JPWO2020206139A5 - - Google Patents
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- JPWO2020206139A5 JPWO2020206139A5 JP2021559067A JP2021559067A JPWO2020206139A5 JP WO2020206139 A5 JPWO2020206139 A5 JP WO2020206139A5 JP 2021559067 A JP2021559067 A JP 2021559067A JP 2021559067 A JP2021559067 A JP 2021559067A JP WO2020206139 A5 JPWO2020206139 A5 JP WO2020206139A5
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ＲＮＡｓｅｑ。ＲＮＡ抽出および配列決定ライブラリー調製のプロトコルは、以前に記載されたものと同様であった（Ａｔａｎａｓｉｏ ｅｔ ａｌ．（２０１６）Ｓｃｉ．Ｒｅｐ．６：２３２０４、すべての目的のためにその全体が参照により本明細書に組み込まれる）。鎖特異的ＲＮＡ－ｓｅｑライブラリーは、ＫＡＰＡ鎖ｍＲＮＡ－Ｓｅｑキット（ＫＡＰＡ Ｂｉｏｓｙｓｔｅｍｓ）を使用して５００ｎｇ ＲＮＡから調製した。ライブラリーは１２サイクルのＰＣＲによって増幅された。配列決定は、イルミナＨｉＳｅｑ（登録商標）２０００（イルミナ）で、マルチプレックスシングルリードラン（ｉＰＳヒトニューロン、マウス初代ニューロン、およびマウス組織の場合）、またはペアリードラン（ＳＨ－ＳＹ５ＹおよびＮｅｕｒｏ－２Ａ細胞株の場合）によって実行された。結果のＦＡＳＴＱファイルは、十分なデータ品質を確保するためにＦａｓｔＱＣを介して分析された。読み取りは、２つのミスマッチが許容される商用ソフトウェアＡｒｒａｙＳｔｕｄｉｏ（ＯｍｉｃＳｏｆｔ）を使用して、ヒトゲノム（ｈｇ１９）またはマウスゲノム（ｍｍ１０）にマッピングされた。サンプルでは、８３％～９０％の読み取りが一意にマッピングされた。遺伝子発現は、ＯｍｉｃＳｏｆｔ ＡｒｒａｙＳｔｕｄｉｏ ＲＮＡＳｅｑ分析パイプライン（Ｑｉａｇｅｎ Ｉｎｃ．）を使用した生の配列決定読み取りから導き出された。各遺伝子について、遺伝子のエクソンに一意にマッピングされた読み取りが特定され、カウントされた。得られた読み取りカウントは、生の発現として遺伝子レベルで要約された。
本発明は、例えば、以下の項目を提供する。
(項目１)
ヒト化内因性Ｆ１２遺伝子座をゲノムに含む非ヒト動物であって、前記内因性Ｆ１２遺伝子座のセグメントが削除され、対応するヒトＦ１２配列で置き換えられている、非ヒト動物。
(項目２)
前記ヒト化内因性Ｆ１２遺伝子座が、ヒト凝固因子ＸＩＩ重鎖を含むタンパク質をコードする、項目１に記載の非ヒト動物。
(項目３)
前記ヒト化内因性Ｆ１２遺伝子座が、ヒト凝固因子ＸＩＩ軽鎖を含むタンパク質をコードする、項目１または２に記載の非ヒト動物。
(項目４)
前記ヒト化内因性Ｆ１２遺伝子座が、ヒト凝固因子ＸＩＩシグナルペプチドを含むタンパク質をコードする、先行項目のいずれか一項に記載の非ヒト動物。
(項目５)
コード配列および非コード配列の両方を含む前記内因性Ｆ１２遺伝子座の領域が削除され、コード配列および非コード配列の両方を含む対応するヒトＦ１２配列で置き換えられている、先行項目のいずれか一項に記載の非ヒト動物。
(項目６)
前記ヒト化内因性Ｆ１２遺伝子座が前記内因性Ｆ１２プロモーターを含み、前記ヒトＦ１２配列が前記内因性Ｆ１２プロモーターに作動可能に連結されている、先行項目のいずれか一項に記載の非ヒト動物。
(項目７)
前記内因性Ｆ１２遺伝子座の少なくとも１つのイントロンおよび少なくとも１つのエクソンが削除され、前記対応するヒトＦ１２配列で置き換えられている、先行項目のいずれか一項に記載の非ヒト動物。
(項目８)
前記内因性Ｆ１２遺伝子座のＦ１２コード配列全体が削除され、前記対応するヒトＦ１２配列で置き換えられている、先行項目のいずれか一項に記載の非ヒト動物。
(項目９)
開始コドンから終止コドンまでの前記内因性Ｆ１２遺伝子座の前記領域が削除され、前記対応するヒトＦ１２配列で置き換えられている、項目８に記載の非ヒト動物。
(項目１０)
内因性Ｆ１２の３’非翻訳領域が削除されておらず、前記対応するヒトＦ１２配列で置き換えられていない、先行項目のいずれか一項に記載の非ヒト動物。
(項目１１)
内因性Ｆ１２の５’非翻訳領域が削除されておらず、前記対応するヒトＦ１２配列で置き換えられていない、先行項目のいずれか一項に記載の非ヒト動物。
(項目１２)
前記開始コドンから前記終止コドンまでの前記内因性Ｆ１２遺伝子座の前記領域が削除され、前記対応するヒトＦ１２配列で置き換えられており、
内因性Ｆ１２プロモーターが削除されておらず、前記対応するヒトＦ１２配列で置き換えられていない、先行項目のいずれか一項に記載の非ヒト動物。
(項目１３)
（ｉ）前記ヒト化内因性Ｆ１２遺伝子座の前記ヒトＦ１２配列が、配列番号３１もしくは３２に記載の配列に対して少なくとも９０％、９５％、９６％、９７％、９８％、９９％、もしくは１００％同一の配列を含み、かつ／または
（ｉｉ）前記ヒト化内因性Ｆ１２遺伝子座が、配列番号５もしくは４６に記載の配列に対して少なくとも９０％、９５％、９６％、９７％、９８％、９９％、もしくは１００％同一の配列を含むタンパク質をコードし、かつ／または
（ｉｉｉ）前記ヒト化内因性Ｆ１２遺伝子座が、配列番号１３もしくは４８に記載の配列に対して少なくとも９０％、９５％、９６％、９７％、９８％、９９％、もしくは１００％同一の配列を含むコード配列を含み、かつ／または
（ｉｖ）前記ヒト化内因性Ｆ１２遺伝子座が、配列番号１７もしくは１８に記載の配列に対して少なくとも９０％、９５％、９６％、９７％、９８％、９９％、もしくは１００％同一の配列を含む、先行項目のいずれか一項に記載の非ヒト動物。
(項目１４)
前記ヒト化内因性Ｆ１２遺伝子座が、前記対応するヒトＦ１２配列のイントロン内に選択カセットもしくはレポーター遺伝子を含むか、または前記ヒト化内因性Ｆ１２遺伝子座が選択カセットまたはレポーターを含まない、先行項目のいずれか一項に記載の非ヒト動物。
(項目１５)
前記非ヒト動物が、前記ヒト化内因性Ｆ１２遺伝子座に対してホモ接合である、先行項目のいずれか一項に記載の非ヒト動物。
(項目１６)
前記非ヒト動物がその生殖系列に前記ヒト化内因性Ｆ１２遺伝子座を含む、先行項目のいずれか一項に記載の非ヒト動物。
(項目１７)
前記非ヒト動物が哺乳動物である、先行項目のいずれか一項に記載の非ヒト動物。
(項目１８)
前記非ヒト動物がラットまたはマウスである、項目１７に記載の非ヒト動物。
(項目１９)
前記非ヒト動物がマウスである、項目１８に記載の非ヒト動物。
(項目２０)
前記非ヒト動物が、脳内で短いヒト凝固因子ＸＩＩアイソフォームを発現する、先行項目のいずれか一項に記載の非ヒト動物。
(項目２１)
（ｉ）前記短いヒト凝固因子ＸＩＩアイソフォームが、ヒトＦ１２のエクソン１１～１４に対するプローブによって検出されるが、ヒトＦ１２のエクソン１～６に対するプローブによっては検出されないＦ１２ ｍＲＮＡによってコードされ、かつ／または
（ｉｉ）前記短いヒト凝固因子ＸＩＩアイソフォームがＦＸＩＩの
２９７－５９６
であり、かつ／または
（ｉｉｉ）前記短いヒト凝固因子ＸＩＩアイソフォームが前記脳内のニューロンで発現しており、かつ／または
（ｉｖ）前記短いヒト凝固因子ＸＩＩアイソフォームが、血漿カリクレインによって活性化され得、プロＨＧＦを活性ＨＧＦに変換する、項目２０に記載の非ヒト動物。
(項目２２)
前記非ヒト動物が、対応する野生型非ヒト動物の活性化部分トロンボプラスチン時間（ａＰＴＴ）と顕著に異ならないａＰＴＴを有し、かつ／または前記非ヒト動物が、対応する野生型の非ヒト動物のプロトロンビン時間（ＰＴ）と顕著に異ならないＰＴを有する、先行項目のいずれか一項に記載の非ヒト動物。
(項目２３)
前記非ヒトが少なくとも約５μｇ／ｍＬのヒト凝固因子ＸＩＩ血漿レベルを有する、先行項目のいずれか一項に記載の非ヒト動物。
(項目２４)
ヒト化内因性Ｆ１２遺伝子座をゲノムに含む非ヒト動物細胞であって、前記内因性Ｆ１２遺伝子座のセグメントが削除され、対応するヒトＦ１２配列で置き換えられている、非ヒト動物細胞。
(項目２５)
ヒト化内因性Ｆ１２遺伝子座を含む非ヒト動物ゲノムであって、前記内因性Ｆ１２遺伝子座のセグメントが削除され、対応するヒトＦ１２配列で置き換えられている、非ヒト動物ゲノム。
(項目２６)
ヒト化内因性Ｆ１２遺伝子座を生成するための標的化ベクターであって、前記内因性Ｆ１２遺伝子座のセグメントが削除され、対応するヒトＦ１２配列で置き換えられ、前記標的化ベクターが、前記内因性Ｆ１２遺伝子座の５’標的配列を標的とする５’ホモロジーアームと前記内因性Ｆ１２遺伝子座の３’標的配列を標的とする３’ホモロジーアームとが隣接する前記対応するヒトＦ１２配列を含むインサート核酸を含む、標的化ベクター。
(項目２７)
ヒト化非ヒト動物Ｆ１２遺伝子であって、前記内因性Ｆ１２遺伝子座のセグメントが削除され、対応するヒトＦ１２配列で置き換えられている、ヒト化非ヒト動物Ｆ１２遺伝子。
(項目２８)
インビボでのヒト凝固因子ＸＩＩ標的化試薬の活性を評価する方法であって、
（ａ）前記ヒト凝固因子ＸＩＩ標的化試薬を、項目１～２３のいずれか一項に記載の非ヒト動物に投与することと、
（ｂ）前記非ヒト動物における前記ヒト凝固因子ＸＩＩ標的化試薬の前記活性を評価することと、を含む、方法。
(項目２９)
前記投与することが、アデノ随伴ウイルス（ＡＡＶ）媒介送達、脂質ナノ粒子（ＬＮＰ）媒介送達、流体力学的送達（ＨＤＤ）、または注射を含む、項目２８に記載の方法。
(項目３０)
ステップ（ｂ）が、前記非ヒト動物から血漿を単離することと、前記血漿中の前記ヒト凝固因子ＸＩＩ標的化試薬の活性を評価することと、を含む、項目２８または２９に記載の方法。
(項目３１)
ステップ（ｂ）が、前記非ヒト動物の肝臓または脳における前記ヒト凝固因子ＸＩＩ標的化試薬の前記活性を評価することを含む、項目２８～３０のいずれか一項に記載の方法。
(項目３２)
ステップ（ｂ）が、凝固またはトロンビン生成を評価することを含む、項目２８～３１のいずれか一項に記載の方法。
(項目３３)
ステップ（ｂ）がＨＧＦ－Ｍｅｔシグナル伝達を評価することを含む、項目２８～３２のいずれか一項に記載の方法。
(項目３４)
ステップ（ｂ）が、前記ヒト化内因性Ｆ１２遺伝子座によってコードされるＦ１２メッセンジャーＲＮＡの発現を測定することを含む、項目２８～３３のいずれか一項に記載の方法。
(項目３５)
ステップ（ｂ）が、前記ヒト化内因性凝固因子ＸＩＩ遺伝子座によってコードされる凝固因子ＸＩＩタンパク質の発現を測定することを含む、項目２８～３４に記載のいずれか一項に記載の方法。
(項目３６)
前記ヒト凝固因子ＸＩＩ標的化試薬がゲノム編集剤であり、ステップ（ｂ）が前記ヒト化内因性Ｆ１２遺伝子座の改変を評価することを含む、項目２８～３５のいずれか一項に記載の方法。
(項目３７)
ステップ（ｂ）が、前記ヒト化内因性Ｆ１２遺伝子座内の挿入または欠失の頻度を測定することを含む、項目３６に記載の方法。
(項目３８)
前記ヒト凝固因子ＸＩＩ標的化試薬が、ヒトＦ１２遺伝子の領域を標的化するように設計されたヌクレアーゼ剤を含む、項目２８～３７のいずれか一項に記載の方法。
(項目３９)
前記ヌクレアーゼ剤が、Ｃａｓタンパク質および前記ヒトＦ１２遺伝子のガイドＲＮＡ標的配列を標的化するように設計されたガイドＲＮＡを含む、項目３８に記載の方法。
(項目４０)
前記Ｃａｓタンパク質が、Ｃａｓ９タンパク質である、項目３９に記載の方法。
(項目４１)
前記ヒト凝固因子ＸＩＩ標的化試薬が外因性ドナー核酸を含み、前記外因性ドナー核酸が前記ヒトＦ１２遺伝子を標的化するように設計されており、任意選択で、前記外因性ドナー核酸がＡＡＶを介して送達される、項目２８～４０のいずれか一項に記載の方法。
(項目４２)
前記ヒト凝固因子ＸＩＩ標的化試薬が、ＲＮＡｉ剤またはアンチセンスオリゴヌクレオチドである、項目２８～３５のいずれか一項に記載の方法。
(項目４３)
前記ヒト凝固因子ＸＩＩ標的化試薬が抗原結合タンパク質である、項目２８～３５のいずれか一項に記載の方法。
(項目４４)
前記ヒト凝固因子ＸＩＩ標的化試薬が小分子である、項目２８～３５のいずれか一項に記載の方法。
(項目４５)
インビボでのヒト凝固因子ＸＩＩ標的化試薬の活性を最適化する方法であって、
（Ｉ）ゲノムにヒト化内因性Ｆ１２遺伝子座を含む第１の非ヒト動物において、項目２８～４４のいずれか一項に記載の方法を１回目に実施することと、
（ＩＩ）変数を変更し、ゲノムにヒト化内因性Ｆ１２遺伝子座を含む第２の非ヒト動物において、前記変更された変数を用いてステップ（Ｉ）の方法を２回目に実施することと、
（ＩＩＩ）ステップ（Ｉ）の前記ヒト凝固因子ＸＩＩ標的化試薬の前記活性をステップ（ＩＩ）の前記ヒト凝固因子ＸＩＩ標的化試薬の前記活性と比較し、結果としてより高い活性が得られる前記方法を選択することと、を含む、方法。
(項目４６)
ステップ（ＩＩ）の前記変更された変数が、前記ヒト凝固因子ＸＩＩ標的化試薬を前記非ヒト動物に導入する送達方法である、項目４５に記載の方法。
(項目４７)
ステップ（ＩＩ）の前記変更された変数が、前記ヒト凝固因子ＸＩＩ標的化試薬を前記非ヒト動物に導入する投与経路である、項目４５に記載の方法。
(項目４８)
ステップ（ＩＩ）の前記変更された変数が、前記非ヒト動物に導入された前記ヒト凝固因子ＸＩＩ標的化試薬の濃度または量である、項目４５に記載の方法。
(項目４９)
ステップ（ＩＩ）の前記変更された変数が、前記非ヒト動物に導入された前記ヒト凝固因子ＸＩＩ標的化試薬の形態である、項目４５に記載の方法。
(項目５０)
ステップ（ＩＩ）の前記変更された変数が、前記非ヒト動物に導入された前記ヒト凝固因子ＸＩＩ標的化試薬である、項目４５に記載の方法。
(項目５１)
項目１～２３のいずれか一項に記載の非ヒト動物を作製する方法であって、
（ａ）非ヒト動物宿主胚に、ヒト化内因性Ｆ１２遺伝子座をゲノムに含む遺伝子改変された非ヒト動物胚性幹（ＥＳ）細胞を導入することであって、前記内因性Ｆ１２遺伝子座のセグメントが削除され、対応するヒトＦ１２配列で置き換えられている、導入することと、
（ｂ）前記非ヒト動物宿主胚を代理母において妊娠させることであって、前記代理母が、前記ヒト化内因性Ｆ１２遺伝子座を含むＦ０子孫遺伝子改変非ヒト動物を産生する、妊娠させることと、を含む、方法。
(項目５２)
項目１～２３のいずれか一項に記載の非ヒト動物を作製する方法であって、
（ａ）非ヒト動物胚性幹（ＥＳ）細胞に、前記内因性Ｆ１２遺伝子座の５’標的配列に対応する５’ホモロジーアームと前記内因性Ｆ１２遺伝子座の３’標的配列に対応する３’ホモロジーアームとが隣接する前記ヒトＦ１２配列を含む核酸インサートを含む標的化ベクターを導入することであって、
前記標的化ベクターが前記内因性Ｆ１２遺伝子座を組換えて、前記ヒトＦ１２配列を含む前記ヒト化内因性Ｆ１２遺伝子座をゲノムに含む遺伝子改変された非ヒトＥＳ細胞を産生する、導入することと、
（ｂ）前記遺伝子改変された非ヒトＥＳ細胞を非ヒト動物宿主胚に導入することと、
（ｃ）前記非ヒト動物宿主胚を代理母において妊娠させることであって、前記代理母が、前記ヒトＦ１２配列を含む前記ヒト化内因性Ｆ１２遺伝子座をゲノムに含むＦ０子孫遺伝子改変非ヒト動物を産生する、妊娠させることと、を含む、方法。
(項目５３)
前記標的化ベクターが、少なくとも１０ｋｂの長さの大きな標的化ベクターであるか、または前記５’ホモロジーアームおよび前記３’ホモロジーアームの合計が少なくとも１０ｋｂの長さである、項目５２に記載の方法。
(項目５４)
項目１～２３のいずれか一項に記載の非ヒト動物を作製する方法であって、
（ａ）非ヒト動物１細胞期胚に、前記内因性Ｆ１２遺伝子座の５’標的配列に対応する５’ホモロジーアームと前記内因性Ｆ１２遺伝子座の３’標的配列に対応する３’ホモロジーアームとが隣接する前記ヒトＦ１２配列を含む核酸インサートを含む標的化ベクターを導入することであって、
前記標的化ベクターが前記内因性Ｆ１２遺伝子座を組換えて、前記ヒトＦ１２配列を含む前記ヒト化内因性Ｆ１２遺伝子座をゲノムに含む遺伝子改変された非ヒト動物１細胞期胚を産生する、導入することと、
（ｂ）前記遺伝子改変された非ヒト動物１細胞期胚を代理母において妊娠させて、前記ヒトＦ１２配列を含む前記ヒト化内因性Ｆ１２遺伝子座をゲノムに含む遺伝子改変されたＦ０世代非ヒト動物を産生することと、を含む、方法。
(項目５５)
ステップ（ａ）が、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ことをさらに含み、前記ヌクレアーゼ剤が、前記内因性Ｆ１２遺伝子座の標的配列を標的化する、項目５２～５４のいずれか一項に記載の方法。
(項目５６)
前記ヌクレアーゼ剤が、Ｃａｓタンパク質およびガイドＲＮＡを含む、項目５５に記載の方法。
(項目５７)
前記Ｃａｓタンパク質が、Ｃａｓ９タンパク質である、項目５６に記載の方法。
(項目５８)
ステップ（ａ）が、前記内因性Ｆ１２遺伝子座内の第２の標的配列を標的化する第２のガイドＲＮＡを導入することをさらに含む、項目５６または５７に記載の方法。
(項目５９)
前記非ヒト動物がマウスまたはラットである、項目５１～５８のいずれか一項に記載の方法。
(項目６０)
前記非ヒト動物がマウスである、項目５９に記載の方法。
(項目６１)
生体サンプルにおける短いＦ１２ ｍＲＮＡアイソフォームの発現を評価する方法であって、
（ａ）Ｆ１２遺伝子のエクソン９～１４のうちの１つ以上に対するプライマーおよび／またはプローブを使用してＲＮＡ転写産物を検出および定量することと、
（ｂ）前記Ｆ１２遺伝子のエクソン１～６のうちの１つ以上に対するプライマーおよび／またはプローブを使用してＲＮＡ転写産物を検出および定量することと、を含み、
前記短いアイソフォームが、エクソン９～１４に対するプライマーおよび／またはプローブによって検出されるが、エクソン１～６に対するプライマーおよび／またはプローブによっては検出されない、方法。
RNAseq. The protocol for RNA extraction and sequencing library preparation was similar to that previously described (Atanasio et al. (2016) Sci. Rep. 6:23204, incorporated by reference in its entirety for all purposes). incorporated herein). A strand-specific RNA-seq library was prepared from 500 ng RNA using the KAPA strand mRNA-Seq kit (KAPA Biosystems). The library was amplified by 12 cycles of PCR. Sequencing was performed on an Illumina HiSeq® 2000 (Illumina) on multiplex single read runs (for iPS human neurons, mouse primary neurons, and mouse tissues) or paired read runs (SH-SY5Y and Neuro-2A cells). for stocks). The resulting FASTQ files were analyzed via FastQC to ensure adequate data quality. Reads were mapped to the human genome (hg19) or mouse genome (mm10) using the commercial software ArrayStudio (OmicSoft), which tolerates two mismatches. In the sample, 83%-90% of reads were uniquely mapped. Gene expression was derived from raw sequencing reads using the OmicSoft ArrayStudio RNASeq analysis pipeline (Qiagen Inc.). For each gene, reads that uniquely mapped to gene exons were identified and counted. The read counts obtained were summarized at the gene level as raw expression.
The present invention provides, for example, the following items.
(Item 1)
A non-human animal comprising a humanized endogenous F12 locus in its genome, wherein a segment of said endogenous F12 locus has been deleted and replaced with the corresponding human F12 sequence.
(Item 2)
2. The non-human animal of item 1, wherein said humanized endogenous F12 locus encodes a protein comprising human coagulation factor XII heavy chain.
(Item 3)
3. The non-human animal of items 1 or 2, wherein said humanized endogenous F12 locus encodes a protein comprising the human coagulation factor XII light chain.
(Item 4)
12. The non-human animal of any one of the preceding items, wherein said humanized endogenous F12 locus encodes a protein comprising the human coagulation factor XII signal peptide.
(Item 5)
Any one of the preceding items, wherein a region of said endogenous F12 locus containing both coding and non-coding sequences has been deleted and replaced with a corresponding human F12 sequence containing both coding and non-coding sequences. Non-human animals as described in .
(Item 6)
4. The non-human animal of any one of the preceding items, wherein said humanized endogenous F12 locus comprises said endogenous F12 promoter, and said human F12 sequence is operably linked to said endogenous F12 promoter.
(Item 7)
5. The non-human animal of any one of the preceding items, wherein at least one intron and at least one exon of said endogenous F12 locus have been deleted and replaced with said corresponding human F12 sequence.
(Item 8)
3. The non-human animal of any one of the preceding items, wherein the entire F12 coding sequence of said endogenous F12 locus has been deleted and replaced with said corresponding human F12 sequence.
(Item 9)
9. The non-human animal of item 8, wherein said region of said endogenous F12 locus from start codon to stop codon is deleted and replaced with said corresponding human F12 sequence.
(Item 10)
3. The non-human animal of any one of the preceding items, wherein the 3' untranslated region of endogenous F12 has not been deleted and replaced with said corresponding human F12 sequence.
(Item 11)
3. The non-human animal of any one of the preceding items, wherein the 5' untranslated region of endogenous F12 has not been deleted and replaced with said corresponding human F12 sequence.
(Item 12)
said region of said endogenous F12 locus from said start codon to said stop codon is deleted and replaced with said corresponding human F12 sequence;
3. The non-human animal of any one of the preceding items, wherein the endogenous F12 promoter has not been deleted and replaced with said corresponding human F12 sequence.
(Item 13)
(i) said human F12 sequence of said humanized endogenous F12 locus is at least 90%, 95%, 96%, 97%, 98%, 99% relative to the sequence set forth in SEQ ID NO: 31 or 32; contain 100% identical sequences and/or
(ii) said humanized endogenous F12 locus is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 5 or 46; and/or encodes a protein comprising
(iii) said humanized endogenous F12 locus is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 13 or 48; and/or
(iv) said humanized endogenous F12 locus is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 17 or 18; A non-human animal according to any one of the preceding items, comprising
(Item 14)
of the preceding item, wherein said humanized endogenous F12 locus comprises a selection cassette or reporter gene within an intron of said corresponding human F12 sequence, or said humanized endogenous F12 locus does not comprise a selection cassette or reporter. A non-human animal according to any one of claims.
(Item 15)
3. The non-human animal of any one of the preceding items, wherein said non-human animal is homozygous for said humanized endogenous F12 locus.
(Item 16)
3. The non-human animal of any one of the preceding items, wherein said non-human animal comprises said humanized endogenous F12 locus in its germline.
(Item 17)
A non-human animal according to any one of the preceding items, wherein said non-human animal is a mammal.
(Item 18)
18. The non-human animal of item 17, wherein said non-human animal is a rat or mouse.
(Item 19)
19. The non-human animal of item 18, wherein said non-human animal is a mouse.
(Item 20)
A non-human animal according to any one of the preceding items, wherein said non-human animal expresses the short human clotting factor XII isoform in the brain.
(Item 21)
(i) said short human clotting factor XII isoform is encoded by an F12 mRNA that is detected by a probe to exons 11-14 of human F12 but not by a probe to exons 1-6 of human F12; and/or
(ii) said short human coagulation factor XII isoform is 297-596 of FXII , and/or
(iii) said short human clotting factor XII isoform is expressed in neurons in said brain; and/or
(iv) The non-human animal of item 20, wherein said short human clotting factor XII isoform can be activated by plasma kallikrein to convert pro-HGF to active HGF.
(Item 22)
said non-human animal has an aPTT that is not significantly different from the activated partial thromboplastin time (aPTT) of the corresponding wild-type non-human animal; A non-human animal according to any one of the preceding items, having a PT not significantly different from the prothrombin time (PT).
(Item 23)
The non-human animal of any one of the preceding items, wherein said non-human has a human clotting factor XII plasma level of at least about 5 μg/mL.
(Item 24)
A non-human animal cell comprising a humanized endogenous F12 locus in its genome, wherein a segment of said endogenous F12 locus has been deleted and replaced with the corresponding human F12 sequence.
(Item 25)
A non-human animal genome comprising a humanized endogenous F12 locus, wherein a segment of said endogenous F12 locus has been deleted and replaced with the corresponding human F12 sequence.
(Item 26)
A targeting vector for generating a humanized endogenous F12 locus, wherein a segment of said endogenous F12 locus is deleted and replaced with the corresponding human F12 sequence, said targeting vector generating said endogenous F12 locus an insert nucleic acid comprising the corresponding human F12 sequence flanked by 5' homology arms targeting the 5' target sequence of the locus and 3' homology arms targeting the 3' target sequence of the endogenous F12 locus; A targeting vector, including.
(Item 27)
A humanized non-human animal F12 gene, wherein a segment of said endogenous F12 locus has been deleted and replaced with the corresponding human F12 sequence.
(Item 28)
A method for evaluating the activity of a human coagulation factor XII targeting reagent in vivo, comprising:
(a) administering the human coagulation factor XII targeting reagent to the non-human animal of any one of items 1-23;
(b) evaluating said activity of said human coagulation factor XII targeting reagent in said non-human animal.
(Item 29)
29. The method of item 28, wherein said administering comprises adeno-associated virus (AAV)-mediated delivery, lipid nanoparticle (LNP)-mediated delivery, hydrodynamic delivery (HDD), or injection.
(Item 30)
30. The method of item 28 or 29, wherein step (b) comprises isolating plasma from said non-human animal and assessing the activity of said human coagulation factor XII targeting reagent in said plasma. .
(Item 31)
31. The method of any one of items 28-30, wherein step (b) comprises assessing said activity of said human coagulation factor XII targeting reagent in the liver or brain of said non-human animal.
(Item 32)
32. The method of any one of items 28-31, wherein step (b) comprises assessing coagulation or thrombin generation.
(Item 33)
33. The method of any one of items 28-32, wherein step (b) comprises assessing HGF-Met signaling.
(Item 34)
34. The method of any one of items 28-33, wherein step (b) comprises measuring expression of F12 messenger RNA encoded by said humanized endogenous F12 locus.
(Item 35)
35. The method of any one of items 28-34, wherein step (b) comprises measuring the expression of the coagulation factor XII protein encoded by said humanized endogenous coagulation factor XII locus.
(Item 36)
36. The method of any one of items 28-35, wherein said human coagulation factor XII targeting reagent is a genome editing agent and step (b) comprises assessing alteration of said humanized endogenous F12 locus. .
(Item 37)
37. The method of item 36, wherein step (b) comprises measuring the frequency of insertions or deletions within said humanized endogenous F12 locus.
(Item 38)
38. The method of any one of items 28-37, wherein said human coagulation factor XII targeting reagent comprises a nuclease agent designed to target a region of the human F12 gene.
(Item 39)
39. The method of item 38, wherein said nuclease agent comprises a Cas protein and a guide RNA designed to target a guide RNA target sequence of said human F12 gene.
(Item 40)
40. The method of item 39, wherein the Cas protein is a Cas9 protein.
(Item 41)
said human coagulation factor XII targeting reagent comprises an exogenous donor nucleic acid, said exogenous donor nucleic acid is designed to target said human F12 gene; 41. The method of any one of items 28-40, wherein the method is delivered by
(Item 42)
36. The method of any one of items 28-35, wherein said human coagulation factor XII targeting reagent is an RNAi agent or an antisense oligonucleotide.
(Item 43)
36. The method of any one of items 28-35, wherein said human coagulation factor XII targeting reagent is an antigen binding protein.
(Item 44)
36. The method of any one of items 28-35, wherein said human coagulation factor XII targeting reagent is a small molecule.
(Item 45)
A method of optimizing the activity of a human coagulation factor XII targeting reagent in vivo, comprising:
(I) performing the method of any one of items 28-44 for a first time in a first non-human animal comprising a humanized endogenous F12 locus in its genome;
(II) altering the variables and performing the method of step (I) for a second time using said altered variables in a second non-human animal whose genome contains a humanized endogenous F12 locus;
(III) said method of comparing said activity of said human coagulation factor XII targeting reagent of step (I) with said activity of said human coagulation factor XII targeting reagent of step (II) resulting in a higher activity; A method comprising: selecting a.
(Item 46)
46. The method of item 45, wherein the modified variable of step (II) is a delivery method that introduces the human coagulation factor XII targeting reagent to the non-human animal.
(Item 47)
46. The method of item 45, wherein the altered variable of step (II) is the route of administration by which the human coagulation factor XII targeting reagent is introduced to the non-human animal.
(Item 48)
46. The method of item 45, wherein said modified variable of step (II) is the concentration or amount of said human coagulation factor XII targeting reagent introduced into said non-human animal.
(Item 49)
46. The method of item 45, wherein said altered variable of step (II) is in the form of said human coagulation factor XII targeting reagent introduced into said non-human animal.
(Item 50)
46. The method of item 45, wherein said altered variable of step (II) is said human coagulation factor XII targeting reagent introduced into said non-human animal.
(Item 51)
A method for producing a non-human animal according to any one of items 1 to 23,
(a) introducing into a non-human animal host embryo a genetically modified non-human animal embryonic stem (ES) cell containing a humanized endogenous F12 locus in its genome, wherein said endogenous F12 locus introducing, wherein the segment has been deleted and replaced with the corresponding human F12 sequence;
(b) impregnating said non-human animal host embryo in a surrogate mother, said surrogate mother producing F0 offspring genetically modified non-human animals comprising said humanized endogenous F12 locus; , including, methods.
(Item 52)
A method for producing a non-human animal according to any one of items 1 to 23,
(a) in a non-human animal embryonic stem (ES) cell, a 5' homology arm corresponding to the 5' target sequence of said endogenous F12 locus and a 3' corresponding to the 3' target sequence of said endogenous F12 locus; introducing a targeting vector comprising a nucleic acid insert comprising said human F12 sequence flanked by homology arms,
introducing said targeting vector recombines said endogenous F12 locus to produce a genetically modified non-human ES cell whose genome contains said humanized endogenous F12 locus comprising said human F12 sequence; ,
(b) introducing the genetically modified non-human ES cells into a non-human animal host embryo;
(c) impregnating said non-human animal host embryo in a surrogate mother, wherein said surrogate mother comprises an F0 progeny genetically modified non-human animal whose genome contains said humanized endogenous F12 locus comprising said human F12 sequence; A method comprising producing a , impregnating .
(Item 53)
53. The method of item 52, wherein said targeting vector is a large targeting vector of at least 10 kb in length, or the sum of said 5' homology arm and said 3' homology arm is at least 10 kb in length.
(Item 54)
A method for producing a non-human animal according to any one of items 1 to 23,
(a) a 5' homology arm corresponding to the 5' target sequence of the endogenous F12 locus and a 3' homology arm corresponding to the 3' target sequence of the endogenous F12 locus in a non-human animal 1-cell stage embryo; introducing a targeting vector comprising a nucleic acid insert comprising said human F12 sequence flanked by
said targeting vector recombines said endogenous F12 locus to produce a genetically modified non-human animal 1-cell stage embryo whose genome contains said humanized endogenous F12 locus comprising said human F12 sequence; and
(b) a genetically modified F0 generation non-human animal comprising in its genome said humanized endogenous F12 locus comprising said human F12 sequence by impregnating said genetically modified non-human animal 1-cell stage embryo in a surrogate mother; and producing a.
(Item 55)
Any of items 52-54, wherein step (a) further comprises introducing a nuclease agent or a nucleic acid encoding said nuclease agent, said nuclease agent targeting a target sequence of said endogenous F12 locus. The method according to item 1.
(Item 56)
56. The method of item 55, wherein the nuclease agent comprises Cas protein and guide RNA.
(Item 57)
57. The method of item 56, wherein the Cas protein is a Cas9 protein.
(Item 58)
58. The method of item 56 or 57, wherein step (a) further comprises introducing a second guide RNA targeting a second target sequence within said endogenous F12 locus.
(Item 59)
59. The method of any one of items 51-58, wherein said non-human animal is a mouse or rat.
(Item 60)
60. The method of item 59, wherein said non-human animal is a mouse.
(Item 61)
A method of assessing the expression of short F12 mRNA isoforms in a biological sample, comprising:
(a) detecting and quantifying RNA transcripts using primers and/or probes to one or more of exons 9-14 of the F12 gene;
(b) detecting and quantifying RNA transcripts using primers and/or probes to one or more of exons 1-6 of said F12 gene;
A method, wherein said short isoform is detected by primers and/or probes to exons 9-14, but not by primers and/or probes to exons 1-6.
Claims (21)
コード配列および非コード配列の両方を含む前記内因性Ｆ１２遺伝子座の領域が削除され、コード配列および非コード配列の両方を含む対応するヒトＦ１２配列で置き換えられており、 a region of the endogenous F12 locus containing both coding and non-coding sequences is deleted and replaced with a corresponding human F12 sequence containing both coding and non-coding sequences;
開始コドンから終止コドンまでの前記内因性Ｆ１２遺伝子座の前記領域が削除され、前記対応するヒトＦ１２配列で置き換えられており、 the region of the endogenous F12 locus from the start codon to the stop codon is deleted and replaced with the corresponding human F12 sequence;
前記げっ歯類が、脳において短いヒト凝固因子ＸＩＩアイソフォームを発現し、 the rodent expresses a short human coagulation factor XII isoform in the brain;
（ｉ）前記短いヒト凝固因子ＸＩＩアイソフォームが、ヒトＦ１２のエクソン１１～１４に対するプローブによって検出されるが、ヒトＦ１２のエクソン１～６に対するプローブによっては検出されないＦ１２ ｍＲＮＡによってコードされる、および／または (i) said short human coagulation factor or
（ｉｉ）前記短いヒト凝固因子ＸＩＩアイソフォームが、ＦＸＩＩ (ii) said short human coagulation factor XII isoform is FXII ２９７－５９６297-596 である、is,
げっ歯類。rodents.
（ｉｉ）前記内因性Ｆ１２の５’非翻訳領域は削除されておらず、前記対応するヒトＦ１２配列で置き換えられていない、 (ii) the 5' untranslated region of said endogenous F12 has not been deleted and replaced with said corresponding human F12 sequence;
請求項１～２のいずれかに記載のげっ歯類。The rodent according to any one of claims 1 to 2.
（ＩＩ）前記ヒト化内因性Ｆ１２遺伝子座が、配列番号５もしくは４６に記載の配列に対して少なくとも９０％、９５％、９６％、９７％、９８％、９９％もしくは１００％同一である配列を含むタンパク質をコードする、および／または (II) a sequence in which the humanized endogenous F12 locus is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 5 or 46; encodes a protein comprising and/or
（ＩＩＩ）前記ヒト化内因性Ｆ１２遺伝子座が、配列番号１３もしくは４８に記載の配列に対して少なくとも９０％、９５％、９６％、９７％、９８％、９９％もしくは１００％同一である配列を含むコード配列を含む、および／または (III) a sequence in which the humanized endogenous F12 locus is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 13 or 48; and/or
（ＩＶ）前記ヒト化内因性Ｆ１２遺伝子座が、配列番号１７もしくは１８に記載の配列に対して少なくとも９０％、９５％、９６％、９７％、９８％、９９％もしくは１００％同一である配列を含む、 (IV) a sequence in which the humanized endogenous F12 locus is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 17 or 18; including,
請求項１～３のいずれかに記載のげっ歯類。The rodent according to any one of claims 1 to 3.
（ａ）請求項１～１１のいずれか一項に記載のげっ歯類に前記ヒト凝固因子ＸＩＩ標的化試薬を投与するステップと、(a) administering the human coagulation factor XII targeting reagent to a rodent according to any one of claims 1 to 11;
（ｂ）前記げっ歯類における前記ヒト凝固因子ＸＩＩ標的化試薬の前記活性を評価するステップと、(b) assessing the activity of the human coagulation factor XII targeting reagent in the rodent;
を含む、方法。including methods.
（ＩＩ）ステップ（ｂ）は、前記げっ歯類の肝臓もしくは脳における前記ヒト凝固因子ＸＩＩ標的化試薬の前記活性を評価することを含む、および／または (II) step (b) comprises assessing the activity of the human coagulation factor XII targeting reagent in the liver or brain of the rodent; and/or
（ＩＩＩ）ステップ（ｂ）は、凝固もしくはトロンビン生成を評価することを含む、および／または (III) step (b) comprises assessing coagulation or thrombin generation; and/or
（ＩＶ）ステップ（ｂ）は、ＨＧＦ－Ｍｅｔシグナル伝達を評価することを含む、および／または (IV) step (b) comprises assessing HGF-Met signaling, and/or
（Ｖ）ステップ（ｂ）は、前記ヒト化内因性Ｆ１２遺伝子座によってコードされるＦ１２メッセンジャーＲＮＡの発現を測定することを含む、および／または (V) step (b) comprises measuring the expression of F12 messenger RNA encoded by said humanized endogenous F12 locus, and/or
（ＶＩ）ステップ（ｂ）は、前記ヒト化内因性凝固因子ＸＩＩ遺伝子座によってコードされる凝固因子ＸＩＩタンパク質の発現を測定することを含む、および／または (VI) step (b) comprises measuring the expression of a coagulation factor XII protein encoded by said humanized endogenous coagulation factor XII locus, and/or
（ＶＩＩ）前記ヒト凝固因子ＸＩＩ標的化試薬はゲノム編集剤であり、ステップ（ｂ）は前記ヒト化内因性Ｆ１２遺伝子座の改変を評価することを含み、任意選択で、ステップ（ｂ）は、前記ヒト化内因性Ｆ１２遺伝子座内の挿入もしくは欠失の頻度を測定することを含む、 (VII) said human coagulation factor determining the frequency of insertions or deletions within the humanized endogenous F12 locus.
請求項１２または１３に記載の方法。The method according to claim 12 or 13.
（ＩＩ）前記ヒト凝固因子ＸＩＩ標的化試薬は外因性ドナー核酸を含み、前記外因性ドナー核酸は、前記ヒトＦ１２遺伝子を標的化するように設計され、任意選択で、外因性ドナー核酸は、ＡＡＶを介して送達されるか、 (II) said human coagulation factor XII targeting reagent comprises an exogenous donor nucleic acid, said exogenous donor nucleic acid is designed to target said human F12 gene; delivered via or
（ＩＩＩ）前記ヒト凝固因子ＸＩＩ標的化試薬は、ＲＮＡｉ剤もしくはアンチセンスオリゴヌクレオチドであるか、 (III) the human coagulation factor XII targeting reagent is an RNAi agent or an antisense oligonucleotide;
（ＩＶ）前記ヒト凝固因子ＸＩＩ標的化試薬は抗原結合タンパク質であるか、または (IV) said human coagulation factor XII targeting reagent is an antigen binding protein; or
（Ｖ）前記ヒト凝固因子ＸＩＩ標的化試薬は小分子である、 (V) said human coagulation factor XII targeting reagent is a small molecule;
請求項１２～１４のいずれか一項に記載の方法。A method according to any one of claims 12 to 14.
（Ｉ）ヒト化内因性Ｆ１２遺伝子座をそのゲノム中に含む第１のげっ歯類において初めて請求項１２～１５のいずれか一項に記載の方法を実施するステップと、(I) carrying out the method of any one of claims 12 to 15 for the first time in a first rodent comprising a humanized endogenous F12 locus in its genome;
（ＩＩ）変数を変更し、そのゲノム中にヒト化内因性Ｆ１２遺伝子座を含む第２のげっ歯類において、前記変更された変数を用いてステップ（Ｉ）の方法を２回目に実施するステップと、(II) modifying the variable and performing the method of step (I) a second time using said modified variable in a second rodent that includes a humanized endogenous F12 locus in its genome; and,
（ＩＩＩ）ステップ（Ｉ）の前記ヒト凝固因子ＸＩＩ標的化試薬の活性をステップ（ＩＩ）の前記ヒト凝固因子ＸＩＩ標的化試薬の活性と比較し、結果としてより高い活性が得られる方法を選択するステップと、(III) Comparing the activity of the human coagulation factor XII targeting reagent of step (I) with the activity of the human coagulation factor XII targeting reagent of step (II) and selecting a method that results in higher activity. step and
を含む、方法。including methods.
（ＩＩ）ステップ（ＩＩ）で前記変更された変数は、前記ヒト凝固因子ＸＩＩ標的化試薬を前記げっ歯類に導入する投与経路であるか、 (II) the variable modified in step (II) is the route of administration by which the human coagulation factor XII targeting reagent is introduced into the rodent;
（ＩＩＩ）ステップ（ＩＩ）で前記変更された変数は、前記げっ歯類に導入された前記ヒト凝固因子ＸＩＩ標的化試薬の濃度もしくは量であるか、 (III) the altered variable in step (II) is the concentration or amount of the human coagulation factor XII targeting reagent introduced into the rodent;
（ＩＶ）ステップ（ＩＩ）で前記変更された変数は、前記げっ歯類に導入された前記ヒト凝固因子ＸＩＩ標的化試薬の形態であるか、または (IV) said modified variable in step (II) is in the form of said human coagulation factor XII targeting reagent introduced into said rodent; or
（Ｖ）ステップ（ＩＩ）で前記変更された変数は、前記げっ歯類に導入された前記ヒト凝固因子ＸＩＩ標的化試薬である、 (V) the modified variable in step (II) is the human coagulation factor XII targeting reagent introduced into the rodent;
請求項１６に記載の方法。17. The method according to claim 16.
（ｂ）前記げっ歯類宿主胚を代理げっ歯類母において妊娠させるステップであって、前記代理げっ歯類母は、前記ヒト化内因性Ｆ１２遺伝子座を含むＦ０子孫遺伝子改変げっ歯類を産生し、前記Ｆ０子孫遺伝子改変げっ歯類が脳において短いヒト凝固因子ＸＩＩアイソフォームを発現する、妊娠させるステップと、 (b) impregnating said rodent host embryo in a surrogate rodent mother, said surrogate rodent mother producing F0 progeny genetically modified rodents comprising said humanized endogenous F12 locus; and causing the F0 offspring genetically modified rodent to express a short human coagulation factor XII isoform in the brain;
を含む、請求項１～１１のいずれか一項に記載のげっ歯類を作製する方法。A method for producing a rodent according to any one of claims 1 to 11, comprising:
前記標的化ベクターが前記内因性Ｆ１２遺伝子座を組み替換えて、そのゲノム中に前記ヒト化内因性Ｆ１２遺伝子座を含む遺伝子改変されたげっ歯類ＥＳ細胞を産生する、導入するステップと、 introducing the targeting vector to recombine the endogenous F12 locus to produce genetically modified rodent ES cells containing the humanized endogenous F12 locus in their genome;
（ｂ）前記遺伝子改変されたげっ歯類ＥＳ細胞をげっ歯類宿主胚に導入するステップと、 (b) introducing the genetically modified rodent ES cells into a rodent host embryo;
（ｃ）前記げっ歯類宿主胚を代理げっ歯類母において妊娠させるステップであって、前記代理げっ歯類母は、前記ヒト化内因性Ｆ１２遺伝子座をそのゲノム中に含むＦ０子孫遺伝子改変げっ歯類動物を産生し、前記Ｆ０子孫遺伝子改変げっ歯類が脳において短いヒト凝固因子ＸＩＩアイソフォームを発現する、妊娠させるステップと、 (c) impregnating said rodent host embryo in a surrogate rodent mother, said surrogate rodent mother carrying an F0 progeny genetically modified rodent containing said humanized endogenous F12 locus in its genome. producing and impregnating a dental animal, wherein said F0 progeny genetically modified rodent expresses a short human coagulation factor XII isoform in the brain;
を含む、請求項１～１１のいずれか一項に記載のげっ歯類を作製する方法。A method for producing a rodent according to any one of claims 1 to 11, comprising:
前記標的化ベクターが前記内因性Ｆ１２遺伝子座を組み替換えて、そのゲノム中に前記ヒト化内因性Ｆ１２遺伝子座を含む遺伝子改変されたげっ歯類１細胞期胚を産生する、導入するステップと、 introducing the targeting vector to recombine the endogenous F12 locus to produce a genetically modified rodent one-cell stage embryo containing the humanized endogenous F12 locus in its genome;
（ｂ）前記遺伝子改変されたげっ歯類１細胞期胚を代理げっ歯類母において妊娠させて、前記ヒトＦ１２配列を含む前記ヒト化内因性Ｆ１２遺伝子座をそのゲノム中に含む遺伝子改変されたＦ０世代げっ歯類を産生するステップであって、前記遺伝子改変されたＦ０世代げっ歯類が脳において短いヒト凝固因子ＸＩＩアイソフォームを発現する、産生するステップと、 (b) gestating said genetically modified rodent one-cell stage embryo in a surrogate rodent mother to produce a genetically modified rodent embryo containing in its genome said humanized endogenous F12 locus comprising said human F12 sequence; producing an F0 generation rodent, the genetically modified F0 generation rodent expressing a short human coagulation factor XII isoform in the brain;
を含む、請求項１～１１のいずれか一項に記載のげっ歯類を作製する方法。A method for producing a rodent according to any one of claims 1 to 11, comprising:
（ａ）Ｆ１２遺伝子のエクソン９～１４のうちの１つ以上に対するプライマーおよび／またはプローブを使用してＲＮＡ転写産物を検出および定量するステップと、(a) detecting and quantifying RNA transcripts using primers and/or probes for one or more of exons 9-14 of the F12 gene;
（ｂ）前記Ｆ１２遺伝子のエクソン１～６のうちの１つ以上に対するプライマーおよび／またはプローブを使用してＲＮＡ転写産物を検出および定量することと、(b) detecting and quantifying RNA transcripts using primers and/or probes for one or more of exons 1 to 6 of the F12 gene;
を含み、前記短いアイソフォームが、エクソン９～１４に対するプライマーおよび／またはプローブによって検出されるが、エクソン１～６に対するプライマーおよび／またはプローブによっては検出されない、wherein the short isoform is detected by primers and/or probes to exons 9-14, but not by primers and/or probes to exons 1-6,
方法。Method.
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| JP7169979B2 (en) | 2017-02-27 | 2022-11-11 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Method for evaluating therapeutic efficacy of candidate therapeutic agents for complement-related nephropathy and liver fibrosis |
| CN111163633B (en) | 2017-09-29 | 2022-09-09 | 瑞泽恩制药公司 | Non-human animals comprising humanized TTR loci and methods of use thereof |
| KR20210004994A (en) | 2018-03-26 | 2021-01-13 | 리제너론 파마슈티칼스 인코포레이티드 | Humanized rodents to test therapeutic agents |
| KR20210031868A (en) | 2018-07-16 | 2021-03-23 | 리제너론 파마슈티칼스 인코포레이티드 | Non-human animal model of DITRA disease and uses thereof |
| WO2020206139A1 (en) | 2019-04-04 | 2020-10-08 | Regeneron Pharmaceuticals, Inc. | Non-human animals comprising a humanized coagulation factor 12 locus |
| JP2022534867A (en) | 2019-06-04 | 2022-08-04 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Non-human animals containing humanized TTR loci with beta slip mutations and methods of use |
| BR112021022722A2 (en) | 2019-06-07 | 2022-01-04 | Regeneron Pharma | Non-human animal, non-human animal cell, non-human animal genome, humanized non-human animal albumin gene, targeting vector, method of evaluating the activity of a reagent, and, method of optimizing the activity of a reagent |
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2020
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- 2020-04-02 US US16/838,519 patent/US11737435B2/en active Active
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- 2020-04-02 KR KR1020217035584A patent/KR102661779B1/en active IP Right Grant
- 2020-04-02 AU AU2020253532A patent/AU2020253532A1/en active Pending
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