JP6317709B2 - 標的細胞の光学に基づく刺激及びそれに対する改変 - Google Patents
標的細胞の光学に基づく刺激及びそれに対する改変 Download PDFInfo
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Description
本特許文書は、米国特許法(35 U.S.C.)第119条(e)に基づき、2008年11月14日に提出された「Systems, Methods and Compositions for Optical Stimulation of Target Cells and for Modifications Thereto」という発明の名称の米国仮特許出願第61/114,781号の利益を主張する。この仮文書は、それとともに提出された付属書類を含むものであり、参照により本明細書にその全体が組み入れられる。
本明細書と同時に提出された、次に示す通りの、コンピュータで読み取り可能なヌクレオチド/アミノ酸配列表は、参照によりその全体が組み入れられる:2009年11月13日に作成された「stfd225pct_ST25」というファイル名の31,298バイトのASCII(テキスト)ファイル1点。
1. C128及びD156のうち少なくとも1つが置換されている、配列番号1の分子またはその変異体。
2. 前記置換が、T、A及びSのうち1つによるC128の置換を含む、実施形態1に記載の分子。
3. 前記置換が、AによりD156を置換することを含む、実施形態1に記載の分子。
4. E123における置換をさらに含む、実施形態1に記載の分子。
5. H134における置換をさらに含む、実施形態1に記載の分子。
6. C128及びD156のうち少なくとも1つが置換されている、配列番号1の分子をコードする配列またはその変異体。
7. 前記置換が、T、A及びSのうち1つによるC128の置換を含む、実施形態6に記載の配列。
8. 前記置換が、A及びNのうち1つによるD156の置換を含む、実施形態6に記載の配列。
9. E123における置換をさらに含む、実施形態6に記載の配列。
10. H134における置換をさらに含む、実施形態6に記載の配列。
11. C123及びD151のうち少なくとも1つが置換されている、配列番号2の分子。
12. 光応答性イオンチャネルを設計する方法であって、
配列番号1の分子またはその変異体を、C128及びD156のうち1つ以上の近傍にて改変すること、
を含む方法。
13. 前記改変に、T、A及びSのうち1つによる、配列番号1のC128の置換が含まれる、実施形態12に記載の方法。
14. 前記改変に、A及びNのうち1つによる、配列番号1のD156の置換が含まれる、実施形態12に記載の方法。
15. 前記改変に、配列番号1の、AによるD156の置換、及び、SによるC128の置換が含まれる、実施形態12に記載の方法。
16. 配列番号1を、E123にて、またはその近傍にて、改変する工程をさらに含む、実施形態12に記載の方法。
17. 配列番号1を、H134にて、またはその近傍にて、改変する工程をさらに含む、実施形態12に記載の方法。
18. 光応答性イオンチャネルを有する分子を設計する方法であって、
配列番号2またはその変異体を、C123及びD151のうち少なくとも1つの近傍にて改変すること、
を含む方法。
19. 前記改変する工程が、T、A及びSのうち1つによる、配列番号2のC123の置換を含む、実施形態18に記載の方法。
20. 前記改変する工程が、A及びNのうち1つによる、配列番号2のD151の置換を含む、実施形態18に記載の方法。
21. C128及びD156のうち1つ以上の近傍にて発現する改変を有する、ChR2またはその相同変異体を発現する核酸配列を送達するためのベクター。
22. 前記核酸配列が、E123及びH134のうち少なくとも1つの近傍にて発現する改変をさらに含む、実施形態21に記載のベクター。
23. 神経集団内の神経細胞の固有の神経活動を促進する方法であって、:
前記神経細胞において、配列番号1及び配列番号2のうち1つまたはその変異体に一致し、かつ、C128、D156、C123及びD151のうち1つ以上の近傍に変異を有する、1つ以上の分子を、設計すること;ならびに、
前記1つ以上の分子を、前記神経集団の固有の神経活動に応答して活動電位の発生を促進するのに十分な、光学的な刺激に曝露すること、
を含む方法。
24. 前記光学的な刺激が、前記固有の神経活動からのさらなる刺激の非存在下では活動電位を引き起こすのに十分ではない、実施形態23に記載の方法。
25. 配列番号1及び配列番号2のうち1つまたはその変異体に一致し、かつ、C128、D156、C123及びD151のうち1つ以上の近傍に変異を有するアミノ酸をコードする分子を、神経学的障害または中枢神経系障害の治療のための医薬の製造において用いること、
を含む方法であって、前記分子が前記アミノ酸を発現可能であり、それにより、電流を生じさせることにより光に応答して、ニューロンの脱分極をもたらす、方法。
26. 配列番号1及び配列番号2のうち1つまたはその変異体に一致し、かつ、C128、D156、C123及びD151のうち1つ以上の近傍に変異を有する分子をコードする前記分子を含有する生成物を含む、神経学的障害またはCNS障害の疾患の治療における使用のための組み合わせ調製物としての、アセンブリ。
27. 光に対する応答性を有する別のオプシン系分子をさらに含む、実施形態26に記載のアセンブリ。
28. 前記生成物が、プロモーター+光遺伝学的プローブを送達するためのウィルス性の運搬システムを含む、実施形態26に記載のアセンブリ。
29. 前記生成物が、プロモーター+光遺伝学的プローブを運搬するためのアデノ随伴ウィルスを含む、実施形態26に記載のアセンブリ。
30. 神経疾患モデルの特徴を決定する方法であって、
配列番号1及び配列番号2のうち1つまたはその変異体に一致し、かつ、C128、D156、C123及びD151のうち1つ以上の近傍に変異を有する分子であって、標的とする神経集団において発現し、前記神経疾患モデルに対する公知の治療効果を有する分子、を発現させること;
前記発現した分子を刺激すること;及び
前記刺激の効果または効力を、前記神経疾患モデルの機能として評価すること、
を含む方法。
Claims (23)
- a)配列番号1又は配列番号2に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含む光応答性イオンチャネルタンパク質であって、当該イオンチャネルが、光パルスへの暴露後に長期化された伝導状態を示し、なおかつ、
前記タンパク質が、
i)配列番号1のアミノ酸128に相当する位置にCysからThr、CysからAla又はCysからSerへの置換、又は、
ii)配列番号1のアミノ酸128に相当する位置にCysからSerへの置換及び配列番号1のアミノ酸156に相当する位置にAspからAla又はAspからAsnへの置換、
を含む、光応答性イオンチャネルタンパク質と、
b)配列番号3に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含む光応答性クロライドイオンポンプ
とを含んでなる組成物。 - 前記光応答性イオンチャネルタンパク質が、配列番号2に示すアミノ酸配列に対して少なくとも95%のアミノ酸配列同一性を持つアミノ酸配列を含む、請求項1に記載の組成物。
- 前記光応答性イオンチャネルタンパク質が、配列番号1のアミノ酸123に相当する位置にGluのアミノ酸置換、又は、配列番号1のアミノ酸134に相当する位置にHisのアミノ酸置換を更に含む、請求項1又は2に記載の組成物。
- 前記光応答性イオンチャネルタンパク質が、光の短い1回のパルスによりゲートが開閉して活性状態になり、それと同時に、その光パルスよりも有意に長いある期間にわたり活性状態が維持される、請求項1又は2に記載の組成物。
- a)配列番号1又は配列番号2に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含む光応答性イオンチャネルタンパク質をコードする第一ヌクレオチド配列を含む第一核酸であって、当該イオンチャネルが、光パルスへの暴露後に長期化された伝導状態を示し、なおかつ、
前記タンパク質が、
i)配列番号1のアミノ酸128に相当する位置にCysからThr、CysからAla又はCysからSerへの置換、又は、
ii)配列番号1のアミノ酸128に相当する位置にCysからSerへの置換及び配列番号1のアミノ酸156に相当する位置にAspからAla又はAspからAsnへの置換、
を含むものである、第一核酸と、
b)配列番号3に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含む光応答性クロライドイオンポンプをコードする第二ヌクレオチド配列を含む第二核酸
とを含んでなる組成物。 - 前記第一及び第二のヌクレオチド配列が制御エレメントと作用可能に結合している、請求項5に記載の組成物。
- a)光応答性イオンチャネルタンパク質をコードするヌクレオチド配列を含む第一組み換え発現ベクターであって、当該光応答性イオンチャネルタンパク質が、配列番号1又は配列番号2に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含み、光パルスへの暴露後に長期化された伝導状態を示し、なおかつ、
前記タンパク質が、
i)配列番号1のアミノ酸128に相当する位置にCysからThr、CysからAla又はCysからSerへの置換、又は、
ii)配列番号1のアミノ酸128に相当する位置にCysからSerへの置換及び配列番号1のアミノ酸156に相当する位置にAspからAla又はAspからAsnへの置換、
を含むものである、第一組み換え発現ベクターと、
b)光応答性クロライドイオンポンプをコードするヌクレオチド配列を含む第二組み換え発現ベクターであって、当該光応答性クロライドイオンポンプが、配列番号3に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含むものである、第二組み換え発現ベクター
とを含む組成物。 - 前記第一及び第二のベクターが、レンチウイルスベクター又はアデノ随伴ウイルスベクターである、請求項7に記載の組成物。
- ニューロンの活動電位をコントロールするためのシステムであって、当該システムは、運搬装置、光源、及び制御装置を含み、
前記運搬装置は、請求項1から8のいずれか一項に記載の組成物を含み、
前記光源は光応答性イオンチャネルタンパク質と光応答性クロライドイオンポンプを刺激するための光を生成し、
前記制御装置は光源による光の生成を制御する、システム。 - 前記光源が発光ダイオード又はレーザーである、請求項9に記載のシステム。
- 前記運搬装置が請求項5又は6に記載の組成物を含み、前記第一及び/又は第二の核酸が遺伝子導入ベクターに含まれる、請求項9に記載のシステム。
- 前記遺伝子導入ベクターがレンチウイルスベクターである、請求項11に記載のシステム。
- 前記運搬装置が埋め込み可能である、請求項9に記載のシステム。
- 前記光源が埋め込み可能である、請求項9に記載のシステム。
- 前記運搬装置が請求項5又は6に記載の組成物を含み、前記核酸が、生物学的部分中の水溶性の材料又は脱水された材料で供される、請求項9に記載のシステム。
- インビボで標的細胞を刺激する方法で使用するための請求項1から4のいずれか一項に記載の組成物であって、当該方法は、光応答性イオンチャネルタンパク質と光応答性クロライドイオンポンプを発現する標的細胞を光刺激に暴露することにより標的細胞を刺激することを含む、組成物。
- 前記標的細胞がニューロンである、請求項16に記載の組成物。
- 前記刺激が、ニューロンの活動電位をコントロールすることを含む、請求項17に記載の組成物。
- インビボで標的細胞を刺激する方法で使用するための請求項5又は6に記載の組成物であって、当該方法は、コードされた光応答性イオンチャネルタンパク質及び光応答性クロライドイオンポンプタンパク質を発現する標的細胞を光刺激に暴露することにより標的細胞を刺激することを含む、組成物。
- 前記標的細胞がニューロンである、請求項19に記載の組成物。
- 前記刺激が、ニューロンの活動電位をコントロールすることを含む、請求項20に記載の組成物。
- a)配列番号1又は配列番号2に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含む光応答性イオンチャネルタンパク質であって、当該イオンチャネルが、光パルスへの暴露後に長期化された伝導状態を示し、なおかつ、
前記タンパク質が、
i)配列番号1のアミノ酸128に相当する位置にCysからThr、CysからAla又はCysからSerへの置換、又は、
ii)配列番号1のアミノ酸128に相当する位置にCysからSerへの置換及び配列番号1のアミノ酸156に相当する位置にAspからAla又はAspからAsnへの置換、
を含む、光応答性イオンチャネルタンパク質と、
b)配列番号3に示すアミノ酸配列に対して少なくとも90%のアミノ酸配列同一性を持つアミノ酸配列を含む光応答性クロライドイオンポンプ
を含む哺乳動物神経細胞。 - 治療に使用するための、請求項22に記載の哺乳動物細胞。
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