JP2007537713A - 第VIII因子及びその誘導体の高発現レベルのための改変されたcDNA - Google Patents
第VIII因子及びその誘導体の高発現レベルのための改変されたcDNA Download PDFInfo
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- JP2007537713A JP2007537713A JP2006534669A JP2006534669A JP2007537713A JP 2007537713 A JP2007537713 A JP 2007537713A JP 2006534669 A JP2006534669 A JP 2006534669A JP 2006534669 A JP2006534669 A JP 2006534669A JP 2007537713 A JP2007537713 A JP 2007537713A
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- factor viii
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Abstract
Description
1.1 pD−L2ベクターの生成
全ての第VIII因子コンストラクトは、pcDNA3.1ベクター(pD)(Invitrogen, Leek オランダ国)中に挿入された。
ウサギのβ−グロビンイントロン2を含むpSG5−プラスミドがPCRのためのマトリクスとして使用された。β−グロビンイントロン2DNAフラグメントは増幅され、NheI及びNotI部位によって囲まれ、オリゴヌクレオチドβ−Glob−NheI−S及びβ−Glob−NotI−ASによってもたらされた。該フラグメントは、両酵素によって消化され、同一酵素によって開裂されたpD−L2又はpD−L2−2l中に挿入された。フラグメントの配列は、シークエンシングによって制御された。
CHO及びCOS−1細胞は、ECACC(Sigma, L’lse d’Abeau, フランス国)から得た。HEK−293及びHKB−11細胞は、LGC Promochem (Molsheim, France)フランスのATCC配給業者から得た。全ての細胞培養試薬(培地、抗生物質、添加物及び血清)は、InVitrogen(Cergy Pontoise,フランス国)からである。細胞は、5%CO2の加湿されたインキュベータ中で37℃でインキュベートされた。CHO及びCOS−1細胞は、10%ウシ胎児血清、2mM L−グルタミン及び1%ペニストレプトマイシン(penistreptomycin)を添加されたIMDM培地において増殖された。HEK−293細胞は、10%ウシ胎児血清、1%NEAA、2mM L−グルタミン及び1%ペニストレプトマイシンが添加されたEMEM培地中で増殖された。HKB−11細胞は、2.5%のウシ胎児血清、2%HAT、2mM L−グルタミン及び1%ペニストレプトマイシンが添加されたRPMI培地中で増殖された。
5.105細胞(又は、HEK−293について1×106)が、9.5cm2のディッシュ中にプレーティングされた。一日後、細胞は、製造者の推奨に従って5μlのFuGENE−6試薬でプレコンプレックスされた1μgのDNAと共に6時間インキュベートされた。次いで、新たな培地が添加された。
FVIII抗原の濃度は、ELISAキット(Asserachrom FVIII, Stago, Asnieres,フランス国)を用いて、細胞コンディションドメディウム中で測定された。
続くコンストラクトは、異なる細胞系においてFuGENE−6を用いてトランスフェクトされた:pD−FVIII−L2(イントロンの無いL2)、pD−FVIII−L2−5’(5’におけるβ−グロビンイントロンを有する5’I)、pD−L2−2l(1及び13の位置においてTFIXI1を有する2l)及びpD−L2−3l(5’におけるβ−グロビンイントロン2及び1及び13の位置におけるTFIXI1を有する3l)。ネガティブコントロールとして、トランスフェクトされていない細胞が等しく処理された。トランスフェクション後2日で、細胞は、1%BSAを含むIMDM中で6時間インキュベートされた。この間に産生されたFVIIIの量は、ELISAを用いて定量化された。
配列番号2は、オリゴヌクレオチドFVIII−L2−2I−ASである。
配列番号3は、オリゴヌクレオチドFVIII−N538−Sである。
配列番号4は、オリゴヌクレオチドFVIII−L0−New−ASである。
配列番号5は、オリゴヌクレオチドb−GIob−NheI−Sである。
配列番号6は、オリゴヌクレオチドb−GIob−NotI−ASである。
配列番号7は、オリゴヌクレオチドF8/AG−kozak.Sである。
配列番号8は、オリゴヌクレオチドF8/ATG−kozak.ASである。
Claims (16)
- ゲノム第VIII因子配列のイントロン1と13が挿入される位置において、第VIII因子のcDNAはまた、1つ以上のスプライシング可能なヌクレオチド配列、又はプレ−mRNAの核からの輸送時にスプライシングされるヌクレオチド配列、さらにプロモーター配列の下流且つ改変された第VIII因子cDNAの上流に挿入された別のスプライシング可能なヌクレオチド配列を含むことを特徴とする、改変第VIII因子のcDNA。
- ゲノム第VIII因子配列のイントロン位置1及び/又は13において1つ以上の完全な又は切断されたイントロンが挿入されたことを特徴とする、請求項1に記載の改変された第VIII因子のcDNA。
- ゲノム第VIII因子配列のイントロン位置1及び/又は13において、スプライシングされる能力を保持する1つ以上の天然又は合成の核酸配列が挿入されたことを特徴とする、請求項1に記載の改変された第VIII因子のcDNA。
- ゲノム第VIII因子配列のイントロン位置1及び13において切断されたFIXイントロンIが挿入されたことを特徴とする、請求項1に記載の改変された第VIII因子のcDNA。
- プロモーターの下流且つFVIIIコーディング配列の上流において、1つの完全な又は切断されたイントロンが挿入されたことを特徴とする、請求項1に記載の改変された第VIII因子のcDNA。
- プロモーターの下流且つFVIIIコーディング配列の上流において、スプライシングされる能力を保持する1つの天然又は合成の核酸配列が挿入されたことを特徴とする、請求項1に記載の改変された第VIII因子のcDNA。
- プロモーターの下流且つFVIIIコーディング配列の上流においてβ−グロビンイントロン2が挿入されたことを特徴とする、請求項1に記載の改変された第VIII因子のcDNA。
- ヒト第VIII因子のアミノ酸1〜740をコードする第一DNAセグメントとヒト第VIII因子のアミノ酸1649〜2332をコードする第二DNAセグメントを含み、前記セグメントがリシン及びアルギニンから選択される少なくとも二つのアミノ酸のリンカーペプチドをコードするリンカーDNAセグメントによって相互に連結されていることを特徴とする、請求項1又は4に記載の改変された第VIII因子のcDNA。
- 請求項1〜5の改変された第VIII因子のcDNA配列を含む転写ユニット、転写プロモーター及びポリアデニル化配列を含有する組換え発現ベクター。
- 請求項9の組換え発現ベクターを用いて形質転換された動物由来の宿主細胞系。
- 生物学的活性のある組換えヒト第VIII因子又はその誘導体の製造方法であって、前記製造は請求項10の動物細胞系をヒト第VIII因子またはその誘導体の発現及び分泌を可能にする栄養培地中で培養し、前記発現産物を該培養培地から回収することによって実施されることを特徴とする、方法。
- 請求項11の方法によって調製されるヒト第VIII因子又はその誘導体。
- 請求項12に記載される第VIII因子を含有する薬学的組成物。
- 請求項1〜5に記載の改変された第VIII因子のcDNAを含むことを特徴とする、ヒト遺伝子治療における使用のためのトランスファーベクター。
- それがヒトの細胞であることを特徴とする、請求項10の宿主細胞。
- 該細胞が人体中にあることを特徴とする、請求項15に記載の宿主細胞。
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FR2913020B1 (fr) | 2007-02-23 | 2012-11-23 | Biomethodes | Nouveaux facteurs viii pour le traitement des hemophiles de type a |
TWI791433B (zh) | 2016-04-15 | 2023-02-11 | 賓州大學委員會 | 治療a型血友病之基因治療 |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS63152986A (ja) * | 1986-09-12 | 1988-06-25 | ジェネンテク,インコーポレイテッド | 改良型の組換え発現 |
JPH06343476A (ja) * | 1993-06-10 | 1994-12-20 | Miles Inc | 改良された生産性を有するベクターおよび哺乳動物細胞系 |
EP1048726A2 (en) * | 1999-04-27 | 2000-11-02 | Centeon Pharma GmbH | Modified factor VIII cDNA |
JP2003070491A (ja) * | 2001-08-08 | 2003-03-11 | Aventis Behring Gmbh | 第VIII因子およびその誘導体の高発現レベル改変cDNA |
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EP1038959A1 (en) * | 1999-03-17 | 2000-09-27 | Aventis Behring Gesellschaft mit beschränkter Haftung | Factor VIII without B-domain, comprising one or more insertions of a truncated intron I of factor IX |
EP1233064A1 (en) * | 2001-02-09 | 2002-08-21 | Aventis Behring Gesellschaft mit beschränkter Haftung | Modified factor VIII cDNA and its use for the production of factor VIII |
EP1283263A1 (en) * | 2001-08-08 | 2003-02-12 | Aventis Behring GmbH | Modified cDNA for high expression levels of factor VIII and its derivatives |
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JPS63152986A (ja) * | 1986-09-12 | 1988-06-25 | ジェネンテク,インコーポレイテッド | 改良型の組換え発現 |
JPH06343476A (ja) * | 1993-06-10 | 1994-12-20 | Miles Inc | 改良された生産性を有するベクターおよび哺乳動物細胞系 |
EP1048726A2 (en) * | 1999-04-27 | 2000-11-02 | Centeon Pharma GmbH | Modified factor VIII cDNA |
JP2003070491A (ja) * | 2001-08-08 | 2003-03-11 | Aventis Behring Gmbh | 第VIII因子およびその誘導体の高発現レベル改変cDNA |
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