JP3779966B2 - Mpmv及びhivに基づく欠損パッケージング非オンコウイルスベクター - Google Patents
Mpmv及びhivに基づく欠損パッケージング非オンコウイルスベクター Download PDFInfo
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Description
本発明はベクター及び遺伝子転移でのそれらの用途に関する。ベクターは、レトロウイルスに基づき、それゆえ、それらはそれら自身のRNAをパッケージできず、そして例えば体細胞遺伝子治療用に外来遺伝子を転移するための感染因子として用いることができる。
レトロウイルスは幾つもの方法で分類される。それらは、それらの形態に基づいて種々のグループに分けられる。それらのグループは、A、B、C及びD型ウイルスである。それらは又、3のつ亜科、即ち、オンコウイルス、スプマウイルス及びレンチウイルスの一つに属するように分類される。
本発明の第一の局面によれば、RNAはビリオン内にパッケージできないから、プロウイルスは、MPMV蛋白を生成しうるが複製コンピテントを生成し得ない。このパッケージング欠損プロウイルスベクターを用いると、パッケージング欠損細胞系が創造でき、ウイルスのパッケージング機構を研究し、このパッケージング機構を損なう戦略を開発するのに用いることができる。このようなパッケージング陰性プロウイルスにより産生されたビリオンは、ワクチンとして、又、所望の遺伝子を哺乳動物細胞に効果的に導入するためのシステムとして用いうる。これは、又、今や本発明の第二の局面により、HIVについて、その結果他のレンチウイルスについて首尾よく達成された。
本発明の第一の局面は、MPMVに関する。
MPMVは、これまで用いられ、ヒトにおいて潜在的使用のための遺伝子ベクターとして開発されている他のレトロウイルスに比べ、レトロウイルスベクターとして幾つかの潜在的利点を有する。特に、
1)それは、アッセーされる全てのヒト細胞に感染性である。
2)それは、ヒトに非病原性である。
3)それは、補体仲介溶解に感受性のエンベロープを有する。これはありそうもないこと、例えば組換えがヒト使用でのレトロウイルスベクターの生成で起きても、ウイルスはそれが細胞外空間に現れるとすぐにインビボで死滅されるので再構成MPMVからの危険がないことを意味する。
4)分子生物学及びウイルス組立て機能がよく理解されている。
5)それは、パッケージング機能にとって大きく余分であるように見え、又、従って所望の遺伝子配列により置換できる8キロ塩基よりも大のゲノムを有する。遺伝子治療の候補者である遺伝子をコードするRNAのほとんどのDNA複写は、これらの次元のウイルスゲノムに適合する。
6)内因性ヒトレトロウイルス配列との明白な検出可能な類似性がなく、従って治療用ベクターとインビボに現れる複製コンピテントウイルスに導く内因性配列との組換えの可能性を最少にする。
7)MPMVが、マウスレトロウイルスベクターによりカプシド化され、マウスレトロウイルスベクターシステムにより感染した全ての細胞に伝達される例えばVL30科のような異種配列をカプシド化するという証拠はない。
(1) リー等(1990)ジャーナル・オブ・バイロロジィ 64、3844−3854。
(2) ソニゴ等(1986)セル 45、375−385。
(3) ターウィリガー等(1989)PNAS USA 86、3857−3861。
(4) ローセン等(1986)ジャーナル・オブ・バイロロジィ 57、379−384。
(5) マドン等(1986)セル 47、333−348。
(6) フィッシャー等(1985)ネイチャー(ロンドン) 316、262−265。
(7) ロサルド等(1990)ジャーナル・オブ・バイロロジィ 64、1756−1763。
(8) モーゲンスターン等(1980)ヌクレイック・アシッズ・リサーチ 18、3587−3596。
(9) ソーグ等(1983)ジャーナル・オブ・バイロロジィ 48、667−675。
(10) コムジンスキー等(1987)アナリティカル・バイオケミストリィ 162、156−159。
(11) ファィンバーグ等(1983)アナリティカル・バイオケミストリィ 132、6−13。
(12) キングストン(1987)イン・カレント・プロトコールス・イン・モレキュラー・バイオロジィ、1巻、グリーン・パブリッシング・アソシエイツ・アンド・ウィリィ・インターサイエンス、ニューヨーク。
(13) ポッツ(1990)テクニクス・イン HIVリサーチ.ストックトン・プレス,ニューヨーク、103−106頁。
(14) コワルスキィ等(1987)サイエンス 237、1351−1355。
(15) シマダ等(1991)ジャーナル・オブ・クリニカル・インベスティゲーション 88、1043−1047。
(16) レーバー等(1989)ジャーナル・オブ・バイロロジィ 63、4085−4087。
(17) セルドン(1987)トランスフェクション・ユージング,DEAE−デキストラン、ユニット 9、2。 カレント・プロトコールス・イン・モレキュラー・バイオロジィ、1巻、グリーン・パブリッシング・アソシエイツ・アンド・ウィリィ・インターサイエンス,ニューヨーク。
実施例は、又、添付の図面を引用する。
本実施例では、既に記述したプロウイルスとスクローンpSHRM15(図3)をヘルパーウイルスとして用い、transに、MPMVビリオンの全ての構造及び酵素蛋白を与えた。MPMV5' LTR配列、gag開いた読み取り枠中位置1320にBall制限部位の下流の5'リーダー配列、続いてベクターに対しアンチセンス定位にハイグロマイシンB耐性遺伝子、ポリアデニル化配列を存在させることなく、そして最後に3'LTRの末端に延びる7440にドロール部位からのMPMVプロウイルスの3'末端を含むベクターを構築した(図3)。ヘルパーウイルス及び/又はベクターを、種々の欠失を既に記載したそれらに、即ちMPS1、MPS2、MPS3又はMPS4に導入した。ベクター及びヘルパーはCos−1細胞に同時形質転換し、得られるビリオンを集めて、Hela細胞を感染するのに用いた。次いでヘラ細胞は抗生物質ハイグロマイシンを含む媒体に保持し、そしてベクターが取り込まれなければならない薬物に対し耐性のコロニーの数をカウントした。これは、ベクターのパッケージ性の措置及びRNAビリオン粒子へのカプシド化で津失した配列の役割を与える。
細胞及びウイルス
細胞系ジャルカット、ジャルカット−tat(4)及びヘラT4(5)を、10%ウシ胎児血清、ペニシリン及びストレプトマイシンを補足したRPMI 1640中で生育した。これらの細胞系及びHIV−1分離HTLV−III8はメディカル・リサーチ・カウンシル(UK)エイズ・リエイジェント・プログラムより供給された。
全ベクターはpSVC21に由来し、最初にプラスミド(pHXBc2)からのHTLV−IIIB分離物の感染プロウイルスクローンは、R.ガロ及びF.ウォングーストール博士(6)により提供された。pSVC21は複製のSV40オリジンを組込む。ベクターは図4に示す。あるベクターでは、gag開始コドンをオリゴヌクレオチド指定突然変異導入法によりGAGATGGGTからGAGTATACTに変更した。与えられた制限部位は、HXBc2ゲノムの位置を示す(ロス・アンゼルス・データベース番号付け、ここで位置1は5'LTRの最初の塩基である)。
(i) pSVC21、これは完全感染プロウイルスクローンHXBc2及び複製のSV40オリジンを含む。
(ii) pSVΔP1、主要スプライスドナーとgag開始コドンの間の19bp欠失を有し、その結果、損傷ウイルス複製となるpSVC21の既に記載されたミュータント(16)。
(iii) pSVΔP2、これはスプライスドナーとpSVC21のgag開始コドンとの間の36bp欠失を有する。本欠失は、結果としてよりひどい複製障害となる。
(iv) pSVΔPlΔLTR、Xhal部位(8897)の下流の配列を除去することにより得られたpSVΔPlの非感染誘導体。これは、ウイルス蛋白システムに影響を及ぼすことなく全3'LTRを除く。
(v) pSVΔPlΔenv、env遺伝子中、BglII切片(7041−7621)を欠損しているpSVΔPlの非感染ミュータント。無傷のビリオンを生成するため、本プラスミドは、env発現プラスミドpLenvと同時形質転換され、そしてそれは3'LTRの除去によりpIIIenv3から誘導されたものであり、その結果、下流SV40ポリアデニル化シグナルが用いられる。
puro及びhygro選択可能マーカーを含むベクタープラスミドをエレクトロポレーションによりT細胞系ジャルガット及びジャルガットtatに移入した。ベクター含有細胞の選択のため、プロマイシンをml当り0.5μgで用い、ハイグロマイシンはml当り500μgで用いた。ジャルガットtat細胞は移入neo遺伝子を既に含むので、LCNLベクターはジャルガット細胞だけにエレクトロポレーションを付した。G418は選択のため、ml当り2mgで用いた。移入システムに無傷のベクターが存在することをノーザン及びサウザンプロッティングにより、並びにCAT活性及び適当な薬剤耐性の証明により確認した。
100×(puro試料×plo ref)/(puro ref×POL試料)
を用いて計算した。
(細胞中のパッケージされたFLV×III8ゲノム)/(細胞中のFLV×パッケージされたIII8ゲノム)
を用い計算した。
a 104cpm/μlのRT活性に正常化した
b 3又はそれ以上の実験の意味
c 野生型ウイルスに対応するカプシド化レベル
d ジャルカット標的細胞
e 試験せず
f 検出できず
g LCPL.CX、LCPL.HX、LCPL、PX、LCPL2M
h LCPL.CX、LPL.HX、LPL.PX
b ビリオン中に存在するヘルパーウイルスRNAのパーセントとして表した
★ 形質導入力価から評価された値
ND 検出できず
b 1000cpm/μlのRT活性に正常化した
c ジャルカットtat細胞に関する感染ウイルス力価
d 実施せず
e 適用できず
Claims (9)
- レンチウイルスのパッケージングヌクレオチドに対応するヌクレオチド、異種遺伝子および当該ヌクレオチドと当該異種遺伝子に隣接して、パッケージング、ベクターの標的細胞への逆転写および組み込み並びに異種遺伝子の発現に十分なレンチウイルスLTRの内部および近傍のものに対応する配列を含むベクターであって、前記ヌクレオチドがレンチウイルス5’−リーダー配列とREV−反応部分に少なくとも実質的に対応するものを含むベクター;ただし、上記レンチウイルスはHIVである。
- パッケージングヌクレオチドを含む複製欠失HIV−基本ベクターであって、当該パッケージングヌクレオチドがHIV5’リーダー配列とHIVのcis−作用REV反応要素、異種遺伝子および当該ヌクレオチドと当該異種遺伝子に隣接して、パッケージング、ベクターの標的細胞への逆転写および組み込み並びに異種遺伝子の発現のためのHIV配列を含んでいるベクター。
- さらにgag遺伝子に対応するヌクレオチドを含んでおり、異種遺伝子によって分解されない、請求項1または2に記載のベクター。
- 異種遺伝子に対するプロモータとして、MPMVまたはHIVLTR配列を含む、請求項1〜3のいずれかに記載のベクター。
- 異種遺伝子が治療に有用な生成物のためのものである、請求項1〜4のいずれかに記載のベクター。
- 外来遺伝子の伝達による治療に使用する、請求項5に記載のベクター。
- HIVビリオン産生細胞を1またはそれ以上のパッケージング欠失HIVで形質転換することを含む方法によりHIVビリオンを産生するために使用される、請求項1〜6のいずれかに記載のベクター。
- 哺乳細胞を請求項7の方法によって産生されたHIVビリオンと接触させることを含む、異種遺伝子を哺乳細胞に伝達する方法。
- 遺伝子伝達による治療方法で使用するための請求項7の方法により産生されたベクター。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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GB929204350A GB9204350D0 (en) | 1992-02-28 | 1992-02-28 | Dtv agents |
GB929208489A GB9208489D0 (en) | 1992-04-16 | 1992-04-16 | Ctv agents |
GB929219935A GB9219935D0 (en) | 1992-04-16 | 1992-09-21 | Ctv agents |
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JP51467993A Division JP3765103B2 (ja) | 1992-02-28 | 1993-03-01 | Mpmv及びhivに基づく欠損パッケージング非オンコウイルスベクター |
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JP2004000280A JP2004000280A (ja) | 2004-01-08 |
JP3779966B2 true JP3779966B2 (ja) | 2006-05-31 |
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JP51467993A Expired - Fee Related JP3765103B2 (ja) | 1992-02-28 | 1993-03-01 | Mpmv及びhivに基づく欠損パッケージング非オンコウイルスベクター |
JP2003309935A Expired - Fee Related JP3779966B2 (ja) | 1992-02-28 | 2003-09-02 | Mpmv及びhivに基づく欠損パッケージング非オンコウイルスベクター |
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JP51467993A Expired - Fee Related JP3765103B2 (ja) | 1992-02-28 | 1993-03-01 | Mpmv及びhivに基づく欠損パッケージング非オンコウイルスベクター |
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US (2) | US5747307A (ja) |
EP (2) | EP1262554A3 (ja) |
JP (2) | JP3765103B2 (ja) |
AT (1) | ATE228569T1 (ja) |
AU (1) | AU671101B2 (ja) |
CA (2) | CA2557882C (ja) |
DE (1) | DE69332519T2 (ja) |
DK (1) | DK0630409T3 (ja) |
ES (1) | ES2187504T3 (ja) |
PT (1) | PT630409E (ja) |
WO (1) | WO1993017118A2 (ja) |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2269175A (en) * | 1992-07-31 | 1994-02-02 | Imperial College | Retroviral vectors |
WO1995025806A2 (en) * | 1994-03-24 | 1995-09-28 | Syngenix Limited | Packaging-deficient lentiviruses |
US5714353A (en) * | 1994-05-24 | 1998-02-03 | Research Corporation Technologies, Inc. | Safe vectors for gene therapy |
GB9510272D0 (en) * | 1995-05-22 | 1995-07-19 | Isis Innovation | Retroviral vectors |
US6013516A (en) * | 1995-10-06 | 2000-01-11 | The Salk Institute For Biological Studies | Vector and method of use for nucleic acid delivery to non-dividing cells |
FR2747046B1 (fr) * | 1996-04-05 | 1998-06-19 | Univ Paris Curie | Nouveaux vaccins issus de plasmovirus |
US5994136A (en) * | 1997-12-12 | 1999-11-30 | Cell Genesys, Inc. | Method and means for producing high titer, safe, recombinant lentivirus vectors |
US6218181B1 (en) | 1998-03-18 | 2001-04-17 | The Salk Institute For Biological Studies | Retroviral packaging cell line |
US6797512B1 (en) | 1998-11-13 | 2004-09-28 | Cell Genesys, Inc. | Selection system for generating efficient packaging cells for lentiviral vectors |
DE69936531T2 (de) * | 1998-11-13 | 2008-04-03 | Cell Genesys, Inc., South San Francisco | Auswahlverfahren zur erzeugung effizienter pack-zellen für lentivirale vektoren |
US6790657B1 (en) * | 1999-01-07 | 2004-09-14 | The United States Of America As Represented By The Department Of Health And Human Services | Lentivirus vector system |
AU778698B2 (en) * | 1999-04-29 | 2004-12-16 | Miltenyi Biotec B.V. & Co. KG | Method and means for producing high titer, safe, recombinant lentivirus vectors |
WO2000072886A1 (en) | 1999-05-26 | 2000-12-07 | Dana-Farber Cancer Institute, Inc. | Episomally replicating lentiviral vectors |
US20020164800A1 (en) * | 2001-01-31 | 2002-11-07 | Smith Clayton A. | Retroviral vector |
GB0108065D0 (en) * | 2001-03-30 | 2001-05-23 | Syngenix Ltd | Viral vectors |
DE60233047D1 (de) | 2001-05-14 | 2009-09-03 | Gbp Ip Llc | Lentivirale vektoren kodierend für gerinnungsfaktoren für die gentherapie |
ES2582028T3 (es) * | 2001-06-29 | 2016-09-08 | Sloan-Kettering Institute For Cancer Research | Vector que codifica el gen de la globulina humana y uso del mismo en el tratamiento de hemoglobinopatías |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5604114A (en) * | 1986-05-20 | 1997-02-18 | Dana-Farber Cancer Institute | Cis-acting repression sequences, cis-acting antirepression sequences, vectors, methods of preparation and use |
US5716826A (en) * | 1988-03-21 | 1998-02-10 | Chiron Viagene, Inc. | Recombinant retroviruses |
JPH05501201A (ja) * | 1989-10-16 | 1993-03-11 | ホワイトヘツド・インスチチユート・フオー・バイオメデイカル・リサーチ | 非感染性hiv―1粒子およびそれらの使用 |
US5861282A (en) * | 1989-10-16 | 1999-01-19 | Whitehead Institute For Biomedical Research | Non-infectious HIV particles and uses therefor |
US5981276A (en) * | 1990-06-20 | 1999-11-09 | Dana-Farber Cancer Institute | Vectors containing HIV packaging sequences, packaging defective HIV vectors, and uses thereof |
CA2084659A1 (en) * | 1990-06-20 | 1991-12-21 | Joseph G. Sodroski | Vectors containing hiv packaging sequences, packaging defective hiv vectors, and uses thereof |
US6174666B1 (en) * | 1992-03-27 | 2001-01-16 | The United States Of America As Represented By The Department Of Health And Human Services | Method of eliminating inhibitory/instability regions from mRNA |
-
1993
- 1993-03-01 JP JP51467993A patent/JP3765103B2/ja not_active Expired - Fee Related
- 1993-03-01 ES ES93905485T patent/ES2187504T3/es not_active Expired - Lifetime
- 1993-03-01 AT AT93905485T patent/ATE228569T1/de not_active IP Right Cessation
- 1993-03-01 PT PT93905485T patent/PT630409E/pt unknown
- 1993-03-01 AU AU36394/93A patent/AU671101B2/en not_active Ceased
- 1993-03-01 DK DK93905485T patent/DK0630409T3/da active
- 1993-03-01 EP EP02014431A patent/EP1262554A3/en not_active Withdrawn
- 1993-03-01 DE DE69332519T patent/DE69332519T2/de not_active Expired - Fee Related
- 1993-03-01 EP EP93905485A patent/EP0630409B1/en not_active Expired - Lifetime
- 1993-03-01 CA CA2557882A patent/CA2557882C/en not_active Expired - Lifetime
- 1993-03-01 WO PCT/GB1993/000417 patent/WO1993017118A2/en active IP Right Grant
- 1993-03-01 US US08/295,737 patent/US5747307A/en not_active Expired - Fee Related
- 1993-03-01 CA CA002117607A patent/CA2117607A1/en not_active Abandoned
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1997
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Also Published As
Publication number | Publication date |
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CA2557882C (en) | 2010-05-18 |
AU3639493A (en) | 1993-09-13 |
JP2004000280A (ja) | 2004-01-08 |
ATE228569T1 (de) | 2002-12-15 |
PT630409E (pt) | 2003-04-30 |
WO1993017118A3 (en) | 1993-10-14 |
DE69332519D1 (de) | 2003-01-09 |
EP0630409B1 (en) | 2002-11-27 |
US5747307A (en) | 1998-05-05 |
DK0630409T3 (da) | 2003-03-24 |
CA2557882A1 (en) | 1993-09-02 |
CA2117607A1 (en) | 1993-09-02 |
WO1993017118A2 (en) | 1993-09-02 |
ES2187504T3 (es) | 2003-06-16 |
AU671101B2 (en) | 1996-08-15 |
JP3765103B2 (ja) | 2006-04-12 |
EP1262554A2 (en) | 2002-12-04 |
EP1262554A3 (en) | 2007-08-29 |
JPH07504322A (ja) | 1995-05-18 |
US6294165B1 (en) | 2001-09-25 |
EP0630409A1 (en) | 1994-12-28 |
DE69332519T2 (de) | 2003-07-17 |
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