JP4855504B2 - レトロウイルスベクター - Google Patents
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- JP4855504B2 JP4855504B2 JP2009183045A JP2009183045A JP4855504B2 JP 4855504 B2 JP4855504 B2 JP 4855504B2 JP 2009183045 A JP2009183045 A JP 2009183045A JP 2009183045 A JP2009183045 A JP 2009183045A JP 4855504 B2 JP4855504 B2 JP 4855504B2
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16051—Methods of production or purification of viral material
- C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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Description
第1図は、293T細胞の3-プラスミド同時トランスフェクションを用いた、本発明のベクター作製システムを示す:
第2図は、本発明において使用したHIVに基づくベクターゲノムを示す;
第3図は、本発明において使用したHIV-1 gag-pol遺伝子発現プラスミドを示す;
第4図は、5つの補助因子を欠いた本発明のベクターの形質導入効率を示す。
プラスミドの構築
pGP-RRE1はpW13(Kimら、1989)由来のgag-pol vif発現プラスミドである。SmaIによって切断されたpBluescript KS+に、pW13のRRE(受入れ番号:U26942)を、StyI/StyI断片(7720-8050)を平滑末端にすることによって挿入し、pBSRREを作製した。pW13のNarI/Eco R1断片(637-5743)をClaIおよびEco RIで切断したpBSRREに挿入し、pBSGPRRE1を作製した。XhoI/NotI断片(gagpolおよびRRE含有)を発現プラスミドpCl-Neoに挿入し、pGR-RRE1を作製した。vifコード領域を除去するために、pBSGPRRE1をNdeIおよびSmaIで切断して平滑末端とし、pBSGPRRE2を作製した。gagpol遺伝子およびRREをXhoIおよびNotIサイト内のpCl-neoに挿入し、pGPRRE2を作製した。
上記の増幅物をpLIGATOR(R & D Systems)にクローニングした。vprコード領域を含有するMluIおよびXhoI断片をpCI-Neo(Promega)に挿入することによって、発現プラスミドpCI-vprを作製した。
293T(293ts/Al609)(DuBridgeら、1987)細胞は、ダルベッコ改変イーグル培地(GIBCO)で維持し、HeLa細胞および208F細胞はMEM(GIBCO)で維持した。
レトロウイルスベクター系は、本発明者らのすでに出版されたプロトコール(Soneokaら、1995)にしたがって予め作製しておいた。簡単に述べると、ヒト腎臓293T細胞(1.5x106)を10-cmプレートにプレーティングし、15mgの各プラスミド(ベクタープラスミドとともにgag-polおよびenv発現プラスミド)を用いて、リン酸カルシウムDNA沈澱法(ChenおよびOkayama,1987)によって一過性のトランスフェクションを行なった。培養上清を36時間後に回収し、0.45mmを通して濾過し、ただちに使用するか、または-70℃で冷凍した。8mg/mlポリブレンの存在下で、ウイルスを標的細胞に添加することによって形質導入を2時間行ない、次いで新鮮な培地を添加した。既述のように(Soneokaら、1995)、5-ブロモ-4-クロロ-3-インドリルーβ-D-ガラクトシド(X-Gal)を用いて、β−ガラクトシダーゼの48時間後の発現を測定した。1mlあたりのlacZ(青色の点)形成単位の数を計数することによって力価が得られた(1.f.u./ml)。感染の24時間前、次いで実験の間を通じて毎日、アフィジコリン(5mg/ml)を添加することによって、Gl/S期停止培養物を調製した。
HIVベクター作製
H3Z(tat陽性)およびH4Z(tat陰性)は、293T細胞への3プラスミド同時トランスフェクションによって作製されるように設計された、HIV-1を基にしたベクターである(第2図)。HIVコアによる効率のよいパッケージングのために、上記ベクターはgagの最初の778塩基を含有するが、ATG開始コドンから40bpに導入されたフレームシフト変異によりgagタンパク質の発現は妨げられる。RREは、パッケージング効率を高めるために包含されており、revはHIV mRNAの輸送を支えるためにベクターから発現される。レポーター遺伝子として働くように、内在するCMVプロモーターで駆動されるβ−ガラクトシダーゼ遺伝子が挿入された。どちらについても、ベクターゲノムの転写は、5'LTR U3を置き換えるために用いられたCMVプロモーターによって駆動される。これによってベクターゲノムはtat非依存性となる。二つのHIV-1gagpol構築物が作製された(第3図);pGP-RREI(vif陽性)およびpGP-RRE2(vif陰性)。CMVで駆動される発現プラスミドであるpCI-neoにgagpol遺伝子が挿入されているため、gagpol発現はtatに依存しない。pRV67、VSV糖タンパク質構築物をシュードタイピングのために使用した。異なるプラスミドに別の遺伝子を載せることによって、組換えにより複製コンピテントウイルスを生じる可能性を最小にすることができる。
上記の3プラスミドを用いてヒト腎臓293T細胞を一過性同時トランスフェクションすることによって、複製能欠損レトロウイルス粒子を作製し、これをただちに使用するか、または-70℃で冷凍した。異なるベクター構築物を用いてウイルスを作製した。最小構築物(H4ZおよびpGP-RRE2)が、vif,vpr,nefおよびtat陽性ウイルスと同等の力価を与えることが明らかになった(第1表)。
非分裂細胞形質導入へのvprの影響を調べるために、pH4Z,pGP-RRE2およびpRV67プラスミドをともに使ったpCI-vprの同時トランスフェクションによってvprをパッケージングシステム内に入れた。生成したウイルス粒子の形質導入効率は、293T細胞およびHeLa細胞の増殖細胞および増殖停止細胞についてアッセイされた(第4図)。MLV由来のパッケージングおよび形質導入ベクター(Soneoka、1995)を対照として用いた。HeLa細胞および293T細胞は、アフィジコリン処理によってGl/S期で増殖停止状態となった。最小HIVベクターH4Zは増殖中のHeLaおよび293T細胞と比較して、Gl/S期停止細胞の形質導入に際しても同等の有効性を示したが、MLVを基にしたベクターは、わずかに0.002%しか有効ではなかった。
発明者らは、HIV-1を基にしたベクター作製システムを確立した。このシステムはvpr,vpu,nef,vifおよびtatを含有せず、3プラスミド同時トランスフェクション法に基づく。このベクターは、増殖細胞を3.2x105l.f.u./mlまでの力価で形質導入することができ、この力価はほかのMLVを基にしたベクターに匹敵するが、さらに超遠心を用いた濃縮によって容易に増加することができる(データ示さず)。ヘルパーウイルスは検出されなかった(データ示さず)。
Claims (18)
- 遺伝子治療のためにレンチウイルスに由来する複製能欠損ベクター粒子を作製するためのレトロウイルスベクター作製システムであって、前記ベクター粒子は哺乳動物の非分裂標的細胞に感染及び形質導入することができ、前記システムは前記ベクターの成分をコードする一組の核酸配列を含んでなり、前記一組の核酸配列は、前記ベクターのRNAゲノム、GagおよびPolタンパク質、Envタンパク質またはその機能的代替物をコードし、補助遺伝子vpu,vpr,vif,tat,revおよびnef、または補助遺伝子vpu,vpr,vif,tat,revおよびnefと同一のもしくは同様の機能を果たすそれらに類似した補助遺伝子をコードする核酸配列が、これら補助遺伝子が機能的補助タンパク質をコードできないように破壊されているか、または前記システムより除去されている、上記レトロウイルスベクター作製システム。
- ベクターのRNAゲノムをコードする核酸配列が、1以上の治療上活性を有する遺伝子を含んでなる、請求項1に記載のレトロウイルスベクター作製システム。
- ベクターが、HIV-1、HIV-2、SIV、FIV、BLV、EIAV、CEVまたはビスナレンチウイルスに由来する、請求項1または2に記載のレトロウイルスベクター作製システム。
- 宿主細胞中の、請求項1〜3のうちいずれか1項に記載のレトロウイルスベクター作製システム。
- 機能し得るようにプロモーターに結合した、パッケージ可能なRNAベクターゲノムをコードし、機能し得る補助遺伝子vpu,vpr,vif,tat,revおよびnef、または補助遺伝子vpu,vpr,vif,tat,revおよびnefと同一のもしくは同様の機能を果たすそれらに類似した機能し得る補助遺伝子を含まない、請求項1〜4のいずれか1項に記載のシステムにおいて使用されるDNA構築物。
- プロモーターが非レンチウイルス性のプロモーターである、請求項5に記載のDNA構築物。
- 請求項5または6に記載のDNA構築物並びにGagおよびPolタンパク質をコードするDNA構築物を含んでなる、請求項1〜4のいずれか1項に記載のシステムにおいて使用されるDNA構築物の組合わせ物。
- Envタンパク質またはその代替物をコードするDNA構築物をさらに含んでなる、請求項7に記載のDNA構築物の組合わせ物。
- 1以上の発現ベクター中に、請求項5または6に記載のDNA構築物を含んでなる、請求項1〜4のいずれか1項に記載のシステムにおいて使用されるDNA構築物。
- 遺伝子治療のためにレンチウイルスに由来する複製能欠損ベクター粒子を作製するためのレトロウイルスベクター作製システムであって、前記ベクター粒子は哺乳動物の非分裂標的細胞に感染及び形質導入することができ、前記システムは前記ベクターの成分をコードする一組の核酸配列を含んでなり、前記一組の核酸配列は、前記ベクターのRNAゲノム、GagおよびPolタンパク質、Envタンパク質またはその機能的代替物をコードしている、上記システムを作製するための方法であって、補助遺伝子vpu,vpr,vif,tat,revおよびnef、または補助遺伝子vpu,vpr,vif,tat,revおよびnefと同一のもしくは同様の機能を果たすそれらに類似した補助遺伝子を、前記一組の核酸配列から除去するかまたは破壊して、これら補助遺伝子が機能的補助タンパク質をコードできなくさせるステップを含む、上記方法。
- ベクターが、HIV-1、HIV-2、SIV、FIV、BLV、EIAV、CEVまたはビスナレンチウイルスに由来する、請求項10に記載の方法。
- 請求項10または11に記載の方法によって作製されるレトロウイルスベクター作製システム。
- レトロウイルスベクター粒子を作製するための方法であって、請求項1〜9のいずれか1項に記載される一組の核酸配列またはDNA構築物を宿主細胞に導入すること、およびレトロウイルスベクター粒子を回収すること、を含む、上記方法。
- 請求項1〜4または10〜13のいずれか1項に記載のシステムまたは方法によって作製されるレトロウイルスベクター粒子。
- 非分裂細胞に形質導入することができる高力価のレトロウイルスベクター粒子を作製するための、請求項1〜4もしくは12のいずれか1項に記載のレトロウイルスベクター作製システムまたは請求項5〜9のいずれか1項に記載のDNA構築物の使用。
- 請求項14に記載のレトロウイルスベクター粒子を含む医薬組成物。
- 請求項14に記載のレトロウイスベクター粒子に感染したまたは該粒子を用いて形質導入された、単離された標的細胞。
- 標的の感染および形質導入による遺伝子治療のための医薬の製造における、請求項14に記載のレトロウイルスベクター粒子の使用。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9621680.9A GB9621680D0 (en) | 1996-10-17 | 1996-10-17 | Lentiviral vectors |
GB9621680.9 | 1996-10-17 | ||
GBGB9624457.9A GB9624457D0 (en) | 1996-11-25 | 1996-11-25 | Retroviral vectors |
GB9624457.9 | 1996-11-25 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP51908698A Division JP4418536B2 (ja) | 1996-10-17 | 1997-10-17 | レトロウイルスベクター |
Publications (2)
Publication Number | Publication Date |
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JP2009291205A JP2009291205A (ja) | 2009-12-17 |
JP4855504B2 true JP4855504B2 (ja) | 2012-01-18 |
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Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
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JP51908698A Expired - Lifetime JP4418536B2 (ja) | 1996-10-17 | 1997-10-17 | レトロウイルスベクター |
JP2009183045A Expired - Lifetime JP4855504B2 (ja) | 1996-10-17 | 2009-08-06 | レトロウイルスベクター |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
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JP51908698A Expired - Lifetime JP4418536B2 (ja) | 1996-10-17 | 1997-10-17 | レトロウイルスベクター |
Country Status (23)
Country | Link |
---|---|
US (2) | US6312682B1 (ja) |
EP (2) | EP0904392B1 (ja) |
JP (2) | JP4418536B2 (ja) |
KR (1) | KR20000049251A (ja) |
CN (1) | CN1195863C (ja) |
AT (1) | ATE198910T1 (ja) |
AU (1) | AU725143B2 (ja) |
BG (1) | BG103397A (ja) |
CA (1) | CA2267636A1 (ja) |
CZ (1) | CZ137399A3 (ja) |
DE (1) | DE69703974T2 (ja) |
DK (1) | DK0904392T3 (ja) |
ES (1) | ES2153654T3 (ja) |
GB (1) | GB2325003B (ja) |
GR (1) | GR3035600T3 (ja) |
HK (2) | HK1014993A1 (ja) |
HU (1) | HUP0000421A2 (ja) |
IL (1) | IL129017A0 (ja) |
NO (1) | NO991781L (ja) |
NZ (1) | NZ334860A (ja) |
PL (1) | PL332875A1 (ja) |
PT (1) | PT904392E (ja) |
WO (1) | WO1998017815A1 (ja) |
Families Citing this family (216)
Publication number | Priority date | Publication date | Assignee | Title |
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US20070213290A1 (en) * | 1996-10-17 | 2007-09-13 | Kingsman Alan J | Neurite regeneration |
GB2325003B (en) * | 1996-10-17 | 2000-05-10 | Oxford Biomedica Ltd | Rectroviral vectors |
US7198784B2 (en) * | 1996-10-17 | 2007-04-03 | Oxford Biomedica (Uk) Limited | Retroviral vectors |
JP4312265B2 (ja) * | 1997-04-09 | 2009-08-12 | ラング−ジ チャン | ワクチン評価のための動物モデル |
EP0981636B1 (en) | 1997-05-13 | 2005-03-23 | University Of North Carolina At Chapel Hill | Lentivirus-based gene transfer vectors |
US5994136A (en) * | 1997-12-12 | 1999-11-30 | Cell Genesys, Inc. | Method and means for producing high titer, safe, recombinant lentivirus vectors |
US6121021A (en) * | 1997-12-16 | 2000-09-19 | Connaught Laboratories Limited | Constitutive expression of non-infectious HIV-like particles |
EP1895010B1 (en) * | 1997-12-22 | 2011-10-12 | Oxford Biomedica (UK) Limited | Equine infectious anaemia virus (eiav) based vectors |
GB9803351D0 (en) | 1998-02-17 | 1998-04-15 | Oxford Biomedica Ltd | Anti-viral vectors |
CA2328404C (en) | 1998-05-13 | 2007-07-24 | Genetix Pharmaceuticals, Inc. | Novel lentiviral packaging cells |
CA2331782A1 (en) | 1998-06-24 | 1999-12-29 | Musc Foundation For Research Development | Tissue-specific and target rna-specific ribozymes |
US6958226B1 (en) | 1998-09-11 | 2005-10-25 | The Children's Medical Center Corp. | Packaging cells comprising codon-optimized gagpol sequences and lacking lentiviral accessory proteins |
GB9906177D0 (en) | 1999-03-17 | 1999-05-12 | Oxford Biomedica Ltd | Anti-viral vectors |
US6271359B1 (en) | 1999-04-14 | 2001-08-07 | Musc Foundation For Research Development | Tissue-specific and pathogen-specific toxic agents and ribozymes |
EP1171624B1 (en) * | 1999-04-29 | 2007-07-25 | Cell Genesys, Inc. | Method and means for producing high titer, safe, recombinant lentivirus vectors |
CA2388337C (en) | 1999-10-22 | 2013-01-08 | Aventis Pasteur Limited | Method of inducing and/or enhancing an immune response to tumor antigens |
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