JP7390694B2 - 随時尿中細胞を用いた筋系細胞の誘導方法 - Google Patents
随時尿中細胞を用いた筋系細胞の誘導方法 Download PDFInfo
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Description
(1)尿中細胞から筋管を作製する方法であって、
尿中細胞にMYOD1遺伝子を導入する導入ステップと、
前記尿中細胞を少なくとも1種のエピジェネティクス制御化合物に曝露する曝露ステップと、
を含む方法。
(2)前記導入ステップ及び前記曝露ステップ後の前記尿中細胞が筋芽細胞及び筋管からなる群より選択される少なくとも1種を含む、(1)に記載の方法。
(3)エピジェネティクス制御化合物が、ヒストンメチル基転移酵素阻害剤、ヒストン脱メチル化酵素阻害剤、ヒストン脱アセチル化酵素阻害剤、SIRT2阻害剤、及びPARP阻害剤からなる群より選択される少なくとも1種を含む、(1)又は(2)に記載の方法。
(4)ヒストンメチル基転移酵素阻害剤が、3-デアザネプラノシンA及び3-デアザネプラノシンA塩酸塩(DZNep)、GSK343、SGC707、フラミジン二塩酸塩、UNC2327、E7438、及びMI-2(メニン-MLL阻害剤)からなる群より選択される少なくとも1種を含む、(3)に記載の方法。
(5)ヒストン脱メチル化酵素阻害剤が、IOX 1及びGSK-J1からなる群より選択される少なくとも1種を含む、(3)に記載の方法。
(6)ヒストン脱アセチル化酵素阻害剤が、LMK-235、CAY10603、BRD73954、及びVORINOSTATからなる群より選択される少なくとも1種を含む、(3)に記載の方法。
(7)SIRT2阻害剤がSirReal 2を含む、(3)に記載の方法。
(8)PARP阻害剤がEB47を含む、(3)に記載の方法。
(9)MYOD1遺伝子の導入が、誘導性プロモーターの制御下にあるMYOD1遺伝子を含む発現ベクターの導入により行われる、(1)~(8)のいずれかに記載の方法。
(10)発現ベクターが選択マーカー遺伝子をさらに含む、(9)に記載の方法。
(11)尿中細胞が、筋疾患患者又は筋ジストロフィー患者由来のものである、(1)~(10)のいずれかに記載の方法。
(12)尿中細胞から筋管を作製するためのキットであって、
尿中細胞にMYOD1遺伝子を導入するための導入手段と、
少なくとも1種のエピジェネティクス制御化合物と、
を備えるキット。
(13)前記導入手段が、MYOD1遺伝子を尿中細胞へ導入するための発現ベクターである、請求項(12)に記載のキット。
(14)エピジェネティクス制御化合物が、ヒストンメチル基転移酵素阻害剤、ヒストン脱メチル化酵素阻害剤、ヒストン脱アセチル化酵素阻害剤、SIRT2阻害剤、及びPARP阻害剤からなる群より選択される少なくとも1種を含む、(12)又は(13)に記載のキット。
(15)エピジェネティクス制御化合物が、3-デアザネプラノシンA及び3-デアザネプラノシンA塩酸塩(DZNep)、GSK343、SGC707、フラミジン二塩酸塩、UNC2327、E7438、及びMI-2(メニン-MLL阻害剤)、IOX 1及びGSK-J1、LMK-235、CAY10603、BRD73954、及びVORINOSTAT、SirReal 2、並びにEB47からなる群より選択される少なくとも1種を含む、(12)~(14)のいずれかに記載のキット。
(16)筋ジストロフィー患者に対するエクソン・スキップ治療薬の検定方法であって、
(1)~(11)のいずれかに記載の方法により筋ジストロフィー患者の尿中細胞から筋管を作製する作製ステップと、
前記筋管にエクソン・スキップ治療薬を適用する適用ステップと、
前記筋管におけるジストロフィンmRNA及び/又はタンパク質の回復を検出する検出ステップと、を含む方法。
(17)前記検出ステップにおいて、ジストロフィンmRNA及び/又はタンパク質の回復が、RT-PCR、ウエスタンブロット、及び免疫細胞染色からなる群より選択される少なくとも1つの方法により検出される、(16)に記載の方法。
(18)エクソン・スキップ治療薬が、エクソン44スキップ薬、エクソン45スキップ薬、エクソン50スキップ薬、エクソン51スキップ薬及びエクソン53スキップ薬からなる群より選択される少なくとも1種を含む、(16)又は(17)に記載の方法。
(19)骨格筋障害を引き起こす状態の治療薬又は予防薬候補のスクリーニング方法であって、
骨格筋障害を引き起こす状態を有する患者の尿中細胞から(1)~(11)のいずれかに記載の方法により筋管を作製する作製ステップと、
前記筋管に被験物質又は因子を適用する適用ステップと、
前記適用ステップ後の前記筋管における変化をモニターすることにより前記被験物質又は因子を治療薬又は予防薬候補として同定する同定ステップと、
を含む方法。
本発明は、非侵襲的かつ効率的に尿中細胞から尿細胞由来筋管を作製するための方法及びキット、並びにかかる尿細胞由来筋管の用途に関する。
骨格筋障害を引き起こす状態を有する患者の尿中細胞から上述した方法により筋管を作製する作製ステップと、
前記筋管に治療薬を適用する適用ステップと、
前記筋管における骨格筋障害の状態の改善を検出する検出ステップと、
を含む。具体的な実施形態において、本発明は、筋ジストロフィー患者に対するエクソン・スキップ治療薬の検定方法であって、
上述した方法によって筋ジストロフィー患者の尿中細胞から筋管を作製する作製ステップと、
前記筋管にエクソン・スキップ治療薬を適用する適用ステップと、
前記適用ステップ後の前記筋管におけるジストロフィンmRNA及び/又はタンパク質の回復を検出する検出ステップと、
を含む方法に関する。
骨格筋障害を引き起こす状態を有する患者の尿中細胞から上述した方法によって筋管を作製する作製ステップと、
前記筋管に被験物質又は因子を適用する適用ステップと、
前記適用ステップ後の前記筋管における変化をモニターすることにより前記被験物質又は因子を治療薬又は予防薬候補として同定する同定ステップと、
を含む。
滅菌したプラスチックボトル(Corning Incorporated, NY, USA; 430281)に対して対象に排尿させることによって尿を採取した。Zhouらの方法(Zhou, T. et al. Nature protocols vol.7, pp.2080-2089, 2012)を若干変更して、採取した尿の初代細胞培養を採取後数時間以内に行った。
In-Fusion HD Cloning Plus(Clontech; 638909)を用いてMYOD1配列(CCDS 7826.1)をpRetroX-TetOne-Puroベクター(Clontech; 634307)に挿入した。GP2-293細胞(Clontech; 631458)をコラーゲンコートした細胞培養用プレート上で、10%ウシ胎児血清を含むDMEM培地で培養した。GP2-293細胞に、pVZV-Gカプシドベクターと作製したMYOD1挿入後のpRetroX-TetOne-Puroベクターを、Xfect trasfection reagent(Clontech; 631317)を用いてGP2-293細胞に導入した。GP2-293細胞が産生したレトロウイルスベクター(以下「MYOD1ウイルスベクター」という)(図2)を、24時間後と48時間後に培養上清から回収し、-80℃フリーザー内に保存した。図2に示すレトロウイルスベクターは、MYOD1遺伝子がTRE3GSプロモーターの制御下にあるため、ドキシサイクリン(Dox)によりMYOD1遺伝子の発現を誘導可能である。また、選択マーカーとしてピューロマイシン耐性遺伝子を含む。
尿中細胞を培養用ディッシュ又はプレート上に播種し(例えば、3,000~5,000個/cm2)、増殖培地内で培養後(例えば24時間後)にポリブレンなどを用いてMYOD1ウイルスベクターを感染させることにより尿中細胞にMYOD1を導入した。これにより、導入ステップが行われた。感染の一定期間後にピューロマイシンを培地に添加し数日間培養することで、MYOD1陽性尿中細胞を選択した。
MYOD1陽性尿中細胞をコラーゲンコートした培養用ディッシュ又はプレート上に播種し、ドキシサイクリン(例えば1μg/mL)を添加した分化培地(高グルコース含有DMEM with GlutaMAX-I(Thermo Fisher Scientific; 10569-010)、5%ウマ血清、ITS Liquid Media Supplement(Sigma; I3146)、1μg/mL ドキシサイクリンを含む)で培養し、筋管を誘導した。その際、化合物ライブラリー(Sigma; S990043-EPI1)を用いた低分子化合物を分化培地に添加することにより、筋分化を促進し得るかを検討した。低分子化合物は最終濃度を0.1、1又は10μMとして添加した。筋管誘導の評価は免疫細胞染色及びウエスタンブロットで行った。
DMD患者の尿中細胞から誘導された筋管(尿細胞由来筋管)を対象に、エクソン・スキップ治療薬であるアンチセンス・オリゴヌクレオチド(AON)の治療効果を検定可能か検討するために以下の実験を行った。DMD遺伝子にエクソン45欠損を有するDMD患者(1名)から尿を採取し、実施例1~3に記載の手順で尿中細胞から筋管を誘導した。筋分化誘導後7日時点で、エクソン・スキップ治療薬であるAONと6μMエンドポーター(Gene Tools, Philomath, OR, USA)を含む分化培地に変更した。さらに3日後に分化培地のみの培地に変更し、筋分化誘導後14日時点で細胞を回収した。使用したAONの詳細は、Wilton, S. D. et al. Mol Ther 15, 1288-1296 (2007)に従った。これにより、適用ステップが行われた。
エクソン・スキップ効率 =
エクソン・スキップあり/(エクソン・スキップなし+エクソン・スキップあり)
なお、図6のBに示すグラフは、求めたエクソン・スキップ効率を平均値±標準誤差で示し、***はP<0.001、****はP<0.0001を表す。
DMD遺伝子にエクソン45-54欠損を有するDMD患者から尿を採取し、尿細胞由来筋管を誘導した。筋分化誘導後7日時点で配列の異なるアンチセンスオリゴヌクレオチド(AON)と6μMエンドポーター(Gene Tools, Philomath, OR, USA)を含む分化培地に変更した。さらに3日後に分化培地のみの培地に変更し、筋分化誘導後14日時点で、実施例4に記載の方法と同様にジストロフィンタンパク質の発現を免疫細胞染色により半定量化した。使用したAONは、エクソン44スキップ薬であり、コントロールとしてエクソン45スキップ薬、エクソン50スキップ薬及びエクソン51スキップ薬を使用した。これらのAONの詳細は、エクソン44スキップ薬とエクソン45スキップ薬はWilton, S. D. et al. Mol Ther 15, 1288-1296 (2007)、エクソン50スキップ薬はWu, B. et al. PLoS One 6, e19906 (2011)に従い、エクソン51スキップ薬はeteplirsen(AVI-4658)を使用した。
Claims (9)
- 骨格筋障害を引き起こす状態の治療薬の検定方法であって、
尿中細胞から筋管を作製する方法によって、骨格筋障害を引き起こす状態を有する患者の尿中細胞から筋管を作製する作製ステップと、
前記筋管に治療薬を適用する適用ステップと、
前記筋管における骨格筋障害を引き起こす状態の改善を検出する検出ステップと、
を含み、
前記尿中細胞から筋管を作製する方法が、前記骨格筋障害を引き起こす状態を有する患者の尿中細胞にMYOD1遺伝子を導入する導入ステップと、前記尿中細胞を少なくとも1種のエピジェネティクス制御化合物に曝露する曝露ステップとを含む、方法(但し、前記骨格筋障害を引き起こす状態の治療薬は、筋ジストロフィー患者に対するエクソン・スキップ治療薬ではない)。 - 前記エピジェネティクス制御化合物が、ヒストンメチル基転移酵素阻害剤、ヒストン脱メチル化酵素阻害剤、ヒストン脱アセチル化酵素阻害剤、SIRT2阻害剤、及びPARP阻害剤からなる群より選択される少なくとも1種を含む、請求項1に記載の方法。
- ヒストンメチル基転移酵素阻害剤が、3-デアザネプラノシンA及び3-デアザネプラノシンA塩酸塩(DZNep)、GSK343、SGC707、フラミジン二塩酸塩、UNC2327、E7438、及びMI-2(メニン-MLL阻害剤)からなる群より選択される少なくとも1種を含む、請求項2に記載の方法。
- ヒストン脱メチル化酵素阻害剤が、IOX 1及びGSK-J1からなる群より選択される少なくとも1種を含む、請求項2に記載の方法。
- ヒストン脱アセチル化酵素阻害剤が、LMK-235、CAY10603、BRD73954、及びVORINOSTATからなる群より選択される少なくとも1種を含む、請求項2に記載の方法。
- SIRT2阻害剤がSirReal 2を含む、請求項2に記載の方法。
- PARP阻害剤がEB47を含む、請求項2に記載の方法。
- 前記導入ステップにおいて、MYOD1遺伝子の導入が、誘導性プロモーターの制御下にあるMYOD1遺伝子を含む発現ベクターの導入により行われる、請求項1~7のいずれか1項に記載の方法。
- 発現ベクターが選択マーカー遺伝子をさらに含む、請求項8に記載の方法。
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