JP2019517279A - Chrimsonの変異型光誘導性イオンチャネル - Google Patents
Chrimsonの変異型光誘導性イオンチャネル Download PDFInfo
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Abstract
Description
Cys - Arg - Met - Val - Val - Lys - Leu - Met - Ala - Tyr - Ala - Xaa12 - Phe - Ala - Ser - Trp - Gly - Xaa18 - Xaa19 - Pro - Ile - Leu - Trp - Ala - Val
のヘリックス6モチーフ、
ここでXaa12はPhe又はTyrである、好ましくはここでXaa12はPheである;
ここでXaa18はMet又はSerである、好ましくはここでXaa18はMetである; および
ここでXaa19はTyr又はPheである、好ましくはここでXaa19はPheである、
を含む。
配列番号1(Chrimson;受託番号KF992060;ヘリックス6は太字で強調されている)
Cys - Arg - Met - Val - Val - Lys - Leu - Met - Ala - Tyr - Ala - Xaa12 - Phe - Ala - Ser - Trp - Gly - Xaa18 - Xaa19 - Pro - Ile - Leu - Trp - Ala - Val、
ここでXaa12はPhe又はTyrである、好ましくはここでXaa12はPheである;
ここでXaa18はMet又はSerである、好ましくはここでXaa18はMetである; かつ
ここでXaa19はPhe又はTyrである、好ましくはここでXaa19はPheである。
Xaa1-Asp-Xaa3-Xaa4-Xaa5-Lys-Xaa7-Xaa8-Xaa9
ここでXaa1は、Leu、Ile、Ala、又はCysである;
ここでXaa3、Xaa4、Xaa5、Xaa7、及びXaa8は、独立して任意のアミノ酸である;
ここでXaa9は、Thr、Phe、又はTyrである。
Chrimson-YFP及びChrimson-YFP変異体を一過的に発現するNG108-15細胞を、-60mVのクランプ電位で全細胞構成におけるパッチクランプ測定によって調べた。バス溶液は、140 mM NaCl、2 mM CaCl2、2 mM MgCl2、10 mM HEPES、pH 7.4を含有し、ピペット溶液は、110 mM NaCl、2 mM MgCl2、10 mM EGTA、10 mM HEPES、pH 7.4を含有していた。光電流は、23mW/mm 2の強度および594nmの波長を有する3 ms光パルスに応答させて測定した。τoff値は、照明の停止後の電流を単指数関数へフィットさせることによって決定した。電流密度(J-60 mV)は、23mW/mm2の強度および594 nmの波長を有する500msの光パルスに応答する定常電流を、細胞の静電容量で除算することによって決定した。
ChrimsonおよびChrimson変異体の作用スペクトルを、-60 mVのクランプ電位で全細胞構成におけるパッチクランプ測定により調べた。バス溶液は、140 mM NaCl、2 mM CaCl2、2 mM MgCl2、10 mM HEPES、pH 7.4を含有し、ピペット溶液は、110 mM NaCl、2 mM MgCl2、10 mM EGTA、10 mM HEPES、pH 7.4を含有していた。Opolette 355可変レーザシステム(OPTOPRIM)を用いて、パルス長7nsの光パルスを発生させた。作用スペクトルの記録のために、様々な波長でのパルスエネルギーを、Chrimson野生型では1018光子/m2、Chrimson変異体では1019光子/m2の等光子数に対応する値に設定した。
神経細胞への適用に対するChrimson変異体の適合性を検査するために、構築物を培養ラット海馬神経細胞において発現させた。
生後のP1 Sprague-Dawleyラット(Jackson Laboratory)から海馬を単離し、37℃で20分間パパイン(20 U ml-1)で処理した。この海馬を10%ウシ胎児血清を添加したDMEM(Invitrogen/Gibco、高グルコース)で洗浄し、少量の当該溶液を用いて粉砕した。〜75,000個の細胞を、24ウェルプレート中のポリ-D-リジン/ラミニン被覆ガラスカバースリップ上にプレーティングした。3時間後、プレート培地を培養培地(2%B-27 supplement、2mM GlutamaxIを含むNeurobasal A)で置換した。
rAAV2/1ウイルスは、Chrimson-YFP野生型またはChrimson-YFP変異体を含むヒトシナプシンプロモーター(16)を保持するpAAV2ベクターを使用して、Dr. Botond Roska, FMI, Baselの研究室で調製した。ウイルス力価は名目上1×1012〜1×1013 GC/mlであった。
培養海馬神経細胞における全細胞記録のために、3〜8MΩの抵抗を有するパッチピペットを、129mMグルコン酸カリウム、10mM HEPES、10mM KCl、4mM MgATPおよび0.3mM Na3GTPを含みpH7.2に滴定した溶液で満たした。Tyrode溶液を細胞外溶液(125mM NaCl、2mM KCl、2mM CaCl2、1mM MgCl2、30mMグルコースおよび25mM HEPES、pH7.4に滴定)として用いた。興奮性シナプス伝達遮断薬、1,2,3,4-テトラヒドロ-6-ニトロ-2,3-ジオキソ-ベンゾ[f]キノキサリン-7-スルホンアミド(NBQX、10 μM、Sigma)およびD(-)-2-アミノ-5-ホスホノペンタン酸(AP-5、50 μM、Sigma)の存在下で記録を行った。τoff及びJ-70mVの測定は、1μM TTXの存在下で行った。
聴覚神経の光遺伝学的な刺激は、蝸牛インプラントや聴性脳幹インプラントのような聴覚プロテーゼによるサウンドコーディングの大きな前進を約束するものである。光が簡便に集束できるので、光学的な聴覚プロテーゼは、コーディングの周波数分解能の大幅な改善を約束する。現在利用可能な光遺伝学的なツールの制限事項の1つは、聴覚信号処理の速度と比較してキネティクスが遅いことである。ここでは、Hernandez et al. J Clin Invest. 2014;124(3):1114-29に従前に記載されたシステムを適用し、マウスにおけるSGNの出生後のアデノ随伴ウィルス(AAV)による形質導入を用いて、蝸牛の光遺伝学に関し、急速にゲーティングするChrimson-YFP Y261F/S267Mの能力を検査した。AAV2/6-hSyn-Chrimson-YFP Y261F/S267Mを、p3マウスの蝸牛窓を介して鼓室内に注入し、注射後4〜8週間の発現および機能を分析した。注入されたマウスは、コロニーで明白な表現型を示さなかった。Chrimson-YFP Y261F/S267Mの発現およびSGN密度を、注入を行った耳および注入を行わなかった耳の凍結切片の免疫組織化学的解析並びにYFPおよびカルレチニン免疫蛍光の共焦点画像を用いて分析した(図5A,5B)。注入を行った全ての耳で、Chrimson-YFP Y261F/S267Mはすべての蝸牛転でSGNの約90%が注入耳に導入され(非注入耳では5%未満であった)、SGN密度は有意に変化しなかった(図1B)。形質導入率は、AAV2/6-hSyn-CatCh-YFPの子宮内注射を用いた我々の以前の研究で達成されたもの(Hernandez et al., 2014)よりもはるかに高く、それとは異なり、トノトピック位置から独立していた。後方鼓室切開術を行い、50 μm光ファイバーを円形窓を介して挿入し、595nm連続波レーザーの光を基底回転のSGNに投射した。光聴覚脳幹反応(optical auditory brainstem responses:oABR, 図6A)を容易に誘発でき、それは振幅及び波形が音響ABR(acoustic ABR:aABR)に類似していた。光強度が増すほどoABR振幅が増大しoABR待ち時間は短くなった(図6B,6E)。0.5 mWほどの低い刺激(図6C、刺激持続時間:1 ms、刺激レート:10 s-1)及び80 μsほどの短い刺激(図6D、刺激強度:11 mW、刺激レート:10 s-1)でoABRを駆動するのに十分であった。oABRの振幅および波形は動物によって異なる(図6A)が、通常は1桁を超える光強度の変化で変化する(図6C、出力ダイナミックレンジ> 20 dB)。刺激持続時間の減少または刺激速度の増加に従いoABR振幅は減少したが(図6D)、200HzまではoABRが見られた(図6F)。oABRは、5〜7msにわたって時間が延びる集団応答を反映することが注目され、そのために、応答がその後の刺激間の相互作用によって減弱されたのではと仮定される。Chrimson-YFP Y261F/S267Mが介在する蝸牛の光遺伝学的手法(Chrimson-YFP Y261F/S267M mediated cochlear optogenetics)により175Hz以上でSGNを駆動することができると結論づけられ、それは生理学的音声駆動SGN発火率の範囲内である(Liberman, M.C., J. Acoust. Soc. Am. 63, 442-455 (1978); Winter, et al., Hear. Res. 45, 191-202 (1990))。要約すると、SGNの出生後形質導入が効率的に確立され、そして低光強度および短時間の刺激でSGN発火を達成した。十分な時間的忠実度を提供し、また、光がより深部へ浸透し、組織へ散乱が少なく、光毒性のリスクが低い点でブルーチャネルロドプシンよりも優れているChrimson-YFP Y261F/S267Mは、聴覚回復および聴覚研究に関する光遺伝学の貴重なツールになると予想される。
Mace et al. Mol Ther. 2015;23(1):7-16は、アデノ随伴ウイルス(AAV)遺伝子治療による網膜神経細胞の光遺伝学的再活性化を記載する、本発明者らの一部によって執筆された初期の刊行物である。ほとんどの遺伝性網膜ジストロフィーでは、重度の視覚障害を引き起こす進行性の光受容体細胞変性を示す。アデノ随伴ウイルス(AAV)遺伝子治療による網膜神経細胞の光遺伝学的再活性化は、患者特異的な変異にかかわらず視力を回復させる可能性がある。臨床的な翻訳可能性への挑戦は、脆弱な変性網膜のために外科的に安全な送達経路を用いながらできるだけ自然な視力に近い視力を回復することである。網膜内側の視覚処理を維持するために、疾患の後期の段階でも依然として存在するON双極細胞が標的とされる。安全な遺伝子送達のために、眼に容易にアクセス可能な硝子体液への注入後に双極細胞を形質導入することができる近年設計されたAAV変異体が使用される。ON双極細胞特異的プロモーター下でチャネルロドプシンをコードするAAVは、硝子体内投与後にON双極細胞に限定的な長期の遺伝子送達を仲介することが示されている。ON双極細胞におけるチャネルロドプシン発現は、網膜レベルおよび皮質レベルでのONおよびOFF応答の回復をもたらす。さらに、光誘発性の運動器官の挙動が、治療を受けた盲目マウスにおいて回復される。
US 2016/0039902
WO 03/084994
WO 2012/032103
WO 2013/071231
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Claims (16)
- 配列番号1(Chrimson)の全長配列に対し少なくとも90%の類似性及び/又は少なくとも72%の同一性を有するアミノ酸配列を含む変異型光誘導性イオンチャネルであって、
該変異型光誘導性イオンチャネルは、親光誘導性イオンチャネルと、配列番号1のY261、Y268、及びS267に相当する位置から選択された1つまたは複数の位置における置換のみ異なっており、
前記置換は、-60 mVのクランプ電位、140 mM NaCl, 2mM CaCl2, 2MgCl2, 10 mM HEPES, pH 7.4のバス溶液、及び110 mM NaCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.4のピペット溶液で、全細胞構成におけるパッチクランプ測定により比較したとき、前記親チャネルと比べて、前記変異型チャネルのオフキネティクスを加速する、前記変異型光誘導性イオンチャネル。 - 前記変異型光誘導性イオンチャネルは、配列番号1(Chrimson)の全長に対し、少なくとも91%、好ましくは少なくとも92%、より好ましくは少なくとも93%、より好ましくは少なくとも94%、より好ましくは少なくとも95%、より好ましくは少なくとも96%、より好ましくは少なくとも97%、より好ましくは少なくとも98%、より好ましくは少なくとも99%の類似性を有する;及び/又は
前記変異型光誘導性イオンチャネルは、配列番号1(Chrimson)の全長に対し、少なくとも74%、好ましくは少なくとも75%、より好ましくは少なくとも76%、より好ましくは少なくとも78%、より好ましくは少なくとも80%、より好ましくは少なくとも85%、より好ましくは少なくとも90%、より好ましくは少なくとも91%、より好ましくは少なくとも92%、より好ましくは少なくとも93%、より好ましくは少なくとも94%、より好ましくは少なくとも95%、より好ましくは少なくとも96%、より好ましくは少なくとも97%、より好ましくは少なくとも98%、より好ましくは少なくとも99%の同一性を有する、
請求項1に記載の変異型光誘導性イオンチャネル。 - 前記変異型光誘導性イオンチャネルは、
位置Y261における置換、好ましくはここで置換はY261Fである;又は
位置Y268における置換、好ましくはここで置換はY268Fである;又は
位置S267における置換、好ましくはここで置換はS267Mである;を含む、
特に、ここで、前記変異型光誘導性イオンチャネルは、配列番号1の位置176に相当する位置においてArgを更に含む、請求項1又は2に記載の変異型光誘導性イオンチャネル。 - 前記変異型光誘導性イオンチャネルは、位置Y261及び位置S267における置換を含む、好ましくはここで位置Y261における置換はY261Fである、そして好ましくはここで位置S267における置換はS267Mである;又は
前記変異型光誘導性イオンチャネルは、位置Y268及び位置S267における置換を含む、好ましくはここで位置Y268における置換はY268Fである、そして好ましくはここで位置S267における置換はS267Mである、
請求項1〜3のいずれか1項に記載の変異型光誘導性イオンチャネル。 - 前記変異型光誘導性イオンチャネルは、位置Y261、位置Y268、及び位置S267における置換を含む、好ましくはここで位置Y261における置換はY261Fである、好ましくはここで位置Y268における置換はY268Fである、そして好ましくはここで位置S267における置換はS267Mである、請求項4に記載の変異型光誘導性イオンチャネル。
- 配列番号1の位置176に相当する位置においてArgを更に含む、請求項4又は5に記載の変異型光誘導性イオンチャネル。
- 前記変異型チャネルは、配列番号3:
Cys - Arg - Met - Val - Val - Lys - Leu - Met - Ala - Tyr - Ala - Xaa12 - Phe - Ala - Ser - Trp - Gly - Xaa18 - Xaa19 - Pro - Ile - Leu - Trp - Ala - Val
のモチーフ、
ここでXaa12はPhe又はTyrである、好ましくはここでXaa12はPheである;
ここでXaa18はMet又はSerである、好ましくはここでXaa18はMetである;および
ここでXaa19はPhe又はTyrである、好ましくはここでXaa19はPheである、
を含む、請求項1〜6のいずれか1項に記載の変異型光誘導性イオンチャネル。 - 前記変異型光誘導性イオンチャネルは、配列番号1(Chrimson)のアミノ酸配列を含む、好ましくは配列番号1(Chrimson)のアミノ酸配列からなる、
但し、位置Y261、Y268、及びS267における前記置換、及び場合により配列番号1の位置176に相当する位置におけるArgを除く、
請求項1〜7のいずれか1項に記載の変異型光誘導性イオンチャネル。 - 前記変異型光誘導性イオンチャネルは、赤色光吸収性チャネルロドプシンである、請求項1〜8のいずれか1項に記載の変異型光誘導性イオンチャネル。
- 請求項1〜9のいずれか1項に記載の変異型光誘導性イオンチャネルをコードするヌクレオチド配列を含む核酸構築物。
- 請求項1〜9のいずれか1項に記載の光誘導性イオンチャネルをコードするヌクレオチド配列又は請求項9に記載の核酸構築物を含む発現ベクター。
- 請求項10に記載の核酸構築物又は請求項11に記載の発現ベクターを含む細胞であって、
特にここで前記細胞は哺乳類細胞であり、好ましくはここで前記細胞は、
(a)海馬細胞、光受容体細胞、網膜桿状細胞、網膜錐状細胞、網膜神経節細胞、双極神経細胞、神経節細胞、偽単極性神経細胞、多極神経細胞、錐体神経細胞、プルキンエ細胞、又は顆粒細胞;あるいは、
(b)神経芽腫細胞、特にNG108-15細胞、HEK293細胞;COS細胞;BHK細胞;CHO細胞;骨髄腫細胞;又はMDCK細胞である、前記細胞。 - ハイスループットスクリーニングにおける、請求項1〜9のいずれか1項に記載の光誘導性イオンチャネル又は請求項12に記載の細胞の使用。
- 神経細胞の光刺激のための、請求項1〜9のいずれか1項に記載の光誘導性イオンチャネルの非治療的使用。
- 医薬における使用のための請求項1〜8のいずれか1項に記載の光誘導性イオンチャネル。
- 請求項1〜9のいずれか1項に記載の光誘導性イオンチャネル、請求項10に記載の核酸構築物、請求項11に記載の発現ベクター、又は請求項12に記載の細胞、を含む非ヒト動物。
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