JP7199492B2 - Factor VIII又はFactor IX遺伝子がノックアウトされたウサギ、その製造方法及びその用途 - Google Patents
Factor VIII又はFactor IX遺伝子がノックアウトされたウサギ、その製造方法及びその用途 Download PDFInfo
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Description
(a)上記の本発明に係るCRIPSR/Cas9システムを転写(transcription)してsgRNA及びCas9mRNAを製造する段階;
(b)受精卵に前記(a)段階で製造したmRNAを導入して培養する段階;及び
(c)前記(b)段階で得た受精卵を代理母に移植して分娩させる段階;を含む方法で製造されることを特徴とし得る。
(a)前記本発明に係るCRIPSR/Cas9システムを転写(transcription)してsgRNA及びCas9mRNAを製造する段階;
(b)受精卵に前記(a)段階で製造したmRNAを導入して培養する段階;及び
(c)前記(b)段階で得た受精卵を代理母に移植して分娩させる段階を含む方法は、第8因子又は第9因子がノックアウトされた形質転換ウサギの製造方法に関する。
1-1.sgRNA設計
第8因子の場合、エクソン1領域で下記の配列番号1で表される配列を用いて配列番号3及び配列番号4の塩基配列で表されるsgRNAを設計した(図1)。
配列番号1:
ATGCAAATAGAGCTCTCCACCTGTTTCTTTGTGTGTATTTTACAATTGAGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGAACTGTCCTGGGACTATATGCACAGTGAC CTGCTCAGTGA
配列番号3:sgRNA1(+Strand)
5’- GCCACCAGAAGATACTACCTGGG -3’
配列番号4:sgRNA2(-Strand)
5’- GTCACTGTGCATATAGTCCCAGG -3’
配列番号2:
TTTTTCTTGATCATGAAAATGCCACCAAAATTCTGAATCGGGCAAAGAGGTACAATTCAGGTAAACTGGAAGAGTTTGTTTCAGGGAACCTTGAGAGAGAATGTATAGAAGAAAGGTGTAGTTTTGAAGAAGCTCGAGAAGTTTTTGAAAACACTGAAAAAACT
配列番号5:sgRNA1(+Strand)
5’- ATGCCACCAAAATTCTGAATCGG -3
配列番号6:sgRNA2(+Strand)
5’ CGGGCAAAGAGGTACAATTCAGG -3’
実施例1-1で設計したsgRNA配列に合うオリゴヌクレオチドをデザインしてpUC57-T7ベクター(Addgene ID 51306)にクローニングした後、完成されたsgRNA及びその前に位置したT7プロモーターを配列番号7及び8のプライマーでPCRによって増幅した。
配列番号7:T7-F
5’- GAAATTAATACGACTCACTAT -3’
配列番号8:T7-R
5’- AAAAAAAGCACCGACTCGGTGCCAC -3’
公知の方法(Sci Rep.2016;6:222024)でウサギ受精卵に、実施例1で得たCas9/FVIII sgRNA又はCas9/FIX sgRNAをそれぞれ導入した。
3-1.第8因子ノックアウトウサギの遺伝型分析
図2の(a)に表示されたアンプリコンを配列番号9及び10のプライマーを用いて増幅した後、2次及び3次PCRによってアダプター及びタグ(tag)を付着した後、MiSeq(Illumina,MiSeq Reagent Kit V)を用いて深いシーケンシング(deep sequencing)を行った後、その結果をCas-Analyzer(Bioinformatics,2017Jan15;333(2):286-288)を用いて分析した。
配列番号9:F8-F
5’- gagccatgcaaatagagctc -3’
配列番号10:F8-R
5’- atctttctccagccagagtc -3’
その結果、表1及び図3に示すように、第2個体及び第3個体のFVIII遺伝子からインデルが検出されることが確認できた。
図2の(b)に表示されたアンプリコンを配列番号11及び12のプライマーを用いて増幅した後、2次及び3次PCRによってアダプター及びタグを付着した後、MiSeq(Illumina,MiSeq Reagent Kit V)を用いて深いシーケンシングを行った後、その結果をCas-Analyzer(Bioinformatics,2017Jan15;333(2):286-288)を用いて分析した。第5個体の場合、配列番号13のF9-R2プライマーを用いて1次増幅した。
配列番号11:F9-F
5’- ttggctttgggattagttgg -3’
配列番号12:F9-R
5’- tcaaaaacttctcgagcttc -3’
配列番号13:F9-R2
5‘- tctctgtctgtaactctacc -3’
4-1.APTT(Activated Partial Thromboplastin Time)分析
APTT分析を行うために、Start 4 Hemostasis Analyzer(Stago Inc.,USA)を用いて実時間でAPTTクロット(clot)のウェーブフォームを観察した。すなわち、50μlのAPTT試薬(Dade(登録商標) Actin FSL,Siemens Medical Solutions Inc.,USA)溶液と形質転換ウサギから採取した50μlの血漿をキュベットで混ぜた後、37゜Cで3分間培養した後、50μlの塩化カルシウム(CaCl2)水溶液(最終濃度:25mM)をキュベットに注入した後、クロットができる時間を測定した。
トロンビン生産は、Fluoroskan Ascent(Thermo Scientific)蛍光プレートリーダーをThrombinoscope BVソフトウェアで分析して測定した。すなわち、形質転換ウサギの血漿80μlと組織因子(tissue factor)及びリン脂質(phospholipids)を含有した2μlのPPP-reagent LOWを混ぜて37℃のImmulon microtiter2HB-High Binding 96ウェルプレート(Thermo Nunc)で培養した。コントロールウェルには80μlのウサギ血漿と20μlのトロンビン校正試薬(Thrombin Calibrator reagent)を混ぜて培養し、反応を開始する前に、蛍光を持つトロンビン基質とあらかじめ温めておいたFlu-Ca試薬を注入してよく混ぜた。20μlのFlu-Ca試薬を注入して反応を開始し、生産されるトロンビンの量はThrombinoscope Analysis Version 3.0で分析した。
実施例2で製造した血友病ウサギをP(又はF0)といい、その子孫に該当するF1とF2を得るために次を進行した。第8因子ノックアウト或いは第9因子をノックアウトさせた形質転換ウサギの子孫を得るためには、図8に開示されたように個体を繁殖させた。すなわち、血友病は、血友病遺伝子がX染色体上に存在する伴性遺伝病に該当するので、実施例2で製造した第8因子ノックアウト及び第9因子ノックアウト形質転換ウサギ父体(P,X’Y)を正常ウサギ雌(XX)と交差させて子孫を生成した。最初の交差から得られた子孫雌(F1)の遺伝子分析をして、保因者(X’X)を確認した後、正常雄(XY)と交差させて第8因子ノックアウト及び第9因子ノックアウトをさせた形質転換ウサギの雄(F2,X’Y)を製造した。
6-1.第8因子ノックアウトウサギ保因者の遺伝型分析
表1の4bpの核酸が欠失した突然変異が検出された#2個体である雄ウサギ(X’Y)をSamtakoから購入した体重2kg以上及び10~12週齢である正常雌(XX)と交配させて子孫を得、これを実施例3-1のような方法で遺伝子分析を行い、F1世代である雌保因者(X’X)を確認した。表3及び図9に示すように、第1個体と第5個体のFVIII遺伝子からインデルパターンが検出されることが確認できた。
表2で6bpの核酸が欠失した突然変異が検出された#6個体である雄(X’Y)を正常雌(XX)と交配させて子孫(F1)を得、実施例3-2のような方法で遺伝子分析を行って雌保因者(X’X)を確認した。表4及び図10に示すように、#4個体のFIX遺伝子からインデルパターンが検出されることが確認でき、形態は-6bp欠失(deletion)と1bp挿入(insertion)である。
7-1.第8因子ノックアウトウサギの遺伝型分析
F1世代である保因者雌(X’X)をSamtakoから購入した体重2kg以上及び10~12週齢である正常雄(XY)と交配させて得たF2雄(X’Y)に対して、実施例3-1のような方法で遺伝子分析を実施した。表5及び図11に示すように、#1-4個体と#5-1個体のFVIII遺伝子からインデルパターンが検出されることが確認できた。
F1世代である保因者雌(X’X)を正常雄(XY)と交配させて得たF2雄(X’Y)に対して、実施例3-2のような方法で遺伝子分析を実施した。表6及び図12に示すように、#4-1個体のFIX遺伝子からインデルパターンが検出されることが確認でき、形態は6bp、7bp、27bp欠失の形態であるが、7bp、27bp欠失は、その比率がそれぞれ0.023%、0.001%であり、配列分析エラーと判断され、その形態は6bp核酸欠失変異であることを確認した。
8-1.爪出血モデル
ウサギにおける爪出血モデルを誘導するために、体重2kg以上及び12週齢以上になった実施例7の血友病ウサギとSamtakoから購入した体重2kg以上及び10~12週齢である正常ウサギにジアゼパム(Diazepam)0.4mg/kgとペントバルビタールナトリウム(pentobarbital sodium)25mg/kgを耳介静脈から注射して麻酔した。麻酔されたウサギの片前足をバリカン(hair clipper)で除毛し、デジマチックキャリパ(digimatic caliper)を用いて第3足指の爪の遠位部の端から2mm近位部をクイック油性ペンを用いて表示した後、ワイヤカッター(wire cutter)を用いて切断した(図13)。爪を切断すると直ちに37゜Cに維持させた滅菌生理食塩水が入っている50mL円錐管(conical tube)に入れて60分間血液を集めた後、1500xgで5分間遠心分離して上澄液を除去した後、10mLピペットを用いて円錐管に3次蒸留水を20mLまで入れた後、vortexを用いて血液を完全に溶血させた。
実施例8-1で溶血したサンプルを用いてヘモグロビン分析キット(Sigma and Aldrich,MAK115-1KT,#BF03A26V)の方法で血液中のヘモグロビンの量を定量して失血(blood loss)を測定した。正常ウサギの場合、ヘモグロビンの濃度が1,014nM(Range:502-1503)であることを確認し、第8因子ノックアウトウサギのヘモグロビンの濃度が45,787nM、第9因子ノックアウトウサギのヘモグロビンの濃度が4,620nMであることを確認した(図14)。
Claims (14)
- 第9因子(Factor IX,FIX)のエクソン領域に相補的に結合するターゲッティングドメインを含むsgRNAであって、前記sgRNAが配列番号5又は6の塩基配列で表される、sgRNA。
- 請求項1に記載のsgRNAが挿入されたベクター。
- 請求項1に記載のsgRNA及びCas9タンパク質又はそれをコードするDNAを含むCRISPR/Cas9システム。
- 請求項3に記載のCRISPR/Cas9システムを用いて製造された形質転換ウサギ。
- (a)ウサギ受精卵に請求項3に記載のCRISPR/Cas9システムを導入して培養する段階;及び
(b)前記(a)段階で得た受精卵を代理母に移植して形質転換ウサギを分娩させる段階
を含む方法で製造されることを特徴とする、請求項4に記載の形質転換ウサギ。 - 前記形質転換ウサギは分娩後に形質転換の有無を確認する段階をさらに含む方法で製造されることを特徴とする、請求項5に記載の形質転換ウサギ。
- 請求項5に記載の形質転換ウサギを交配して子孫形質転換ウサギを生産する段階を含む方法で製造されることを特徴とする、子孫形質転換ウサギであって、前記子孫形質転換ウサギが、第9因子がノックアウトされて血友病表現型を示す、子孫形質転換ウサギ。
- 前記形質転換ウサギの交配は、請求項5に記載の形質転換ウサギ又は正常ウサギと交配することを特徴とする、請求項7に記載の子孫形質転換ウサギ。
- 請求項4に記載の形質転換ウサギ又は請求項7に記載の子孫形質転換ウサギから分離した副産物であって、
前記形質転換ウサギ又は子孫形質転換ウサギが、第9因子がノックアウトされて血友病表現型を示し、
前記副産物が血液、血清、尿、糞便、唾液、臓器及び皮膚からなる群から選択される、前記副産物。 - 請求項4に記載の形質転換ウサギ又は請求項7に記載の子孫形質転換ウサギから分離した細胞であって、
前記形質転換ウサギ又は子孫形質転換ウサギが、第9因子がノックアウトされて血友病表現型を示す、細胞。 - 請求項4に記載の形質転換ウサギ又は請求項7に記載の子孫形質転換ウサギから分離した組織であって、
前記形質転換ウサギ又は子孫形質転換ウサギが、第9因子がノックアウトされて血友病表現型を示す、組織。 - 次の段階を含む形質転換ウサギの製造方法。
(a)ウサギ受精卵に請求項3に記載のCRISPR/Cas9システムを導入して培養する段階;及び
(b)前記(a)段階で得た受精卵を代理母に移植して形質転換ウサギを分娩させる段階。 - 請求項12に記載の方法で製造された形質転換ウサギを交配して子孫形質転換ウサギを生産する段階を含む子孫形質転換ウサギの製造方法。
- 前記形質転換ウサギの交配は、請求項12に記載の方法で製造された形質転換ウサギ又は正常ウサギと交配することを特徴とする、請求項13に記載の子孫形質転換ウサギの製造方法。
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WO2017077386A1 (en) | 2015-11-06 | 2017-05-11 | Crispr Therapeutics Ag | Materials and methods for treatment of glycogen storage disease type 1a |
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WO2017077386A1 (en) | 2015-11-06 | 2017-05-11 | Crispr Therapeutics Ag | Materials and methods for treatment of glycogen storage disease type 1a |
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