: 7).
前記オリゴＤＮＡは、５’ＴＴＧＧＧＴＧＧＧＧＡＡ３’（配列番号８）または５’ＴＴＧＧＧＴＧＡＧＧＧＧＡＡ３’（配列番号１０）で表されてもよい。 The oligo DNA may be represented by 5'TTGGGTGGGGAA3' (SEQ ID NO: 8) or 5'TTGGGTGAGGGGAA3' (SEQ ID NO: 10).
前記オリゴＤＮＡは、５’ＴＴＴＧＧＧＴＧＧＧＧＡＡＡ３’（配列番号９）または５’ＴＴＴＧＧＧＴＧＡＧＧＧＧＡＡＡ３’（配列番号１１）で表されてもよい。 The oligo DNA may be represented by 5'TTTGGGTGGGGAAA3' (SEQ ID NO: 9) or 5'TTTGGGTGAGGGGAAA3' (SEQ ID NO: 11).
前記筋分化促進剤は、哺乳類もしくは鳥類の細胞または個体に対して適用してもよい。 The muscle differentiation promoting agent may be applied to cells or individuals of mammals or birds.
前記細胞はマウス筋芽細胞またはニワトリ筋芽細胞またはヒト筋芽細胞であってもよい。 The cells may be mouse myoblasts or chicken myoblasts or human myoblasts.
前記個体はマウスまたはニワトリまたはヒトであってもよい。 Said individual may be a mouse or a chicken or a human.
本開示の一態様に係る筋分化促進方法は、前記筋分化促進剤を使用する。 A method for promoting muscle differentiation according to one aspect of the present disclosure uses the muscle differentiation promoting agent.
本開示の一態様に係る筋分化促進オリゴＤＮＡは、３連グアニン配列と４連グアニン配列がアデニンかチミンのいずれか又は両方を含む１乃至３個の塩基を介して結合した塩基配列（配列番号２０または配列番号２１または配列番号２２）を有し、前記３連グアニン配列と前記４連グアニン配列がそれぞれ対向する立体構造を有する。 The muscle differentiation-promoting oligo DNA according to one aspect of the present disclosure has a base sequence (SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22), and has a three-dimensional structure in which the triple guanine sequence and the quadruple guanine sequence face each other.
前記筋分化促進オリゴＤＮＡの塩基長は８以上１６以下であってもよい。 The base length of the muscle differentiation-promoting oligo DNA may be 8 or more and 16 or less.
前記筋分化促進オリゴＤＮＡの塩基長は１４以下であってもよい。 The muscle differentiation promoting oligo DNA may have a base length of 14 or less.
前記筋分化促進オリゴＤＮＡは、５’ＧＧＧＴＧＧＧＧ３’（配列番号４）または５’ＧＧＧＴＧＡＧＧＧＧ３’（配列番号５）のいずれかで表される塩基配列を有してもよい。 The muscle differentiation promoting oligo DNA may have a base sequence represented by either 5'GGGTGGGG3' (SEQ ID NO: 4) or 5'GGGTGAGGGG3' (SEQ ID NO: 5).
前記筋分化促進オリゴＤＮＡは、５’ＴＴＧＧＧＴＧＧＧＧＡＡ３’（配列番号６）または５’ＴＴＧＧＧＴＧＡＧＧＧＧＡＡ３’（配列番号７）で表される塩基配列を有してもよい。 The muscle differentiation promoting oligo DNA may have a base sequence represented by 5'TTGGGTGGGGAA3' (SEQ ID NO: 6) or 5'TTGGGTGAGGGGAA3' (SEQ ID NO: 7).
前記筋分化促進オリゴＤＮＡは、５’ＴＴＧＧＧＴＧＧＧＧＡＡ３’（配列番号８）または５’ＴＴＧＧＧＴＧＡＧＧＧＧＡＡ３’（配列番号１０）で表されてもよい。 The muscle differentiation-promoting oligo DNA may be represented by 5'TTGGGTGGGGAA3' (SEQ ID NO: 8) or 5'TTGGGTGAGGGGAA3' (SEQ ID NO: 10 ).
前記筋分化促進オリゴＤＮＡは、５’ＴＴＴＧＧＧＴＧＧＧＧＡＡＡ３’（配列番号９）または５’ＴＴＴＧＧＧＴＧＡＧＧＧＧＡＡＡ３’（配列番号１１）で表されてもよい。 The muscle differentiation-promoting oligo DNA may be represented by 5'TTTGGGTGGGGAAA3' (SEQ ID NO: 9 ) or 5'TTTGGGTGAGGGGAAA3' (SEQ ID NO: 11).
本開示の一態様によれば、塩基長１２～１６という、さらに好ましくは塩基長１４以下という、比較的短くて安定な、化学合成に適した非テロメア型のオリゴＤＮＡを用い、筋分化を強力に促進することが可能となる。 According to one aspect of the present disclosure, a relatively short and stable non-telomeric oligo DNA having a length of 12 to 16 bases, more preferably 14 bases or less, and suitable for chemical synthesis is used to strongly promote muscle differentiation. This makes it possible to promote
（本開示の実施形態）
以下、本開示の一態様に係る実施の形態（以下、本実施の形態）について図面を参照して詳細に説明する。
(Embodiments of the present disclosure)
Hereinafter, an embodiment (hereinafter referred to as the present embodiment) according to one aspect of the present disclosure will be described in detail with reference to the drawings.
その前に、まず骨格筋の再生メカニズムについて図１を参照しながら説明する。図１は、筋肉組織の一部に損傷もしくは劣化が生じたときの、骨格筋組織の再生および恒常性維持機構を示す説明図である。骨格筋は、衛星細胞と呼ばれる骨格筋幹細胞の増殖と分化によって、組織としての恒常性が保たれる。筋再生時には、衛星細胞は筋芽細胞と呼ばれる筋前駆細胞へと活性化される。筋芽細胞は数回の細胞分裂によって増殖した後、筋細胞へと分化する。筋細胞は互いに融合して多核の筋管を形成し、骨格筋組織を再生する。このように、筋芽細胞は骨格筋の維持・再生において中心的な役割を担っている。しかし、老化や様々な疾患によって筋芽細胞の分化能が低下することが報告されており、これが骨格筋萎縮の原因の一つと考えられていた。 Before that, the regeneration mechanism of skeletal muscle will first be explained with reference to FIG. 1. FIG. 1 is an explanatory diagram showing the regeneration and homeostasis mechanism of skeletal muscle tissue when damage or deterioration occurs in a part of the muscle tissue. Skeletal muscle tissue homeostasis is maintained through the proliferation and differentiation of skeletal muscle stem cells called satellite cells. During muscle regeneration, satellite cells are activated into muscle precursor cells called myoblasts. Myoblasts proliferate through several rounds of cell division and then differentiate into muscle cells. Muscle cells fuse with each other to form multinucleated myotubes and regenerate skeletal muscle tissue. Thus, myoblasts play a central role in the maintenance and regeneration of skeletal muscle. However, it has been reported that the differentiation ability of myoblasts decreases with aging and various diseases, and this was thought to be one of the causes of skeletal muscle atrophy.
そこで、発明者らは以前、乳酸菌Ｌａｃｔｏｂａｃｉｌｌｕｓ ｒｈａｍｎｏｓｕｓ ＧＧのゲノム配列に由来するオリゴＤＮＡライブラリを用いて筋分化促進作用の検証実験を行った（特許文献２）。その結果、配列式１（５’ＡＧＡＴＴＡＧＧＧＴＧＡＧＧＧＴＧＡ３’）で示される１８塩基のオリゴＤＮＡ（ｉＳＮ０４）が筋芽細胞の分化を顕著に促進することが明らかとなった。なお、本実施の形態において、オリゴＤＮＡとは６から２５の塩基からなる短いＤＮＡを意味する。好ましくは９から１８の塩基からなる。また、配列式１においてＴＴＡＧＧＧはテロメア配列と呼ばれ、その繰り返し配列（ＴＴＡＧＧＧ）ｎは、真核生物の染色体の末端部に存在する。テロメアは細胞分裂すなわちＤＮＡ複製とともに短くなり、細胞の老化が進むとされる（特許文献１）。 Therefore, the inventors previously conducted an experiment to verify the muscle differentiation promoting effect using an oligo DNA library derived from the genome sequence of lactic acid bacterium Lactobacillus rhamnosus GG (Patent Document 2). As a result, it was revealed that the 18 base oligo DNA (iSN04) represented by the sequence formula 1 (5'AGATTAGGGTGAGGGGTGA3') significantly promoted myoblast differentiation. Note that in this embodiment, oligo DNA means short DNA consisting of 6 to 25 bases. Preferably it consists of 9 to 18 bases. Furthermore, in sequence formula 1, TTAGGG is called a telomere sequence, and its repeating sequence (TTAGGG)n is present at the end of a eukaryotic chromosome. It is said that telomeres become shorter with cell division, that is, DNA replication, and cell aging progresses (Patent Document 1).
ｉＳＮ０４による筋芽細胞の分化促進効果については以下のように考えられる。分子シミュレーションによるｉＳＮ０４の立体構造解析（図２）ならびに塩基置換ないし欠失を導入した変異ｉＳＮ０４の活性測定（図３）から、１３～１５番目の塩基（ＧＧＧ）が分子内で近接（スタック）しており、ここが活性に必須の部位であることがわかった。 The effect of iSN04 on promoting myoblast differentiation is thought to be as follows. Three-dimensional structural analysis of iSN04 by molecular simulation (Figure 2) and activity measurement of mutant iSN04 with base substitutions or deletions (Figure 3) revealed that the 13th to 15th bases (GGG) are close together (stacked) within the molecule. This was found to be an essential site for activity.
図３の実験結果について、より詳細に説明する。ここで、図中のオリゴＤＮＡの配列において、「－」は塩基が削除され、その両端の塩基が直に結合していることを表す。本実験では、マウス骨格筋から採取した衛星細胞を初代培養して得られた筋芽細胞を用いた。コラーゲンコートした９６－ｗｅｌｌプレートに、１万個／ｗｅｌｌのマウス筋芽細胞を播種する。翌日、１０μＭのｉＳＮ０４および変異ｉＳＮ０４、または陰性対象（純水、図中Ｃｏｎｔｒｏｌ）を投与する。４８時間後に、骨格筋の最終分化マーカーであるミオシン重鎖（ＭＨＣ）の抗体を用いて免疫染色する。このとき、ＤＡＰＩ染色によって細胞核を可視化する。イメージアナライザーを用いて、細胞の染色画像の撮影と、ＭＨＣ陽性細胞の割合（ＭＨＣ陽性細胞内に存在するＤＡＰＩ陽性細胞核の数を、全てのＤＡＰＩ陽性細胞核の数で除したもの）の算出を自動的に行う。 The experimental results shown in FIG. 3 will be explained in more detail. Here, in the oligo DNA sequence in the figure, "-" indicates that a base has been deleted and the bases at both ends are directly bonded. In this experiment, myoblasts obtained by primary culturing satellite cells collected from mouse skeletal muscle were used. 10,000 mouse myoblasts/well are seeded onto a collagen-coated 96-well plate. The next day, 10 μM of iSN04 and mutant iSN04, or a negative control (pure water, Control in the figure) is administered. After 48 hours, immunostaining is performed using an antibody for myosin heavy chain (MHC), which is a marker for terminal differentiation of skeletal muscle. At this time, cell nuclei are visualized by DAPI staining. An image analyzer is used to automatically capture stained images of cells and calculate the percentage of MHC-positive cells (the number of DAPI-positive cell nuclei present in MHC-positive cells divided by the number of all DAPI-positive cell nuclei). Do it on purpose.
実験の結果、陰性対象では約２０％であるＭＨＣ陽性細胞率が、ｉＳＮ０４投与群では５０％を超えていた。このことから、ｉＳＮ０４が筋芽細胞からＭＨＣ陽性の筋細胞ないし筋管への分化を促進していることがわかる。一方、ｉＳＮ０４の１５番目のＧを削除した変異ｉＳＮ０４（Δ１５）、１４～１５番目のＧＧを削除したΔ１４－１５、１３～１５番目のＧＧＧを削除したΔ１３－１５の順に、筋分化促進活性が低下した。これらの結果から、ｉＳＮ０４の１３～１５番目のＧＧＧが筋分化促進活性に不可欠であることがわかった。 As a result of the experiment, the MHC positive cell rate was approximately 20% in the negative control group, but exceeded 50% in the iSN04 administration group. This shows that iSN04 promotes the differentiation of myoblasts into MHC-positive myocytes or myotubes. On the other hand, mutant iSN04 (Δ15) in which the 15th G of iSN04 was deleted, Δ14-15 in which the 14th to 15th GGs were deleted, and Δ13-15 in which the 13th to 15th GGG were deleted showed muscle differentiation promoting activity in the following order: decreased. These results revealed that GGG at positions 13 to 15 of iSN04 is essential for muscle differentiation promoting activity.
以上のｉＳＮ０４を用いた結果から、ｉＳＮ０４の立体構造の特徴を有していれば、１８塩基より短いオリゴＤＮＡでも筋分化促進活性を発揮する可能性が考えられる。筋分化促進オリゴＤＮＡの臨床応用を考えるとき、より短い（分子量の小さい）オリゴＤＮＡの開発は、合成コストの低下や、摂取・吸収率の向上につながると期待される。また、安定性についても分子量が小さいほど有利であることが一般的に期待される。 From the above results using iSN04, it is considered possible that even oligo DNA shorter than 18 bases can exhibit muscle differentiation promoting activity if it has the three-dimensional structure characteristics of iSN04. When considering the clinical application of muscle differentiation-promoting oligo DNA, the development of shorter (lower molecular weight) oligo DNA is expected to lead to lower synthesis costs and improved uptake and absorption rates. In addition, it is generally expected that the smaller the molecular weight, the more advantageous it will be in terms of stability.
表１に本実施の形態におけるオリゴＤＮＡの配列（配列番号８～１５）を示す。なお、本実施形態における前記オリゴＤＮＡは一本鎖であってもよい。また、核酸分解酵素に対して耐性を高めるためにホスホジエステル結合を有するオリゴＤＮＡのリン酸基の酸素原子を硫黄原子で置換したもの（例えばホスホロチオエート結合）であってもよいが、これらに限定されない。
表１のオリゴＤＮＡ（ｉＭｙｏ０１～０８）の塩基配列は、上記の分子シミュレーションおよび実験結果を踏まえたものである。すなわちｉＳＮ０４の１３～１５番目のＧＧＧの立体構造の特徴を保持すべく設計されたものである。ただし、いずれのオリゴＤＮＡにもテロメア配列（ＴＴＡＧＧＧ）は含まれていない。特に、ｉＭｙｏ０１～０４は、３連グアニン配列（ＧＧＧ）と４連グアニン配列（ＧＧＧＧ）がアデニン（Ａ）かチミン（Ｔ）のいずれか又は両方を含む１乃至３個の塩基を介して結合した塩基配列を有する、という特徴を有している。当該塩基配列はＧＧＧＷＧＧＧＧ（配列番号２０）またはＧＧＧＷＷＧＧＧＧ（配列番号２１）またはＧＧＧＷＮＷＧＧＧＧ（配列番号２２）と表すこともできる。ここでＷはアデニン（Ａ）かチミン（Ｔ）を、Ｎはアデニン（Ａ）かチミン（Ｔ）かグアニン（Ｇ）かシトシン（Ｃ）のいずれかを表す。さらにｉＭｙｏ０１～０４の塩基長は８以上１６以下となっている。 The base sequences of the oligo DNAs (iMyo01-08) in Table 1 are based on the above molecular simulation and experimental results. In other words, it was designed to maintain the three-dimensional structure characteristics of GGG at positions 13 to 15 of iSN04. However, none of the oligo DNAs contains a telomere sequence (TTAGGG). In particular, in iMyo01 to 04, a triple guanine sequence (GGG) and a quadruple guanine sequence (GGGG) are linked via 1 to 3 bases containing either adenine (A) or thymine (T), or both. It has the characteristic of having a base sequence. The base sequence can also be expressed as GGGWGGGG (SEQ ID NO: 20), GGGWWGGGG (SEQ ID NO: 21), or GGGWNWGGGG (SEQ ID NO: 22). Here, W represents adenine (A) or thymine (T), and N represents either adenine (A), thymine (T), guanine (G), or cytosine (C). Furthermore, the base length of iMyo01 to 04 is 8 or more and 16 or less.
例えば、ｉＭｙｏ０１、０２はコア配列ＧＧＧＴＧＧＧＧ（配列番号４）を、ｉＭｙｏ０３、ｉＭｙｏ０４はコア配列ＧＧＧＴＧＡＧＧＧＧ（配列番号５）を有することにより、より好ましくは、ｉＭｙｏ０１、ｉＭｙｏ０２はコア配列ＴＴＧＧＧＴＧＧＧＧＡＡ（配列番号６）を、ｉＭｙｏ０３、ｉＭｙｏ０４はコア配列ＴＴＧＧＧＴＧＡＧＧＧＧＡＡ（配列番号７）をそれぞれ有することにより、筋分化促進活性に有利な構造を有していると考えられる。 For example, iMyo01 and iMyo02 have the core sequence GGGTGGGG (SEQ ID NO: 4), and iMyo03 and iMyo04 have the core sequence GGGTGAGGGG (SEQ ID NO: 5), and more preferably, iMyo01 and iMyo02 have the core sequence TTGGGTGGGGAA (SEQ ID NO: 6). , iMyo03, and iMyo04 each have a core sequence TTGGGTGAGGGGAA (SEQ.
そこで、ｉＭｙｏ０３（配列番号１０）、ｉＭｙｏ０４（配列番号１１）、および比較例としてｉＭｙｏ０７（配列番号１４）の、それぞれ分子シミュレーションによる立体構造解析を行った。立体構造の算出には単純軌跡和マルチカノニカル分子動力学（ＭｃＭＤ）法（Ｊ． Ｉｋｅｂｅ ｅｔ ａｌ．， Ｊ． Ｃｏｍｐｕｔ． Ｃｈｅｍ．， ２０１１）を用いた。ＭｃＭＤ法は、高温から低温までを構造サンプリングすることで、効率的に室温付近における平衡状態の構造集団を求めることができる手法である。ここでは、３１０Ｋにおける構造集団を求め、解析に用いた。なお、本方法は前述のｉＳＮ０４の立体構造計算と同じである。立体構造解析の結果を、それぞれ図４、図５、図６に示す。 Therefore, three-dimensional structure analysis was performed by molecular simulation of iMyo03 (SEQ ID NO: 10), iMyo04 (SEQ ID NO: 11), and iMyo07 (SEQ ID NO: 14) as a comparative example. A simple trajectory sum multicanonical molecular dynamics (McMD) method (J. Ikebe et al., J. Comput. Chem., 2011) was used to calculate the three-dimensional structure. The McMD method is a method that can efficiently obtain a structural population in an equilibrium state near room temperature by sampling structures from high temperatures to low temperatures. Here, the structural population at 310K was determined and used for analysis. Note that this method is the same as the three-dimensional structure calculation of iSN04 described above. The results of the three-dimensional structure analysis are shown in FIGS. 4, 5, and 6, respectively.
各図の左側に示した立体構造（水素原子を非表示、５’末端から３’ 末端へ、塩基ごとにグラジュエ―ションを施す）は３１０Ｋにおいて最も確率の高い（最安定な）計算構造である。図４のｉＭｙｏ０３（配列番号１０）の立体構造は７～９番目ＴＧＡの部分で曲がった構造を示している。また、１番目のＴ（チミン）が曲がった部分に入り込み、全体的な立体構造の保持に働いているように見える。 The three-dimensional structure shown on the left side of each figure (hydrogen atoms hidden, gradient applied for each base from the 5' end to the 3' end) is the calculated structure with the highest probability (most stable) at 310 K. . The three-dimensional structure of iMyo03 (SEQ ID NO: 10) in FIG. 4 shows a bent structure at the 7th to 9th TGA portions. Additionally, the first T (thymine) seems to enter the curved part and work to maintain the overall three-dimensional structure.
図４の右側に、ｉＭｙｏ０３分子内における各塩基間の接触確率を示す。濃い色の部分は接触確率が高く、塩基同士が近くに位置していることを表している。ｉＭｙｏ０３内では特に４番目と１１番目、５番目と１０番目、６番目と９番目、７番目と９番目の塩基同士付近が濃い色を示し、それらのペアは近くに位置している確率が高い。これは両端が接触したヘアピン様の立体構造を表している。すなわち、ｉＭｙｏ０３の立体構造的な特徴として、塩基配列の７～９番目で折れ曲がり、かつ両側の連続グアニンＧＧＧが互いに逆平行に並んで対向している可能性が高い。 The right side of FIG. 4 shows the probability of contact between each base within the iMyo03 molecule. Dark colored areas have a high probability of contact, indicating that the bases are located close to each other. In iMyo03, the areas around the 4th and 11th, 5th and 10th, 6th and 9th, and 7th and 9th bases show a particularly dark color, and there is a high probability that these pairs are located nearby. . This represents a hairpin-like three-dimensional structure with both ends touching. That is, as a three-dimensional structural feature of iMyo03, it is highly likely that the base sequence is bent at positions 7 to 9, and continuous guanines GGG on both sides are arranged antiparallel to each other and face each other.
図５のｉＭｙｏ０４（配列番号１１）では、ｉＭｙｏ０３で見られたヘアピン様の特徴が明らかではない。図５中の左側において、ｉＭｙｏ０４の最安定な立体構造はヘアピン様構造を形成している。しかし、１番目のチミン（Ｔ）は曲がった部分には入り込んでいない。加えて、図５中の右側の図（ｉＭｙｏ０４内の各塩基間の接触確率）からｉＭｙｏ０３で見られた特徴が薄いことがわかる。 In iMyo04 (SEQ ID NO: 11) in FIG. 5, the hairpin-like feature seen in iMyo03 is not evident. On the left side of FIG. 5, the most stable conformation of iMyo04 forms a hairpin-like structure. However, the first thymine (T) does not enter the curved part. In addition, it can be seen from the diagram on the right side of FIG. 5 (contact probability between each base in iMyo04) that the features observed in iMyo03 are weak.
図６のｉＭｙｏ０７（配列番号１４）立体構造はｉＭｙｏ０３（配列番号１０）およびｉＭｙｏ０４（配列番号１１）のものとは大きく異なる。また、右側の図（ｉＭｙｏ０７内の各塩基間の接触確率）からはヘアピン様の特徴が見られない。つまり、ｉＭｙｏ０７の立体構造的な特徴はｉＭｙｏ０３およびｉＭｙｏ０４のものとは異なり、ｉＳＮ０４とも異なることを示している。 The tertiary structure of iMyo07 (SEQ ID NO: 14) in Figure 6 is significantly different from that of iMyo03 (SEQ ID NO: 10) and iMyo04 (SEQ ID NO: 11). Moreover, no hairpin-like feature is seen in the figure on the right (contact probability between each base in iMyo07). In other words, the three-dimensional structural features of iMyo07 are different from those of iMyo03 and iMyo04, and also different from iSN04.
以上、まとめると、ｉＭｙｏ０３はヘアピン様構造の安定性が高く、かつ、ｉＳＮ０４の３連グアニン（ＧＧＧ）と同様な局所構造を保っている。ｉＭｙｏ０４はｉＭｙｏ０３に比べて構造安定性が低く、立体構造にばらつきがある。ｉＭｙｏ０７の立体構造はｉＳＮ０４、ｉＭｙｏ０３、ｉＭｙｏ０４と異なる。以上の構造的特徴は、各オリゴＤＮＡにおける筋分化促進活性効果と密接に関係している。これについては実施例で詳細に説明する。 In summary, iMyo03 has a highly stable hairpin-like structure and maintains a local structure similar to the triple guanine (GGG) of iSN04. iMyo04 has lower structural stability than iMyo03, and its steric structure has variations. The three-dimensional structure of iMyo07 is different from iSN04, iMyo03, and iMyo04. The above structural features are closely related to the muscle differentiation promoting activity of each oligo DNA. This will be explained in detail in Examples.
なお、本実施の形態におけるオリゴＤＮＡを筋分化促進剤として使用する場合、個体を構成する細胞に対しても培養液で培養された細胞に対しても適用可能である。また、前記筋分化促進剤はマウス、ヒトを含む哺乳類もしくはニワトリを含む鳥類を対象とすることができるが、これらに限定されない。さらに、本実施の形態における筋分化促進剤は、注射薬、塗布薬、錠剤、カプセル剤、シロップ、座薬等に製剤化できるが、これらに限定されない。また、前記筋分化促進剤は、経口、静脈内、筋肉内、関節内、動脈内、髄内、髄腔内、心室内、経皮、皮下、腹腔内、経腸、局所、舌下または直腸手段によって投与することができるが、これらに限定されない。 In addition, when the oligo DNA in this embodiment is used as a muscle differentiation promoting agent, it is applicable to both cells constituting an individual and cells cultured in a culture solution. Furthermore, the muscle differentiation promoting agent can be used for mammals including mice and humans, or birds including chickens, but is not limited thereto. Furthermore, the muscle differentiation promoting agent in this embodiment can be formulated into injections, liniments, tablets, capsules, syrups, suppositories, etc., but is not limited thereto. The muscle differentiation promoting agent may be administered orally, intravenously, intramuscularly, intraarticularly, intraarterially, intramedullarily, intrathecally, intraventricularly, transdermally, subcutaneously, intraperitoneally, enterally, locally, sublingually, or rectally. It can be administered by, but not limited to, means.
以下、本開示の実施例について説明する。なお、以下の実験に用いたオリゴＤＮＡのサンプル（ｉＳＮ０４（配列番号１）、ｉＳＮ１４（配列番号１６）、ｉＳＮ４５（配列番号１７）およびｉＭｙｏ０１～０８（配列番号８～１５）は、核酸分解酵素に対して耐性を高めるために、ヌクレオチド間のホスホジエステル結合をホスホロチオエート結合に置換して合成した後、ＨＰＬＣ精製したものである。
（実施例１）
図７に、市販の健常者ヒト筋芽細胞を用いた試験の結果を示す。まず、コラーゲンコートした９６－ｗｅｌｌプレートに、６千個／ｗｅｌｌのヒト筋芽細胞を播種する。翌日、３０μＭのオリゴＤＮＡまたは陰性対象（純水、図中Ｃｔｒｌ）を投与する。オリゴＤＮＡは、試験対象としてｉＭｙｏ０１～０８（配列番号８～１６）、陽性対照としてｉＳＮ０４（図中ｉＳＮ－０４）（配列番号１）、陰性対照としてｉＳＮ１４（図中ｉＳＮ－１４）（配列番号１６）およびｉＳＮ４５（図中ｉＳＮ－４５）（配列番号１７）を用いた。ｉＳＮ１４およびｉＳＮ４５は従来、筋分化促進活性を有しないことがわかっている（特許文献２）。
Examples of the present disclosure will be described below. The oligo DNA samples (iSN04 (SEQ ID NO: 1), iSN14 (SEQ ID NO: 16), iSN45 (SEQ ID NO: 17), and iMyo01-08 (SEQ ID NO: 8-15) used in the following experiments are nucleolytic enzymes. The phosphodiester bond between nucleotides was replaced with a phosphorothioate bond in order to increase the resistance to phthalate, and the compound was synthesized and purified by HPLC.
(Example 1)
FIG. 7 shows the results of a test using commercially available healthy human myoblasts. First, 6,000 human myoblasts/well are seeded onto a collagen-coated 96-well plate. The next day, 30 μM of oligo DNA or a negative control (pure water, Ctrl in the figure) is administered. The oligo DNAs were iMyo01 to 08 (SEQ ID NOs: 8 to 16) as test targets, iSN04 (iSN-04 in the figure) (SEQ ID NO: 1) as a positive control, and iSN14 (iSN-14 in the figure) (SEQ ID NO: 16) as a negative control. ) and iSN45 (iSN-45 in the figure) (SEQ ID NO: 17) were used. It has been known that iSN14 and iSN45 do not have muscle differentiation promoting activity (Patent Document 2).
４８時間後に、免疫染色によって骨格筋の最終分化マーカーであるミオシン重鎖（ＭＨＣ）の発現を定量化する。このとき、ＤＡＰＩ染色によって細胞核を可視化する。さらにイメージアナライザーを用いて、細胞の染色画像の撮影と、ＭＨＣシグナル強度（画像中のＭＨＣシグナル強度の総計を、ＤＡＰＩ陽性細胞核の数で除したもの）またはＭＨＣ陽性細胞率の算出を自動的に行った。陰性対照（純水群）とオリゴＤＮＡ投与群のＭＨＣシグナル強度の差は、Ｄｕｎｎｅｔｔ’ｓ ｔｅｓｔによって検定した。結果を図７に示す。 After 48 hours, the expression of myosin heavy chain (MHC), a marker of terminal differentiation of skeletal muscle, is quantified by immunostaining. At this time, cell nuclei are visualized by DAPI staining. Furthermore, an image analyzer is used to automatically capture stained images of cells and calculate MHC signal intensity (total MHC signal intensity in the image divided by the number of DAPI-positive cell nuclei) or MHC-positive cell percentage. went. The difference in MHC signal intensity between the negative control (pure water group) and the oligo DNA administration group was tested using Dunnett's test. The results are shown in FIG.
ｉＳＮ０４（配列番号１）、ｉＭｙｏ０１（配列番号８）、ｉＭｙｏ０３（配列番号１０）を投与した群のＭＨＣシグナル強度は、有意な差をもって純水群（Ｃｔｒｌ）のＭＨＣシグナル強度を上回っていた。すなわち、ｉＭｙｏ０１およびｉＭｙｏ０３はｉＳＮ０４と同程度にヒト筋芽細胞の分化を促進することが示された。 The MHC signal intensity of the groups administered with iSN04 (SEQ ID NO: 1), iMyo01 (SEQ ID NO: 8), and iMyo03 (SEQ ID NO: 10) exceeded that of the pure water group (Ctrl) with a significant difference. In other words, iMyo01 and iMyo03 were shown to promote human myoblast differentiation to the same extent as iSN04.
（実施例２）
後日、ヒト筋芽細胞を用いた２回目の試験を、図７の実験と同様の方法で行った。結果を図８に示す。本実施例では、純水群（Ｃｔｒｌ）におけるＭＨＣシグナル強度が低く（実施例１の約１７０に対し本実施例では約６０）、ヒト筋芽細胞が分化誘導されにくい状態であったことがわかる。このような条件下においても、ｉＳＮ０４（配列番号１）およびｉＭｙｏ０３（配列番号１０）を投与した群のＭＨＣシグナル強度は、有意な差をもって純水群（Ｃｔｒｌ）のＭＨＣシグナル強度を上回った。一方、１回目の試験で筋分化促進活性を示したｉＭｙｏ０１（配列番号８）は、本２回目の試験ではＭＨＣシグナル強度を顕著には増強しなかった。以上の結果から、ｉＭｙｏ０３の筋分化促進活性はｉＳＮ０４と同程度であるが、ｉＭｙｏ０１の活性はｉＭｙｏ０３よりも劣っている可能性があると考えられる。
(Example 2)
At a later date, a second test using human myoblasts was conducted in the same manner as the experiment in FIG. The results are shown in FIG. In this example, the MHC signal intensity in the pure water group (Ctrl) was low (approximately 60 in this example compared to approximately 170 in Example 1), indicating that human myoblasts were in a state where it was difficult to induce differentiation. . Even under these conditions, the MHC signal intensity of the groups administered with iSN04 (SEQ ID NO: 1) and iMyo03 (SEQ ID NO: 10) significantly exceeded that of the pure water group (Ctrl). On the other hand, iMyo01 (SEQ ID NO: 8), which showed muscle differentiation promoting activity in the first test, did not significantly enhance MHC signal intensity in the second test. From the above results, it is considered that the muscle differentiation promoting activity of iMyo03 is comparable to that of iSN04, but the activity of iMyo01 may be inferior to that of iMyo03.
（実施例３）
本実施例ではマウス筋芽細胞でのスクリーニング試験とその結果について説明する。本実施例も図７の実験と同様の方法で行った。陰性対照（純水群）とオリゴＤＮＡ投与群のＭＨＣ陽性細胞率の差を、Ｄｕｎｎｅｔｔ’ｓ ｔｅｓｔによって検定した。結果を図９に示す。ｉＳＮ０４（配列番号１）、ｉＭｙｏ０３（配列番号１０）、ｉＭｙｏ０４（配列番号１１）を投与した群のＭＨＣ陽性細胞率は、有意な差をもって純水群（Ｃｔｒｌ）のＭＨＣ陽性細胞率を上回った。すなわち、ｉＭｙｏ０３およびｉＭｙｏ０４はｉＳＮ０４と同様にマウス筋芽細胞の分化を促進することが示された。
(Example 3)
This example describes a screening test using mouse myoblasts and its results. This example was also conducted in the same manner as the experiment shown in FIG. Differences in MHC-positive cell rates between the negative control (pure water group) and the oligo DNA administration group were tested using Dunnett's test. The results are shown in FIG. The MHC-positive cell rate of the groups administered with iSN04 (SEQ ID NO: 1), iMyo03 (SEQ ID NO: 10), and iMyo04 (SEQ ID NO: 11) exceeded the MHC-positive cell rate of the pure water group (Ctrl) with a significant difference. That is, iMyo03 and iMyo04 were shown to promote the differentiation of mouse myoblasts similarly to iSN04.
（実施例４）
本実施例ではニワトリ筋芽細胞でのスクリーニング試験とその結果について説明する。本実施例では、ニワトリ骨格筋から採取した衛星細胞を初代培養して得られた筋芽細胞を用いた。コラーゲンコートした９６－ｗｅｌｌプレートに、５千個／ｗｅｌｌのニワトリ筋芽細胞を播種する。翌日、１０μＭのオリゴＤＮＡまたは陰性対照（純水）を投与する。４８時間後にＭＨＣを免疫染色する。このとき、ＤＡＰＩ染色によって細胞核を可視化する。イメージアナライザーを用いて、細胞の染色画像の撮影と、ＭＨＣシグナル強度の算出を自動的に行った。陰性対照（純水群）とオリゴＤＮＡ投与群のＭＨＣシグナル強度の差を、Ｄｕｎｎｅｔｔ’ｓ ｔｅｓｔによって検定した。結果を図１０に示す。
(Example 4)
This example describes a screening test using chicken myoblasts and its results. In this example, myoblasts obtained by primary culturing satellite cells collected from chicken skeletal muscle were used. 5,000 chicken myoblasts/well are seeded onto a collagen-coated 96-well plate. The next day, administer 10 μM oligo DNA or negative control (pure water). MHC is immunostained after 48 hours. At this time, cell nuclei are visualized by DAPI staining. Using an image analyzer, stained images of cells were photographed and MHC signal intensity was automatically calculated. The difference in MHC signal intensity between the negative control (pure water group) and the oligo DNA administration group was tested using Dunnett's test. The results are shown in FIG.
ｉＳＮ０４（配列番号１）、ｉＭｙｏ０１～０４（配列番号８～１１）を投与した群のＭＨＣシグナル強度は、有意な差をもって純水群（Ｃｔｒｌ）のＭＨＣシグナル強度を上回った。以上の結果から、ｉＭｙｏ０１～０４はｉＳＮ０４と同様にニワトリ筋芽細胞の分化を促進することが示された。 The MHC signal intensity of the groups administered with iSN04 (SEQ ID NO: 1) and iMyo01-04 (SEQ ID NO: 8-11) exceeded that of the pure water group (Ctrl) with a significant difference. The above results showed that iMyo01-04 promoted the differentiation of chicken myoblasts similarly to iSN04.
以上、実施例１～４の結果をまとめると、ｉＳＮ０４は、ヒト、マウス、ニワトリのいずれの筋芽細胞に対しても、安定して顕著な筋分化促進活性を示した。同様に、ｉＭｙｏ０３は全ての試験において、いずれの筋芽細胞に対しても筋分化促進活性を示した。また、ｉＭｙｏ０１はヒト筋芽細胞の、ｉＭｙｏ０４はマウス筋芽細胞の分化を、それぞれ促進した。ニワトリ筋芽細胞に対しては、ｉＭｙｏ０１～０４が筋分化促進作用を示した。一方、ｉＭｙｏ０５～０８はいずれの筋芽細胞に対しても筋分化促進作用を示さなかった。 To summarize the results of Examples 1 to 4, iSN04 stably exhibited significant muscle differentiation-promoting activity against human, mouse, and chicken myoblasts. Similarly, iMyo03 showed muscle differentiation-promoting activity for all myoblasts in all tests. Furthermore, iMyo01 promoted the differentiation of human myoblasts, and iMyo04 promoted the differentiation of mouse myoblasts. For chicken myoblasts, iMyo01-04 showed a muscle differentiation promoting effect. On the other hand, iMyo05 to 08 did not show any muscle differentiation-promoting effect on any myoblasts.
以上の結果から、ｉＭｙｏ０３（配列番号１０）はｉＳＮ０４（配列番号１）と極めて類似した筋分化促進活性を有する、すなわちｉＭｙｏ０３（配列番号１０）とｉＳＮ０４（配列番号１）の構造はよく似た特徴を示すことが考えられる。一方で、ｉＭｙｏ０１（配列番号８）、ｉＭｙｏ０２（配列番号９）、ｉＭｙｏ０４（配列番号１１）の筋分化促進活性には種特異性があることが強く示唆された。 From the above results, iMyo03 (SEQ ID NO: 10) has very similar muscle differentiation promoting activity as iSN04 (SEQ ID NO: 1), that is, the structures of iMyo03 (SEQ ID NO: 10) and iSN04 (SEQ ID NO: 1) have very similar characteristics. It is possible to show that On the other hand, it was strongly suggested that the muscle differentiation-promoting activity of iMyo01 (SEQ ID NO: 8), iMyo02 (SEQ ID NO: 9), and iMyo04 (SEQ ID NO: 11) is species-specific.
以上の実験結果は、先に示した各オリゴＤＮＡの立体構造と密接に関係していると思われる。すなわち、ｉＭｙｏ０３（配列番号１０）の連続グアニン内において隣り合うグアニンＧＧＧは接触確率が高く（図４右の各塩基間接触確率）、ｉＳＮ０４（配列番号１）の連続グアニンＧＧＧ（１３～１５番目）と同様のスタック構造を形成している。つまり、ｉＳＮ０４の活性に必須のＧＧＧと同様な局所構造をｉＭｙｏ０３は有している。 The above experimental results are thought to be closely related to the three-dimensional structure of each oligo DNA shown above. In other words, adjacent guanines GGG in the consecutive guanines of iMyo03 (SEQ ID NO: 10) have a high contact probability (contact probability between each base on the right side of Figure 4), and consecutive guanines GGG (13th to 15th) in iSN04 (SEQ ID NO: 1) It forms a stack structure similar to . In other words, iMyo03 has a local structure similar to GGG, which is essential for the activity of iSN04.
これに対し、ｉＭｙｏ０４（配列番号１１）はｉＭｙｏ０３に比べて、ヘアピン様構造の安定性が低く（図５右の各塩基間接触確率）、両者の構造に大きなばらつきがある。この構造のばらつきのため、ｉＭｙｏ０４はｉＭｙｏ０３に比べて全体的に活性が低く、種特異性が現れたことが考えられる。さらに、ｉＭｙｏ０７（配列番号１４）に至ってはヘアピン様構造状の立体構造を有しておらず、そのため活性がなかったものと考えられる。このように、筋分化促進活性に違いが現れた理由として、ｉＭｙｏシリーズの立体構造的な特徴の違いが考えられる。 On the other hand, iMyo04 (SEQ ID NO: 11) has a hairpin-like structure with lower stability than iMyo03 (contact probability between each base on the right side of FIG. 5), and there is large variation in both structures. Due to this structural variation, iMyo04 has an overall lower activity than iMyo03, and it is thought that species specificity appears. Furthermore, iMyo07 (SEQ ID NO: 14) did not have a hairpin-like three-dimensional structure, and therefore it is thought that it had no activity. Thus, the reason for the difference in muscle differentiation promoting activity is thought to be the difference in the three-dimensional structural characteristics of the iMyo series.
なお、オリゴＤＮＡが種特異性を示すことは珍しくなく、むしろｉＳＮ０４のように、哺乳類と鳥類を含む幅広い種の細胞に活性を示す配列の方が少ない。例えば、ＣｐＧ ＯＤＮ ２００６（配列番号１８）はマウスとヒト両方のＴｏｌｌ様受容体９（ＴＬＲ９）を活性化するが、ＣｐＧ ＯＤＮ １８２６（配列番号１９）はマウスＴＬＲ９特異的でありヒトＴＬＲ９を活性化しない、といった報告がある（Ｐｏｈａｒ ｅｔ ａｌ．，Ｊ Ｉｍｍｕｎｏｌ，２０１５）。 Note that it is not uncommon for oligo DNA to exhibit species specificity; rather, there are fewer sequences like iSN04 that exhibit activity in cells of a wide range of species, including mammals and birds. For example, CpG ODN 2006 (SEQ ID NO: 18) activates both mouse and human Toll-like receptor 9 (TLR9), whereas CpG ODN 1826 (SEQ ID NO: 19) is specific for mouse TLR9 and activates human TLR9. There are reports that it does not (Pohar et al., J Immunol, 2015).
恐らく、ｉＳＮ０４（配列番号１）およびｉＭｙｏ０３（配列番号１０）の共通した構造的特徴は、ヒト、マウス、ニワトリで共通する分子基盤を認識ないし標的とするのに適していると考えられる。他方、ｉＭｙｏ０１、０２、０４の構造はｉＳＮ０４やｉＭｙｏ０３と少し異なるため、その微細な差異により、動物種によっては活性を発揮しないと推測される。 Presumably, the common structural features of iSN04 (SEQ ID NO: 1) and iMyo03 (SEQ ID NO: 10) make them suitable for recognizing or targeting common molecular bases in humans, mice, and chickens. On the other hand, since the structures of iMyo01, 02, and 04 are slightly different from those of iSN04 and iMyo03, it is presumed that due to the minute differences, they do not exhibit activity depending on the animal species.
いずれの種の筋芽細胞において作用を示さなかったｉＭｙｏ０５～０８（配列番号１２～１５）は、塩基配列後半のグアニンが３連であることがｉＭｙｏ０１～０４（配列番号８～１１）とは異なる。したがって、ｉＭｙｏ０１～０４の活性には塩基配列後半の４連グアニン（ＧＧＧＧ）が重要であることが強く示唆される。ｉＭｙｏ０１～０４の４連グアニンは、ｉＳＮ０４の活性に不可欠な１３～１５番目の３連グアニンと類似の立体構造に重要であると考えられる。 iMyo05-08 (SEQ ID NO: 12-15), which did not show any effect on myoblasts of any species, differs from iMyo01-04 (SEQ ID NO: 8-11) in that there are three guanines in the latter half of the base sequence. . Therefore, it is strongly suggested that the quadruple guanine (GGGG) in the latter half of the base sequence is important for the activity of iMyo01-04. The quadruple guanines of iMyo01 to 04 are considered to be important in a steric structure similar to the 13th to 15th triple guanines, which are essential for the activity of iSN04.
以上、本開示によれば、塩基長１２～１６のオリゴＤＮＡ、例えば塩基長１４のｉＭｙｏ０３（配列番号１０）で示されるオリゴＤＮＡにより筋分化を強力に促進することが可能となる。すなわち筋分化を強力に促進する一連のオリゴＤＮＡ群を曝露した筋芽細胞では、ミオシン重鎖（ＭＨＣ）陽性の筋細胞への分化および筋管の形成が促進されることが確認された。しかも、上記オリゴＤＮＡは化学合成が可能で安定構造を有すため、将来的に大量に供給および保存ができる。 As described above, according to the present disclosure, it is possible to strongly promote muscle differentiation using an oligo DNA having a length of 12 to 16 bases, for example, an oligo DNA shown by iMyo03 (SEQ ID NO: 10) having a length of 14 bases. That is, it was confirmed that in myoblasts exposed to a series of oligo DNA groups that strongly promote muscle differentiation, differentiation into myosin heavy chain (MHC)-positive muscle cells and formation of myotubes were promoted. Furthermore, since the oligo DNA can be chemically synthesized and has a stable structure, it can be supplied and stored in large quantities in the future.
本発明は、加齢性筋萎縮症（サルコペニア）、癌悪液質（カヘキシー）による骨格筋量の減少、糖尿病性筋萎縮症などの、加齢性、疾患性および／または代謝性の筋萎縮の予防および／または治療、筋疾患の再生医療、横紋筋肉腫の増殖および／または転移の抑制、多能性幹細胞（ＥＳ細胞やｉＰＳ細胞）または体性幹細胞の筋分化誘導、創薬のスクリーニングに供する骨格筋幹細胞（衛星細胞）、骨格筋前駆細胞（筋芽細胞）および骨格筋細胞からなる群より選択される少なくとも１種の作成、食肉用家畜および／または家禽の飼料の添加物、食肉用家畜および／または家禽の骨格筋の発生、形成および／または生育の促進に利用することができる。
The present invention deals with age-related, disease-related, and/or metabolic muscle atrophy, such as age-related muscular atrophy (sarcopenia), decreased skeletal muscle mass due to cancer cachexia, and diabetic muscular atrophy. prevention and/or treatment of muscle diseases, regenerative medicine for muscle diseases, suppression of rhabdomyosarcoma growth and/or metastasis, induction of muscle differentiation of pluripotent stem cells (ES cells and iPS cells) or somatic stem cells, screening for drug discovery. Creation of at least one selected from the group consisting of skeletal muscle stem cells (satellite cells), skeletal muscle progenitor cells (myoblasts), and skeletal muscle cells, additives for livestock and/or poultry feed, and meat. It can be used to promote the development, formation and/or growth of skeletal muscle in livestock and/or poultry.
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