JPWO2020131632A5 - - Google Patents
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- JPWO2020131632A5 JPWO2020131632A5 JP2021535039A JP2021535039A JPWO2020131632A5 JP WO2020131632 A5 JPWO2020131632 A5 JP WO2020131632A5 JP 2021535039 A JP2021535039 A JP 2021535039A JP 2021535039 A JP2021535039 A JP 2021535039A JP WO2020131632 A5 JPWO2020131632 A5 JP WO2020131632A5
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Description
一部のそのような方法では、１つまたは複数のアッセイは、イントロン含有Ｃ９ｏｒｆ７２ ＲＮＡ転写物を検出するための定量ポリメラーゼ連鎖反応（ｑＰＣＲ）を含む。一部のそのような方法では、１つまたは複数のアッセイがＣ９ｏｒｆ７２センスまたはアンチセンスＲＮＡ転写物を含むＲＮＡ病巣を測定することを含み、必要に応じてＲＮＡ病巣は蛍光ｉｎ ｓｉｔｕハイブリダイゼーションによって測定される。一部のそのような方法では、１つまたは複数のアッセイがジペプチドリピートタンパク質の蓄積を測定することを含み、必要に応じてジペプチドリピートタンパク質はｐｏｌｙＧＡジペプチドリピートタンパク質またはｐｏｌｙＧＰジペプチドリピートタンパク質であり、必要に応じてジペプチドリピートタンパク質の蓄積は免疫組織化学によって測定される。
本発明の実施形態において、例えば以下の項目が提供される。
（項目１）
標的ゲノム遺伝子座においてリピート配列のコピー数が増加している改変された細胞を生成するための方法であって、
（ａ）前記標的ゲノム遺伝子座において前記リピート配列の複数のコピーを含むリピート伸長配列を含む細胞の集団を提供するステップと；
（ｂ）細胞の前記集団の中にヌクレアーゼ剤または前記ヌクレアーゼ剤をコードする核酸を導入するステップであって、前記ヌクレアーゼ剤は、前記リピート伸長配列の５’末端または３’末端の近傍のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成して、改変された細胞の集団を生成するステップと；
（ｃ）改変された細胞の前記集団の中の前記リピート配列のコピー数を定量化し、前記リピート配列のコピー数が増加している改変された細胞を選択するステップと
を含む方法。
（項目２）
外因性の修復鋳型が細胞の前記集団に導入されない、項目１に記載の方法。
（項目３）
前記ヌクレアーゼ標的部位が、前記リピート伸長配列の５’末端もしくは３’末端から約５０、約４０、約３０、約２０もしくは約１０ヌクレオチド以内にあるか、または前記リピート伸長配列の５’末端もしくは３’末端と重なる、項目１または２に記載の方法。
（項目４）
前記ヌクレアーゼ標的部位が、前記リピート伸長配列の５’末端もしくは３’末端から約２５、約２４、約２３、約２２、約２１、約２０、約１９、約１８、約１７、約１６、約１５、約１４、約１３、約１２、約１１、約１０、約９、約８、約７、約６、約５、約４、約３もしくは約２ヌクレオチド以内にあるか、または前記リピート伸長配列の５’末端もしくは３’末端と重なる、項目３に記載の方法。
（項目５）
前記ヌクレアーゼ標的部位が前記リピート伸長配列の５’末端または３’末端と重なる、項目４に記載の方法。
（項目６）
前記ヌクレアーゼ標的部位が前記リピート伸長配列の５’末端の近傍にある、前記項目のいずれかに記載の方法。
（項目７）
前記ヌクレアーゼ標的部位が前記リピート伸長配列の３’末端の近傍にある、項目１から５のいずれか一項に記載の方法。
（項目８）
前記ヌクレアーゼ標的部位が前記リピート伸長配列の外側にある、前記項目のいずれかに記載の方法。
（項目９）
前記ヌクレアーゼ剤が二本鎖切断または一本鎖切断を形成する部位が、前記リピート伸長配列の５’末端または３’末端から約５０、約４０、約３０、約２０または約１０ヌクレオチド以内にある、前記項目のいずれかに記載の方法。
（項目１０）
前記ヌクレアーゼ剤が二本鎖切断または一本鎖切断を形成する部位が、前記リピート伸長配列の５’末端または３’末端から約２５、約２４、約２３、約２２、約２１、約２０、約１９、約１８、約１７、約１６、約１５、約１４、約１３、約１２、約１１、約１０、約９、約８、約７、約６、約５、約４、約３または約２ヌクレオチド以内にある、項目９に記載の方法。
（項目１１）
前記ヌクレアーゼ剤が二本鎖切断または一本鎖切断を形成する部位が、前記リピート伸長配列の５’末端の近傍にある、前記項目のいずれかに記載の方法。
（項目１２）
前記ヌクレアーゼ剤が二本鎖切断または一本鎖切断を形成する部位が、前記リピート伸長配列の３’末端の近傍にある、項目１から１０のいずれか一項に記載の方法。
（項目１３）
前記ヌクレアーゼ剤が二本鎖切断または一本鎖切断を形成する部位が、前記リピート伸長配列の外側にある、前記項目のいずれかに記載の方法。
（項目１４）
前記ヌクレアーゼ標的部位が、前記細胞による前記二本鎖切断または前記一本鎖切断の修復の後にも保持される、前記項目のいずれかに記載の方法。
（項目１５）
前記二本鎖切断または前記一本鎖切断の修復が、前記リピート伸長配列の外側に挿入も欠失ももたらさない、前記項目のいずれかに記載の方法。
（項目１６）
前記ヌクレアーゼ剤が、前記リピート伸長配列の５’末端または３’末端の近傍で二本鎖切断を形成する、前記項目のいずれかに記載の方法。
（項目１７）
前記ヌクレアーゼ剤が、前記リピート伸長配列の５’末端または３’末端の近傍で一本鎖切断を形成するニッカーゼである、項目１から１５のいずれか一項に記載の方法。
（項目１８）
第１のヌクレアーゼ剤または前記第１のヌクレアーゼ剤をコードする核酸および第２のヌクレアーゼ剤または前記第２のヌクレアーゼ剤をコードする核酸が前記細胞に導入され、前記第１のヌクレアーゼ剤は、前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成し、前記第２のヌクレアーゼ剤は、前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する、前記項目のいずれかに記載の方法。
（項目１９）
前記第１のヌクレアーゼ剤が、前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成し、前記第２のヌクレアーゼ剤が、前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する、項目１８に記載の方法。
（項目２０）
前記第１のヌクレアーゼ剤が、前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成し、前記第２のヌクレアーゼ剤が、前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する、項目１８に記載の方法。
（項目２１）
前記ヌクレアーゼ剤が、ジンクフィンガーヌクレアーゼ（ＺＦＮ）、転写活性化因子様エフェクターヌクレアーゼ（ＴＡＬＥＮ）またはＣｌｕｓｔｅｒｅｄ Ｒｅｇｕｌａｒｌｙ Ｉｎｔｅｒｓｐａｃｅｄ Ｓｈｏｒｔ Ｐａｌｉｎｄｒｏｍｉｃ Ｒｅｐｅａｔｓ（ＣＲＩＳＰＲ）結合（Ｃａｓ）タンパク質およびガイドＲＮＡである、前記項目のいずれかに記載の方法。
（項目２２）
前記ヌクレアーゼ剤が前記Ｃａｓタンパク質および前記ガイドＲＮＡである、項目２１に記載の方法。
（項目２３）
前記Ｃａｓタンパク質がＣａｓ９タンパク質である、項目２２に記載の方法。
（項目２４）
前記リピート伸長配列が異種のリピート伸長配列である、前記項目のいずれかに記載の方法。
（項目２５）
前記リピート伸長配列が、少なくとも約１０、少なくとも約２０、少なくとも約３０、少なくとも約４０、少なくとも約５０、少なくとも約６０、少なくとも約７０、少なくとも約８０、または少なくとも約９０コピーの前記リピート配列を含む、前記項目のいずれかに記載の方法。
（項目２６）
前記リピート配列のコピーが前記リピート伸長配列の中で連続している、前記項目のいずれかに記載の方法。
（項目２７）
前記リピート配列が、トリヌクレオチドリピート、テトラヌクレオチドリピート、ペンタヌクレオチドリピート、ヘキサヌクレオチドリピートまたはドデカヌクレオチドリピートである、前記項目のいずれかに記載の方法。
（項目２８）
前記リピート配列が配列番号１～１２のいずれか１つを含む、前記項目のいずれかに記載の方法。
（項目２９）
（ｉ）前記リピート配列が配列番号２を含み、前記標的ゲノム遺伝子座がＨＴＴ、ＡＲ、ＡＴＮ１、ＡＴＸＮ１、ＡＴＸＮ２、ＡＴＸＮ３、ＣＡＣＮＡ１Ａ、ＡＴＸＮ７、ＰＰＰ２Ｒ２ＢまたはＴＢＰ遺伝子座であるか；
（ｉｉ）前記リピート配列が配列番号３を含み、前記標的ゲノム遺伝子座がＦＭＲ１遺伝子座であるか；
（ｉｉｉ）前記リピート配列が配列番号４を含み、前記標的ゲノム遺伝子座がＤＭＰＫ、ＪＰＨ３、ＡＴＸＮ８またはＴＣＦ４遺伝子座であるか；
（ｉｖ）前記リピート配列が配列番号５を含み、前記標的ゲノム遺伝子座がＦＸＮ遺伝子座であるか；
（ｖ）前記リピート配列が配列番号６を含み、前記標的ゲノム遺伝子座がＡＦＦ２遺伝子座であるか；
（ｖｉ）前記リピート配列が配列番号７を含み、前記標的ゲノム遺伝子座がＰＡＢＰＮ１遺伝子座であるか；
（ｖｉｉ）前記リピート配列が配列番号８を含み、前記標的ゲノム遺伝子座がＣＮＢＰ遺伝子座であるか；
（ｖｉｉｉ）前記リピート配列が配列番号９を含み、前記標的ゲノム遺伝子座がＡＴＸＮ１０遺伝子座であるか；
（ｉｘ）前記リピート配列が配列番号１０を含み、前記標的ゲノム遺伝子座がＴＫ２またはＢＥＡＮ１遺伝子座であるか；
（ｘ）前記リピート配列が配列番号１１を含み、前記標的ゲノム遺伝子座がＮＯＰ５６遺伝子座であるか；
（ｘｉ）前記リピート配列が配列番号１を含み、前記標的ゲノム遺伝子座がＣ９ＯＲＦ７２遺伝子座であるか；または
（ｘｉｉ）前記リピート配列が配列番号１２を含み、前記標的ゲノム遺伝子座がＣＳＴＢ遺伝子座である、
項目２８に記載の方法。
（項目３０）
前記リピート配列が配列番号１を含み、前記標的ゲノム遺伝子座がＣ９ＯＲＦ７２遺伝子座である、項目２９に記載の方法。
（項目３１）
前記ヌクレアーゼ剤がＣａｓ９タンパク質およびガイドＲＮＡであり、前記ヌクレアーゼ標的部位が配列番号２８または３３を含む、項目３０に記載の方法。
（項目３２）
前記細胞が非ヒト動物細胞である、前記項目のいずれかに記載の方法。
（項目３３）
前記細胞が、非ヒト動物の胚性幹細胞、胚性幹細胞由来の運動ニューロン、脳細胞、皮質細胞、ニューロン細胞、筋細胞、心臓細胞または胚細胞である、前記項目のいずれかに記載の方法。
（項目３４）
前記細胞が非ヒト動物の１細胞期胚である、項目１から３２のいずれか一項に記載の方法。
（項目３５）
前記細胞がげっ歯動物細胞である、前記項目のいずれかに記載の方法。
（項目３６）
前記細胞がマウス細胞またはラット細胞である、項目３５に記載の方法。
（項目３７）
前記細胞がマウス細胞である、項目３６に記載の方法。
（項目３８）
前記細胞がマウス胚性幹細胞またはマウス１細胞期の胚である、項目３７に記載の方法。
（項目３９）
前記細胞がヒト誘導多能性幹細胞である、項目１から３１のいずれか一項に記載の方法。
（項目４０）
前記細胞がｉｎ ｖｉｔｒｏにある、前記項目のいずれかに記載の方法。
（項目４１）
前記細胞がｉｎ ｖｉｖｏにある、項目１から３８のいずれか一項に記載の方法。
（項目４２）
複数回のステップ（ａ）～（ｃ）を含み、第１回の後の各回のステップ（ａ）における細胞の前記集団が、前回のステップ（ｃ）で選択される前記改変された細胞から増大した細胞のクローン集団である、前記項目のいずれかに記載の方法。
（項目４３）
少なくとも３回または少なくとも４回を含む、項目４２に記載の方法。
（項目４４）
１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目４５）
１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目４６）
１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目４７）
１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目４８）
１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目４９）
１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目５０）
１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目５１）
１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目５２）
１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目５３）
１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目５４）
１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目５５）
１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、項目４２または４３に記載の方法。
（項目５６）
前記第１のヌクレアーゼ標的部位が前記第２のヌクレアーゼ標的部位と同じである、項目４６、４７、５０、５１、５４または５５に記載の方法。
（項目５７）
前記ヌクレアーゼ標的部位が前記リピート伸長配列の外側にあり、前記ヌクレアーゼ標的部位が前記リピート伸長配列の５’末端または３’末端から約５０、約４０、約３０、約２０または約１０ヌクレオチド以内にあり、前記ヌクレアーゼ剤がＣａｓ９タンパク質およびガイドＲＮＡである、前記項目のいずれかに記載の方法。
（項目５８）
前記ヌクレアーゼ剤が前記リピート伸長配列の５’末端または３’末端の近傍で一本鎖切断を形成するニッカーゼであり、前記ヌクレアーゼ標的部位が、前記細胞による前記一本鎖切断の修復の後にも保持され、前記一本鎖切断の修復が前記リピート伸長配列の外側に挿入も欠失ももたらさない、項目５７に記載の方法。
（項目５９）
内因性Ｃ９ｏｒｆ７２遺伝子座に挿入された異種のヘキサヌクレオチドリピート伸長配列をそのゲノムに含む、非ヒト動物または非ヒト動物細胞であって、前記異種のヘキサヌクレオチドリピート伸長配列は、配列番号１で示すヘキサヌクレオチド配列の約１００より多いリピートを含む、非ヒト動物または非ヒト動物細胞。
（項目６０）
前記異種のヘキサヌクレオチドリピート伸長配列が、配列番号１で示すヘキサヌクレオチド配列の少なくとも約３００リピートを含む、項目５９に記載の非ヒト動物または非ヒト動物細胞。
（項目６１）
前記ヘキサヌクレオチドリピート伸長が、配列番号１で示すヘキサヌクレオチド配列の少なくとも約５００リピートを含む、項目６０に記載の非ヒト動物または非ヒト動物細胞。
（項目６２）
前記ヘキサヌクレオチドリピート伸長が、配列番号１で示すヘキサヌクレオチド配列の少なくとも約６００リピートを含む、項目６１に記載の非ヒト動物または非ヒト動物細胞。
（項目６３）
前記リピートが前記異種リピート伸長配列の中で連続している、項目５９から６２のいずれか一項に記載の非ヒト動物または非ヒト動物細胞。
（項目６４）
前記異種のヘキサヌクレオチドリピート伸長配列が前記内因性Ｃ９ｏｒｆ７２遺伝子座の第１の非コード内因性エクソンと第２エクソンの間に位置する、項目５９から６３のいずれか一項に記載の非ヒト動物または非ヒト動物細胞。
（項目６５）
前記内因性Ｃ９ｏｒｆ７２遺伝子座がヒトＣ９ＯＲＦ７２ヌクレオチド配列を含む、項目５９から６４のいずれか一項に記載の非ヒト動物または非ヒト動物細胞。
（項目６６）
前記ヒトＣ９ＯＲＦ７２ヌクレオチド配列が配列番号４６および／または配列番号４７を含む、項目６５に記載の非ヒト動物または非ヒト動物細胞。
（項目６７）
前記非ヒト動物がげっ歯動物であるかまたは前記非ヒト動物細胞がげっ歯動物細胞である、項目５９から６６のいずれか一項に記載の非ヒト動物または非ヒト動物細胞。
（項目６８）
前記げっ歯動物がラットもしくはマウスであるかまたは前記非ヒト動物細胞がラット細胞もしくはマウス細胞である、項目６７に記載の非ヒト動物または非ヒト動物細胞。
（項目６９）
前記異種のヘキサヌクレオチドリピート伸長配列についてヘテロ接合性である、項目５９から６８のいずれか一項に記載の非ヒト動物または非ヒト動物細胞。
（項目７０）
（ａ）野生型Ｃ９ｏｒｆ７２遺伝子座を含む対照非ヒト動物または対照非ヒト動物細胞と比較したイントロン配列を保持するＣ９ｏｒｆ７２転写物の発現の増加；および／または
（ｂ）野生型Ｃ９ｏｒｆ７２遺伝子座を含む対照非ヒト動物または対照非ヒト動物細胞と比較したＲＮＡ病巣の数の増加；および／または
（ｃ）野生型Ｃ９ｏｒｆ７２遺伝子座を含む対照非ヒト動物または対照非ヒト動物細胞と比較したジペプチドリピートタンパク質のレベルの増加
を示す、項目５９から６９のいずれか一項に記載の非ヒト動物または非ヒト動物細胞。
（項目７１）
胚性幹細胞、胚性幹細胞由来の運動ニューロン、脳細胞、皮質細胞、ニューロン細胞、筋細胞、心臓細胞または胚細胞である、項目５９から７０のいずれか一項に記載の非ヒト動物細胞。
（項目７２）
１細胞期の胚である、項目５９から７０のいずれか一項に記載の非ヒト動物細胞。
（項目７３）
ｉｎ ｖｉｔｒｏにある、項目５９から７２のいずれか一項に記載の非ヒト動物細胞。
（項目７４）
ｉｎ ｖｉｖｏにある、項目５９から７２のいずれか一項に記載の非ヒト動物細胞。
（項目７５）
その生殖細胞系列ゲノムに、前記内因性Ｃ９ｏｒｆ７２遺伝子座に挿入された前記異種のヘキサヌクレオチドリピート伸長配列を含む、項目５９から７０のいずれか一項に記載の非ヒト動物。
（項目７６）
内因性Ｃ９ｏｒｆ７２遺伝子座に挿入された異種のヘキサヌクレオチドリピート伸長配列を含む非ヒト動物ゲノムであって、前記異種のヘキサヌクレオチドリピート伸長配列は配列番号１で示すヘキサヌクレオチド配列の約１００より多いリピートを含む、非ヒト動物ゲノム。
（項目７７）
異種のヘキサヌクレオチドリピート伸長配列を含む非ヒト動物Ｃ９ｏｒｆ７２遺伝子であって、前記異種のヘキサヌクレオチドリピート伸長配列は配列番号１で示すヘキサヌクレオチド配列の約１００より多いリピートを含む、非ヒト動物Ｃ９ｏｒｆ７２遺伝子。
（項目７８）
標的ゲノム遺伝子座においてリピート配列の複数のコピーを含むリピート伸長配列を含む、前記標的ゲノム遺伝子座において前記リピート配列のコピー数が増加している改変された細胞を生成するための方法で使用するためのヌクレアーゼ剤であって、前記リピート伸長配列の５’末端または３’末端の近傍のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成するように設計されるヌクレアーゼ剤。
（項目７９）
Ｃ９ｏｒｆ７２遺伝子座のヘキサヌクレオチドリピート伸長配列と関連した疾患または状態の処置のための治療薬候補を評価する方法であって、
（ａ）項目５９から７０および７５のいずれか一項に記載の非ヒト動物または項目５９から７４のいずれか一項に記載の非ヒト動物細胞に候補薬剤を投与するステップと；
（ｂ）前記候補薬剤が前記疾患または状態と関連した１つまたは複数の徴候または症状に影響を及ぼすかどうか決定するために１つまたは複数のアッセイを実行するステップと；
（ｃ）前記疾患または状態と関連した前記１つまたは複数の徴候または症状に影響を及ぼす前記候補薬剤を治療薬候補として識別するステップと
を含む方法。
（項目８０）
前記候補薬剤が前記非ヒト動物にｉｎ ｖｉｖｏ投与され、前記候補薬剤の投与の後に前記非ヒト動物から単離される細胞において前記１つまたは複数のアッセイがｉｎ ｖｉｔｒｏで実行される、項目７９に記載の方法。
（項目８１）
前記候補薬剤が非ヒト胚性幹細胞由来の運動ニューロンにｉｎ ｖｉｔｒｏ投与される、項目７９に記載の方法。
（項目８２）
前記１つまたは複数のアッセイが、イントロン含有Ｃ９ｏｒｆ７２ ＲＮＡ転写物を検出するための定量ポリメラーゼ連鎖反応（ｑＰＣＲ）を含む、項目７９から８１のいずれか一項に記載の方法。
（項目８３）
前記１つまたは複数のアッセイがＣ９ｏｒｆ７２センスまたはアンチセンスＲＮＡ転写物を含むＲＮＡ病巣を測定することを含み、必要に応じて前記ＲＮＡ病巣は蛍光ｉｎ ｓｉｔｕハイブリダイゼーションによって測定される、項目７９から８２のいずれか一項に記載の方法。
（項目８４）
前記１つまたは複数のアッセイがジペプチドリピートタンパク質の蓄積を測定することを含み、必要に応じて前記ジペプチドリピートタンパク質はｐｏｌｙＧＡジペプチドリピートタンパク質またはｐｏｌｙＧＰジペプチドリピートタンパク質であり、必要に応じてジペプチドリピートタンパク質の前記蓄積は免疫組織化学によって測定される、項目７９から８３のいずれか一項に記載の方法。
In some such methods, the one or more assays comprises quantitative polymerase chain reaction (qPCR) to detect intron-containing C9orf72 RNA transcripts. In some such methods, one or more assays comprise measuring RNA foci comprising C9orf72 sense or antisense RNA transcripts, optionally the RNA foci are measured by fluorescence in situ hybridization. be. In some such methods, the one or more assays comprise measuring accumulation of dipeptide repeat proteins, optionally the dipeptide repeat proteins are polyGA dipeptide repeat proteins or polyGP dipeptide repeat proteins, optionally Accumulation of dipeptide repeat proteins is accordingly measured by immunohistochemistry.
In embodiments of the present invention, for example, the following items are provided.
(Item 1)
A method for generating a modified cell having increased copy number of repeat sequences at a target genomic locus, comprising:
(a) providing a population of cells comprising repeat expansion sequences comprising multiple copies of said repeat sequence at said target genomic locus;
(b) introducing into said population of cells a nuclease agent or a nucleic acid encoding said nuclease agent, said nuclease agent having a nuclease target near the 5' or 3' end of said repeat extension sequence; forming a double-strand break or a single-strand break at the site to generate a population of modified cells;
(c) quantifying the copy number of the repeat sequence in the population of modified cells and selecting modified cells with increased copy number of the repeat sequence;
method including.
(Item 2)
2. The method of item 1, wherein no exogenous repair template is introduced into said population of cells.
(Item 3)
The nuclease target site is within about 50, about 40, about 30, about 20 or about 10 nucleotides from the 5' or 3' end of the repeat extension sequence, or the 5' or 3' end of the repeat extension sequence. 3. The method of item 1 or 2, wherein the 'end overlaps.
(Item 4)
The nuclease target site is about 25, about 24, about 23, about 22, about 21, about 20, about 19, about 18, about 17, about 16, about within 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3 or about 2 nucleotides, or said repeat extension A method according to item 3, overlapping the 5' or 3' end of the sequence.
(Item 5)
5. The method of item 4, wherein the nuclease target site overlaps the 5' or 3' end of the repeat extension sequence.
(Item 6)
A method according to any of the preceding items, wherein said nuclease target site is near the 5' end of said repeat extension sequence.
(Item 7)
6. The method of any one of items 1-5, wherein said nuclease target site is near the 3' end of said repeat extension sequence.
(Item 8)
A method according to any of the preceding items, wherein said nuclease target site is outside said repeat extension sequence.
(Item 9)
The site at which the nuclease agent forms a double-strand or single-strand break is within about 50, about 40, about 30, about 20, or about 10 nucleotides from the 5' or 3' end of the repeat extension sequence A method according to any of the preceding items.
(Item 10)
the site at which the nuclease agent forms a double-strand or single-strand break is about 25, about 24, about 23, about 22, about 21, about 20 from the 5' or 3' end of the repeat extension sequence; about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3 or within about 2 nucleotides.
(Item 11)
A method according to any of the preceding items, wherein the site at which the nuclease agent forms a double-strand or single-strand break is near the 5' end of the repeat extension sequence.
(Item 12)
11. The method of any one of items 1-10, wherein the site at which the nuclease agent forms a double-strand or single-strand break is near the 3' end of the repeat extension sequence.
(Item 13)
A method according to any of the preceding items, wherein the site at which the nuclease agent forms a double-strand or single-strand break is outside the repeat extension sequence.
(Item 14)
A method according to any of the preceding items, wherein said nuclease target site is retained following repair of said double-strand break or said single-strand break by said cell.
(Item 15)
The method of any of the preceding items, wherein repair of said double-strand break or said single-strand break does not result in insertions or deletions outside of said repeat extension sequence.
(Item 16)
A method according to any of the preceding items, wherein the nuclease agent forms a double-stranded break near the 5' or 3' end of the repeat extension sequence.
(Item 17)
16. The method of any one of items 1-15, wherein the nuclease agent is a nickase that creates a single-strand break near the 5' or 3' end of the repeat extension sequence.
(Item 18)
A first nuclease agent or nucleic acid encoding said first nuclease agent and a second nuclease agent or nucleic acid encoding said second nuclease agent are introduced into said cell, wherein said first nuclease agent comprises said repeat Forming a double-strand or single-strand break at a first nuclease target site near the 5' end of the extended sequence, said second nuclease agent forming a second nuclease break near the 3' end of said repeat extended sequence; A method according to any of the preceding items, wherein a double-strand break or a single-strand break is formed at the nuclease target site of .
(Item 19)
The first nuclease agent forms a double-stranded break at a first nuclease target site near the 5' end of the repeat extension sequence, and the second nuclease agent forms a double-strand break at the 3' end of the repeat extension sequence. 19. The method of item 18, wherein the double-strand break is formed at the second nuclease target site in the vicinity of .
(Item 20)
The first nuclease agent forms a single-strand break at a first nuclease target site near the 5' end of the repeat extension sequence, and the second nuclease agent forms a single-strand break at the 3' end of the repeat extension sequence. 19. The method of item 18, wherein a single-strand break is formed at the second nuclease target site in the vicinity of .
(Item 21)
Any of the preceding items, wherein the nuclease agent is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) binding (Cas) protein and guide RNA. the method of.
(Item 22)
22. The method of item 21, wherein said nuclease agent is said Cas protein and said guide RNA.
(Item 23)
23. The method of item 22, wherein the Cas protein is a Cas9 protein.
(Item 24)
A method according to any of the preceding items, wherein the repeat-expansion sequence is a heterologous repeat-expansion sequence.
(Item 25)
said repeat extension sequence comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, or at least about 90 copies of said repeat sequence; A method according to any of the preceding items.
(Item 26)
A method according to any of the preceding items, wherein the copies of the repeat sequence are contiguous within the repeat extension sequence.
(Item 27)
The method according to any of the preceding items, wherein the repeat sequence is a trinucleotide repeat, tetranucleotide repeat, pentanucleotide repeat, hexanucleotide repeat or dodecanucleotide repeat.
(Item 28)
The method of any of the preceding items, wherein said repeat sequence comprises any one of SEQ ID NOs: 1-12.
(Item 29)
(i) said repeat sequence comprises SEQ ID NO: 2 and said target genomic locus is HTT, AR, ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B or TBP locus;
(ii) said repeat sequence comprises SEQ ID NO: 3 and said target genomic locus is the FMR1 locus;
(iii) said repeat sequence comprises SEQ ID NO: 4 and said target genomic locus is a DMPK, JPH3, ATXN8 or TCF4 locus;
(iv) said repeat sequence comprises SEQ ID NO: 5 and said target genomic locus is the FXN locus;
(v) said repeat sequence comprises SEQ ID NO: 6 and said target genomic locus is the AFF2 locus;
(vi) said repeat sequence comprises SEQ ID NO: 7 and said target genomic locus is the PABPN1 locus;
(vii) said repeat sequence comprises SEQ ID NO:8 and said target genomic locus is a CNBP locus;
(viii) said repeat sequence comprises SEQ ID NO: 9 and said target genomic locus is the ATXN10 locus;
(ix) said repeat sequence comprises SEQ ID NO: 10 and said target genomic locus is the TK2 or BEAN1 locus;
(x) said repeat sequence comprises SEQ ID NO: 11 and said target genomic locus is the NOP56 locus;
(xi) said repeat sequence comprises SEQ ID NO: 1 and said target genomic locus is the C9ORF72 locus; or
(xii) said repeat sequence comprises SEQ ID NO: 12 and said target genomic locus is the CSTB locus;
29. The method of item 28.
(Item 30)
30. The method of item 29, wherein said repeat sequence comprises SEQ ID NO: 1 and said target genomic locus is the C9ORF72 locus.
(Item 31)
31. The method of item 30, wherein said nuclease agent is Cas9 protein and guide RNA and said nuclease target site comprises SEQ ID NO:28 or 33.
(Item 32)
The method of any of the preceding items, wherein the cells are non-human animal cells.
(Item 33)
The method of any of the preceding items, wherein the cells are non-human animal embryonic stem cells, embryonic stem cell-derived motor neurons, brain cells, cortical cells, neuronal cells, muscle cells, heart cells or embryonic cells.
(Item 34)
33. The method of any one of items 1-32, wherein the cell is a one-cell stage embryo of a non-human animal.
(Item 35)
The method of any of the preceding items, wherein the cells are rodent cells.
(Item 36)
36. The method of item 35, wherein said cells are mouse cells or rat cells.
(Item 37)
37. The method of item 36, wherein said cells are mouse cells.
(Item 38)
38. The method of item 37, wherein said cells are mouse embryonic stem cells or mouse 1-cell stage embryos.
(Item 39)
32. The method of any one of items 1-31, wherein said cells are human induced pluripotent stem cells.
(Item 40)
The method of any of the preceding items, wherein said cell is in vitro.
(Item 41)
39. The method of any one of items 1-38, wherein said cell is in vivo.
(Item 42)
comprising multiple rounds of steps (a)-(c), wherein said population of cells in each round of step (a) after the first round is expanded from said modified cells selected in the previous step (c); The method according to any of the preceding items, which is a clonal population of cells that have been isolated from each other.
(Item 43)
43. The method of item 42, comprising at least 3 times or at least 4 times.
(Item 44)
a first modified cell run with a first nuclease agent that forms a double-strand break or a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence for the first time; said first modified cell with a second nuclease agent that produces and a second time forms a double-strand or single-strand break at a second nuclease target site near the 3' end of said repeat extension sequence; 44. A method according to item 42 or 43, wherein said method is performed on said cell to generate a second modified cell.
(Item 45)
a first modified cell run with a first nuclease agent that forms a double-strand break or a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence for the first time; said first modified cell with a second nuclease agent that produces and a second time forms a double-strand or single-strand break at a second nuclease target site near the 5' end of said repeat extension sequence; 44. A method according to item 42 or 43, wherein said method is performed on said cell to generate a second modified cell.
(Item 46)
a first modified cell run with a first nuclease agent that forms a double-strand break or a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence for the first time; said first modified cell with a second nuclease agent that produces and a second time forms a double-strand or single-strand break at a second nuclease target site near the 5' end of said repeat extension sequence; 44. A method according to item 42 or 43, wherein said method is performed on said cell to generate a second modified cell.
(Item 47)
a first modified cell run with a first nuclease agent that forms a double-strand break or a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence for the first time; said first modified cell with a second nuclease agent that produces and a second time forms a double-strand or single-strand break at a second nuclease target site near the 3' end of said repeat extension sequence; 44. A method according to item 42 or 43, wherein said method is performed on said cell to generate a second modified cell.
(Item 48)
a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 5' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 3' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 49)
a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 3' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 5' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 50)
a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 5' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 5' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 51)
a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 3' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 3' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 52)
a first run with a first nuclease agent that creates a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 3' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 53)
a first run with a first nuclease agent that creates a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 5' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 54)
a first run with a first nuclease agent that creates a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 5' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 55)
a first run with a first nuclease agent that creates a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence to produce a first modified cell; a second modification performed on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 3' end of said repeat extension sequence to 44. A method according to item 42 or 43, wherein the method produces a modified cell.
(Item 56)
56. The method of items 46, 47, 50, 51, 54 or 55, wherein said first nuclease target site is the same as said second nuclease target site.
(Item 57)
said nuclease target site is outside said repeat extension sequence and said nuclease target site is within about 50, about 40, about 30, about 20 or about 10 nucleotides from the 5' or 3' end of said repeat extension sequence; , the method of any of the preceding items, wherein the nuclease agent is Cas9 protein and guide RNA.
(Item 58)
the nuclease agent is a nickase that forms a single-strand break near the 5' or 3' end of the repeat extension sequence, and the nuclease target site is retained after repair of the single-strand break by the cell; 58. The method of item 57, wherein repair of said single-strand break does not result in insertions or deletions outside of said repeat extension sequence.
(Item 59)
A non-human animal or non-human animal cell comprising in its genome a heterologous hexanucleotide repeat extension sequence inserted at the endogenous C9orf72 locus, wherein said heterologous hexanucleotide repeat extension sequence is the hexanucleotide repeat extension sequence shown in SEQ ID NO: 1 A non-human animal or non-human animal cell comprising more than about 100 repeats of a nucleotide sequence.
(Item 60)
60. The non-human animal or non-human animal cell of item 59, wherein said heterologous hexanucleotide repeat extension sequence comprises at least about 300 repeats of the hexanucleotide sequence set forth in SEQ ID NO:1.
(Item 61)
61. The non-human animal or non-human animal cell of item 60, wherein said hexanucleotide repeat extension comprises at least about 500 repeats of the hexanucleotide sequence set forth in SEQ ID NO:1.
(Item 62)
62. The non-human animal or non-human animal cell of item 61, wherein said hexanucleotide repeat extension comprises at least about 600 repeats of the hexanucleotide sequence set forth in SEQ ID NO:1.
(Item 63)
63. The non-human animal or non-human animal cell of any one of items 59-62, wherein said repeats are continuous within said heterologous repeat extension sequence.
(Item 64)
64. The non-human animal of any one of items 59-63, wherein said heterologous hexanucleotide repeat extension sequence is located between the first non-coding endogenous exon and the second exon of said endogenous C9orf72 locus, or non-human animal cells.
(Item 65)
65. The non-human animal or non-human animal cell of any one of items 59-64, wherein said endogenous C9orf72 locus comprises a human C9ORF72 nucleotide sequence.
(Item 66)
66. The non-human animal or non-human animal cell of item 65, wherein said human C9ORF72 nucleotide sequence comprises SEQ ID NO:46 and/or SEQ ID NO:47.
(Item 67)
67. The non-human animal or non-human animal cell of any one of items 59-66, wherein said non-human animal is a rodent or said non-human animal cell is a rodent cell.
(Item 68)
68. The non-human animal or non-human animal cell of item 67, wherein said rodent is a rat or mouse or said non-human animal cell is a rat or mouse cell.
(Item 69)
69. The non-human animal or non-human animal cell of any one of items 59-68, which is heterozygous for said heterologous hexanucleotide repeat extension sequence.
(Item 70)
(a) increased expression of C9orf72 transcripts that retain intronic sequences compared to a control non-human animal or control non-human animal cell containing a wild-type C9orf72 locus; and/or
(b) an increase in the number of RNA foci compared to a control non-human animal or control non-human animal cell containing the wild-type C9orf72 locus; and/or
(c) increased levels of dipeptide repeat proteins compared to control non-human animals or control non-human animal cells containing the wild-type C9orf72 locus;
70. The non-human animal or non-human animal cell of any one of items 59-69, which exhibits
(Item 71)
71. The non-human animal cell according to any one of items 59-70, which is an embryonic stem cell, an embryonic stem cell-derived motor neuron, a brain cell, a cortical cell, a neuronal cell, a muscle cell, a cardiac cell or an embryonic cell.
(Item 72)
71. The non-human animal cell according to any one of items 59-70, which is a 1-cell stage embryo.
(Item 73)
73. The non-human animal cell of any one of items 59-72, in vitro.
(Item 74)
73. Non-human animal cell according to any one of items 59-72, in vivo.
(Item 75)
71. The non-human animal of any one of items 59-70, comprising in its germline genome said heterologous hexanucleotide repeat extension sequence inserted at said endogenous C9orf72 locus.
(Item 76)
A non-human animal genome comprising a heterologous hexanucleotide repeat expansion sequence inserted into an endogenous C9orf72 locus, said heterologous hexanucleotide repeat expansion sequence comprising more than about 100 repeats of the hexanucleotide sequence shown in SEQ ID NO:1. non-human animal genomes, including;
(Item 77)
A non-human animal C9orf72 gene comprising a heterologous hexanucleotide repeat extension sequence, said heterologous hexanucleotide repeat extension sequence comprising greater than about 100 repeats of the hexanucleotide sequence shown in SEQ ID NO:1.
(Item 78)
for use in a method for generating a modified cell having increased copy number of said repeat sequence at said target genomic locus comprising a repeat extension sequence comprising multiple copies of said repeat sequence at said target genomic locus which is designed to form a double-strand or single-strand break at a nuclease target site near the 5' or 3' end of said repeat extension sequence.
(Item 79)
1. A method of evaluating a therapeutic drug candidate for treatment of a disease or condition associated with a hexanucleotide repeat expansion sequence of the C9orf72 locus, comprising:
(a) administering a candidate agent to the non-human animal of any one of items 59-70 and 75 or the non-human animal cell of any one of items 59-74;
(b) performing one or more assays to determine whether said candidate agent affects one or more signs or symptoms associated with said disease or condition;
(c) identifying said candidate agent affecting said one or more signs or symptoms associated with said disease or condition as a candidate therapeutic agent;
method including.
(Item 80)
80. The method of paragraph 79, wherein the candidate agent is administered to the non-human animal in vivo and the one or more assays are performed in vitro on cells isolated from the non-human animal after administration of the candidate agent. the method of.
(Item 81)
80. The method of item 79, wherein said candidate agent is administered in vitro to non-human embryonic stem cell-derived motor neurons.
(Item 82)
82. The method of any one of items 79-81, wherein said one or more assays comprises quantitative polymerase chain reaction (qPCR) to detect intron-containing C9orf72 RNA transcripts.
(Item 83)
of paragraphs 79 to 82, wherein said one or more assays comprise measuring RNA foci comprising C9orf72 sense or antisense RNA transcripts, optionally said RNA foci being measured by fluorescence in situ hybridization. A method according to any one of paragraphs.
(Item 84)
Said one or more assays comprise measuring the accumulation of dipeptide repeat proteins, optionally said dipeptide repeat proteins are polyGA dipeptide repeat proteins or polyGP dipeptide repeat proteins, optionally said 84. The method of any one of items 79-83, wherein accumulation is measured by immunohistochemistry.
Claims (47)
（ａ）前記標的ゲノム遺伝子座において前記リピート配列の複数のコピーを含むリピート伸長配列を含む細胞の集団を提供するステップと；
（ｂ）細胞の前記集団の中にヌクレアーゼ剤または前記ヌクレアーゼ剤をコードする核酸を導入するステップであって、前記ヌクレアーゼ剤は、前記リピート伸長配列の５’末端または３’末端の近傍のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成して、改変された細胞の集団を生成するステップと；
（ｃ）改変された細胞の前記集団の中の前記リピート配列のコピー数を定量化し、前記リピート配列のコピー数が増加している改変された細胞を選択するステップと
を含む方法。 A method for generating a modified cell having increased copy number of repeat sequences at a target genomic locus, comprising:
(a) providing a population of cells comprising repeat expansion sequences comprising multiple copies of said repeat sequence at said target genomic locus;
(b) introducing into said population of cells a nuclease agent or a nucleic acid encoding said nuclease agent, said nuclease agent having a nuclease target near the 5' or 3' end of said repeat extension sequence; forming a double-strand break or a single-strand break at the site to generate a population of modified cells;
(c) quantifying the copy number of said repeat sequence in said population of modified cells and selecting modified cells with increased copy number of said repeat sequence.
（ｉｉ）前記リピート配列が配列番号３を含み、前記標的ゲノム遺伝子座がＦＭＲ１遺伝子座であるか；
（ｉｉｉ）前記リピート配列が配列番号４を含み、前記標的ゲノム遺伝子座がＤＭＰＫ、ＪＰＨ３、ＡＴＸＮ８またはＴＣＦ４遺伝子座であるか；
（ｉｖ）前記リピート配列が配列番号５を含み、前記標的ゲノム遺伝子座がＦＸＮ遺伝子座であるか；
（ｖ）前記リピート配列が配列番号６を含み、前記標的ゲノム遺伝子座がＡＦＦ２遺伝子座であるか；
（ｖｉ）前記リピート配列が配列番号７を含み、前記標的ゲノム遺伝子座がＰＡＢＰＮ１遺伝子座であるか；
（ｖｉｉ）前記リピート配列が配列番号８を含み、前記標的ゲノム遺伝子座がＣＮＢＰ遺伝子座であるか；
（ｖｉｉｉ）前記リピート配列が配列番号９を含み、前記標的ゲノム遺伝子座がＡＴＸＮ１０遺伝子座であるか；
（ｉｘ）前記リピート配列が配列番号１０を含み、前記標的ゲノム遺伝子座がＴＫ２またはＢＥＡＮ１遺伝子座であるか；
（ｘ）前記リピート配列が配列番号１１を含み、前記標的ゲノム遺伝子座がＮＯＰ５６遺伝子座であるか；
（ｘｉ）前記リピート配列が配列番号１を含み、前記標的ゲノム遺伝子座がＣ９ＯＲＦ７２遺伝子座であるか；または
（ｘｉｉ）前記リピート配列が配列番号１２を含み、前記標的ゲノム遺伝子座がＣＳＴＢ遺伝子座である、
請求項２８に記載の方法。 (i) said repeat sequence comprises SEQ ID NO: 2 and said target genomic locus is HTT, AR, ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B or TBP locus;
(ii) said repeat sequence comprises SEQ ID NO: 3 and said target genomic locus is the FMR1 locus;
(iii) said repeat sequence comprises SEQ ID NO: 4 and said target genomic locus is a DMPK, JPH3, ATXN8 or TCF4 locus;
(iv) said repeat sequence comprises SEQ ID NO: 5 and said target genomic locus is the FXN locus;
(v) said repeat sequence comprises SEQ ID NO: 6 and said target genomic locus is the AFF2 locus;
(vi) said repeat sequence comprises SEQ ID NO: 7 and said target genomic locus is the PABPN1 locus;
(vii) said repeat sequence comprises SEQ ID NO:8 and said target genomic locus is a CNBP locus;
(viii) said repeat sequence comprises SEQ ID NO: 9 and said target genomic locus is the ATXN10 locus;
(ix) said repeat sequence comprises SEQ ID NO: 10 and said target genomic locus is the TK2 or BEAN1 locus;
(x) said repeat sequence comprises SEQ ID NO: 11 and said target genomic locus is the NOP56 locus;
(xi) said repeat sequence comprises SEQ ID NO: 1 and said target genomic locus is the C9ORF72 locus; or (xii) said repeat sequence comprises SEQ ID NO: 12 and said target genomic locus is the CSTB locus. be,
29. The method of claim 28.
（ＩＩ）１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（ＩＩＩ）１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（ＩＶ）１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断または一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（Ｖ）１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（ＶＩ）１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（Ｖ）１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（ＶＩ）１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で二本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で二本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（ＶＩＩ）１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（ＶＩＩＩ）１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（ＩＸ）１回目に前記リピート伸長配列の５’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の５’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
（Ｘ）１回目に前記リピート伸長配列の３’末端の近傍の第１のヌクレアーゼ標的部位で一本鎖切断を形成する第１のヌクレアーゼ剤で実行されて第１の改変された細胞を生成し、２回目に前記リピート伸長配列の３’末端の近傍の第２のヌクレアーゼ標的部位で一本鎖切断を形成する第２のヌクレアーゼ剤で前記第１の改変された細胞に対して実行されて第２の改変された細胞を生成する、
請求項４２または４３に記載の方法。 (I) a first modified run with a first nuclease agent that first creates a double-strand break or a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence; said first modification with a second nuclease agent that, a second time, forms a double-strand or single-strand break at a second nuclease target site proximal to the 3' end of said repeat extension sequence; to generate a second modified cell;
(II) a first modified run with a first nuclease agent that forms a double-strand break or a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence for the first time; said first modification with a second nuclease agent that, a second time, forms a double-strand or single-strand break at a second nuclease target site proximal to the 5' end of said repeat extension sequence; to generate a second modified cell;
(III) a first modified run with a first nuclease agent that forms a double-strand break or a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence for the first time; said first modification with a second nuclease agent that, a second time, forms a double-strand or single-strand break at a second nuclease target site proximal to the 5' end of said repeat extension sequence; to generate a second modified cell;
(IV) a first modified run with a first nuclease agent that forms a double-strand break or a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence for the first time; said first modification with a second nuclease agent that, a second time, forms a double-strand or single-strand break at a second nuclease target site proximal to the 3' end of said repeat extension sequence; to generate a second modified cell;
(V) performing a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 5' end of said repeat extension sequence for a first time to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 3′ end of said repeat extension sequence; generating 2 modified cells,
(VI) performing a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 3' end of said repeat extension sequence for a first time to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 5' end of said repeat extension sequence; generating 2 modified cells,
(V) performing a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 5' end of said repeat extension sequence for a first time to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 5' end of said repeat extension sequence; generating 2 modified cells,
(VI) performing a first run with a first nuclease agent that forms a double-strand break at a first nuclease target site near the 3' end of said repeat extension sequence for a first time to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that forms a double-strand break at a second nuclease target site near the 3′ end of said repeat extension sequence; generating 2 modified cells,
(VII) performing a first run with a first nuclease agent that creates a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 3′ end of said repeat extension sequence; generating 2 modified cells,
(VIII) run with a first nuclease agent that first creates a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 5′ end of said repeat extension sequence; generating 2 modified cells,
(IX) performing a first round with a first nuclease agent that creates a single-strand break at a first nuclease target site near the 5' end of said repeat extension sequence to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 5′ end of said repeat extension sequence; generating 2 modified cells,
(X) performing a first run with a first nuclease agent that creates a single-strand break at a first nuclease target site near the 3' end of said repeat extension sequence for the first time to produce a first modified cell; , a second time on said first modified cell with a second nuclease agent that creates a single-strand break at a second nuclease target site near the 3′ end of said repeat extension sequence; generating 2 modified cells,
44. A method according to claim 42 or 43.
the nuclease agent is a nickase that forms a single-strand break near the 5' or 3' end of the repeat extension sequence, and the nuclease target site is retained after repair of the single-strand break by the cell; 47. The method of claim 46 , wherein repair of said single-strand break does not result in insertions or deletions outside of said repeat extension sequence.
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