JP2022157516A - 神経細胞及びその応用 - Google Patents
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Abstract
Description
<1> 導入された外因性野生型タウ遺伝子を有する神経細胞。
<2> ヒト多能性幹細胞に由来する、<1>に記載の神経細胞。
<3> ヒト多能性幹細胞が、健常者由来ヒト多能性幹細胞である、<2>に記載の神経細胞。
<4> ヒト多能性幹細胞から神経細胞への分化後に外因性野生型タウ遺伝子が発現されている、<2>又は<3>に記載の神経細胞。
<5> 外因性野生型タウ遺伝子の発現が条件特異的に制御されている、<1>から<4>のいずれか一に記載の神経細胞。
<6> 外因性野生型タウ遺伝子が過剰発現したときにタウが凝集する、<1>から<5>の何れか一に記載の神経細胞。
<7> タウの凝集が遺伝子発現によるもののみである、<1>から<6>の何れか一に記載の神経細胞。
<8> <1>から<7>の何れか一に記載の神経細胞を含む、タウ過剰発現による変化に作用する作用をもつ薬剤候補物質のスクリーニングキット。
<9> <1>から<7>の何れか一に記載の神経細胞を用いる、タウ凝集または細胞死を抑制する作用をもつ薬剤候補物質のスクリーニング方法。
<10> <9>に記載のスクリーニング方法により得られる薬剤候補物質。
<11> 導入された外因性野生型タウ遺伝子を有する、ヒト多能性幹細胞。
<12> ヒト多能性幹細胞に外因性野生型タウ遺伝子を導入すること、及びヒト多能性幹細胞を神経細胞に分化させることを含む、神経細胞の製造方法。
<13> 外因性野生型タウ遺伝子の発現と、神経細胞の分化のための遺伝子発現の工程とがTETシステムである、<12>に記載の方法。
本明細書において、“NP_”、“NM_”、“NG_”とそれに続く数字は、NCBI(National Center for Biotechnology Information)データベースに標準配列(Reference Sequence)として登録されているアミノ酸配列(NP_~)、転写物のヌクレオチド配列(NM_~)、ゲノムDNA配列(NG_~)のIDを各々表す。
本発明の神経細胞は、導入された外因性野生型タウ遺伝子を有する神経細胞である。
好ましくは、本発明の神経細胞は、多能性幹細胞に由来する細胞である。
タウ(microtubule-associated protein tau、MAPTとも呼ばれる)は、ヒトでは第17番染色体(17q21.1)に存在するMAPT遺伝子(Official full name:microtubule-associated protein tau、Official symbol;MAPT、NG_007398.1)にコードされるタンパク質で、選択的スプライシングによって生じる6つのアイソフォームが同定されている。
ヒトの胎児期の脳では3R型タウのみが発現するが、ヒト成人脳では上記6種類すべてが発現し、正常人では、4R型(4リピートタウ)と3R型(3リピートタウ)の発現比(=4リピートタウ/3リピートタウ)は同程度である。
マウスでは新生仔までは3R型タウのみが発現するが、離乳期以降は4R型タウのみが発現する。
ラットおよびマーモセットでも、脳では新生仔までは3R型タウのみが発現するが、離乳期以降は4R型タウのみが発現する。
本発明では、特に定めがない限り、タウとは、3リピートタウ及び4リピートタウのいずれでもよい。
本発明の神経細胞は、導入された外因性野生型タウ遺伝子を有するヒト多能性幹細胞を神経細胞に分化させることにより製造することができる。導入された外因性野生型タウ遺伝子を有するヒト多能性幹細胞は、ヒト多能性幹細胞に外因性野生型タウ遺伝子を導入することにより製造することができる。即ち、本発明によれば、ヒト多能性幹細胞に外因性野生型タウ遺伝子を導入すること、及びヒト多能性幹細胞を神経細胞に分化させることを含む、神経細胞の製造方法が提供される。
さらに本発明の神経細胞は、立体(3次元)培養された細胞ではないことが好ましく、単層培養された細胞であることがより好ましい。
多能性幹細胞から神経細胞を分化誘導する方法としては、例えば、
(1)無血清培地中で培養して胚様体(神経前駆細胞を含む細胞塊)を形成させて分化させる方法(SFEB法:Watanabe K.,et al, Nat.Neurosci.,8:288-296,2005;SFEBq法:Wataya T.,et al Proc.Natl.Acad.Sci.USA.,105:11796-11801,2008);
(2)ストローマ細胞上で培養して分化させる方法(SDIA法:Kawasaki H., et al,Neuron,28:31-40, 2000);
(3)薬剤を添加したマトリゲル上で培養して分化させる方法(Chambers S.M.,et al,Nat.Biotechnol.,27:275-280,2009);
(4)サイトカインの代替物として低分子化合物を含む培地中で培養して分化する方法(米国特許第5,843,780号);
(5)多能性幹細胞に神経誘導因子(neurogenin2(Ngn2)等)を導入し発現させることで分化させる方法(WO2014/148646;及びZhang Y., et al, Neuron,78:785-98,2013);
(6)多能性幹細胞にmiR-9/9*-124を導入し発現させることで分化させる方法;
及びこれらの方法の組み合わせ等が挙げられる。
多能性幹細胞におけるmiR-9/9*-124の発現は、miR-9/9*-124をコードする核酸(DNA又はRNA)を、多能性幹細胞に導入することによって実施することができる。
また、制御配列及び上記プロモーターの調節因子(rtTA及び/又はCymRリプレッサー等)は、neurogenin2遺伝子またはmiR-9/9*-124を導入したベクターによって供給されてもよい。
(N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester)を添加した培地を使用することが特に好ましい。
本発明は、上記した本発明の神経細胞を含む、タウ過剰発現による変化に作用する作用をもつ薬剤候補物質のスクリーニングキット、並びに上記した本発明の神経細胞を用いる、タウ凝集または細胞死を抑制する作用をもつ薬剤候補物質のスクリーニング方法に関する。本発明はさらに、上記したスクリーニング方法により得られる薬剤候補物質に関する。
本発明の一態様において、野生型タウの過剰発現以外のタウ凝集促進操作を経ずに、タウを凝集させ、細胞死を誘導することができる。タウ凝集促進操作としては例えば、タウフラグメント(K18など)の凝集体、脱リン酸化阻害剤 (オカダ酸など)、タンパク質凝集促進剤 (Congo Redなど)が挙げられる。
本発明において、過剰発現とは操作を行わなかった場合と比較して、対象mRNAまたは対象タンパク質が多く発現されていることを意味する。過剰発現とは、好ましくは内因性タンパク質の等量以上に発現していることを言う。
(1)本発明の神経細胞と被験物質とを接触させる工程;
(2)上記神経細胞におけるタウを過剰発現させる工程;
(3)上記神経細胞におけるタウ凝集又はそれに伴う神経細胞死を測定する工程;
及び
(4)工程(1)において被験物質と接触させた場合の工程(3)で測定したタウ凝集又はそれに伴う神経細胞死が、工程(1)において被験物質と接触させなかった場合のタウ凝集又はそれに伴う神経細胞死よりも低下している場合、上記被験物質を、タウオパチーの予防薬又は治療薬として選択する工程;
を含む、タウオパチーの予防又は治療薬のスクリーニング方法が提供される。
タウオパチーとしては、アルツハイマー型認知症(Alzheimer’s disease;AD)、第17染色体遺伝子に連鎖しパーキンソニズムを伴う前頭側頭型認知症(frontotemporal dementia with Parkinsonism linked to chromosome 17;FTDP-17)、前頭側頭型認知症(Frontotemporal dementia;FTD)、ピック病(Pick’s disease)、進行性核上性麻痺(progressive supranuclear palsy;PSP)、大脳皮質変性症(corticobasal degeneration;CBD)、嗜銀顆粒性認知症(嗜銀性顆粒病)、神経原線維変化型認知症、石灰沈着を伴うび慢性神経原線維変化病(DNTC)、年齢依存的タウタンパク質異常症(Primary age-related tauopathy: PART)等が挙げられる。
非特許文献(Mitsuru Ishikawa,et al.,Cells 2020,9,532;)の方法に従ってレンチウイルスの作製を行った。簡潔に記載すると、パッケージングコンストラクト(pCAG-HIVgp)、VSV-GおよびRev発現コンストラクト(pCMV-VSV-G-RSV-Rev)、self-inactivating (SIN)レンチウイルスベクターコントストラクト(CSIV-miR-9/9*-124-mRFP1-TRE-EF-BsdT、またはCSII-TRE-hTau(1N4R)-IRES-Zeo)の3種類のプラスミドを、HEK293T細胞にポリエチレンイミン(Polysciences)を用いてトランスフェクションすることでレンチウイルスを産生させた。さらに、培養上清を超遠心により濃縮し、レンチウイルスを濃縮した。濃縮後、lenti-Gostix PLUS(タカラバイオ)を用いて力価の測定を行い、実験に用いた。
CSIV-miR-9/9*-124-mRFP1-TRE-EF-BsdT(図1)、およびCSII-TRE-hTau(1N4R)-IRES-Zeo(図2)を示す。CSII-TRE-hTau(1N4R)-IRES-Zeoに関しては、まずはattL1およびattL2とタウ遺伝子配列の両端の配列を含むプライマーを設計し、タウ遺伝子を鋳型としてPCRを行い、attL1およびattL2を両端に持つタウ遺伝子のPCR産物を作成した。その後、Gateway法を用いてプラスミドを作成する為に、pDONR(Thermo Fisher Scientific)ベクターと、上記のPCR産物を鋳型としてreverse PCRを行い、タウ遺伝子を含むpDONRベクターを作成した。その後、そのベクターと、CSIIベクターを、Gateway法を用いて遺伝子を組換え、CSII-TRE-hTau(1N4R)-IRES-Zeoを作成した。
非特許文献(Mitsuru Ishikawa,et al.,Cells 2020,9,532;)の方法に従って、図3に示すようなTransposaseベクター(pCMV-HyPBase-PGK-Puro)、rtTAベクター(PB-CAGrtTA3G-IH)、Neurogenin2(Ngn2)ベクター(PB-TET-PH-lox66FRT-NEUROG2)を作製した。これらベクターをStemFit(登録商標)で培養したiPS細胞に対して、GeneJuice(Novagen社)を用いたリポフェクションで導入し、さらに、miR-9/9*-124遺伝子を含むレンチウイルス(CSIV-miR-9/9*-124-mRFP1-TRE-EF-BsdT)をiPS細胞に導入した。その後、Puromycin、Hygromycin, Blasticidin Sを用いた薬剤セレクションによって、ベクターが安定導入されたiPS細胞株を取得した。
Ngn2およびmiR-9/9*-124遺伝子を導入したiPS細胞をTrypLE selectで剥離し、ポリオルニチンおよびiMatrix(ニッピ)でコーティングした6well plateに播種した。Doxycycline(DOX)を含む神経誘導培地(Neurobasal plus培地に2%のB27 Plus supplement、1%のCulture One supplemet、200μmol/LのdbcAMP、200μmol/LのL-ascorbic acid、10μmol/LのY27632、10μmol/LのDAPT
(N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester)、4μg/mlのDoxを添加したもの)で5日間培養することで神経細胞へ分化誘導した。TrypLE Selectで神経細胞を剥離し、Stem Cell Bankerで凍結細胞ストックにした。
凍結神経細胞を湯浴で解凍し、Poly-D-lysine(PDL)およびiMatrix(ニッピ)によりコーティングした96well plateに約3×104cells/wellまたは12well plateに約30×104cells/wellの細胞数で、DOXを含む神経再播種培地(Neurobasal plus:DMEM/F12 HAM=1:1の培地に、2%のB27 Plus supplement、1%のCulture One supplemet、200μmol/LのdbcAMP、200μmol/LのL-ascorbic acid、2μg/mlのDoxを添加したもの) に播種した。また、後記の(7)に記載の1μmol/LのTau Accell siRNA(Dharmacon、#A-012488-13)、又は後記の(9)に記載の化合物を添加した。37℃ CO2インキュベーター内で2日間培養し、レンチウイルスベクターを処置し、7日間程度培養した後に各種評価を行った。
(4)の方法により神経細胞においてタウ発現レンチウイルスベクターを感染させ、5日培養した後、神経細胞をRIPA bufferを用いて可溶化した。NuPAGEゲル(Thermo Fisher Scientific)を用いてSDS-PAGEを行った。電気泳動後のゲル上のタンパク質をPVDF(ポリフッ化ビニリデン)メンブレン転写し、ブロッキング後、Tau抗体(DAKO社製、2000倍希釈、4℃、一晩)、二次抗体anti-rabbit 800nm(LI-COR社から購入、2000倍希釈、室温、1時間)をそれぞれ反応させ、Odyssey CLx(LI-COR)で蛍光を検出した。結果を図2に示す。内因性タウ以上の量のタウが過剰発現(overexpression; OE)により誘導されたことが確認できた。
(4)の方法により神経細胞においてタウ発現レンチウイルスベクターを感染させ、6日後に4%パラホルムアルデヒドで細胞を固定した。ブロッキング後、一次抗体(1000倍希釈、4℃、一晩)、二次抗体(2000倍希釈、室温、1時間)をそれぞれ反応させ、蛍光顕微鏡で蛍光を検出した。結果を図3に示す。1N4Rタウの過剰発現により、4R Tau、リン酸化タウ(AT-8,PHF-1)のシグナルが増加した。さらにタウ過剰発現によりβIII tubulinの断片化およびMAP2神経突起の減少が観察され、神経突起に変性が起きていることが確認できた。
使用した抗体を以下に示す。
Rabbit 4R Tau抗体(コスモバイオ)
Mouse AT-8抗体(Thermofisher)
Mouse PHF抗体(Dr. Peter Daviesより譲受)
Rabbit βIII-tubulin抗体(Abcam)
Mouse MAP2抗体(Sigma Aldrich)
Anti-Mouse IgG Alexa Fluor 555 (Thermo Fisher Scientific)
Anti-Rabbit IgG Alexa Fluor 647(Thermo Fisher Scientific)
(4)の方法によりタウを過剰発現させたことによる細胞生存率の低下をCellTiter Gloo assay(プロメガ)により確認した。結果を図4に示す。タウ過剰発現によりレンチウイルス量依存的に約20%~90%程度の神経細胞死が起きていることが確認できた。さらに、Tau Accell siRNA(Dharmacon,#D-001910-03)処置により神経細胞死は抑制されたことから、神経細胞死はタウ発現依存的に生じていることが示された。
(4)の方法に従ってタウ過剰発現後の神経細胞において、Tau oligomer抗体(T22)(Millipore)を用いて蛍光染色を行った。結果を図7に示す。神経突起内にTau oligomer陽性の染色像が確認できた。これは、神経細胞内においてタウ凝集が生じていることを示している。さらに、T22抗体陽性突起長をImage Jを用いて定量したところ、非処置群と比較してタウ過剰発現群でT22陽性神経突起長の増加が確認できた。
(4)の方法に従って、タウ過剰発現後の神経細胞においてタウ凝集阻害作用が報告されているmethylene blueおよびisoproterenol、微小管安定化剤epothilone Dの薬効評価を行った。化合物の細胞保護作用はCellTiter Glo assayで行った。タウ凝集への効果は、蛍光染色による神経突起中のTau oligomer抗体陽性突起長を、4R Tau抗体陽性突起長で標準化することで定量した。結果を図8に示す。methylene blue、isoproterenol、epothiloneDにおいて部分的な細胞死保護作用が確認できた。さらに、細胞保護作用のあった化合物濃度においてTau重合を評価した。Tau重合阻害作用の報告されているmethylene blueおよびisoproterenolにおいては部分的なTau凝集阻害作用が確認できたが、一方で微小管安定化剤epothilone DではTau重合は変化させず、Tau重合非依存的に細胞保護作用を示した。
Claims (13)
- 導入された外因性野生型タウ遺伝子を有する神経細胞。
- ヒト多能性幹細胞に由来する、請求項1に記載の神経細胞。
- ヒト多能性幹細胞が、健常者由来ヒト多能性幹細胞である、請求項2に記載の神経細胞。
- ヒト多能性幹細胞から神経細胞への分化後に外因性野生型タウ遺伝子が発現されている、請求項2又は3に記載の神経細胞。
- 外因性野生型タウ遺伝子の発現が条件特異的に制御されている、請求項1から4のいずれか一項に記載の神経細胞。
- 外因性野生型タウ遺伝子が過剰発現したときにタウが凝集する、請求項1から5の何れか一項に記載の神経細胞。
- タウの凝集が遺伝子発現によるもののみである、請求項1から6の何れか一項に記載の神経細胞。
- 請求項1から7の何れか一項に記載の神経細胞を含む、タウ過剰発現による変化に作用する作用をもつ薬剤候補物質のスクリーニングキット。
- 請求項1から7の何れか一項に記載の神経細胞を用いる、タウ凝集または細胞死を抑制する作用をもつ薬剤候補物質のスクリーニング方法。
- 請求項9に記載のスクリーニング方法により得られる薬剤候補物質。
- 導入された外因性野生型タウ遺伝子を有する、ヒト多能性幹細胞。
- ヒト多能性幹細胞に外因性野生型タウ遺伝子を導入すること、及びヒト多能性幹細胞を神経細胞に分化させることを含む、神経細胞の製造方法。
- 外因性野生型タウ遺伝子の発現と、神経細胞の分化のための遺伝子発現の工程とがTETシステムである、請求項12に記載の方法。
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