JP2015523098A - 対立遺伝子置換によるfmdv抵抗性家畜の産生 - Google Patents
対立遺伝子置換によるfmdv抵抗性家畜の産生 Download PDFInfo
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Abstract
Description
本特許出願は、2013年3月15日に出願された米国特許出願第13/836,860号明細書及び2012年7月31日に出願された米国仮特許出願第61/677,904号明細書に対する優先権を主張するものであり、これらの仮特許出願は双方とも、本明細書における参照により本明細書によって援用される。
FMDVは、ポリオウイルス、ライノウイルス(感冒)、及びA型肝炎を含む動物ウイルスのなかで最も小さいピコルナウイルス科(picornaviridae)のアフトウイルス属(Aphthovirus)に属する。複数のサブタイプを含む7種の血清型がある[2]。他のピコルナウイルスと同様に、FMDVゲノムは、直接翻訳され得る(プラス鎖ゲノム)約8,500ヌクレオチドの一本鎖RNAであり、100KDaを上回る単一のポリタンパク質をコードする。FMDV RNAのN末端領域には、2つのアイソフォームLab及びLb(このうちLbが重要な産物である)を有するLproと称されるパパイン様プロテアーゼがコードされる[3]。Lproはいずれも非常に小さく、且つ非常に特異的である。Lpro配列は初めにFMDVポリタンパク質をその合成中に切断し、自らをポリタンパク質から遊離させる。次に遊離Lproは、残りのポリタンパク質をさらに切断して、莫大な数の子孫ウイルスを産生する個々の機能性ポリペプチドにする。Lproは他にいくつかの標的部位を有し、そのなかで最も重要な標的部位は、真核生物翻訳開始因子4G(eIF4G)であるものと思われ[4、5]、これはLproによって切断されると、哺乳類細胞の4mGキャップmRNAの開始を促進することができない。2つのアイソフォームeIF4GI及びeIF4GIIがあるeIF4Gは、キャッピングされ且つポリアデニル化されたあらゆるmRNAの5’末端及び3’末端の構造に結合するいくつかのeIF4 RNA結合タンパク質を一つにまとめる足場タンパク質である。ある種のピコルナウイルス、例えばポリオウイルスでは、2AproプロテアーゼがLproと同等の活性を有する。
染色体修飾が単一対立遺伝子又は二対立遺伝子である動物が、マーカーを所定の位置に残し、それを動物から出して育てることを可能にする方法を用いるか、或いは動物にマーカーを置かない方法により作製され得る。例えば、本発明者らは、相同性依存型組換え(homologous dependent recombination:HDR)の方法を使用して、動物の染色体に対する変化、又はそこへの外来性遺伝子の挿入を作製している。部位特異的(siste−specific)ヌクレアーゼ、例えば、TALEN、ジンクフィンガーヌクレアーゼ(ZFN)、又はCRISPR、及びリコンビナーゼ融合タンパク質などのツール、並びに従来の方法が利用可能である。
TALENは、遺伝子操作ツールである。遺伝子の不活性化は、TALENの多くの用途のうちの一つである。用語TALENは、本明細書で使用されるとき、広義であり、別のTALENの助けなしに二本鎖DNAを切断することのできる単量体TALENを含む。用語TALENはまた、共同して同じ部位のDNAを切断する働きをするように操作されるTALENの対の一方又は両方のメンバーを指しても使用される。共同して働くTALENは、DNAの利き手を参照して、左TALEN(left−TALEN)及び右TALEN(right−TALEN)と称され得る。
ジンクフィンガーヌクレアーゼ(ZFN)は、ジンクフィンガーDNA結合ドメインをDNA切断ドメインと融合することにより生成される人工的な制限酵素である。ジンクフィンガードメインは、所望のDNA配列を標的化するように操作することができ、これによりジンクフィンガーヌクレアーゼが複合的なゲノム内にあるユニーク配列を標的化することが可能になる。内在性DNA修復機構を利用することにより、これらの試薬を使用して高等生物のゲノムを改変することができる。ZFNは、遺伝子を不活性化させる方法において用いられ得る。
クラスターを形成する規則的に間隔が置かれた短いパリンドロームリピート(CRISPR)は、細菌/古細菌適応免疫防御から誘導される。CRISPR活性には、CRISPR遺伝子座への侵入ウイルス又はプラスミドDNAからの「スペーサー」の組込み、スペーサー−リピート単位からなる低分子ガイドCRISPR RNA(crRNA)の発現及びプロセシング、並びにスペーサーと相補的な核酸の切断が関わる。ヌクレアーゼCas9は、crRNAと一致する配列を探して切断する。正しいプロトスペーサー隣接モチーフ(PAM)が3’末端にも存在する場合にのみ、Cas9はDNAを切断する。ゲノム操作ツールとして、gRNA指向性Cas9切断の特異性は極めて有用である。
ノックアウトを目的として、遺伝子の不活性化のため、遺伝子の発現を得るため、又は他の目的で、様々な核酸が偶蹄類細胞又は他の細胞に導入され得る。本明細書で使用されるとき、用語の核酸には、DNA、RNA、及び核酸類似体、及び二本鎖又は一本鎖(即ち、センス又はアンチセンス一本鎖)である核酸が含まれる。核酸類似体を塩基部分、糖部分、又はリン酸骨格で修飾することにより、例えば、核酸の安定性、ハイブリダイゼーション、又は溶解度が向上し得る。塩基部分の修飾には、デオキシチミジンに対するデオキシウリジン、並びにデオキシシチジンに対する5−メチル−2’−デオキシシチジン及び5−ブロモ−2’−ドキシシチジン(doxycytidine)が含まれる。糖部分の修飾には、2’−O−メチル糖又は2’−O−アリル糖を形成するリボース糖の2’ヒドロキシルの修飾が含まれる。デオキシリボースリン酸骨格は、各塩基部分が6員モルホリノ環に連結されているモルホリノ核酸か、又はデオキシリン酸骨格が擬ペプチド骨格によって置換されており、且つ4塩基が維持されているペプチド核酸が生じるように修飾することができる。Summerton and Weller(1997)Antisense Nucleic Acid Drug Dev.7(3):187;及びHyrupら(1996)Bioorgan.Med.Chem.4:5を参照のこと。加えて、デオキシリン酸骨格は、例えば、ホスホロチオエート又はホスホロジチオエート骨格、ホスホラミダイト(phosphoroamidite)、又はアルキルリン酸トリエステル骨格で置換することができる。
動物は、TALEN、ジンクフィンガー、CRISPR/Cas9、又はリコンビナーゼ融合タンパク質、若しくは公知の様々なベクターを含む他の遺伝子操作ツールを使用して修飾され得る。動物を遺伝子修飾する材料及び方法は、2009年11月10日に出願された米国特許出願公開第2012/0222143号明細書、米国特許出願公開第2012/0220037号明細書及び米国特許出願公開第2010/0251395号明細書(これらは、本明細書によってあらゆる目的から参照により本明細書に援用される)にさらに詳述される;矛盾が生じた場合、本明細書が優先する。用語のトランス作用性は、異なる分子から標的遺伝子に(即ち分子間で)作用する過程を指す。トランス作用エレメントは、通常、遺伝子を含むDNA配列である。この遺伝子は、標的遺伝子の調節において使用されるタンパク質(又はマイクロRNA又は他の拡散性分子)をコードする。トランス作用遺伝子は、標的遺伝子と同じ染色体上にあってもよいが、活性は、中間タンパク質又はそれをコードするRNAを介する。ドミナントネガティブを用いた遺伝子の不活性化には、概して、トランス作用エレメントが関わる。用語のシス調節又はシス作用は、タンパク質又はRNAをコードしない作用を意味する;遺伝子不活性化との関連において、これは概して、遺伝子のコード部分、又は機能性遺伝子の発現に必要なプロモーター及び/又はオペレーターの不活性化を意味する。
創始動物は、クローニング及び本明細書に記載される他の方法により産生され得る。創始動物は、接合子又は初代細胞がホモ接合性の修飾を受ける場合のように、遺伝子修飾に関してホモ接合体であり得る。同様に、ヘテロ接合体の創始動物もまた作製され得る。創始動物はゲノム修飾されていてもよく、即ち、そのゲノムにおける細胞の全てが修飾を受けていてもよい。創始動物は、ベクターが胚における複数の細胞のうちの1つに、典型的には胚盤胞期に導入されたときに起こり得るように、修飾に関してモザイクであってもよい。モザイク動物の子孫を試験することにより、ゲノム修飾されている子孫を同定し得る。有性生殖することができる、又は生殖補助技術により生殖できる動物であって、異種又はホモ接合体の子孫が一貫して修飾を発現する動物のプールが作り出されたとき、動物系統が樹立される。
本発明の実施形態は、1つ又は複数のTALEN又はジンクフィンガーヌクレアーゼをリコンビナーゼ又はDNA組換えに関連する他のDNA結合タンパク質と共に投与することを含む。実施形態はまた、リコンビナーゼ融合タンパク質、例えば、米国特許出願公開第2011/0059160号明細書にあるとおりのRecA−gal4融合物の投与により、細胞染色体に二本鎖切断を作成することも含む。
ある場合には、ペプチドと本明細書に示される配列との同一性パーセントの決定が必要となり得る。そのような場合、同一性パーセントは、ペプチド、又はペプチドの一部分の残基の数の点で計測される。例えば90%の同一性のポリペプチドが、より大きいペプチドの一部分であることもあり得る。実施形態には、本明細書に示される配列の指示される同一性及び/又は保存的置換を有するかかるポリペプチドが含まれる。
本明細書における細胞及び胚の修飾方法は、幅広い研究ツールに適用することができる。細胞又は胚それ自体もまた、研究に有用である。例えば、伝統的な育種プログラムは、しばしば、繁殖させる動物の遺伝子成分を理解することに多く頼っている。望ましい形質を有する動物は、品種育成のため繁殖される。これらの形質は、動物の遺伝子及び遺伝子の組み合わせに大きく依存する。遺伝子を細胞又は胚に入れると、又はそれらの遺伝子を修飾すると、遺伝子がどのように相互作用して目的とする形質を生み出すかに関する価値ある情報が提供される。細胞又は胚は好適な時間にわたり培養され、次に試験され得る。胚は、任意の意味のある発生段階に達する前に破壊されるが、それにもかかわらず有用な洞察を提供する。
以下の参考文献は、全て本明細書によって参照により本明細書に援用される。本書に示される特許及び特許出願もまた、本明細書によって参照により本明細書に援用される。矛盾が生じた場合には、本明細書が優先する。1. Thompson, D., et al., Economic costs of the foot and mouth disease outbreak in the United Kingdom in 2001. Rev. Sci. Tech., 2002. 21: p. 657−687. 2. Grubman, M.J. and B. Baxt, Foot−and−mouth disease. Clin. Microbiol. Rev., 2004. 17: p. 465−493. 3. Cao, X., et al., Functional analysis of the two alternative translation initiation sites of foot−and−mouth disease virus. J. Virol., 1995. 69: p. 560−563. 4. Glaser, W. and T. Skern, Extremely efficient cleavage of eIF4G by picornaviral proteinases L and 2A in vitro. FEBS Lett., 2000. 480: p. 151−155. 5. Gradi, A., et al., Cleavage of eukaryotic translation initiation factor 4GII within foot−and−mouth disease virus−infected cells: identification of the L−protease cleavage site in vitro. J. Virol., 2004. 78: p. 3271−3278. 6. nton, T.M., et al., Functional analysis of individual binding activities of the scaffold protein eIF4G. J. biol. Chem., 2007. 282: p. 1695−1708. 7. Aitken, C.E. and J.R. Lorsch, A mechanistic overview of translation initiation in eukaryotes. Nature Rev. Struc. Mol. Biol., 2012. 19: p. 568−576. 8. Kerekatte, V., et al., Cleavage of Poly(A)−binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? J. Virol., 1999. 73: p. 709−717. 9. Foeger, N., et al., The binding of foot−and−mouth disease virus leader proteinase to eIF4GI involves conserved ionic interactions. FEBS J., 2005. 272: p. 2602−2611. 10. Prevot, D., J.L. Darlix, and T. Ohlmann, Conducting the initiation of protein synthesis: the role of eIF4G. Biol. Cell., 2003. 95: p. 141−156. 11. Lloyd, R.E., Translational control by viral proteinases. Virus Res., 2006. 119: p. 76−88. 12. Truniger, V. and M.A. Aranda, Recessive resistance to plant viruses. Adv. 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Virol., 2007. 81: p. 12803−12815. 18. Borman, A.M., et al., elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES−containing mRNAs. RNA, 1997. 3: p. 186−196. 19. Zhao, X., et al., Protection of cap−dependent protein synthesis in vivo and in vitro with an eIF4G−1 variant highly resistant to cleavage by Coxsackievirus 2A protease. J. Biol. Chem., 2003. 278: p. 4449−4457. 20. Lopez de Quinto, S. and E. Martinez−Salas, Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo. RNA, 2000, 6: p. 1380−1392. 21. Saleh, L., et al., Functional interaction of translation initiation factor eIF4G with the foot−and−mouth disease virus internal ribosome entry site. J. Gen. Virol., 2001. 82: p. 757−763. 22. Hinton, T.M., et al., Conservation of L and 3C proteinase activities across distantly related aphthoviruses. J. Gen. Virol., 2002. 83: p. 3111−3121. 23. 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図2を参照すると、ブタEIF4GIの一部がパネルAに示される。野生型配列はLpro切断部位(矢印)と比べてマイナス3位及び2位にアスパラギン及びロイシン残基を有する。この例では、HDR鋳型がマイナス3位及び2位の残基をアスパラギン酸及びフェニルアラニンに置き換えることで、修飾EIF4GIをLpro切断に対して抵抗性にする。野生型EIF4GIを切断して相同組換えを刺激するように2対のTALEN(上)を設計した。パネルB:各TALEN対をトランスフェクトしたブタ線維芽細胞のサーベイヤー(Cel−I)アッセイ。パネルC:相同組換え効率を決定するためのRFLPアッセイ。
コロニー単離については、細胞を計数し、1〜20細胞/cm2の密度範囲で10cmディッシュ上に播き得る。細胞は、直径3〜4mmの個々のコロニーが存在するまで、10〜15日間培養し得る。個々のコロニーをp−200ピペッターで穏やかな吸引下に吸引し、500μlの成長培地を含む24ウェルプレートのウェルに吐き出し得る(Carlson,Garbeら2011)。明確に画定されたコロニー(約10〜30/プレート)を含むプレートをコロニー吸引用に選択し、複数のコロニーから細胞が吸引される可能性を抑え得る。コロニーが24ウェルディッシュにおいて70〜90パーセントコンフルエントに達したところで、RFPL分析用に一部を回収し、残りを凍結保存し得る。目的の特徴の獲得に成功したことが決定されたコロニーから細胞を取り出し、それをクローニングに使用して創始動物を作製し得る。
Claims (27)
- eIF4G遺伝子に対する修飾又は外来性eIF4G遺伝子を発現する核酸を含むインビトロ細胞。
- 前記eIF4G遺伝子が、動物の野生型eIF4Gタンパク質と比べて改変されたeIF4Gタンパク質を発現し、前記eIF4Gタンパク質が、口蹄疫ウイルス酵素のプロテイナーゼによる切断に抵抗性である、請求項1に記載の細胞。
- ブタ、魚、ウサギ、雌ウシ、ニワトリ、ヤギ、及びヒツジからなる群から選択される、請求項1又は2に記載の細胞。
- 前記修飾eIF4G遺伝子によって発現されるeIF4Gタンパク質をさらに含む、請求項1〜3のいずれか一項に記載の細胞。
- 口蹄疫ウイルス酵素のプロテイナーゼによる切断に抵抗性であるeIF4Gタンパク質のアイソフォームをコードする単離核酸。
- 口蹄疫に対する抵抗性の存在に関するスクリーニング方法であって、細胞、胚、又は動物を調べて、それが修飾eIF4G遺伝子及び/又は修飾eIF4Gタンパク質を含むかどうかを決定するステップを含む方法。
- 遺伝子修飾生物の作出方法であって、
a.細胞、初代細胞、又は胚の天然eIF4G遺伝子をインビトロで改変するステップ、及び前記初代細胞をクローニングするか又は母動物に前記胚を移植するステップであって、前記eIF4G遺伝子が、口蹄疫プロテアーゼによるタンパク質分解に抵抗するeIF4Gアイソフォームを発現するように改変される、ステップ、又は
b.外来性eIF4G遺伝子の発現を初代細胞又は胚にインビトロで加えるステップ、及び前記初代細胞をクローニングするか又は母動物に前記胚を移植するステップであって、前記外来性eIF4G遺伝子が、口蹄疫プロテアーゼによるタンパク質分解に抵抗するeIF4Gアイソフォームを発現する、ステップ、又は
c.(a)及び(b)の両方
を含む方法。 - 前記初代細胞又は前記胚に:
a.前記天然eIF4G遺伝子のある部位を特異的に切断する部位特異的ヌクレアーゼをコードする核酸と、
b.前記eIF4G遺伝子の少なくとも一部分を含む核酸鋳型であって、前記鋳型が前記天然eIF4G遺伝子の代替の対立遺伝子を提供し、前記代替の対立遺伝子が、口蹄疫ウイルス酵素のプロテイナーゼによる切断に抵抗性のeIF4Gアイソフォームをコードする、核酸鋳型と
を導入するステップを含む、請求項7に記載の方法。 - 前記部位特異的ヌクレアーゼが、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)及びクラスターを形成する規則的に間隔が置かれた短いパリンドロームリピート(CRISPR)からなる群から選択される、請求項7又は8に記載の方法。
- 請求項7〜9のいずれか一項に記載の方法により作製される動物。
- eIF4G遺伝子にゲノム修飾を含む遺伝子修飾された家畜動物。
- 前記修飾が、前記eIF4G遺伝子の1つ以上の塩基の挿入、欠失、又は置換を含む、請求項11に記載の動物。
- 前記eIF4G遺伝子が、前記動物の野生型eIF4Gタンパク質と比べて、口蹄疫ウイルス酵素のプロテイナーゼによる切断に抵抗性となるように改変されたeIF4Gタンパク質を発現する、請求項11又は12に記載の動物。
- 前記eIF4G遺伝子が、前記動物の野生型eIF4Gタンパク質と比べて、口蹄疫ウイルス酵素のリーダープロテイナーゼ(Lpro)による切断に抵抗性となるように改変されたeIF4Gタンパク質を発現する、請求項11〜13のいずれか一項に記載の動物。
- 前記eIF4G遺伝子が、前記動物の野生型eIF4Gタンパク質と比べて、口蹄疫ウイルス酵素のウイルスコード3Cプロテアーゼ(3Cpro)による切断に抵抗性となるように改変されたeIF4Gタンパク質を発現する、請求項11〜14のいずれか一項に記載の動物。
- ブタ、魚、ウサギ、雌ウシ、ニワトリ、ヤギ、及びヒツジからなる群から選択される、請求項11〜15のいずれか一項に記載の動物。
- 前記動物が第1の品種の動物であり、前記ゲノム修飾が、その動物の別の品種に見出されるeIF4G遺伝子の天然の対立遺伝子である、請求項11〜16のいずれか一項に記載の動物。
- 前記動物が第1の種の動物であり、前記ゲノム修飾が、第2の種の別の品種におけるeIF4G遺伝子の対立遺伝子である、請求項11〜16のいずれか一項に記載の動物。
- 前記動物が前記修飾されたeIF4G遺伝子に関してホモ接合体である、請求項11に記載の動物。
- 創始動物である、請求項11〜19のいずれか一項に記載の動物。
- 前記修飾eIF4G遺伝子によって発現される前記eIF4Gタンパク質をさらに含む、請求項11〜20のいずれか一項に記載の動物。
- 口蹄疫に抵抗性である、請求項11〜21のいずれか一項に記載の動物。
- 前記修飾eIF4Gタンパク質が、Lpro及び/又はCproの結合を防ぐように修飾される、請求項11〜22のいずれか一項に記載の動物。
- 口蹄疫の治療又は予防に使用するための、口蹄疫ウイルス酵素のプロテイナーゼによる切断に抵抗性のeIF4Gタンパク質をコードする単離核酸化合物。
- 外来性eIF4G遺伝子を発現する核酸又は前記動物の野生型eIF4Gタンパク質と比べて改変されたeIF4Gタンパク質を発現する核酸である、請求項24に記載の核酸。
- 請求項24又は25に記載の核酸をコードするベクター。
- 口蹄疫を治療するための、又はそれを予防するための、請求項24〜26のいずれか一項に記載の核酸又はベクターの使用。
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JPWO2019216338A1 (ja) * | 2018-05-08 | 2021-07-15 | 国立大学法人大阪大学 | ホモ接合型細胞の作製方法 |
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CL2015000248A1 (es) | 2015-10-02 |
US20140041066A1 (en) | 2014-02-06 |
BR112015002280A2 (pt) | 2019-01-15 |
RU2015106818A (ru) | 2016-09-27 |
EP3540050A1 (en) | 2019-09-18 |
JP6446360B2 (ja) | 2018-12-26 |
CN104837989A (zh) | 2015-08-12 |
US20180310536A1 (en) | 2018-11-01 |
CA2880616A1 (en) | 2014-02-06 |
AU2013296901A1 (en) | 2015-02-12 |
AU2013296901B2 (en) | 2019-05-09 |
EP2880153A4 (en) | 2016-03-30 |
WO2014022120A1 (en) | 2014-02-06 |
KR20150036792A (ko) | 2015-04-07 |
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