JP2004524813A - Vectors replicating with improved conditioning, methods for their production and use - Google Patents

Abstract

The present invention provides replication competent vectors or vectors that replicate under improved conditions with improved safety against the production of viruses. Methods for making, propagating and selectively packaging, modifying and using such vectors are also disclosed. Improved helper constructs for use with improved vectors, host cells, pharmaceutical compositions and host cells comprising said vectors, use of vector-containing host cells for screening drugs, and gene function Methods of using the vector to determine The method also includes prophylactic and therapeutic treatment of diseases, especially viral and HIV infections.

Description

【０３２２】
表から見出されるように、細胞残骸の透明化の後、ベクター上清液サンプルにおける合計のタンパク質は３０２７ｍｇであり、そしてその段階でのベクター力価は、５．９×１０^６である。次に、サンプルは、限外濾過による濃縮を受け、この時までに、このサンプルにおける合成タンパク質は７２２ｍｇであり、そしてベクター力価は８．２×１０^７に上昇し、６１．７倍の精製を伴なう。限外濾過による濃縮の後、サンプルをダイアフィルトレーションされ、この時までに、このサンプルにおける合計タンパク質はさらに、２２４ｍｇに低められ、そしてベクター力価は６．８×１０^８／ｍｌに高められ、１６５０倍の精製を伴なう。これは、非ベクター外因性タンパク質が除去され、そしてＶＳＶ−Ｇにより偽性型分類されたレンチウィルスベクターが上記プロトコールを用いて精製され得ることを示す。
【０３２３】
上記のことは、図１６に示されるように、約１０^１０の力価を生成するＨｅｌａ−ｔａｔ細胞系において生成されるベクターと組合して使用され得る。
【０３２４】
例９．
条件付で複製するベクターを偽性型分類するための他のヘルパー構成体は、Ｒｅｖ−依存性制御下にＶＳＶ−Ｇ発現を配置することによって開発されて来た。ＲＲＥ−含有ｍＲＮＡはＲｅｖの不在下で発現されないので、それらは、ｇａｇ／ｐｏｌ， ｐｏｌ及びｅｎｖ遺伝子内に存在するシス抑制配列（ＣＲＳ）の存在のために欠陥性であることが推定された。ＣＲＳは、核におけるｍＲＮＡを閉じ込め、ｍＲＮＡを不安定化するか、又はリボソームとｍＲＮＡとの結合を妨げることが報告されている。従って、ｍＲＮＡスプライシングと組合される、トランスで供給されるＣＲＳ及びＲｅｖの存在は、遺伝子発現を調節するために使用され得る。
【０３２５】
Ｒｅｖ／ＲＲＥ／ＣＲＳシステムを用いて、Ｒｅｖ−依存性態様でＶＳＶ−Ｇ発現を制御した。このアプローチのためのヘルパー構造体は、５’スプライスドナー部位；ＨＩＶ−２ＲＲＥ及び３’スプライス受容体部位（好ましくは、ｔａｔ／ｒｅｖのための）；ＨＩＶ−１に基づくベクターとの組換えの傾向を低めるためのＨＩＶ−２ ｐｏｌ領域からのＣＲＳフラグメント；及びに任意には、いずれかの誘発性プロモーターの代わりの非誘発性プロモーター、例えばＣＭＶ直前（ＩＥ）プロモーターを含む。ＶＳＶ−ＧコードのＲＮＡは、誘発を伴なわないで生成されるが、スプライシングされていないＲＮＡのみがＲｅｖを伴なわないで細胞質に輸送され、そして発現され得るので、Ｒｅｖの存在により調節される。ＶＳＶ−Ｇ発現は、ＲｅｖがｍＲＮＡスプライシング及びＣＲＳにより制御される転写体の核残留を防げるので、Ｒｅｖ介在性のまま存在する。
【０３２６】
好ましくは、スプライスドナー部位は、合成５’β−グロビンドナー部位ではないが、しかし生来のＨＩＶ、又はＨＩＶ類似体、スプライスドナー部位である。ＨＩＶ−２ ＲＲＥは、ＨＩＶ−２ ＲＲＥ， 及びＴａｔ／Ｒｅｖのためのスプライス受容体部位の両者を含む２８０ｂｐのフラグメントであり；そしてＣＲＳはＨＩＶ−２ ｐｏｌ配列からの５５０ｂｐの内部フラグメントである。ＨＩＶ−２からのＣＲＳ及びＲＲＥ要素は、ＰＣＲにより調製され得る。そのようなヘルパー構成体の非制限例は、ｐＣＧＣＲＳＲＲＦ， ｐＣＧＣＲＳＲｚＲＲＥ， 及び図１５Ａ−１５Ｅに示される他のものを包含する。最終プラスミド構成体は、多重制限酵素消化により確かめられた。ｐＥＨ−ＧＰ及びｐＣＭＶ−ＧＰは、ｇａｇ−ｐｏｌポリタンパク質を構成的に発現する構成体であるが、レンチウィルス又はＨＩＶプロモーターを包含する他のプロモーターが、所望により使用され得る。
【０３２７】
図１７は、ＶＳＶ−ＧのＲｅｖ依存性発現のためへの多くの異なったスプライスドナー組合せの使用からの結果を示す。示されるように、テトラサイクリンの除去は、Ｒｅｖ依存性発現をもたらす。
スプライス−ドナー部位組合せ及びコンセンサス配列の追加の例が下記に提供される。すべてが使用され得るが、ＨＩＶ主要、ＨＩＶ−１ｅｎｖ、ＨＩＶ−２主要、及び類似体スプライス−ドナー組合せが好ましい。
【０３２８】

【０３２９】
例 １０．
使用の前、ベクターの貯蔵のための適切な条件を同定するために、種々の貯蔵緩衝液及び条件を試験した。その結果は図１８に示されている。ベクター回収の有意な損失を伴なわないで、−２０℃で及び医薬的許容できる緩衝液においてベクターを貯蔵する能力は、使用の前、ベクターの維持において莫大な利点のものであろう。もちろん、他の医薬的に許容できる組成物における、約−８０℃、約−２０℃、及び約４℃でのベクターの貯蔵はまた容易に実施され得る。
【０３３０】
例 １１．
この例は、本発明の実施への使用のためのキメラＨＩＶ ＣＴＬエピトープのアミノ酸配列を記載する。配列は、翻訳を開始するための第１メチオニン（Ｍ）、続いて、それぞれｐ１７， ｐ２４， ｐ１５， ｐｏｌ， Ｒｅｖ， ｇｐ１２０ｅｎｖ， ｇｐ４１ｅｎｖ及びｎｅｆに対応する種々の連続副配列を含む。
【０３３１】
【表２】

【０３３２】
例 １２．
この例は、上記に記載されるような本発明の態様の要素を提供する。本発明は、発現される１つの遺伝子（又は他の核酸）、及び１又は複数の第１ヌクレオチド配列を含んで成る、条件付で複製するウィルスベクターを包含し、ここで前記ベクターは、（ａ）野生型ウィルス株、ヘルパーウィルス、又はヘルパーベクターとの相補性に基づいてのみ宿主細胞において複製し、それらの個々は前記１又は複数の第１ヌクレオチド配列の存在に対して敏感であり；（ｂ）前記１又は複数の第１ヌクレオチド配列に対して耐性であり、そして（ｃ）複製コンピテントベクターを生成する傾向を低める、１又は複数の置換、挿入又は欠失を含む。
【０３３３】
複製コンピテントベクターを生成する傾向を低めるために、本発明は、ヘルパー構成体、宿主細胞の核酸含有物、又は他の核酸の１又は複数の配列に関しての縮重された配列を含むベクターを提供する。さらに、ヘルパー構成体はまた、ベクター、宿主細胞の核酸含有物又は他の核酸の１又は複数の配列に関しての縮重された配列も含むことができる。
【０３３４】
本発明のベクターはまた、ヘルパー構成体を標的化する１又は複数の配列を含むことができる。そのような配列は、遺伝的抗ウィルス剤を包含し、そして好ましくは、リボザイム又はアンチセンス核酸である（又はコードする）。好ましくはリボザイム及びアンチセンス配列は、標準のＡ−Ｔ、Ａ−Ｕ及びＧ−Ｃ塩基対合の他に、Ｇ−Ｕ塩基対合を通して、それらのそれぞれの標的物との塩基対相補性を受けることができる。同様に、本発明のヘルパー構成体はまた、ベクターを標的化するために同じタイプの配列を含むことができる。
【０３３５】
本発明のヘルパーベクターはまた、ベクターに比較してヘルパーにより発現されるＲＮＡの局在化、又は細胞トラフィキングに影響を及ぼす１又は複数の配列を含むことができる。例えば、ヘルパーベクターは、ベクターに比較して、イントロン、異種ＲＲＥ、又はベクターに比較して異種ｇａｇ配列を含むことができる。ヘルパーベクターはまた、ヘルパー及びベクターＲＮＡがたぶん、同時局在化されるよう、ベクターに比較して、縮重されたｇａｇ及び／又はｐｏｌ配列を含むことができる。ヘルパーベクターはまた、ペッケージング及びＲＲＥシグナル（配列）が、ベクターＲＮＡとのそれらの同時局在化を妨げるためにＲＮＡから除去されるよう、スプライス−ドナー部位を含むことができる。そのようなスプライス−ドナー部位はまた、ヘルパーベクター又は野生型ウィルスに比較して、ベクターの示差的追跡をもたらすためにヘルパーベクターに存在することができる。
【０３３６】
本発明のヘルパーベクターはまた、本発明のベクターを偽性型分類することができる。典型的な例は、ＶＳＶ−Ｇ、ＲＤ１１４、狂犬病ウィルス、ＧＡＬＶ及びキメラ性エンベロープタンパク質を包含する。本発明のヘルパーベクターはまた、好ましくは、ベクターに関して、異種ウィルスに由来する。例としては、ＨＩＶ−２由来のヘルパーベクターである。本発明の方法は、本発明のベクターを複製するためへのそのようなヘルパーベクターの使用を包含する。
【０３３７】
本発明の典型的なベクターは、ＧＦＰ配列の除去又は置換を伴なって、図１Ａ〜１Ｋに示される一連のｐＮ１及びｐＮ２のそれらを包含する。好ましくは、ベクターは、少なくともアンチセンス配列及びｃＰＴ包含配列を保持する。本発明の典型的なヘルパー構成体は、リボザイムを伴なって及び伴なわないで、図６Ａ−６Ｇに示されるような一連のｐＶｉｒＰａｃのそれらを包含する。
【０３３８】
本発明のすべてのベクター及びヘルパーは、上記のようにして、又は必要なら、構成体の他の部分において縮重され得る。好ましい縮重は、ヒト細胞において選択的に使用されるコドンに対してである。ベクター及びヘルパーはまた、それが異なった天然に存在する核酸及び合成核酸に由来するよう、天然においてキメラ性であり得る。しかしながら、特に好ましいベクターは、ＨＩＶ−１由来するか、又はＨＩＶ−１ Ｔａｔタンパク質をコードする。他方では、ベクターは、ヘルパーベクター、野生型ウィルス又は宿主細胞により供給される、ｔａｔタンパク質をコードしない。
【０３３９】
本発明のベクターはまた、好ましくは、ウィルス補助タンパク質をコードせず、又は発現しない。ベクターはまた、宿主細胞に存在する場合、ウィルス感染を低め、最少にするか又は妨げられるか、又は毒性剤に対して前記細胞を耐性にする核酸を含んで成ることができる。そのような核酸の例は、細胞表面タンパク質又は切断されたＣＣＲ５タンパク質、好ましくはＣＣＲ５Δ３２Ｔタンパク質を包含する。他の例は、薬剤又はＤＮＡ損傷剤に対する耐性、好ましくは多重薬剤耐性、又はＭＧＭＴ変異体、特にＧ１５６Ａ変異体をコードする核酸を包含する。追加の例は、トランスドミナント表現型をコードする核酸を包含する。典型的な例は、トランスドミナントＨＩＶ−１ｇａｇ配列である。
【０３４０】
本発明のベクターはまた、異種配列の発現を助ける配列を含むことができる。典型的な例は、インスレーター配列である。ベクターはまた、細胞中へのベクターの形質導入を改良する配列を含むことができる。典型的な例は、本明細書に記載されるｃＰＴを含む５４５塩基対の配列であるが、但しｃＰＴを含むより小さなフラグメント又はより大きなフラグメントでも使用され得る。ベクターはまた、そこに位置する転写結合部位において修飾されたＬＴＲを包含する、遺伝子及び核酸を発現するためにＬＴＲを含むことができる。好ましくは、そのような修飾されたＬＴＲは、レンチウィルスベクターに含まれる。本発明の方法は、そのようなベクターの使用を包含する。
【０３４１】
本発明のベクターはまた、適切に配置されたスプライス部位又はＩＲＥＳ要素を用いて、少なくとも２種の遺伝子を発現することができる。ベクターはまた、形質導入のために標的物又は宿主細胞を刺激する１又は複数のキメラ性エンベロープタンパク質によりウィルス粒子中にパッケージングされ得る。さらに、ベクターは、細胞にパッケージングされ得、ここでヘルパーはタンパク質を含むか、又はヘルパーは遺伝子発現を調節するためにＲｅｖ／ＲＲＥ／ＣＲＳシステムを含む。本発明の方法は、そのようなベクターの使用を包含する。
【０３４２】
本発明のベクターはまた、形質導入されていない細胞に対して通常、毒性でない、投与される剤と組合して、形質導入された細胞を選択的に殺害するために使用され得る。好ましくは、そのようなベクターは、タンパク質よりもむしろ核酸分子の生成により作用する。典型的な例は、抗−ＭＧＭＴアンチセンス又はリボザイム核酸分子の生成である。本発明の方法は、そのようなベクターの使用を包含する。
【０３４３】
本発明はまた、本発明のベクターの貯蔵方法を提供する。
本明細書に引用されるすべての引例、例えば特許、特許出願及び出版物は、引用により本明細書に組み込まれている。本明細書において使用される場合、単数形で表されるすべての用語は、単数形及び複数形の両者を包含するものである。 本発明は好ましい態様に基づいての強調により記載されて来たが、好ましい態様における変動が調製され、そして使用され得、そして本発明が特に本明細書に記載されるよりも他の方法で実施され得ることは、当業者に明らかであろう。本発明は、そのような変動及び他の実施の包含を意図する。従って、本発明は、本発明の範囲内に包含されるすべての修飾を包含する。
【図面の簡単な説明】
【図１】
図１Ａ−１Ｋは、本発明のにより包含される特定の改良された、条件付で複製するベクター：ｐＮ１（ｃＰＴ）ＡＳｅｎｖＧＦＰ（４５２）， ｐＮ１（ｃＰＴ２）ＡＳｅｎｖＧＦＰ， ｐＮ１（ｃＰＴ）ｃＧＦＰ， ｐＮ１ＧＦＰ（ｃＰＴ）Ｔ， ｐＮ１（ｃＰＴ）ＧＦＰＴＡＲ， ｐＮ１ＧＦＰ（ｃＰＴ）ＶＴ， ｐＮ２ＧＦＰ， ｐＮ２ＡＳｅｎｖＧＦＰ（４１８）及びｐＮ２（ｓｐｅ）ＡＳｅｎｖＧＦＰの図である。もちろん、緑色蛍光タンパク質（ＧＦＰ）のためのマーカー遺伝子は、本明細書に記載される用途へのベクターの使用の前、除去され得る。表示：Ｎ１， ｇａｇ／ｐｏｌ配列を有さないが、しかしｇａｇ’又はｇａｇ’’として示されるｇａｇからのパッケージング配列、及びｇａｇ配列のＡＴＧから約４０塩基対の位置に配置される終結コドンを有する、最少ＨＩＶ−１由来のベクター；Ｎ２， ｇａｇ／ｐｏｌ配列を発現できる、ＨＩＶ−１由来のベクター；ＡＳ、アンチセンス；ＡＳｅｎｖ， アンチセンス配向に存在するｅｎｖ配列；ｇａｇ， ｐｏｌ及びｅｎｖ， それぞれウィルスコアー、逆転写酵素及びエンベロープを形成するタンパク質についてのコード配列；ｔａｔ， ｒｅｖ， ｒｒｅ及びｎｅｆ， 追加のウィルス遺伝子；ｃＰＴｃ，最少中心ポリピリミジン系配列；ｃＰＴ，約５４８個の塩基対の大きな挿入体を含む中心ポリピリミジン系；ｃＰＴ２，約４３８個の塩基対の配列を含む中心ポリピリミジン系（ｃＰＴと同様ｇａｇ配列を包含しない）；ｓｐｅ， ｇａｇ／ｐｏｌ配列が翻訳されない；ＧＦＰ、コードされる縁色蛍光タンパク質。
【図２】
図２は、野生型ＨＩＶ Ｕ５ ＲＮＡ配列番号１（Ａ）及び修飾されたｃｒＨＩＶ Ｕ５ ＲＮＡ配列番号（Ｂ）のＤＮＡ配列を示す。番号は、転写から下流の塩基の数を言及する。
【図３】
図３Ａ−３Ｅは、生成されるｃｒベクターの力価に対する、ヘルパーベクター：条件付で複製する（ｃｒ）ベクターの比の効果を示す。図３Ａは、ｐＮ１（ｃＰＴｃ）ＧＦＰについて最高力価をもたらすための１：０．５のモル比を示し；図３Ｂ及び３Ｃは、それぞれｐＮ１（ｃＰＴ）ＧＦＰ及びｐＮ１（ｃＰＴ２）ＡＳｅｎｖＧＦＰについて最高力価を提供するための１：０．７５のモル比を示し；そして図３Ｄ及び３Ｅは、それぞれｐＮ１ｃＧＦＰ及びｐＮ２ｃＧＦＰについての最高力価を提供するための１：１．５のモル比を示す。ベクターは図１に示されているが、但しアンチセンスｅｎｖ配列を欠いているｐＮ１（ｃＰＴ）ＧＦＰ， 及びＧＦＰを発現するために、挿入されたサイトメガロウィルス（ＣＭＶ）プロモーターを有するｐＮ１ｃＧＦＰ又はｐＮ２ｃＧＦＰを除く。図は、少なくとも１．５×１０^７の形質導入単位／ｍｌの力価の生成のための条件が２種のプラスミドシステムにより達成されたことを示す。
【図４】
図４Ａ及び４Ｂは、２種のＨＩＶ−２に基づく条件付で複製するベクター；ｐＳ１ｃＧＦＰ及びｐＳ２ｃＧＦＰの地図を示す。表示ｐＳ１及びｐＳ２は、ｐＮ１及びｐＮ２について上記に記載されるようにｇａｇ／ｐｏｌ配列の不在又は存在を言及する。表示ｃは、ＧＦＰの発現を指図するためのＣＭＶプロモーターの存在を示す。
【図５】
図５Ａ及び５Ｂは、ｐＳ１ｃＧＦＰ及びｐＳ２ｃＧＦＰのパッケージングに対する異なったヘルパーベクター構成体の効果を示す。ヘルパーベクターと条件付で複製するベクターとの間の異なったモル比の効果が、ヘルパーベクターに対する異なったＲＲＥの効果と共に試験された。示されるように、１：１の比のヘルパーベクター：条件付で複製するベクターの使用は、試験された他の比よりも、機能的ベクター粒子の生成で、より効果的であった。
【図６】
図６Ａ−６Ｇは、次の種々ヘルパーベクター構成体、すなわち本発明により包含されるようなｐＶＩＲＰＡＣ−１， ｐＶＩＲＰＡＣ−２， ｐＶＩＲＰＡＣ−１．１Ｒｚ， ｐＶＩＲＰＡＣ−１．２， ＶｉｒＰａｃ１．２Ｒｚ， ｐＶＩＲＰＡＣ−１．２Ｒｚ２及びｐＶＩＲＰＡＣ１．２ＲｚＩｎの構造を示す。Ｒｚは抗−Ｕ５リボザイムの存在を言及し、そして１．１及び１．２は、それぞれＨＩＶ−１及びＨＩＶ−２に由来するＲＲＥを有するヘルパーを言及する。
【図７】
図７は、ウィルスベクターの力価に対する１又は複数のリボザイムの影響を示す。ｐＮ１（ｃＰＴ）ＧＦＰは、ｐＶｉｒＰａｃ１．２（リボザイムを含まない）、ｐＶｉｒＰａｃ１．２ＲｚＩｎ（１つのリボザイム及びイントロンを含む）、ｐＶＰ１．２Ｒｚ（１つのリボザイムを含む）又はｐＶＰ１．２Ｒｚ２（２種のリボザイムを含む）の存在下でＨｅｌａ−ｔａｔ細胞にパッケージングされた。グラフにより示されるように、１つのリボザイム（ｐＶｉｒＰａｃ１．２Ｒｚ）、又はリボザイム、及びヘルパーＲＮＡ（ｐＶｉｒＰａｃ１．２ＲｚＩｎ）の細胞トラフィッキングに影響を及ぼすよう企画されたイントロンの存在は、ベクター力価に対して有意な効果を有さなかった。同時パッケージングされたヘルパー構成体についての力価サンプルのＰＣＲ分析は、リボザイムの不在下での高い同時パッケージングに対するリボザイムの存在下での低い同時パッケージングを示した。インジケーター“ＶｉｒＰａｃ”はまた、“ＶＩＲＰＡＣ”又は“ＶＰ”として示され得る。
【図８】
図８は、Ｔ細胞における野生型ＨＩＶ複製に対する一連のｐＮ１及びｐＮ２からのベクターの阻害効果を示す。Ｔ細胞は、最初にベクターにより形質導入され、そしえ次に、形質導入された１００％の細胞に関して、０．１の多重感染で野生型ウィルスにより攻撃された。ウィルスの複製は、ｐ２４ ＥＬＩＳＡ抗原捕獲アッセイによりアッセイされた。ｐＮ２ＡＳｅｎｖＧＦＰは、野生型ウィルス複製の十分な阻害を示した。
【図９】
図９Ａ及び９Ｂは、ｐＮ１及びｐＮ２に基づくベクターによる野生型ＨＩＶ複製の有力な阻害を示す。図９Ａは、野生型ＨＩＶによる感染に基づく対照腫瘍細胞に比較して、ｐＮ１ＧＦＰ（ｃＰＴ）ＶＴ及びｐＮ２ＡＳｅｎｖＧＦＰによるヒトＴ細胞における野生型ＨＩＶの有力な阻害を示す。図９Ｂは、ｐＮ１（ｃＰＴ）ＡＳｅｎｖＧＦＰ及びｐＮ２ＡＳｅｎｖＧＦＰに関して、Ｔ細胞において類似する結果を示す。
【図１０】
図１０Ａ及び１０Ｂは、形質導入された細胞を選択するためのベクター及びそれらの使用を示す。図１０Ａは使用されるベクターの機構を示し、ここでｐＮ１ＣＭＩＧ及びｐＮ１ＭＣＧは内部ＣＭＶプロモーターを含み、そしてｐＮ１ＭＩＧ及びｐＮ１ＭＩＧ−ＷはＨＩＶ−ＬＴＲプロモーターを通してＭＧＭＴ遺伝子を発現する。図１０Ｂは、上記ベクターにより形質導入され、そして下記例５に記載のようにして、ＢＣ及びＢＣＮＵにより選択されたＳｕｐＴ１細胞の拡張のグラフを示す。
【図１１】
図１１（パネルＡ−Ｆ）は、ＢＧ及びＢＣＮＵによる、形質導入された一次ＣＤ４＋細胞の選択を示す。ｐＮ２ＭＩＧにより形質導入されたＣＤ４^＋細胞が、示される濃度のＢＣＮＵと共に、０， ０．５， ２，５， １０及び１０μＭのＢＧ（それぞれ、パネルＡ−Ｆに示される）において培養された。パネルＦはさらに、形質導入された細胞の１：５倍での希釈、続いて、１０μＭのＢＧ及び示される濃度のＢＣＮＵによる処理の結果を示す。パネルＦにおける３％ＧＦＰ＋細胞の開始レベルは、少なくとも３２倍、９７％に上昇した（これまでに、インビトロでは見出されていない）。
【図１２】
図１２は、ベクター調製物中へのヘルパーＲＮＡの同時パッケージングの防止に対するリボザイムの効果を示す。ウィルス含有上清液が、“ブーム抽出”により抽出され、そしてＤＮアーゼＩ消化、続いて、ヘルパー構成体に見出されるＨＩＶ−１ ｇａｇ−ｐｏｌ領域の定性逆転写酵素−ＰＣＲ（ＲＴ−ＰＣＲ）検出にゆだねられた。示されるように、使用されるヘルパー構成体における１又は２種のリボザイム（ｐＶＰ１．２Ｒｚ又はｐＶＰ１．２Ｒｚ２）の存在は、リボザイム⁻ヘルパー（ｐＶＰ１．２）よりも同時パッケージングされたヘルパーベクターの量を有意に低めた。
【図１３】
図１３Ａ及び１３Ｂは、ＣＤ３４^＋幹細胞から腫瘍細胞をパージする能力を示す。図１３Ａは、１０のＭＯＩで、ｐＮ１（ｃＰＴ）ＡＳｅｎｖＧＦＰが、１回の形質導入の後、ＳｕｐＴ１腫瘍細胞の９８．５２％を形質導入したことを示す。図１３Ｂは、ＣＤ３４^＋細胞の形質導入において、有意な形質導入が、１回の形質導入の後、見出されなかったことを示す。３回の後でのみ、有意に形質導入が観察された。表示：１／２／Ａは、１回の形質導入、２のＭＯＩ及び形質導入されたベクターでのウィルス補助タンパク質の存在を言及し、そして３／５０は、３回の形質導入及び５０のＭＯＩを言及する。
【図１４】
図１４Ａは、示される細胞外、トランスメンブラン及び細胞ドメインを有する、ＶＳＶ−Ｇ野生型、ＲＤ１１４野生型及びキメラエンベロープタンパク質の構造を示す。図１４Ｂは、異なったエンベロープタンパク質（ＶＳＶ−Ｇ野生型、狂犬病ウィルスＧ， ＲＤ１１４野生型及びＲＤ１１４Ｅ、キメラＶＳＶ−Ｇ及びＲＤ１１４構成体）による、ＨＴ１０８０において偽性型分類されたＨＩＶ−１ベクターの力価を示す。
【図１５】
図１５Ａ−１５Ｅは、本発明により包含される特定のパッケージング系構成体の図である：ｐ（ＣＧＣＲＳＲＲＥ）， ｐ（ＴＲＥｔＴＡｐｕｒｏ）， ｐ（ＢＩ−ＲｅｖＴａｔ）， ｐ（ＥＨ−ＧＰ）及びｐ（ＣＭＶ−ＧＰ）。図１５Ｆは、ｒｅｖ依存性ＶＳＶ−Ｇ構成体の構成を示す。
【図１６】
図１６は、Ｈｅｌａ−ｔａｔ細胞における、濃縮の前及び後、細胞製造体当たりのｐＮ１（ｃＰＴ）ＧＦＰベクターの収量を示す。示されるように、その収量は、いずれかの組の条件下で約１０^１０で存続する。
【図１７】
図１７は、ＶＳＶ−Ｇ発現を制御するためにＲｅｖ／ＲＲＥ／ＣＲＳシステムを用いることからの結果を示す。
【図１８】
図１８は、異なった温度での３〜５週間の貯蔵の後、ベクター回収率に対する貯蔵緩衝液の影響を示す。示されるように、Ｄ−ＰＢＳ又はＨＢＳのいずれかにおける、１０％トレハロース又は１０％グルコースの存在は、−２０又は−８０℃での貯蔵の後、ベクターの良好な回収率を提供した。[0001]
TECHNICAL FIELD OF THE INVENTION:
The present invention provides methods for producing, propagating, and selectively packaging, modifying and using conditionally replicating, conditionally replicating and lentiviral vectors. I do. Importantly, the vector has a reduced ability to become replication-competent and, therefore, has increased safety for use. Also determine the appropriate specified nucleotide and amino acid sequences for the vector, pharmaceutical compositions and host cells comprising the vector, the use of such host cells to screen drugs, and gene function. There is provided a method of using said vector to
[0002]
The method also includes prophylactic and therapeutic treatment of diseases, especially viral infections and especially HIV infections. The present invention is also directed to direct and indirect methods for generating novel vaccines, for treating infectious diseases, cancer and other gene-related diseases, and for diagnostic methods. Other methods and compositions of the invention include the use of conditionally replicating and lentiviral vectors for gene therapy and other uses.
[0003]
Background of the Invention:
The expression of the human immunodeficiency virus (HIV), a lentivirus, as a cause of acquired immunodeficiency syndrome (AIDS) has facilitated overresearch into the viral infection cycle and the underlying mechanisms of viral pathogenesis. Studies on those mechanisms have led researchers to continually increase the number of targets for the development of antiviral agents that are effective not only against HIV, but also against HIV products, their genomes and other viruses. Provided to you. Those antivirals, especially those directed against HIV, can be divided into groups depending on the mode of action. Such groups include inhibitors of reverse transcriptase, competitors of virus entry into cells, vaccine and protease inhibitors, and the more recent, referred to herein as "genetic antiviral agents". Include groups.
[0004]
In general, each type of antiviral agent has its own associated advantages and limitations and should be evaluated for the urgency of a particular treatment situation. Antiviral agents, such as zidovudine (3'-azido-3'-deoxythymidine, also known as AZT), protease inhibitors and the like, can be relatively easily delivered into cells of the patient's body, and Has been extensively studied. Although targeting one particular factor in the viral infection cycle, such agents have been found to be relatively ineffective against HIV.
[0005]
This is mainly due to the fact that strains of HIV change rapidly and become resistant to agents with a single position of effect (Richman, AIDS Res. And Hum. Retrovir., 8, 1065). -1071 (1992)). Thus, the problem of genetic variation and rapid mutations in the HIV genome has forced consideration of new antiviral methods to treat HIV infection. Among these strains, genetic antiviral agents are advantageous because they act intracellularly at many different levels.
[0006]
Genetic antiviral agents differ from other therapeutic agents in that they are transferred as molecular entities into target cells that protect cells from viral infection (Baltimore, Nature, 325, 395-396 (1988)). And Dropulic et al., Hum. Gene. Ther., 5, 927-939 (1994)). Genetic antiviral agents can be any gene sequence and include, but are not limited to, antisense molecules, RNA decoys, transdominant variants, interferons, toxins, immunogens and ribozymes . In particular, ribozymes are antisense-like genetic antiviral agents that degrade target RNA, including HIV RNA, in a sequence-specific manner.
[0007]
The specificity of ribozyme-mediated degradation of target RNA suggests a possible use of ribozymes as therapeutic inhibitors of viral replication, including HIV replication. Different types of ribozymes, such as hammerhead and hairpin ribozymes, have been used in anti-HIV methods (eg, US Pat. Nos. 5,144,019, 5,180,818 and 5,272,262). And PCT patent applications WO 94/01549 and WO 93/23569). Both hammerhead and hairpin ribozymes can be constructed to degrade any target RNA containing GUC sequences (Haseloff et al., Nature, 334, 585-591 (1988); Uhlenbeck, Nature, 334, 585 ( 1987); Hampel et al., Nuc. Acids Res., 18, 299-304 (1990); and Simons, Ann. Rev. Biochem., 61, 641-671 (1992)).
[0008]
Generally speaking, hammerhead ribozymes have two types of functional domains, a conserved catalytic domain flanked by two hybridization domains. The hybridization domain binds to sequences surrounding the GUC sequence, and the catalytic domain degrades RNA targets 3 ′ to the GUC sequence (Uhlenbeck (1987), supra; Haseloff et al. (1988), supra; and Symons). (1992), supra).
[0009]
Numerous studies have confirmed that ribozymes may be at least partially effective in inhibiting the growth of HIV in tissue culture cells (eg, Sarver et al., Science, 247, 1222-1225 (1990); Sarver , NIH Res., 5, 63-67 (1993a); Droplic et al., J. Viral., 66, 1432-1441 (1992); Dropulic et al., Methods: Comp. Meth. Enzymol., 5, 42. -49 (1993); Ojwang et al., PNAS, 89, 10802-10806 (1992); Yu et al., PNAS, 90, 6340-6344 (1993); and Weerasinghe et al., J. Viro. ., 65, see 5531-5534 (1991)). In particular, Sarver et al. (1990), supra) show that hammerhead ribozymes designed to degrade within the transcribed region of the HIV gag gene, ie, anti-gag ribozymes, specifically degrade HIV gag RNA in vitro. I have.
[0010]
Furthermore, when cell lines expressing anti-gag ribozyme were challenged with HIV-1, a 50-100 fold inhibition of HIV replication was observed. Similarly, Weerossinghe et al. ((1991), supra) disclose that a retroviral vector encoding a ribozyme designed to degrade within the U5 sequence of HIV-1 RNA can transform HIV-1 transduced cells into transduced cells based on subsequent challenge with HIV. This indicates that resistance is imparted. Different clones of the transduced cells showed different levels of resistance to attack as determined by the promoter system used to drive ribozyme expression, but most ribozyme expressing cell lines Succumbed to HIV expression after a limited time in culture.
[0011]
Transduction of tissue culture cells by provirus into a ribozyme, a ref gene (not essential for virus replication in tissue culture) into which the hybridization domain specific for the U5 region of HIV has been introduced. Has been shown to inhibit virus replication in transduced cells 100-fold compared to cells transduced by wild-type provirus (eg, Dropulic et al. (1992) and (1992) 1993), supra). Similarly, hairpin ribozymes have been transduced with vectors containing the U5 hairpin ribozyme and have been shown to inhibit IHV replication in T-cells challenged with HIV (Ojwang et al. (1992), supra). Other studies have shown that vectors containing ribozymes expressed from the tRNA promoter also inhibit various HIV strains (Yu et al. (1993), supra).
[0012]
Cellular targets of HIV infection (eg, CD4⁺  The supply of ribozymes or other genetic antiviral agents to T-cells and monocytic macrophages) has been a major hurdle for effective genetic therapeutic treatment of AIDS. Current approaches to target cells of the hematopoietic system (ie, primary targets for HIV infection) are based on differentiation, into precursor multifunctional stem cells that give rise to mature T-cells, or On the other hand, it requires the introduction of a therapeutic gene into mature CD4 + T lymphocytes.
[0013]
However, targeting stem cells is problematic because the cells are difficult to culture and transduce in vitro. The targeting of circulating T lymphocytes is also problematic because their cells are so scattered that it is difficult to reach all target cells using current vector delivery systems. In addition, macrophages need to be considered as cellular targets because they are the major reservoir for virus spread to other organs. However, since macrophages are ultimately differentiated and therefore do not undergo cell division, they are not easily transduced by commonly used vectors.
[0014]
Therefore, the preferred current approach to HIV treatment is to specifically interfere with viral replication or prevent the death of infected cells in cells that are susceptible to viral infection (eg, HIV infection). Utilize replication-defective viral vectors and packaging (ie, "helper") cell lines to introduce foreign genes that cause (Buchscher (1993), reviewed above) (eg, Buchscher, JAMA, 269). (22), 2880-2886 (1993); Anderson, Science, 256, 808-813 (1992); Miller, Nature, 357, 455-460 (1992); Mulligan, Science, 260, 926-931 (1993). Friedman, Science, 244, 1275-1281 (1989); and Cournoyer et al., Ann. Rev. Immunol., 11, 297-329 (1993)).
[0015]
Such replication-defective viral vectors contain, in addition to the foreign gene of interest, cis-acting sequences required for viral replication, which are not sequences encoding essential viral proteins. Consequently, such vectors are unable to complete the viral replication cycle and contain the viral gene in its genome, and a helper cell line that continuously expresses the viral gene propagates it. Used for Following the introduction of the replication-defective viral vector into a helper cell line, the proteins required for virus particle formation are supplied to the vector in trans and infect target cells, where virus replication occurs. A vector virus particle is produced that is capable of expressing a gene that prevents infection or causes death of cells infected with the virus.
[0016]
Such replication-defective retroviral vectors are known as adenoviruses and adeno-medullary half viruses, and those retroviral vectors used in HIV gene therapy clinical trials, and in particular, Moloney murine leukemia virus (MuLV). Includes a mouse amphotropic retroviral vector. These defective viral vectors are used to generate CD4 by a genetic antiviral agent, such as an anti-HIV ribozyme.⁺Cells have been used to transduce cells to varying degrees of success (Sarver et al. (1990), supra; Weerasinghe et al. (1991), supra; Dropulic et al. (1993), supra; Ojwang et al. (1992) supra; and Yu et al. (1993) supra). However, those vectors are inherently limited for HIV gene therapy applications.
[0017]
For example, high transduction occasionally results in a rare CD34 that is already almost infected by HIV during the clinical "latent" phase of the disease.⁺Precursor hematopoietic stem cells or widespread target DC⁺ Of particular importance in the treatment of HIV, where the T-cells should be transduced by the vector. However, MuLV vectors are difficult to obtain at high titers and thus result in poor transduction. In addition, long-term expression of transduced DNA, especially after differentiation into mature T lymphocytes,⁺It has never been obtained in precursor stem cells. Furthermore, the use of defective viral vectors requires an ex vivo gene transfer method (see, eg, US Pat. No. 5,399,346) which is expensive and more than the cost of the general subject population.
[0018]
Their shortcomings with regard to the use of currently available vectors for AIDS gene therapy treatment have led researchers to explore new viral vectors. One such vector is HIV itself. HIV vectors have been used for infectivity studies (Page et al., J. Virol., 64, 5270-5276 (1990)), and for the transfer of genes (eg, suicide genes) into CD4 + cells, particularly CD4 + HIV-infected cells. Introduction (e.g., Buchschacher et al., Hum. Gener. Ther., 3, 391-397 (1992); Richardson et al., J. Virol, 67, 3997-4005 (1993); Carroll et al., J. Virol, 68). , 6047-6051 (1994); and Parolin et al., J. Virol., 68, 3888-3895 (1994)). Their research strategy is CD4⁺ The use of HIV vectors to introduce genes into T-cells and monocyte cells.
[0019]
However, to date, these vectors are very complex. The use of these vectors carries the risk of producing wild-type HIV through intracellular recombination. It has been observed that co-transfection / co-infection of defective vector sequences and helper virus results in recombination between homologous regions of the viral genome (Inouc et al., PNAS, 88, 2278-282 (1991)). The observed complementation in vitro indicates that similar replication-defective HIV vectors can recombine in vivo and thus exacerbate pre-existing HIV infection. The fact that the retrovirus packages two RNA genomes in one virion is that the retrovirus carries two viral RNAs to avoid any genetic defects caused by complementation and / or recombination The proposal was led to researchers (Inoue et al. (1991), supra).
[0020]
In addition to the risk of intracellular recombination, thereby resulting in wild-type HIV, HIV vectors have the associated risk of mutations in vivo, which increases the pathogenesis of viral vectors. This is described by Sarver et al. For the development of a second generation recombinant HIV vector that is replication-competent and non-pathogenic. (AIDS Res. And Hum. Retrovir., 9, 483-487 (1993b)). Such vectors continue to replicate in patients compared to preferentially used non-replicating vectors (ie, replication defective vectors), thus providing a constant competition with wild-type HIV. However, to date, no such vector has been obtained.
[0021]
Ideally, the best opportunity to treat an infected individual exists at the time of inoculation, before the virus infects the host. However, this is difficult to achieve unless many individuals fully understand that they have become infected by HIV until the clinical latency of the disease. On this basis, the stage where antiviral intervention is strongly required is during clinical latency. Treatment at this stage will result in a large number of already infected CD4s with the viral genome.⁺It is imperative that the attack provided by lymphocytes be confronted.
[0022]
This is not a boring provocation, as evidenced by the fact that, to date, HIV is an incurable disease and is hardly treatable with currently available treatments. Effective vaccines do not appear to be available soon, and inhibitors of reverse transcriptase and protease have been shown to prevent HIV replication in this tissue culture, but the development of virus resistance in vivo has led to treatment failure. Has led. Thus, HIV gene therapy to date has been 3 × 10⁷These are almost ineffective against most HIV-infected individuals.
[0023]
In view of the above, it has become increasingly important to develop a long-lasting and sustained immunological response to certain pathogens, especially in AIDS and cancer. Survival-attenuated (LA) vaccines using replication-competent but non-pathogenic viruses have been considered (Daniel et al., Science, 258, 1938-1941 (1992)). And Desrosiers, AIDS Res. & Human Retrovir., 10, 331-332 (1994)). However, such non-pathogenic viruses, which differ from their corresponding wild-type virus by deletion of one or more genes, have the following characteristics: (i) the antigen is not persistent (the LA-virus does not replicate efficiently; ), Unable to elicit a protective immune response, or (ii) as indicated by the ability of the LA-virus to cause disease in a young animal model (Baba et al., Science, 267, 1823, 1823-1825 (1995) )), The LA-virus replicates and has other pathogenic potential.
[0024]
For the above reasons, there remains a need for a mode of treatment for the prevention and treatment of viral infections, especially in AIDS and cancer. The present invention provides such other methods by providing a conditionally replicating vector. The present invention also provides additional methods by which such vectors can be used. A helper vector construct that complements the conditionally replicating vector to enable its replication and packaging as viral particles and virions is further provided by the present invention. Such helper vectors are modified so that conditionally replicating vectors are minimized. Various embodiments of such helper-vector constructs are described. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
[0025]
BRIEF SUMMARY OF THE INVENTION:
The present invention provides improved conditionally replicating vectors and improved compositions, and methods of making and using such vectors. Conditionally replicable vectors are characterized by their ability to replicate only in host cells that allow the vector to replicate. The improved vector provided thereby has increased safety by presenting the ability to restore replication at a reduced risk. As such, the vector may also be referred to as a "reduced recombinant" vector.
[0026]
In one aspect of the invention, a vector that replicates under improved conditioning comprises at least one nucleic acid sequence, the presence, transcription or translation of which is derived from the vector in a replication-permissive host cell. It confers selective advantages over the wild-type strain of the virus corresponding to the virus. Preferably, the vector that replicates under improved conditioning comprises at least one nucleic acid sequence that confers a selective advantage on the vector to replication over any other competing genome or genomic element.
[0027]
A genomic element as used herein is not derived from either the vector or any wild-type virus, and may compete with, prevent or affect vector replication in a host cell. Defined as a nucleic acid sequence in a host cell. A genomic element is also not the complete genome, for example, the genome of a host cell or another vector or virus. The selective advantage for replication is conferred by the presence, transcription or translation of said nucleic acid sequence.
[0028]
In another preferred embodiment, the conditionally replicating vector is a retrovirus, especially a lentivirus, and comprises at least one nucleic acid sequence, the presence, transcription or translation of which is determined by the host infected by the vector. The cell confers a selection advantage over cells infected by a wild-type strain of the virus corresponding to the virus from which the vector is derived. On the other hand, the vector is not completely derived from the wild-type strain, but is chimeric, containing components derived from more than one wild-type virus. More than one wild-type virus can be a different (or heterologous) virus, or a different strain or isolate of one virus. Preferably, the nucleic acid sequence can be expressed to provide a protective effect in the host cell. This results in cells being conferred with a survival advantage because the nucleic acid sequence prevents the replication of any infectious virus to levels that cause cell death.
[0029]
Another preferred embodiment is an improved conditionally replicating plasmid vector comprising at least one nucleic acid sequence that confers a selective advantage on replication over the vector over any other competing vector or plasmid molecule. is there. For example, such a vector, when co-localized with the vector, degrades or destroys, or results in, or degrades, a competing vector or plasmid, i. Ribozymes or antisense sequences). If the competition vector and helper are to be destroyed, the vectors of the invention are constructed to include other sequences to provide it with an overall selective advantage for replication.
[0030]
For example, a vector of the present invention can include an antisense sequence that disrupts both a competition vector and a helper, and further, the vector is a vector for which it further conditionally replicates the overall selective advantage. A second 1-nucleotide sequence (e.g., a promoter that produces vector RNA more than competing vector RNA (the copy number of the vector in transduced cells is higher than the competing genome)) or is present in the vector But will have selective advantages for replication as they contain packaging signals that are not present in the helper).
[0031]
Thus, the at least first nucleotide sequence further comprises two first nucleotide sequences (ie, the first 1-nucleotide sequence targets a competition vector and / or a helper, and the second 1-nucleotide sequence). In this embodiment, the replication of our vector of the overall selective advantage is performed because the sequences provide a selective advantage for replication) work together to achieve an overall selective advantage effect. provide. Thus, the synergistic effect of the replication 1st nucleotide sequence is that sufficient selection conditions for a conditionally replicating vector, where any individual 1st nucleotide sequence provides a differential selection over competing genomes. Can be induced.
[0032]
Degradation or disruption of the competing vector or plasmid also reduces the chance of full length recombination with the vector, and thus the vector has "reduced recombination" properties. The vector may be protected from degradation by degenerating its sequence so that it is not targeted by ribozyme or antisense sequences. The vector may be further protected from degradation by constructing the vector or a genomic portion of the vector to specifically track the RNA so that its genomic vector RNA is not significantly degraded by a competing vector or helper.
[0033]
On the other hand, helpers are used to construct a vector genomic RNA that contains a first nucleic acid sequence, for example, that is present only in unspliced but unspliced vector RNA, and to force the helper into the splicesome. It can be constructed similarly or alone by inserting a splicing element. Such non-limiting examples of improved vectors include during generation of viral or plasmid vectors, and cloning or subcloning of nucleic acid sequences into such vectors, as well as mutagenesis, inducible gene expression and the like. It is particularly advantageous when used during their use for things.
[0034]
Pharmaceutical compositions comprising the conditionally replicating vectors of the invention and a pharmaceutically acceptable carrier are also provided by the invention. Also provided is a host cell comprising a conditionally replicating viral vector. A vector, wherein said vector, when present in DNA, comprises SEQ ID NOs: 2,3,4,5,6,14 (where at least one N is mutagenized), 15 and 16 The selected nucleotide sequence, and the vector, when present in RNA, comprises SEQ ID NOs: 2, 3, 4, 5, 6, 14 (where at least one N is mutagenized), 15 And a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of: and 16) is also provided as an isolated and purified nucleic acid molecule as set forth herein. Also provided are methods for constructing vectors with ribozymes, modifying vectors, and methods for propagating and selectively packaging conditionally replicating vectors without using a packaging cell line.
[0035]
In yet another aspect of the present invention, there is provided a method of treating host cells therapeutically and prophylactically for viral infection. In a particularly preferred embodiment, such treatment is effected in said host by imparting a dominant phenotype that inhibits viral infection or by imparting a phenotype that inhibits infection by other viruses. Preferably, the other viruses to be inhibited include a wide range of virus strains.
[0036]
Such methods further include, where appropriate, the use of helper-inducing vectors, also referred to as "helper vectors" or "helper vector constructs", cytotoxic agents, proteins / factors, or protease / reverse transcriptase inhibitors. Can be included. The methods include, for example, to treat diseases (eg, cancer, genetic, infectious, vascular and other diseases), to inhibit viral replication, to perform effective ex vivo and in vivo gene transfer. In addition, it can be used to enable safe vector production, to determine the function of a gene, or to express a gene of interest in a host cell.
[0037]
In yet another aspect of the present invention, there is provided a method of using a host cell comprising a conditionally replicating vector of the present invention to detect an interaction between a drug / agent and a protein. Such methods allow for protein characterization and screening of drugs, agents or other proteins for activity or physical interaction with a given protein encoded by the vector. The method further allows for the identification and characterization of proteins functionally related to a given protein encoded by the vector. If, for example, the encoded protein is a transcription factor, its expression by the vector of the invention allows the identification of genes regulated by the transcription factor.
[0038]
The present invention also provides compositions and conditions for storage of the vector under various conditions prior to their use. Examples of such conditions include storage at -80C, -20C and 4C in the presence of various carriers.
[0039]
A further aspect of the invention reduces, minimizes, or eliminates recombination of helper-vectors with common lentiviral vectors, i.e., replication competent vectors or vectors that conditionally replicate to produce the virus. Modification of the helper-vector construct that acts to Accordingly, the present invention includes a helper-vector that acts to prepare a "reduced recombinant" vector. The helper vectors of the invention also preferably have a conditionally replicating vector titer of 10%.⁷To the level of transduction units / ml or more. Other embodiments include high speed (but not ultra) centrifugation for concentration and purification, and concentration of the vector using chromatography, ultrafiltration and diafiltration methods.
[0040]
The helper vector modifications of the present invention include the insertion of an antisense sequence that degrades or results in disruption or inactivation of a ribozyme, eg, an anti-U5 ribozyme, or a conditionally replicating vector, The helper vector and the conditionally replicating vector are co-localized or co-packaged. In one embodiment, the helper component comprises 10⁷To a total of> 5 transduction units / ml of supernatant, to completely create one plasmid construct to create two plasmid functional vector-helper systems that effectively produce unenriched vectors. Incorporated (see FIG. 3 herein).
[0041]
In another embodiment, the nucleotide sequence of the helper-vector is degenerate to minimize recombination with a conditionally replicating vector. In yet another embodiment, the helper-vector comprises a heterologous trans-acting element for use in packaging a conditionally replicating vector. Examples of such elements include, but are not limited to, vesicular stomatitis virus envelope protein VSV-G, RD114 envelope protein, rabies virus envelope protein, gibbon ape leukemia virus envelope protein. (GALV) and chimeric envelope proteins. Further modifications also include a heterologous rev-responsive element (RRE), a post-transcriptional regulatory element (PRE) or a constitutive transport element (CTE).
[0042]
In yet another embodiment, the helper-vector construct further comprises a splice site, a splice acceptor site, or a site for a degenerate or humanized nucleotide sequence. For example, the splice site may be located such that the packaging signal and / or RRE can be removed from the transcribed RNA by a splicing event. In addition, sites for intron insertion into the vector or helper construct may be used to dimerize, co-localize, or conditionally replicate the vector and helper, or other competing genome or genomic element. Supplied to minimize recombination. Insertion of introns can be directed to result in trafficking of helper RNA into splicesomes and from vector RNA. Surprisingly, it has been found that some helper vector constructs enhance the titer of conditionally replicating vectors.
[0043]
Description of the preferred embodiment:
The present invention provides improved conditionally replicating vectors, lentiviral vectors and uses thereof. In addition to being selectively replicated, the vector contains certain modifications to reduce the tendency for recombination to result in the vector becoming replication competent. Methods for inhibiting viral replication of a wild-type strain and methods for delivering genes into cells comprising such improved vectors are included. The method comprises providing a host, which can be infected, is present at risk of being infected, or is, in fact, infected by a virus of the wild-type strain, and a vector (ie, a nonpathogenic or non-disease-producing condition). Contacting with an improved vector that is propagated only in a host that allows the replication of the (cr) vector to be replicated.
[0044]
As further described herein, a particular purpose of the method is to establish a competitive infection in the host with a non-pathogenic conditionally replicating vector. Generally, the conditionally replicating vector of the present invention comprises at least one nucleic acid sequence that confers a selective advantage for replication and expansion on the conditionally replicating vector as compared to a wild-type virus, and And / or comprises at least one nucleic acid sequence that confers a selective advantage for the propagation of viral particles to a host cell comprising a conditionally replicating vector as compared to a host cell comprising a wild-type virus.
[0045]
In a preferred embodiment of the invention, the vector comprises an HIV sequence and is used for the treatment of an HIV infection. Thus, the vector, or a host cell containing the vector, provides (1) a selective advantage over the wild-type HIV genome for packaging into progeny virions (ie, in cells in which they are both present). And / or (2) providing selective advantages to host cells producing conditionally replicating vectors (virions) with respect to the production of crHIV virions as compared to host cells producing wild-type virus. At least one nucleic acid sequence. One method (without limiting the invention) is to provide the crHIV genome with one or more ribozymes capable of isolating wild-type HIV genomes, thereby providing selective advantages for packaging to those genomes. It is to give.
[0046]
In another aspect of the invention, the vector is conferred with a reduced propensity for recombination with wild-type and helper constructs to provide a replication competent vector. In addition, helper vectors, cells and packaging systems that also contribute to reduced recombination are provided to further reduce the opportunity for productive recombination events, since the vector no longer conditionally replicates.
[0047]
Wild-type virus:
According to the present invention, a "virus" is an infectious agent that contains proteins and nucleic acids and uses the host cell genetic machinery to produce the viral product specified by the viral nucleic acids. "Nucleic acid" refers to DNA or RNA, including synthetic, non-natural or modified nucleotides, which are single-stranded or double-stranded, linear or circular, and optionally can be incorporated into DNA or DNA polymers. Of polymers. DNA polynucleotides are preferably composed of genomic or cDNA sequences.
[0048]
A “wild-type strain of a virus” is a strain that does not include any of the artificial mutagenesis as described herein, ie, any virus that can be isolated from nature. On the other hand, wild-type strains have been cultured in the laboratory, but in addition, in the absence of any other virus, the progeny genome is capable of producing virions, such as those isolated from nature. The virus.
[0049]
For example, the pNL4-3 HIV-1 molecular clone described in the following example is a wild-type strain available from AIDS Research and Reference Reagent Program Catalog through the national Institutes of Health (Adachi. 59, 284-291 (1986)). pPSXB is available from Dr. National Institutes of Health, Bethesda, Maryland. Suresh Arya and from Arya et al. , Human Immunodeficiency virus type 2 lentivirs vectors for gene transfer expression and potential for helper virus-free packing. Hum. Gene Ther. 1998 Jun 10; 9 (9): 1371-80.
[0050]
Generally, the method of the present invention is preferably used to treat a viral disease resulting from a viral infection. If desired, the virus (and the vector as discussed below) is an RNA virus, but can also be a DNA virus. RNA viruses are a diverse group that infect prokaryotes (eg, bacteriophages) and many eukaryotes, eg, mammals and especially humans. Most RNA viruses, at least one family, have double-stranded RNA as genetic material, but have single-stranded RNA as their genetic material.
[0051]
RNA viruses are divided into three main groups: positive-strand viruses (ie, the virus causes the transferred genome to be translated into protein, and the deproteinized nucleic acid to be sufficient to initiate infection). And negative-strand viruses (ie, those whose genome transferred by the virus is complementary to the message sense and should be transcribed by virion-related enzymes before translation occurs), and Single-stranded RNA virus. The method of the present invention is preferably used to treat positive-strand viruses, negative-strand viruses and double-stranded RNA viruses.
[0052]
As used in the present invention, an RNA virus is a Sindbis-like virus (eg, a dogavirus, a bromovirus, a cucumovirus, a dobamovirus, an iral virus, a dobravirus, and a potex virus), a picornavirus-like virus (eg, a picornavirus). , Caliciviruses, comoviruses, nepoviruses and potyviruses), negative chain viruses (eg, paramyxovirus, rhabdovirus, orthomyxovirus, bunyavirus and arenavirus), double-stranded viruses (eg, reovirus and birnavirus) Virus), flavivirus-like viruses (eg, flaviviruses and pestiviruses), retrovirus-like viruses (eg, retroviruses), coronaviruses, and other groups of viruses, such as nodawil Encompasses is, but not limited only to them.
[0053]
Preferred RNA viruses of the present invention are viruses of the Flaviviridae family, for example viruses of the genus Filovirus, and in particular Marburg or Ebola virus. Preferably, the Flaviviridae virus is a Flaviviridae virus, such as yellow fever virus, dengue virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis virus, Rossio virus, tick-borne encephalitis virus and the like. Virus.
Preferred are viruses of the Picomaviridae family, preferably hepatitis A virus (HAV), hepatitis B virus (HBA) or non-A or non-B hepatitis viruses.
[0054]
Another preferred RNA virus is the retroviridae (ie, retrovirus), particularly viruses of the genus or subfamily Oncovirus, spumavirus and lentivirus. The RNA virus of the subfamily Oncoviridae is preferably human T-lymphotropic virus type 1 or 2 (ie, HTLV-1 or HTLV-2) or bovine leukemia virus (BLV), avian leukemia-sarcoma virus. (E.g., Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), avian erythroblastosis virus (AEV) and Rous-related virus (RAV; RAV-0 to RAV-50), mammalian type C Viruses (eg, Moloney murine leukemia virus (MuLV), Harvey murine sarcoma virus (HaMSV), Abelso murine leukemia virus (A-MuLV), AKR-MuLV, feline leukemia virus (FeLV), simian sarcoma virus, reticulovirus ( REV), spleen necrosis virus (SNV), type B virus (eg , Mouse mammary tumor virus (MMTV)), and D-type virus (e.g., Mason-Pfizer monkey virus (MPMV) is and "SAIDS" viruses).
[0055]
Lentiviral subfamily RNA viruses are preferably human immunodeficiency virus types 1 or 2 (ie, HIV-1 or HIV-2; where HIV-1 has previously been lymphadenopathy-associated virus 3 (HTLV). -III) and acquired immunodeficiency syndrome (AIDS) -related virus (ARV)), or HIV-1 or HIV-2 associated with AIDS or AIDS-like disease One virus. The acronym "HIV" or the term "AIDS virus" or "human immunodeficiency virus" is generally used in the present invention to refer to those HIV viruses and HIV-associated and linked viruses. Further, the lentivirus subfamily RNA viruses are preferably Visna / Maedivirus (eg, infecting sheep), feline immunodeficiency virus (FIV), bovine lentivirus, simian immunodeficiency virus (SIV), equine infectious Anemia virus (EIAV) and goat arthritis-encephalitis virus (CAEV).
[0056]
The virus of the present invention is also, if desired, a DNA virus. Preferably, the DNA virus is Epstein-Barr virus, adenovirus, herpes simplex virus, papilloma virus, vaccinia virus and the like.
Many of these viruses are classified as "Biosafety Level 4" (i.e., World Health Organization (WHO) "Danger Group 4"), where the largest contamination facility is required for all laboratory work. You. However, those skilled in the art are familiar with these viruses and can carefully consider this safety for them.
[0057]
A “host cell” can be any cell, and is preferably a eukaryotic cell. Desirably, the host cell is a lymphocyte (eg, a T lymphocyte) or a macrophage (eg, a monocyte macrophage), or a precursor of any of those cells, eg, a hematopoietic stem cell. Preferably, the cell is CD4 on the cell surface.⁺Comprising glycoproteins, ie CD4⁺It is. However, if desired, CD4 infected by the AIDS virus⁺ T lymphocytes have not yet been activated (ie, preferably, expression of nef has not yet occurred, and even more preferably, CD4 gene expression is down-regulated, as discussed further below. Never been regulated).
[0058]
Further, the host cell is preferably a cell that lacks the CD4 marker and can be further infected by the virus of the present invention. Such cells include, but are not limited to: astrocytes, dermal fibroblasts, intestinal epithelial cells, endothelial cells, epithelial cells, dendritic cells, Langerhans cells, monocytes Spheres, hematopoietic stem cells, embryonic stem cells, cells that produce sperm or ova, stromal cells, mucosal cells and the like. Preferably, the host cell is eukaryotic, of a multicellular species (eg, as opposed to a unicellular yeast cell), and more preferably, is a mammalian cell, eg, a human cell.
[0059]
A cell can exist as a single entity or can be part of a large aggregate of cells. Such “large aggregates of cells” include, for example, cell cultures (mixed or pure), tissues (eg, endothelial, epithelial, mucosal or other tissues, such as those containing the CD4-deficient cells described above). , Organs (eg, heart, lung, liver, muscle, gallbladder, bladder, gonads, eyes, and other organs), organ systems (eg, circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, rind system, Or other organ systems), or organisms (eg, birds, mammals, or the like).
[0060]
Preferably, the organ / tissue / cell targeted is circulatory (eg, including, but not limited to, heart, blood vessels, and blood, such as white blood cells and red blood cells), respiratory system (eg, , Nose, pharynx, larynx, trachea, bronchi, bronchiole, lungs and the like), gastrointestinal system (eg, mouth, pharynx, esophagus, stomach, intestine, salivary glands, pancreas, liver, gallbladder, and others), The urinary system (eg, kidney, ureter, bladder, urethra and the like), the nervous system (eg, brain and spine, and certain sensory organs, eg, eyes), and the integumentary system (eg, skin, epithelium, and subcutaneous) Or percutaneous tissue cells).
[0061]
Even more preferably, the targeted cells are selected from the group consisting of heart, blood vessels, lung, liver, gallbladder, bladder and eye cells. The target cells need not be normal cells and can be diseased cells. Such diseased cells can be, but are not limited to, tumor cells, genetically abnormal cells, or abnormal tissue, such as cells near or adjacent to tumor vascular endothelial cells.
[0062]
vector:
A "vector" is a nucleic acid molecule (typically, DNA or RNA) that acts to transfer a passenger nucleic acid sequence (ie, DNA or RNA) into a host cell. The three common types of vectors include plasmids, phages and viruses. Preferably, the vector is a virus containing an encapsulated form of the vector nucleic acid, and a viral particle in which the vector nucleic acid is packaged.
[0063]
Desirably, the vector is not a wild-type strain of the virus, so long as it contains an artificial mutation or modification. Thus, the vector is typically derived from a wild-type virus strain by genetic engineering (ie, by deletion) to include a conditionally replicating virus, as further described herein. Optimally, the viral vector includes a virus strain that is of the same type as the wild-type virus causing the infection to be treated (preferably, one of said wild-type viruses). Thus, preferably, the vector is derived from an RNA virus, even more preferably, the vector is derived from a retrovirus, and optimally, the vector is derived from a human immunodeficiency virus. Such vectors derived from a human immunodeficiency virus are generally referred to herein as "crHIV" vectors.
[0064]
The vector is also preferably a "chimeric vector", eg, a combination of a viral vector and other sequences, eg, an HIV sequence and one or more other viruses (desirably wild-type, including conditionally replicating vectors. (Induced from a virus strain). In particular, the HIV sequence is preferably an adenovirus, an adeno-associated virus, a virus from Alphaviridae, a virus from Flaviviridae, a virus from Hepadnaviridae, an empty virus, a virus from Papovaviridae, Papovaviridae. (Parvoviridae), a virus from Herpesviridae, a virus from Poxviridae, a virus from Paramyxoviridae, a virus from Rhabdoviridae, or a virus from Rhabdobiridae (Rhabdoviridae, from Rhabdaviridae). , For example, onco-retroviral, Spuma- retroviruses and Lenti- modified retroviral (i.e., non-wild-type) may be linked by a sequence of lines. Viruses or virus-like genomes from or related to those virions are also encompassed.
[0065]
A preferred chimeric vector is where a vector sequence from a non-lentivirus (ie, a sequence encoding a protein, fragment or non-coding sequence) is inserted into a lentiviral vector. Preferably, the non-HIV sequences are inserted into an HIV-derived vector.
As encompassed herein, a vector can comprise either DNA or RNA. For example, either DNA or RNA vectors can be used to induce a virus. Similarly, cDNA copies can be produced from the viral RNA genome. On the other hand, cDNA (or viral genomic DNA) components can be transcribed in vitro to produce RNA. Those techniques are well known to those skilled in the art and are also described in the following examples.
[0066]
A "conditionally replicating virus" is a replication-defective virus that is defective only under certain conditions. In particular, a virus can complete its replication cycle in an acceptable host cell, and cannot in a restricted host cell. A "host cell" can be infected or, in fact, can be present before or after infection by a wild-type virus or a virus that is infected by a pseudotype vector. On the other hand, a "host cell" is a cell that encodes a wild-type virus or helper gene product necessary for virus replication. Thus, the conditionally replicating vector of the present invention may only be used on the basis of complementation with a wild-type virus strain (or helper), or if the wild-type virus infects cells containing the vector genome that conditionally replicates, A replicating virus (preferably the same type of virus as the infection being treated).
[0067]
In a preferred embodiment, the vector comprises an RNA virus introduced in the form of DNA (eg, a conditionally replicating HIV virus). This preferred embodiment provides a replicative HIV-1 (crHIV) vector method that confers selective advantages to a non-pathogenic crHIV-1 vector genome over a pathogenic wild-type HIV genome. In particular, in cells containing both wild-type HIV and crHIV genomes, the crHIV RNAs are selected for packaging into virions because they contain ribozymes that degrade wild-type RNAs, for example, but not crHIV RNAs. It has a strategic advantage. Such non-pathogenic crHIV can spread to uninfected cells (eg, CD + cells) that are susceptible to HIV infection in the presence of wild-type helper virus. In this aspect, selective packaging and expansion of crHIV prevents wild-type HIV replication.
[0068]
Further preferred embodiments are any of the viral accessory protein sequences that will render the vector pathogenic, such as, but not limited to, Vif, Vpu, Vpr or Nef, or combinations or fragments thereof. A non-pathogenic crHIV vector that is non-pathogenic because it does not include the combination. On the other hand, the sequence can be present, but is not transcriptionally silent or translated. Optionally, the vector does not include any combination of regulatory protein sequences that will render the vector pathogenic, such as, but not limited to, Tat or Rev, or fragments thereof. On the other hand, those sequences are present, but are not transcriptionally silent or translated.
[0069]
However, the vector is preferably a structural protein sequence (eg, gag, or fragment thereof), an enzyme protein sequence (eg, pol, or fragment thereof), and may be transcriptionally active or silent. And / or any combination of envelope protein sequences (eg, env, or a fragment thereof). Thus, a conditionally replicating vector is able to replicate, but not to a level that is pathogenic in a given host.
[0070]
Alternatively, the vector may comprise a helper component (eg, a helper vector) containing the necessary sequences from the wild-type virus to replicate to a level sufficient to elicit the required therapeutic, prophylactic, or biological effect. Requires the complementarity of Determining the exact combination of proteins or nucleotide sequences present in the vector and helper to provide optimal biological effects involves the addition and subtraction of various combinations of nucleotide sequences into the vector or helper construct. And involves only the direct application of simple screening methods common to those skilled in the art.
[0071]
In addition, the nucleotide sequences can be modified or mutagenized to improve biological effects or to reduce the opportunity for recombination with, for example, helper constructs. Many protocols for modifying or mutagenizing a vector or helper construct to obtain more optimized constructs are well known in the art (eg, Current Protocol, in Molecular Biology, Harcour Brace). Molecular Cloning, Sambrook et al., Cold Spring Harbor Press, 1989; and Song et al., Nature Genetics 25: 436-439, 2000; incorporated herein by reference).
[0072]
The approach enabled by the vector differs from the use of live-attenuated (LA) vaccines using replication-competent viruses lacking accessory proteins (Daniel et al., Science, 258, 1938-2941). (1992); and Desrosiers, AIDS Res. & Human Retrovir., 10, 331-332 (1994)). For the LA vaccine, no effort is made to compensate for the deficiency, which prevents the effective generation of an effective immune response and still maintains safety. For example, multiple deleted LA SIV vaccines cannot elicit an effective immune response, whereas single deleted (Nef-negative) LA SIV vaccines can be pathogenic in young rhesus monkeys. It is known (Baba et al., 1995).
[0073]
As mentioned above, another approach to the LA HIV vaccine provided by the present invention is to use at least two vectors, where at least one vector is a multiple deleted HIV vector. Yes, and the second (helper) vector expresses all accessory proteins except Nef. Thus, the multiple attenuated HIV vectors will replicate in a controlled manner and in a conditional manner without causing disease, while being supplemented by Vif, Vpr and Vpu from the helper vector.
[0074]
The first vector can be configured to include a genetic antivirus to prevent wild-type HIV replication and expansion. On the other hand, such a first vector can be used to elicit an effective immune response against the wild-type virus. A simple screening method is that the actual combination of sequences that are deleted from the first vector, but present in the helper vector, provide an optimal therapeutic or prophylactic response of said first vector, and furthermore It will be apparent to those skilled in the art that it will allow for an assessment that allows for maintenance of safety by being non-pathogenic.
[0075]
A further preferred embodiment of the conditionally replicating vector-helper system uses an HIV-1 vector to express gag, pol, env, tat and rev, and uses Vif, Vpu, Vpr and optionally the Nef gene. Will use an HIV-2 derived helper vector. The present invention is based on the fact that any combination of the above genes arranged in any compatible vector combination, including inverted HIV-1 and HIV-2, or chimeric HIV format, is possible at the same time. Not limited by example.
[0076]
Expression of Tat from the HIV-1 scaffold complements both HIV-1 and HIV-2 LTRs with each other to generate vector particles containing either the HIV-1 vector genome or the HIV-2 vector genome. Transactivate for However, the two genomic RNAs do not dimerize effectively, thus preventing the co-localization of both vector and helper genomes packaged into one virion particle. When co-packaged in the same virion particle, recombination between the vector and the helper genome to generate a replication-competent vector (RCV) results in reverse transcription of the target cell following infection of the particle with the helper genome. While it can occur.
[0077]
To further address the possible generation of co-packaged constructs, the vectors can further include one or more nucleic acid sequences that reduce any recombination risk. For example, the first vector can include a ribozyme (or antisense / ribozyme) specific for helper vector degradation. Co-localization of such vectors in the same cellular, non-cellular or extracellular location results in an inactive genome after recombination and is safe by disrupting the dimerization or co-localization of the vector In order to enhance the production of, or, on the other hand, prevent the production of emerging RCVs, resulting in degradation of the helper vector. This approach can alternatively be modified by including a ribozyme on the helper vector, or can be further improved by including the ribozyme in both vectors to provide additional safety.
[0078]
On the other hand, antisense molecules are used to displace the ribozyme so that the formation of double-stranded RNA hybrids will be rapidly degraded by cellular endonucleases. A further preferred embodiment is that the antisense sequence will be longer than about 16 base positions of the ribozyme RNA used for targeting.
[0079]
In a modified approach, the helper vectors of the above examples are derived from a heterologous virus (eg, but preferably as described above, but preferably from an adenovirus, adeno-associated virus, murine choretrovirus or a non-HIV lentivirus and virus family). ), Whereby the protein can be expressed constitutively. On the other hand, a heterologous helper vector can be constructed using the HIV-LTR for inducible or autoregulated expression of Tat to transactivate HIV-1 vector expression. An advantage provided by a heterologous viral helper vector is the restricted association between the genomes of the first and second vectors, thus further reducing the chance of recombination to generate a potential RCV.
[0080]
In another aspect, the vector comprises an antisense sequence present on or inserted into the helper genome. A non-limiting example is similar to the pN1cptASenv vector, except that the antisense sequence is directed to the gag sequence present on the helper vector in addition to, or instead of, the env sequence present on wild-type HIV. Found by the pN1cptASgag vector. The anti-gag antisense sequence is located upstream of the splice acceptor site located downstream of the RRE sequence present on the vector. Thus, the gag sequence is genomic and non-genomic of the vector RNA, or is packaged only in spliced species. Thus, helper genomes of introns, including helpers, such as those of the VIRPAC system, are selectively targeted to the splicesomes of cells, whereas genomic vector RNA containing anti-gag antisense sequences selectively direct the splicing machinery. Bypass to.
[0081]
Differential trafficking of vector and helper genomes in cells should have minimal effect on vector titers, and if vector and helper genomes are to be unexpectedly co-localized, vector and helper Genomic base-pairing hybridization should result in inactivation or disruption of such vectors and helper genome-containing particles, and should prevent vector-helper recombination to produce RCV.
[0082]
In other embodiments, the helper can be constructed to include an anti-U5 vector antisense sequence directed to a U5 sequence found in a conditionally replicating vector. Without limiting the nature of this aspect of the invention, an anti-U5 antisense sequence may be inserted distal to the helper coding sequence, but before the transcription termination site. Thus, if the vector and helper RNA are unexpectedly co-localized and undergo co-packaging and possibly recombination, the antisense sequence will destroy or inactivate such co-packaged viral particles. And will prevent recombination.
[0083]
In yet another aspect, the helper can include a target sequence for the first nucleotide sequence present on the vector. A non-limiting example is by placing the sense env fragment in a helper of a two-plasmid pair containing the pN1captASenv vector. Thus, if the vector and helper RNA are to be co-localized, the env sense sequence in the helper will undergo base-pairing with the env antisense sequence in the vector to prevent recombination.
[0084]
The examples provided above are not meant to limit the invention to one or two types of genetic antiviral sequences. It will be apparent to those skilled in the art that many genetic antiviral sequences and their cognate target sequences can be inserted into vectors and / or helper constructs. As a further non-limiting example, the pN1cptASgagASenv vector and helper, including the sense gag and env fragment sequences, can be paired with each other to increase safety by reducing the likelihood of co-packaging and recombination to produce RCV. Can be used instead.
[0085]
In another preferred embodiment for the expression of the vector or helper component, said component is transiently expressed. One means of transiently expressing the genome or genomic components of a vector or helper is to construct the vector or helper vector, for example, using an integrase negative Pol gene. Such lentiviral integrase variants are known in the art and have been reported to be non-infectious (Hirsch et al., 1989, Nature 341; 573-574). Thus, vectors and / or helper genomes produced from integrase-negative producer cells do not integrate, but can be expressed in a transient manner to regulate the amount of vector or helper replication. Yet another means for transient expression from either the vector or helper is to destroy the AAT site in the vector LTR. These sites can be responsible for active integration of the reverse transcribed genomic vector DNA into the chromosome of the host cell.
[0086]
A further means of achieving the above uses a fusion protein to package a functional integrase molecule into a viral particle. In this embodiment, the conditionally replicating vector, such as a lentivector or a helper vector, does not encode any integrase, but the functional integrase fusion protein is replaced by another plasmid construct, such as a helper vector, or a helper vector. During production or packaging of the construct, and thus supply of functional integrase activity based on infection, it will be available by expression from the integrated copy in the packaging cell used.
[0087]
On the other hand, the integrase protein is provided in trans form through expression from a helper construct that encodes it. In this aspect of the invention, the conditionally replicating vector or lentivector will not encode integrase. The fusion protein includes, but is not limited to, a vpr-integrase fusion protein that includes a protease cleavage site at the junction between vpr and the integrase amino acid sequence.
[0088]
The above description of the two vector-helper systems should in no way limit the invention to two vector or helper constructs. Any combination of two, three or more vectors and / or helpers can be used to provide the necessary components for the production of the vector. Dividing the required genomic elements into more vectors or helper components will have the effect of increasing safety as multiple vectors and helper genomes are more difficult to produce RCV through recombination . Safety can be further enhanced by configuring them to contain little or no region of homology between the vector and the helper, a further preferred embodiment.
[0089]
Splitting a required genomic element into multiple components is also conditional, as it is rare for more than two genomes to be present simultaneously in a host cell compared to only two. The replication of the replicating vector can be further restricted. The optimal number of vectors and helpers required can be readily determined by simple screening of different combinations, and can vary depending on the particular viral vector system used.
[0090]
One simple and non-limiting means of limiting or eliminating regions of homology between the vector and the helper is known in the art, but by simply degenerating the nucleotide sequence. is there. Techniques for degenerating sequences are known in the art. One preferred method is to human-match the sequence when the therapeutic use is in humans. Codon usage is tabulated for the primers, and Wada et al. , Nucleic Acids Research vol. 18 Supplement: 2367-2411, 1990.
[0091]
Degeneracy can also be used to protect helper constructs from the effects of agents designed to target helper-packaged vectors. This can be achieved simply by degenerating the cognate target sequence found on the helper construct, if present. In addition, the degeneracy of both the vector and the helper construct may be designed to reduce recombination with other viral sequences, such as those in the cell, of other wild-type sequences that may be found simultaneously with the vector or helper sequence.
[0092]
For example, the use of a vector and helper pair in a packaging system based on HIV-1 and HIV-2 can be modified such that the vector and helper pair are degenerate with respect to the endogenous retro-element. Thus, the degeneracy of the nucleotide sequence of either the vector or the helper reduces the co-localization of putative recombination, and therefore, between the vector and the helper genome, or with the vector or genome and the competing genome, For example, it reduces the risk of generating replication competent viruses with the endogenous retro-element.
[0093]
In particular, the crHIV genome is introduced into substantially infected or uninfected cells. Infected cells supply the crHIV genome with the proteins required for the encapsulation and production of progeny virions. The crHIV genome can result from transduction (eg, this can be done by liposome-mediated transduction of crHIV DNA or by using a chimeric viral vector) or by transfection of wild-type HIV-infected cells. It is introduced directly into uninfected cells by infection of crHIV particles.
[0094]
Uninfected cells containing the improved crHIV vector of the present invention do not produce large amounts of crHIV particles that are pathogenic to the host. crHIV vector cells that have not been superinfected with wt-HIV will produce crHIV particles at a level that is somewhat but not pathogenic to the host. Cells containing any of the vectors of the invention can remain susceptible to infection by a wild-type virus that supplies the proteins required for further production of crHIV particles.
[0095]
In this case, the conditionally replicating vector of the present invention in the presence of a complementary wild-type superinfection also functions as one type of "virus-supplying vector", whereby, for example, multiple rounds of crHIV infection ( That is, in the presence of co-infection with wild-type HIV). Such vectors provide one or more viral replications, and thus multiple replications to the level that elicits infection of other cells, or a biological response, such as an immune response, to the viral source. This improvement is used by other vectors, such as standard packaging cell lines, and is not sufficient to elicit a single replication, or an appropriate biological response, or In contrast to those vectors that provide multiple replications that are pathogenic.
[0096]
If desired (eg, to facilitate the use of the vector in vitro), the wild-type viral gene can be co-supplied to cells infected by the conditionally replicating vector. The wild-type viral gene product not only results from co-infection with a wild-type virus strain (or a provirus of a cDNA or RNA virus), but also confers sequence (regulatory or structural) transcription or translation to a host cell. Or by supplying them to cells in the form of their genes subcloned by an expression vector, eg, a helper expression vector ("helper" or "helper vector"), or, on the other hand, the gene The product may be supplied exogenously, ie, by adding the protein product to the cell.
[0097]
For example, a crHIV vector can be constructed that contains all proteins from wild-type HIV, except for the Tat coding sequence. Instead of using a helper expression construct that expresses Tat, the Tat protein itself can be used as a helper. An advantage of using a protein instead of a helper construct is that there is no theoretical possibility to generate a wild-type virus, since the nucleic acid sequence is not present for recombination. Thus, crHIV can be propagated by using the Tat protein rather than by the helper nucleic acid construct itself. Tat1 or Tat2 (corresponding to the first exon of Tat, or the first and second exons, respectively) can be used as a helper. Methods for producing and purifying such proteins are well known in the art.
[0098]
In one preferred embodiment, a chimeric Tat protein is used. Tat is produced in a fusion protein (eg, Tat-VP16) and has been shown to preserve its transactivating activity of the HIV-LTR promoter. Other chimeric Tat proteins can be created to enhance the desired biological effect. For example, if the desired biological effect is to enhance an immune response, a Tat-GM-CSF fusion protein can be constructed. This approach is not limited to one type of chimeric typology, and it is the second chimeric (or chimeric) Tat protein to be added (eg, Tat-IFN-α, Tat-TNF-α, Tat- (IL-4, Tat-G-CSF, TNF-α, GM-CSF, IL-4, G-CSF, IFN-α).
[0099]
It will be readily apparent to one of skill in the art to construct and test other equivalent chimeric proteins and screen them for the ability to enhance the immune response while simultaneously providing helper functions to the vector. . Another preferred embodiment is a Tat-Rev fusion protein construct, or a native HIV Tat-Rev fusion protein, eg, Trev. Thus, the above description of the chimeric Tat protein is not limited to the inclusion of cytokines or cellular proteins, but viral proteins (which produce homologous or heterologous viral fusion proteins) also depend on the desired biological effect. Can be used in the present invention.
[0100]
With respect to a "helper vector", its expression may be cell-specific or non-cell-specific, and may be associated with a conditionally replicating viral vector, as defined herein. It can be introduced into a host cell, and thereby allows for the continuous replication of a conditionally replicating viral vector.
[0101]
A "helper vector" of the invention can provide the necessary gene product for replication, either from a single vector or multiple vectors. Such vectors are preferably used in transient transfection systems. On the other hand, the gene product may also be integrated into the genome of the host cell or cell line. According to the present invention, a single vector or plasmid, which may be mono-, bi- or polycistronic in the host cell, is preferably co-transferred to a helper vector into the same cell and conditionally replicates the vector. Due to the increased vector titer due to the enhanced tendency of the
[0102]
The reduced tendency to co-transfect more than two different vectors into the same cell makes the use of a single helper vector more desirable. The reduction in the number of vectors or plasmids included in the transfection system is also costly if fewer plasmids are produced compared to the three conventional plasmid transfection systems for packaging retroviral constructs. effective.
[0103]
As used in the present invention, "complementarity" refers to non-genetic interactions of viral gene products from different sources in a cell. In particular, with mixed infection, complementation involves an increase in virus yield of one or both parent genomes, and the genotype of the parent genome remains unchanged. Complementarity can be non-allelic (ie, intergenic, where variants that are defective in different functions help each other in viral replication by providing functions that are missing in other viruses), or It can be allelic (ie, endogenous, where both parents are defective in different domains of the multimeric protein).
[0104]
If desired, the cell type that can be transfected with crHIV DNA (ie, as described above, by liposomes, or by using a heterologous vector to produce a chimeric vector) is HIV-infected or infected. Not any of the cells. HIV-infected cells can be activated or remain inactive. When they are activated, they will immediately transcribe wild-type and crHIV RNA, resulting in the selective packaging of crHIV RNA into progeny virions.
[0105]
If the HIV-infected cells are not activated, crHIV DNA is present in them until they are activated (eg, through stimulation with mitogens, antigens and the like) and the crHIV RNA is transferred into progeny virions. Will again bring selective packaging. Activated and unactivated, uninfected cells transfected with crHIV DNA do not produce virions until they are superinfected with wild-type HIV and activated by stimulation. Will again result in the selective packaging of crHIV RNA into progeny virions.
[0106]
One embodiment of a host cell transduced with a vector of the present invention is where said cell contains a maximum number of vector copies that are not significantly toxic to the host cell or host organism containing it. Another aspect of a host cell transduced with a vector is that the cell contains at least one copy of the vector, and preferably contains the minimum number of vector copies required to produce a biological effect. Is the case. Copy number in cells can be determined, for example, by TaqMan PCR, and an acceptable copy number range appropriate for both the desired biological effect and loss of toxicity can be readily determined by one of skill in the art. . Acceptable copy number will vary depending on the cell type to be transduced, the nature of the vector (eg, viral origin) and factors including the desired biological effect, but will be readily determined by routine screening by those skilled in the art. Can be determined.
[0107]
Superinfection of cells containing the crHIV genome (as a result of transfection or infection) exists because the crHIV genome does not encode viral proteins (eg, env and net) that prevent superinfection. The resulting crHIV virions can infect uninfected cells. Because viral particles contain reverse transcriptase molecules that all HIV particles carry, so that they can create a DNA provirus from their genomic RNA. This step is called reverse transcription. As soon as crHIV virions infect uninfected cells, they undergo reverse transcription and produce provirus from their genomic RNA.
[0108]
Thus, those cells correspond to those uninfected cells directly transduced by crHIV DNA. They are unable to produce crHIV particles until their cells have been superinfected with wild-type HIV and activated, and then again, selective packaging of crHIV RNA into progeny virions. crHIV particles can also infect some cells that have already been infected by HIV (eg, Yunoki et al., Arch. Virol. 116, 143-158 (1991); Winslow et al., Virol., 196, 849-854 (1993); Chen et al., Nuc. Acids Res., 20, 4581-4589 (1992); and Kim et al., AIDS Res. & Hum. Retrovir., 9, 875-882 (1993). checking).
[0109]
However, those HIV-infected cells should not express proteins that down-regulate CD4 expression. This would protect crHIV virions from infection of those cells. Activated, HIV-infected cells generally down-regulate CD4 expression. Thus, non-activated, HIV-infected cells are substantially susceptible to crHIV superinfection and may therefore be another source for crHIV particle production.
[0110]
With respect to the preferred crHIV vectors of the present invention, said vectors comprise sequences required for RNA transcription, tRNA primer binding, dimerization and packaging, and prevent superinfection with wild-type HIV Lacks or comprises sequences encoding proteins (eg, the nef or nev proteins), but they are functional proteins, since their expression appears to be "siren" Not transcribed or translated.
[0111]
Even more preferably, the vector lacks a region or sequence encoding a region of wild-type HIV from within the gag coding sequence to the nef gene. However, optimally, the vector comprises a rev response element (RRE) into which the vector is cloned, in the region of the deletion or some other convenient region. Such preferred HIV vectors are "lacking the region, or sequences encoding the region, as long as the vector is administered as described above, either in its RNA manifestation or, alternatively, as DNA. "It is said.
[0112]
According to the present invention, in order to achieve favorable complementation in a system utilizing a helper vector with a conditionally replicating vector, it is necessary for viral packaging, but from a conditionally replicating vector. Any viral components that are not expressed should be supplied by a helper vector. Thus, as long as multiple different vectors have sufficient complement of the necessary viral components, the composition of each vector will undergo many hypermutations. Each of these hypermutations is encompassed within the present invention. By way of example, the gag and pol genes required for convenient packaging and replication are both integrated on a helper vector or a conditionally replicating vector, or the two genes are in one of the individual vectors. Above can be individually present.
[0113]
In addition, a single helper vector construct may contain the gag / pal and env sequences, including heterologous env sequences, under the control of a set of transcriptional regulatory elements, including a promoter, or under the control of separate promoter elements. Both can be included. For example, LTRs can be used to express gene products with the vectors of the present invention. The use of different promoters, including inducible promoters, allows for the possibility of differential regulation of gag / pol and env sequences. Thus, env sequences, particularly heterologous env sequences, such as the VSV-G envelope sequence, can be expressed at high levels.
[0114]
Vector construction is well known to those skilled in the art. These techniques can be used to construct both conditionally replicating vectors and helper vectors. Thus, as used herein, the term "vector" can refer to both vector types. Furthermore, the term vector does not necessarily need to be a conditionally replicating vector, but encompasses any lentiviral vector. Such a vector may also be referred to as a genetic lentiviral vector. However, in a preferred embodiment, the vector is a conditionally replicating lentiviral vector.
[0115]
For example, and as described in Example 1, DNA specification of an RNA virus, such as HIV, uses restriction enzymes to cut the HIV coding sequence from within the gag coding region to the U3 region following the nef gene. Is decomposed. A cloning cassette consisting of a polylinker containing multiple restriction sites is inserted into the region of the deletion before ligation to provide convenient restriction sites for cloning into the vector. The DNA fragment containing the RRE is subcloned into one of those sites. The resulting vector produces a truncated gag transcript and does not produce wild-type Gag protein or any other wild-type HIV protein. In addition, the vector need not express the truncated gag protein, as long as the gag translation initiation sequence can be mutagenized to prevent its translation.
[0116]
Using the same approach, crHIV sequences can be ligated to other sequences, such as those of a virus or other vector, to derive a chimeric vector, as described above. For example, crHIV sequences can be linked to those of a Sindbis virus, AAV, adenovirus or an omnidirectional retrovirus. Additional viral sequences that can be used include those of the herpes, pox and other viruses described herein, including any virus that can be used to supply crHIV sequences. Such chimeric vectors can be introduced into cells using a coupled viral mechanism for cell entry (eg, receptor-mediated endocytosis for adenovirus), or other means, eg, using liposomes. .
[0117]
Preferably, according to the present invention, the vector (ie, the conditionally replicating virus, which is preferably a crHIV vector) comprises at least one nucleic acid, whose possession (ie, presence, transcription or translation) is , Confer selective benefits. There are two types of such nucleic acid sequences that are designed for inclusion in a vector: (1) the nucleic acid sequence, whose possession is optimally the wild-type virus strain (ie, preferably, Confer selective advantages for viral replication and expansion to vectors comprising such sequences over wild-type strains from which the vectors are derived and do not contain said sequences; and
[0118]
(2) the nucleic acid sequence, possession thereof, optimally enhances cell presence, enhances vector particle production and / or proliferation, and enhances apoptosis, to achieve the desired prophylactic, therapeutic or biological result. A wild-type virus strain (ie, preferably, a vector (e.g., a vector (B)) that enhances the production of crHIV vector virions from crHIV vector-producing cells, including, And also a helper expression vector that promotes vector replication and / or function in an uninfected host cell) and is compared to cells infected or uninfected by a wild-type strain that does not contain the sequence). Thus, it confers a selective advantage on cells infected with a vector comprising said sequence.
[0119]
Individual of those sequences, or of many of these sequences, ie alone or in combination with another factor, promotes the propagation of the vector and / or has favorable prophylactic, therapeutic and / or biological consequences. A sequence that promotes a particular host cell function to allow for the sequence may be included in the vector in the absence or presence of other sequences, ie, the vector may comprise "at least one nucleic acid sequence" and "at least one additional The nucleic acid sequence of
[0120]
A third type of nucleic acid sequence is a sequence that confers a phenotype based on the host cell, which reduces, minimizes, or prevents viral infection. Such nucleic acid sequences are those sequences that, when expressed, encode a gene product that protects the host cell from viral infection. For example, individuals homozygous for the Δ32 allele of the CCR5 protein will lack major co-receptors for HIV-1 entry into cells expressing the CCR5 protein truncated on their cell surface. Because of the loss, they are protected against HIV-1 infection. Studies on heterozygous individuals have shown that the Δ32 allele has a transdominant effect on wild-type (wt) CCR5 cell surface expression. This may be due to the ability of the Δ32 allele to duplex with the wt protein and prevent its correct expression on the cell surface.
[0121]
The Δ32 allele contains a deletion of 32 nucleotides that causes a frameshift mutation to result in a truncated CCR5 protein. The present invention produces 31 amino acids at the C-terminus of the Δ32 allele, which is not present in the wt protein and, when present on the cell surface, can produce an unwanted immune response. Involves the expression of a truncated CCR5Δ32 mutant protein (CCR5Δ32T), which experimentally lacks 31 amino acids of antigenicity. This protein retains its ability to confer a transdominant effect to provide protection to cells for HIV infection. The sequence encoding CCR5Δ32T is readily generated by introducing a stop codon immediately after the last amino acid common to both wt CCR5 and CCRR5Δ32 (after tyrosine at position 184).
[0122]
In a further aspect, another third type of nucleic acid sequence is a sequence that further enhances the conferred phenotype that reduces, minimizes, or prevents viral infection. Such a nucleic acid sequence can encode, for example, a genetic antiviral agent targeted to a cellular gene. For example, a vector expresses the CCR5Δ32T mutant protein and is further targeted to a wild-type CCR5 molecule, from the U1 promoter and U1 snRNA (described in US Pat. No. 5,814,500). Expresses the antisense or ribozyme molecule contained therein. An antisense region targeting wild-type CCR5 renders the transdominant mutant RNA resistant to the effects of anti-CCR5 antisense or ribozyme molecules, but with the correct amino acid sequence to produce a functional CCR5Δ32T protein. It is degenerated by a vector carrying the CCR5Δ32T sequence so that it can be translated.
[0123]
The above method of co-expressing a ribozyme or antisense targeted to a gene product and degenerating the gene at the targeted site to prevent ribozyme or antisense binding or degradation comprises CCR5, Or, furthermore, it is not limited to molecules that minimize viral replication. Another non-limiting example for therapeutic use is the targeting of oncogene proteins, such as the Bcr-Abl fusion protein, a pathogen for Chronic Myelogenous Leukemia.
[0124]
Vector constructs containing the Bcr or Ab1 gene, or both, can co-express those genes with the expression of an antisense or ribozyme targeted at either site along the Bcr-Ab1 transcription. did it. Thus, the abnormal Bcr-Ab1 fusion transcript is destroyed, whereas native Bcr or Ab1, or both, are expressed to rescue the abnormal defect. Thus, Bcr or Ab1 genes have selective advantages for amplification over wild-type or abnormal Bcr-Abl fusion genes. Preferred vectors express the Bcr and / or Ab1 construct from their native promoter, whereas the anti-Bcr-Abl ribozyme or antisense will be expressed from the U1 promoter and will be contained within the U1 snRNA sequence. .
[0125]
The Bcr-Abl example above does not limit the invention to targeting oncogenes, and this approach may be useful if any of the expressed abnormal genes, unwanted genes, or even desired genes of interest are of interest. It will be apparent to those skilled in the art that it can be used for therapeutic or prophylactic replacement. The target gene can be homologous or heterologous to a modified gene that is resistant to the effects of the genetic antiviral molecule. Other disease states that can be targeted by this method will be apparent to those skilled in the art. For example, molecular targets for treating blood disorders according to the practice of the invention disclosed herein are described in “The Molecular Basis of Blood Diseases” (Stamatoyanopoulos, Majorus, Perlmutter & Varnus, 3).^rd ed, Philadelphia WB Saunders Co. , Copyrighted 1987, 1994 and 2001; all are incorporated herein by reference).
[0126]
The present invention should not be limited to clinical use. The approach can be used to determine the function of a gene sequence of interest. For example, a modified gene sequence of interest in a domain of interest may have an antisense or target that inhibits or destroys the expression of any unmodified gene sequence by targeting the corresponding unmodified region. It can be co-expressed with a ribozyme. Thus, the modified gene sequence of interest has a selective advantage with respect to amplified expression over the unmodified gene sequence, and the modified gene sequence or the modified gene sequence according to assay methods known to those of skill in the art. It allows the function of the encoded gene product to be determined.
[0127]
"Nucleic acid" is as described above. In particular, a “nucleic acid sequence” refers to any gene or coding sequence (ie, DNA or RNA) of substantially any size (ie, of course, limited by any packaging constraints imposed by the vector). ), Which possess optional advantages, as further defined herein. A "gene" is any nucleic acid sequence that encodes a protein or a nascent mRNA molecule, whether or not the sequence is transcribed and / or translated. A gene comprises coding and non-coding sequences (eg, regulatory sequences), but "coding sequence" does not include any non-coding DNA.
[0128]
1. The nucleic acid sequence, its possession, confers a selective advantage in a host cell on a wild-type virus strain, or a vector comprising such a sequence, over competing genomic sequences that prevent or affect vector amplification.
[0129]
A nucleic acid sequence that confers a selective advantage on a vector in a host cell over a wild-type virus strain is preferably a nucleic acid sequence that selectively produces or selectively produces viral particles propagated from a vector as compared to viral particles propagated from a wild-type virus. Any array that allows for packaging. Such sequences include, but are not limited to, sequences that result in an increased number of vector genomes produced intracellularly as compared to the wild-type genome, and antiviral nucleic acid sequences. Such sequences also include, but are not limited to, sequences that result in an increase in the number, nature or state of vectors produced in a cell as compared to a wild-type genome that is not due to a homologous wild-type virus.
[0130]
Said sequence that confers a selective advantage in a host cell on a vector containing a nucleic acid sequence of the first category compared to a wild-type virus strain is a sequence such as a promoter. A "promoter" is a sequence that directs the binding of RNA polymerase, and thereby promotes RNA synthesis, and comprises one or more enhancers. An "enhancer" is a cis-acting element that stimulates or inhibits the transcription of an adjacent gene. Enhancers that inhibit transcription are called "silencers". The silencer can also be an “insulator” element, as found at the DNase-sensitive site of the chicken erythroblast insulator element. An enhancer is a promoter (also referred to as a "promoter element") in that it functions in any orientation and over a distance of several kilobase pairs (kb) from a position downstream of the transcribed region. And DNA-binding sites for sequence-specific DNA binding proteins found only in
[0131]
Thus, and preferably, the promoter of a conditionally replicating HIV vector (eg, a long terminal repeat (LTR)) is modified such that the vector is more responsive to certain cytokines than a wild-type HIV strain. Is done. For example, modified HIV promoters are available that exhibit enhanced transcriptional activity in the presence of interleukin-2. The introduction of this promoter into the vector and the introduction of the vector into wild-type HIV-infected cells are preferably with enhanced production and packaging of progeny virions from the vector genome as compared to the wild-type HIV genome Bringing
[0132]
Other cytokines and / or chemokines (including, but not limited to, interferon-α, tumor necrosis factor-α, RANTES and the like) also select for virions encoded by the vector. Can be used to facilitate strategic packaging. The arrangement of elements that render the HIV-LTR responsive to cytokines does not in any way limit the scope of the invention to such embodiments. Either nucleotide sequence can be inserted into the HIV-LTR to modify this promoter response. A series of factors that bind to DNA sites that can be inserted in the HIV-LTR are described in http://transfac.com, accessed September 8, 2000, which is incorporated herein by reference. gbf. de / tRANSFAC / lists / browse. html. A series of DNA binding factors for humanity is available at http://transfac.com, accessed September 8, 2000, also incorporated herein by reference. gbf. de / TRANSFAC / lists / factor / species / humanhomesapiens. html.
[0133]
Similarly, a silencer or insulator can also be inserted into the promoter or enhancer of a native or modified retroviral LTR, such as the HIV-LTR, to strongly regulate expression from this promoter. In some cases, insertion of the element into the HIV-LTR can place the vector with selective disadvantages for infection by the wild-type virus. However, the application of this type of vector is not to compete with the wild-type virus for packaging into progeny virions, but rather is interesting, for example, to inhibit HIV replication without competition ( Or "payload gene") nucleic acid sequences. In this case, the first nucleic acid sequence does not provide a selective advantage to the vector, but should very minimally inhibit recombination between the vector and the helper during the step of vector generation.
[0134]
The second category of nucleic acid sequences that confers selective advantages on vectors containing the second category of nucleic acid sequences as compared to a wild-type virus strain include antiviral nucleic acid sequences as preferred nucleic acid sequences. “Antiviral agents” are categorized by their mode of action, eg, inhibitors of reverse transcriptase, competitors for virus entry into cells, vaccines, protease inhibitors and genetic antiviral agents. “Genetical antiviral agents” are transferred into cells and to their intracellular targets, either directly (ie, when introduced intracellularly) or to either RNA or proteins. DNA or RNA molecules that affect them after their conversion (Dropulic et al., (1994), reviewed supra).
[0135]
Genetic antiviral sequences are also preferred nucleic acid sequences. Genetic antiviral agents include, but are not limited to, antisense molecules, RNA decoys, transdominant variants, toxins, modifiers and modulators of RNA and protein splicing, EGS (alone , Or by direct binding to M1, U1, PRE or CTE elements), immunogens, RNA1 (see Zamore et al., Cell 101: 25-33, 2000), modifies splicing, or Regulatory nucleotide sequences or molecules, constitutive transport elements (CTEs), nuclear import and export factors, DNA integration factors, RNA stabilizing or destabilizing elements, post-transcriptional regulatory elements (PREs), proteins that interfere with viral replication, interferons, Toxin, immunogen (any immunologically relevant (A complete antibody, single-chain antibody or ligand-indicating molecule), a ribozyme, or any element that affects any point of vector or wild-type virus replication.
[0136]
Desirably, the genetic antiviral agents are antisense molecules, transdominant variants, immunogens and ribozymes. Thus, a preferred nucleic acid sequence that confers a selective advantage on a vector over a wild-type virus strain is the sequence of a genetic antiviral agent selected from the group consisting of antisense molecules, immunogens and ribozymes.
[0137]
Genetic antiviral agents as used in the present invention are limited to only targeting viral sequences, especially if they are not placed in a conditionally replicating viral vector. As a non-limiting example, a genetic antiviral sequence can be placed on a lentiviral vector such that the agent is directed to a viral or non-viral target, such as a cell, bacterial or parasitic target. In this case, the lentiviral vector comprises an agent of interest operably linked to the unmodified or modified HIV-LTR (to the expressed unspliced or spliced RNA). And further comprise a genetic antiviral agent or sequence linked to a promoter sequence that is not operably linked to the HIV-LTR. Preferred sequences are antisense or ribozymes that chimerize with snRNA, preferably with U1, U2, U3, U4, U5 or U6 snRNA.
[0138]
Also, the genetic antiviral agent is not limited to a single or single type sequence present in either the vector or the helper sequence. The multiple agents can be simultaneously present in the vector or helper construct, located remotely, or preferably linked in tandem. Preferably, the plurality of different genetic antiviral agents are linked in tandem. Additional genetic antiviral agents for use in the present invention are any agents that link two different types of genetic antiviral agents.
[0139]
An “antisense molecule” is a molecule that mirrors based on the base pairing rules and the availability of “wobble” base pairs, ie, short segments of the gene whose expression is blocked. Antisense molecules directed against HIV hybridize to wild-type HIV RNA, allowing its selective degeneracy by cellular nucleases. The antisense molecule is preferably about 20 to about 200 base pairs in length, preferably about 20 to about 50 base pairs in length, and optimally no more than 25 base pairs in length. DNA oligonucleotide. An antisense molecule can be expressed from crHIV RNA that selectively binds to, and thereby binds, genomic wild-type RNA and provides crHIV RNA with selective advantages for packaging into progeny virions.
[0140]
Antisense molecules expressed in a vector as RNA are preferably at least 20 base pairs to any size, but preferably up to 2000 base pairs in length, more preferably about 50-500 base pairs. Base pair length. Such antisense molecules preferably bind to genomic wild-type RNA and do not bind to vector RNA, providing a vector with selective advantages over wild-type virus. HIV-derived vectors supply, for example, the envelope region (env), Tat or Rev protein. HIV-derived vectors include, for example, an envelope region (env), a Tat or Rev protein region, an auxiliary gene region (Viv, Nef, Vpr, Vpu), and a 3 'end to an NsiI restriction enzyme site on pNL4-3. And the pol region which does not include the 545 base region in the pol as shown below.
[0141]
"Immunogen" refers to any immunologically related gene and its encoded gene product. Traditionally, the term "immunogen" has been used in connection with adjuvants used for vaccines, but the terms "immunity-" and "-gen" have been used to describe immunologically relevant genes and their encoded gene products. Used to refer to. For example, the immunogen can encode a single-chain antibody (scAb) directed to a viral structural protein, or it can encode an antibody that is extracellularly secreted by the cell. The immunogen is transferred as a nucleic acid and expressed intracellularly. Similarly, the immunogen can also encode any antigen, surface protein (including those that are classically restricted), or display-like antibodies that facilitate vector and / or host cell selection.
[0142]
In a preferred vector, the nucleic acid sequence comprises a scAb coding sequence that binds to a wild-type HIV Rev protein. This preferably prevents mutation of the Rev protein by effecting its suppression in the endoplasmic reticulum. In particular, the Rev protein, alone or in combination with other cellular factors, binds to RRE (a highly structured cis-acting RNA target sequence for Rev, called a Rev responsive element), and The unspliced and solely spliced HIV RNA from the nucleus is then transported to the cytoplasm by oligomerizing by surrounding the HIV RNA. HIV RNA complexed with Rev is transported into the cytoplasm and bypasses the cellular splicing machinery. Thus, if wild-type Rev does not bind to wild-type RRE, wild-type HIV RNA is transported into the cytoplasm and is not encapsulated in progeny virions.
[0143]
Optimally, the vector comprising the scAb nucleic acid sequence further comprises a modified RRE sequence and encodes a mutagenized Rev protein that recognizes a modified but not wild-type RRE. . Thus, in cells containing wild-type HIV and a vector comprising the scAb nucleic acid sequence, the vector is preferably packaged in virions. A similar method is preferably used in which the protein of the wild-type HIV matrix or nucleocapsid (ie, or any protein involved in the entrapping and influencing protein / RNA interaction of the viral RNA) is the target of the scAb. .
[0144]
“Ribozymes” are antisense molecules with catalytic activity, ie, instead of binding RNA and inhibiting translation, ribozymes bind RNA and reduce the site-specific degradation of the bound RNA molecule. Bring. In general, there are four ribozyme groups: Tetrahimera group I intervening sequences, ESG (external guide sequence) and hammerhead and hairpin ribozymes. However, additional catalytic motifs are also present in other RNA molecules, such as ribosomal RNA in hepatitis delta virus and fungal mitochondria.
[0145]
Preferred ribozymes have a catalytic domain in which the 3′-nucleotide NUH sequence (where N is any nucleotide (ie, G, A, U and C) and H is any of A, C or U) ) Is a ribozyme that degrades. However, as long as the sequence most effectively degraded by such ribozymes is a GUC site, preferably the NUH sequence comprises a GUC site.
[0146]
Desirably, such ribozymes degrade in the region of the wild-type virus strain or its transcript, but not in the region of the vector or its transcript. Ribozymes degrade viruses or their transcripts in such a manner that such a virus or vector can be either RNA or DNA, as previously described. By "in the field" degradation is meant degradation in the targeted area, ie, preferably the area of the virus that is required for virus propagation. If desired, the vector was modified such that this particular region to be targeted (ie, if present in the vector at all) was degraded by the ribozyme. Optionally, the ribozyme is capable of degrading the vector, as long as the degradation is not in a region required for the growth of the virus, eg, crHIV particles.
[0147]
Optimally, the ribozyme is encoded by a sequence selected from the group consisting of SEQ ID NO: 3 (ie, CACACAACACTGATGAGGCCGAAAGGCCGAAACGGGCACA) and SEQ ID NO: 4 (ie, ATCTCTTAGTCTGATGAGGCCGAAAGGCCGAAACCAGAGTC). SEQ ID NO: 3 comprises a ribozyme targeted to the +115 site (ie, by the number of bases downstream from the start of transcription) of the wild-type HIV U5 region, whereas SEQ ID NO: 4 comprises a ribozyme of the wild-type HIV U5 region. Comprising a ribozyme targeted at the +133 site.
[0148]
Such ribozymes may be used as long as the vector U5 sequence is modified by in vitro site-directed mutagenesis, as known in the art and as described in Example 1. It can be degraded in its transcript, but not in the vector genome (or its transcript). In particular, the vector sequences are preferably those wherein the vector is SEQ ID NO: 2 (ie, GTGTCCCCACTGTTGTGTGACTCTGGCAGCTAGAGAAC); Induced), comprising a sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 16.
[0149]
In the form of RNA, the vector is preferably selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14 (where at least one N is mutagenized), SEQ ID NO: 15 and SEQ ID NO: 16 Comprising a sequence encoded by the rendered sequence. In contrast, wild-type HIV comprises the U5 sequence encoded by the sequence of SEQ ID NO: 1 (ie, GTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATC). A comparison to the entire modified and wild type U5 sequence (in the form of DNA) is shown in FIG.
[0150]
In addition, viruses and especially other ribozymes targeted to other regions of the HIV genome can be used alone or in combination. For example, ribozymes can be found in other RNA sequences required for viral replication, such as in reverse transcriptase, protease, or transactivator proteins, in Rev, or other necessary as described. It can be broken down within the sequence. Preferably, the vector comprises a plurality of ribozymes targeted, for example, to a plurality of sites. In such cases, similar sequences in the vector may require site-directed mutagenesis, or some other method known in the art, to derive a vector that is resistant to such ribozyme degradation. Modified by means.
[0151]
If the vector is a human immunodeficiency virus, preferably the vector lacks the tat gene and its 5 'splice site, and comprises in its position a ternary anti-Tat ribozyme cassette, wherein said ternary The catalytic domain in each ribozyme of the ribozyme cassette degrades different sites on the wild-type human immunodeficiency virus nucleic acid molecule, especially at tat. Preferably, the catalytic domain of an individual ribozyme degrades the nucleotide sequence in the region of the nucleic acid molecule of the wild-type human immunodeficiency virus where there is no ribozyme-sensitive counterpart in the vector itself.
[0152]
Further aspects of ribozymes as genetic antiviral agents are available not only by Watson-Crick base pairing but also by ribozymes that target sequences by GU wobble base pairing. Previous studies have shown that GU fluctuations in double-stranded RNA have characteristics similar to Watson-Sockock base pairing. Given the high mutation rate, the ability to design ribozymes targeting multiple strains provides a dramatic advantage to the present invention, even in highly conserved regions, i.e., in HIV producing different HIV strains. I do.
[0153]
For example, a highly conserved target region in the Tat gene, which can be used as a target sequence for a ribozyme of the present invention, has either "G" or "A" in 40 well-defined HIV-1 strains. Have. Thus, ribozymes targeting this region will include "U" instead of "C" in the appropriate targeting sequence to allow for pairing with either "G" or "A" residues. Can be planned. This can be compared to the use of "C" for a targeting sequence that reduces the ability of the ribozyme to target HIV-1 strains having "A" at their corresponding positions. This approach, using "U" where the G, A mutation is present at the target position for ribozyme recognition, significantly enhances the utility of the ribozyme of the present invention.
[0154]
A further aspect for practicing the disclosed invention uses a degenerate ribozyme cassette (containing more than one ribozyme in tandem ligation) so that it is not a directly repeated sequence in a vector. That is. The presence of degenerate sequences prevents deletion of repeated sequences, as observed in retroviral vectors and other nucleic acids. Thus, ribozyme cassettes composed of direct repeat sequences arranged in tandem can be deleted during multiple replication cycles. Degenerate ribozyme sequences can be used to overcome this problem.
[0155]
This is because sequences in the hammerhead ribozyme ocatalytic domain can be replaced by other nucleotides based on wobble base pairing without significant loss of activity. Mutagenesis of the hammerhead ribozyme has been performed and the sites that can be degenerate have been determined (Ruffner DE, Stormo GD, Uhlenbeck OC, Sequence requirements of the hammerhead RNAi techie gaseline biotechnology, ref. Nov. 27; 29 (47): 10695-702). Another means of reducing the likelihood of deletion is by directing individual ribozymes in the cassate to target different sites on the target RNA. Thus, the cassette will not include the presence of direct repeats.
[0156]
A "transdominant" or "dominant negative" nucleic acid sequence confers its encoded phenotype, even in the presence of a wild-type (wt) sequence. The discussion of truncated CCR5 protein (above) is one example. Other examples include any mutant retroviral or lentiviral sequence that confers a transdominant phenotype. Examples include the rev and gag genes.
[0157]
One example for selective advantage, and for use in providing inhibition of infectious wild-type virus, is trans-translation, which has been shown to inhibit HIV-1 replication, perhaps by preventing capsid assembly. For the dominant gag sequence. Accordingly, such sequences are preferably used in combination with non-HIV-1 based retroviral vectors to allow their replication and packaging without inhibition from the transdominant sequence. .
[0158]
For example, a retroviral vector containing the HIV-2 gag and pol genes can include a transdominant HIV-1 gag sequence. Such vectors can be replicated and packaged through complementation with the appropriate HIV-2 helper vector construct. However, after growth in cells susceptible to HIV-1 infection, the transdominant gag sequence is available for HIV-1 infection-based expression to block the transfer of the HIV-1 virus. However, retroviral vectors encoding the HIV-2 gag and pol genes are still available to encapsulate and transfer the vector.
[0159]
2. The possession of a nucleic acid sequence, which confers a selective advantage on cells infected or uninfected by a vector comprising said sequence, as compared to cells infected by a wild-type virus strain.
[0160]
A nucleic acid sequence that confers a selective advantage on cells containing a vector comprising the sequence over cells containing or lacking the wild-type virus strain (ie, lacking the sequence) is preferably wild-type. Any sequence that allows the virus particle (ie, a crHIV virus particle) to survive and grow by a cell containing the vector, as compared to a cell containing the virus, or a cell without the nucleic acid sequence. Such a sequence may be any sequence that allows avoidance of destruction by the cell or a vector contained therein, a sequence that promotes cell viability, a sequence that induces apoptosis, a sequence that promotes protein production, or Includes, but is not limited to, sequences that promote function or targeting.
[0161]
For example, preferably, such a nucleic acid sequence contained on a vector encodes a gene for multiple drug resistance (eg, Ueda et al., Biochem. Biophys. Res. Commun., 141, 956-962 (1986). Ueda et al., J. Biol. Chem., 262,505-508 (1987); and Ueda et al., PNAS, 84, 3004-3008 (1987)). In the presence of added cytotoxic drugs (eg, as used for cancer chemotherapy), this allows for the presence of cells containing the vector, but where a wild-type virus, eg, HIV, The containing cells are not. Such cytotoxic drugs include actinomycin D, vincristine sulfate, vincristine sulfate, BCNU (with or without BG conditioning), daunomycin, adriamycin, VP-16 and AMSA. They are not limited to them.
[0162]
Another example of such a nucleic acid sequence is O⁶-Methylguanine-DNA-methyltransferase (MGMT). MGMT can be used to protect hematopoietic precursors from the toxicity of alkylating agents. Wild-type MGMT provides O⁶-Inhibited by benzylguanine (BG). MGMT variants that are resistant to BG-mediated inactivation and that can be protected against alkylating agents can be conferred on any cell type by the present invention. An example of such a mutant is G156A MGMT.
[0163]
For example, hematopoietic progenitor cells transduced with such mutants throughout the present invention are BG and O⁶-May be selected based on the administration of a guanine methylating agent or a chloroethylating agent. Examples of such methylating agents for use in the present invention include, but are not limited to: clinically relevant temozolomide, nitrosoureas, tetrazines, triazines, dacabazines. , Temozolomide, streptozotocin, procarbazine. Examples of chloroethylating agents include, but are not limited to, BCNU (1,3-bis (2-chloroethyl) -1-nitrosourea), CCNU (3-cyclohexyl-1-). Chloroethyl-nitrosourea), ACNU (1- (4-amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloro) -3-nitrosourea), and MeCCNU (1- (2-chloroethyl)- 3- (4-methylchlorohexyl) -1-nitrosourea, preferably the alkylating agent is BCNU.
[0164]
BCNU has been shown to form permanent covalent interstrand DNA crosslinking damage in both quiescent and cell cycle cells. Those damages are cytotoxic to cells during DNA replication. MGMT is a major mechanism of DNA repair of BCNU damage. Given its toxicity to quiescent cells, selection with the MGMT mutant provided by the lentiviral vector of the present invention will also result in cell cycle G₀But can allow selection for totipotent stem cells (HSCs) that circulate intermittently for the generation of more stem cells and blood progenitor cells. Stem cells are generally resistant to retroviral transduction, but they₀If not present alone in the cycling state, they can be transduced with a retroviral vector.
[0165]
Like oncolytic retroviruses, the lentiviral vectors of the invention can be used to transduce hematopoietic stem cells, such as hematopoietic ELTC-IC and NOD-SCID or SCID-hu repopulated cells that can be assayed. However, unlike oncolytic retroviruses, the cytokine conditions required to transduce stem cells without their differentiation are such that lentiviruses (eg, HIV) do not actively divide at the time of infection May prefer lentiviral vectors because they can infect cells. HSC selection does not allow other means to select for cell cycle hematopoietic precursors. Thus, selection mediated by the MGMT mutant and the lentiviral vectors of the present invention may enhance the presence of transduced totipotent HSCs without the need for extended drug administration for selection. it can.
[0166]
The above discussion is discussed in hematopoietic cells in vitro and ex vivo, as well as in vivo. Thus, in addition to transducing cells in vivo with the MGMT variant, the hematopoietic cells of interest are transduced ex vivo, followed by drug treatment ex vivo or in vivo. Such ex vivo treated cells can then be injected into a subject, optionally as part of a repopulation of bone marrow with the transduced cells.
[0167]
Prior to the above implementation, the alkylating agent BCNU was also used in HIV-positive subjects for HIV-infected CD4⁺Can be used to generally reduce cells. As mentioned above, BCNU (after exhaustion of endogenous MGMT) forms cytotoxic damage in both quiescent and proliferating cells. Such damage forms even without forced depletion of MGMT when the drug lowers the level of functional AGT protein. Repetitive histological treatment with BCNU causes cumulative myelosuppression and pancytopenia. Thus, in retrovirally infected subjects, eg, HIV-infected patients, administration of BCNU at low doses to avoid myelosuppression desirably lowers the overall CD4 + cell population, and Reduce the load. This approach can be followed by an injection of hematopoietic precursors, as discussed above.
[0168]
Since BCNU can act in a “stealth” manner by forming cytotoxic lesions, even in resting cells, this approach has the added advantage of reducing the need for repeated processing. Having. Of course, this approach can be combined with the use of BG to enhance the effects of BCNU. On the other hand, this approach involves transduced CD4⁺It can be used in conjunction with the introduction of a MGMT variant with a vector of the invention to provide protection for cells.
[0169]
This latter means, as discussed above, can be independent of or exist with the treatment of hematopoietic cells. Therefore, a preferred embodiment of the present invention is to express a mutated MGMT (second nucleic acid) by a vector containing an anti-HIV antisense or ribozyme (first nucleic acid sequence), resulting in wild-type HIV infection, respectively. Provides selective advantages over isolated cells and over wild-type HIV virus. In a further preferred embodiment, the vector may express the MGMT gene (or the second nucleic acid sequence) from the HIV-LTR promoter as a spliced mRNA.
[0170]
Ultimately, the vector will express an anti-HIV ribozyme or antisense sequence, but the present invention provides those skilled in the art with the ability to easily incorporate any inhibitory nucleotide sequence into the vector for a particular therapeutic, prophylactic or biological effect. Is not limited. A preferred method of expressing the inhibitory sequence is to include it in the U1 snRNA / promoter cassette, as described by Dietz (US Pat. No. 5,814,500).
[0171]
On the other hand, such nucleic acid sequences may desirably contain the sequence of the mutated (ie, mutated) protease (or the sequence encoding it) and the mutated (ie, mutated) reverse transcriptase. A sequence selected from the group consisting of sequences (or sequences encoding the same). Preferably, the mutagenized reverse transcriptase is constructed to be resistant to nucleoside and non-nucleoside reverse transcriptase inhibitors, and the mutagenized protease is resistant to commonly used protease inhibitors. Is constructed to be
[0172]
Administration of those protease or reverse transcriptase inhibitors to the host along with the vector is used to select for cells that produce the vector, as opposed to cells that produce wild-type virus. Similarly, this approach is modified for use with any agent that inhibits viral replication, such that the virus can be mutagenized to avoid inhibition. Thus, for HIV treatment, the selective nucleic acid sequence incorporated into the vector preferably comprises the mutated HIV sequence. However, optimally, those sequences do not prevent superinfection with wild-type HIV.
[0173]
Preferably, the vector is one of those set forth above, and in particular, the improved conditionally replicating vector set forth in FIGS. 1A-1K. Of course, when used in the methods of the invention, the GFP coding sequence can be deleted or replaced by another sequence. The GFP coding sequence is present in those typical vector embodiments as a marker or indicator of the presence of the vector or gene expression from the vector.
[0174]
Preferred vectors of the present invention can include the various elements described in this disclosure. In particular, lentiviral vectors can lack the tat gene and can include at least one antisense sequence. In addition, the vector can include one sequence, such as the central polypyrimidine system of HIV, or a large nucleic acid fragment containing it.
[0175]
Optimally, the vector is compatible with the cell into which it is introduced, for example, and may confer expression on a cell of a nucleic acid sequence encoded by the vector. If desired, the vector comprises an origin of replication functional in the cell. Optimally, when the nucleic acid sequence is transferred in the form of its DNA coding sequence (eg, in the form of a complete gene comprising its own promoter), the vector also directs the expression of the coding sequence. And include a promoter operably linked to the coding sequence. A coding sequence is "operably linked" to a promoter if the promoter is capable of directing transcription of the coding sequence (e.g., both the coding sequence and the promoter together make up the native or recombinant gene). ).
[0176]
According to the recombinant vectors of the present invention, preferably all suitable transcription (eg, start and stop signals), translation (eg, ribosome entry or binding sites and the like), processing signals (eg, splices, if necessary, Donor or acceptor sites, and polyadenylation signals), translocation, assembly, integration sites, ribonucleic acid complex entry, stability and translocation elements (either in cis or trans) can be any gene or coding sequence, It is correctly placed on the vector so that it is properly transcribed (and / or translated, if desired) in the cell into which it is introduced. Such signal manipulation to ensure proper expression in a host is well within the knowledge and expertise of those skilled in the art.
[0177]
Preferably, the vector contains a Pol sequence, defined as an integrated copy of the vector in the host cell genome, that stably enhances transduction. As a central DNA flap (178 base pair fragment from positions 4793-4971 on pLAI3, corresponding to positions 4775-4735 on pNL4-3), see Zennow et al. , (Cell 101: 173-185 (2000)) have been reported to enhance transduction efficiency, but are insufficient to significantly increase the efficiency of stable transduction. Was found. The present invention states that although this small fragment is not sufficient to increase the efficiency of stable transduction, as described in US Pat. No. 5,885,806, a large 545 base pair descending fragment (pNL4-3) At positions 4551 to 5096) or still larger fragments containing them include the discovery that they could enhance stable transduction as part of the present invention. A stable increase in transduction efficiency was detected by GFP expression and FACS analysis of the integrated copy of the vector genome, and Taqman analysis.
[0178]
A further means of increasing transduction efficiency is a co-pending U.S. patent application filed Aug. 31, 2000, US Patent Application No. 397272000400 (still filed), which is incorporated herein by reference. It is described in.
[0179]
The viral vectors used in the present invention can also result from "pseudotyped" formation, wherein co-infection of cells with different viruses is carried out in the outer layer containing one or more envelope proteins of another virus. To generate progeny virions that contain the genome of one virus that is encapsulated in the virus. This phenomenon is caused by the co-transfection or co-infection of packaging cells with both the viral vector of interest and the genetic material encoding at least one envelope protein or cell surface molecule of another virus, resulting in a "pseudo- Type "virions" have been used to package viral vectors of interest. See U.S. Patent No. 5,512,421.
[0180]
Such a mixed virus can be neutralized by antisera against one or more heterologous envelope proteins used. One virus commonly used for pseudotyping is the rhabdovirus vesicular stomatitis virus (VSV). The use of pseudotyping broadens the host cell range of the virus by including elements of the virus entry mechanism of the heterologous virus of the invention used. Pseudotyping of viral vectors and VSV for use in the present invention results in viral particles containing viral vector nucleic acids encapsulated in nucleocapsids surrounded by a membrane containing the VSV G protein.
[0181]
The nucleotide capsid preferably contains a protein normally associated with a viral vector. The surrounding VSV G protein-containing membrane forms part of the viral particle based on its appearance from the cells used to package the viral vector. Examples of packaging cells are described in US Pat. No. 5,739,018. In a preferred embodiment of the invention, the viral particles are attributable to HIV and are pseudotyped by the VSV G protein. Pseudotyped viral particles containing the VSV G protein are able to infect a variety of cell types with higher efficiencies than ambiotropic viral vectors. The range of host cells includes mammalian and non-mammalian species, such as humans, rodents, fish, amphibians and insects.
[0182]
Preferably, the vector also comprises some means by which said vector or its contained subcloned sequence is identified and selected. Vector identification and / or selection is achieved using various approaches known to those skilled in the art. For example, a vector containing a particular gene or coding sequence is identified by hybridization, the presence or absence of a so-called "marker" gene function encoded by a marker gene present on the vector, and / or the expression of the particular sequence.
[0183]
In the first approach, the presence of a particular sequence in the vector is detected by hybridization (eg, by DNA-DNA hybridization) with a probe comprising a sequence that is homologous to the appropriate sequence. . In a second approach, the recombinant vector / host system is used to generate certain marker gene functions, such as resistance to antibiotics, thymidine kinase activity, and the like, caused by specific genes encoding those functions present on the vector. Identified and selected based on the presence or absence of the same. In a third approach, a vector is identified by assaying for a particular gene product encoded by the vector. Such assays are based on the physical, immunological or functional properties of the gene product.
[0184]
Accordingly, the present invention also comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 15, 15, 16, 17 and 18 when it is DNA, and the sequence when it is RNA. A vector comprising a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of Nos. 4, 5, 6, 7 is provided.
[0185]
The present invention further provides a method of producing a vector which is derived from a wild-type human immunodeficiency virus and is capable of replicating a ribozyme only in a host cell which is permissible for replication of said vector. Ribozymes contained within or encoded by the vector degrade the nucleic acid of the wild-type human immunodeficiency virus, but do not degrade the vector itself and its transcript. The method is derived from a wild-type human immunodeficiency virus and is capable of replicating only in a host cell that is permissible for vector replication, and a ribozyme whose catalytic domain degrades the nucleic acid of the wild-type human immunodeficiency virus, However, if present, it comprises obtaining the vector, which does not degrade the vector itself and its transcript) or the nucleic acid sequence encoding it can be incorporated into the vector.
[0186]
In such a method, the nucleotide sequence comprising or encoding the U5 sequence of the wild-type human immunodeficiency virus is deleted from the vector, and if the vector is DNA, SEQ ID NOs: 2,5,5. 6, 14 (where at least one N has been mutagenized), a nucleotide sequence selected from the group consisting of 15 and 16, and if the vector is RNA, SEQ ID NOs: 2, 5, 6, 14 ( Wherein at least one N has been mutagenized), replaced by a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of 15 and 16. Preferably, the vector replicates more than once in a host cell allowing the vector to replicate.
[0187]
A method for modifying a vector is also provided by the present invention. The method obtains one vector and, if the vector is DNA, SEQ ID NOs: 2, 3, 4, 5, 6, 14, where at least one N has been mutagenized, 15 And 16, if the vector is RNA, selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, 15, 16, 16, 17 and 18. Introducing the nucleotide sequence encoded by the nucleotide sequence into the vector.
[0188]
The present invention further provides a method of propagating and selectively packaging a vector that replicates conditionally without using a packaging cell line. The method is a conditionally replicating vector, a vector of the same type as the conditionally replicating vector, and which differs from the conditionally replicating vector by being wild-type with respect to its replication ability. Contacting a cell that can be infected by one vector; subsequently contacting the cell with another vector; and then contacting the cell under conditions that support the growth of the conditionally replicating vector. Culturing. Helper vectors, discussed above and below in more detail, are one such complementary vector.
[0189]
A DNA molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 6, 14, wherein at least one N has been mutagenized, 15 and 16, and SEQ ID NOs: 2, 6 Isolated and purified nucleic acid molecule selected from the group consisting of an RNA molecule comprising a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of: 7, 7, 15, 16, 17, 17 and 18; Also provided.
[0190]
how to use:
The vector is preferably introduced into a host cell for prevention and treatment of viral infection, for ease of vector maintenance, and for other reasons. Accordingly, the present invention provides a host cell comprising the vector of the present invention. The isolation of host cells and / or the maintenance of such cells or cell lines derived therefrom in culture is routine and the person skilled in the art is well familiar.
[0191]
In particular, a conditionally replicating viral vector, or preferably a lentiviral vector, as described above, is preferably for prophylactic and therapeutic treatment of a viral infection, preferably wherein said infection is a wild-type virus, preferably a wild-type virus. It is used when it is from an RNA virus, even more preferably a wild type retrovirus and optimally wild type HIV.
[0192]
The method is performed only in host cells that can be infected by the wild-type virus and in host cells that are permissive for replication of the vector, whose presence, transcription or translation, inhibits replication of the wild-type virus strain in the host cell. Contacting with a conditionally replicating vector capable of replicating. Desirably, the vector replicates more than once and comprises at least one nucleic acid sequence, whose possession (ie, presence, transcription or translation) is optimally the strain from which the vector is derived. Vectors confer selective advantages on host cells over wild-type virus strains.
[0193]
According to this method, the nucleic acid sequence preferably comprises a genetic antiviral agent which advantageously affects the replication and / or expression of a virus other than said vector, or comprises a nucleotide sequence encoding it. Become. If desired, the genetic antiviral agent is selected from the group consisting of an antisense molecule, a ribozyme and an immunogen. Optimally, the genetic antiviral agent is a ribozyme, and preferably the catalytic domain degrades 3 'nucleotide NUH sequences (i.e., especially GUC sequences). Optionally, the ribozyme is at least partially encoded by a sequence selected from the group consisting of SEQ ID NOs: 3 and 4.
[0194]
Desirably, the ribozyme degrades in the region of the wild-type virus strain or its transcript, but not in the region of the vector or its transcript. Preferably, this is for the reason that the wild-type virus strain comprises the sequence encoded by SEQ ID NO: 1, whereas when the vector is DNA, SEQ ID NOs: 2, 5, 6, 14, 14 (where SEQ ID NOs: 2, 5, 6, 14 where at least one N is mutagenized and is a nucleotide sequence selected from the group consisting of 15 and 16; Induced nucleotide sequence), a nucleotide sequence encoded by a nucleotide sequence selected from the group consisting of 15 and 16.
[0195]
A method wherein the vector comprises at least one nucleic acid sequence is also optionally carried out, wherein the possession (ie presence, transcription or translation) is optimally the wild-type virus strain from which the vector is derived Confers a selective advantage on host cells infected with the vector over cells infected with the vector. In this regard, the vector may comprise at least one nucleic acid sequence that confers a selective advantage on a host cell infected by the virus, and a selective advantage of the vector over a wild-type strain of a virus corresponding to the virus from which the vector is derived. At least one nucleic acid sequence that provides
[0196]
Therefore, a method in which the nucleic acid sequence comprises a nucleotide sequence encoding a multidrug resistance gene is preferably performed. On the other hand, if the nucleic acid sequence is treated prophylactically or therapeutically and the viral infection is a retrovirus, the nucleotide sequence encoding the mutated (mutated) protease and the mutated (mutated) A method comprising a nucleotide sequence encoding a reverse transcriptase is performed.
The method preferably further comprises administering to the host cell an agent selected from the group consisting of a cytotoxic agent, a protease inhibitor and a reverser enzyme inhibitor (ie, in addition to administering the vector).
[0197]
Thus, the vectors are not only useful for treating viral infections therapeutically, but also for protecting potential host cells from viral infections, i.e. methods for treating viral infections therapeutically or of interest. It can be used according to "vaccination" against viruses, such as RNA viruses, especially retroviruses, such as HIV. The method substantially inhibits replication of the wild-type virus strain before the host cell contacts the wild-type virus strain. In this regard, the vector may comprise or encode a protein that inhibits superinfection with a wild-type virus. The method comprises contacting a host cell with a conditionally replicating vector as described above, and a "helper expression vector", ie, a viral genome that facilitates replication of the "vector" in an uninfected host. Comprising.
[0198]
Said conditionally replicating vector comprises selective advantages for packaging and / or propagation. Further, the vector may include sequences that enhance cell viability, facilitate virus purification, induce apoptosis, promote protein production, and / or promote immune function and / or targeting. The "helper-expression vector" construct is any expression vector that assists in disabling replication of the "vector". Such helper-expression vectors are conventional and are readily constructed by those skilled in the art. The helper-expression vector can be packaged in a virion, such as a vector, or can be expressed without the packaging requirements.
[0199]
Because "vectors" have selective advantages for packaging and / or propagation, this system provides for high replication of the virus without the possible pathogenic effects that a live attenuated virus could potentially cause. Provide a secure means to achieve In addition, the vectors can be mixed with non-specific adjuvants to increase immunogenicity. Such adjuvants are known to those of skill in the art and include, but are not limited to, Freund's complete or incomplete adjuvant, emulsions composed of cells and mycobacterial cell wall components, and the like.
[0200]
The conditionally replicating viral or preferably lentiviral vectors of the present invention can also be used for immunotherapy in the treatment of viral infections, or for the treatment of oncogene diseases. Dendritic cells or their precursors (eg, CD34⁺Hematopoietic cells or blood monocytes, but not limited to those cell types), and other antigen-providing cells may contain HIV proteins or protein sequences containing major epitopes that cause the host to mount an immune response against a foreign agent, such as a virus. It can be transduced with the expressing vector.
[0201]
Such protein epitopes for HIV are described in “HIV molecular immunology database”, 1998, National Institutes of Allergy and Infections Diseases, incorporated by Maryland & Loss, Inc., in a copy of the book from Maryland & Loss, Inc. ing. A preferred embodiment for an HIV epitope protein is the integrated or component CTL sequence (or fragment thereof) shown in Example 14 below. Other epitopes that can also be expressed are derived from other viruses, bacteria, fungi, parasites, tumor cells and genetically modified cells.
[0202]
More than one epitope is integrated into one protein sequence, so that non-immunogenic sites are vectored with increased safety as the protein coding sequence is severely interrupted. Be eliminated to create. A preferred embodiment of the discontinuous sequence is a modified discontinuous sequence encoding an immunologically relevant epitope (with or without a suitable linker amino acid sequence to stabilize the protein structure, if necessary). Not). The use of degenerate sequences reduces the risk of recombination. Another preferred embodiment is the expression of a sequence of the protein described above (or a fragment thereof) through a spliced message driven by a retroviral LTR, for example the HIV-LTR.
[0203]
However, a concern with the use of retroviral vectors in the treatment of human infections and diseases is the potential for the generation of replication competent viruses (RCV). Modifications of the helper vector construct are provided to minimize or eliminate the possibility of homologous recombination between the helper vector and the conditionally replicating vector. Any modifications that act to reduce the potential for dimerization, co-packaging, and / or recombination of helper vectors and conditionally replicating vectors are contemplated by the present invention.
[0204]
An aspect of the invention is that the 3 'end of the helper gene coding sequence, but 5' of the transcription termination site, is tandemly linked to one or more anti-vector ribozymes or antisense sequences (optionally for ribozymes). A cassette defined as at least two such sequences, and for antisense sequences, defined in tandem and defined as at least two sequences targeted to a discontinuous region of the targeted vector nucleic acid. In the form of), is to insert. There is non-specific packaging of the helper genome into the vector, but such packaging is probably vector dependent. Thus, a number of methods can be used to reduce the packaging of helper vectors into vector particles, which on the other hand is the first step in the possible generation of RCV.
[0205]
Preferred helper constructs include an anti-vector ribozyme or antisense molecule at the 3 'end of the structural protein coding sequence or the envelope (homologous or heterologous) coding sequence. More preferred helper constructs include an anti-vector ribozyme or antisense molecule at the 3 'end of the structural protein coding sequence and the envelope coding sequence. If the nucleic acid sequence is an antisense molecule, it acts through cell nucleases in both cells to destroy the co-localized double-stranded vector-helper molecule. If helpers are packaged with vector RNA in viral particles, those molecules cannot undergo full reverse transcription to produce RCV.
[0206]
Another preferred embodiment is to place, on the helper structure, away from the nucleic acid of the vector, a sequence that promotes the differential tracking or localization of the helper nucleic acid. For example, there is, but is not limited to, the inclusion of a heterologous intron and polyA sequence in a helper construct, such as the construct shown in FIG. These sequences facilitate the differential tracking of vector and helper amino acids to different cellular or non-cellular locations. Preferably, the titer of the resulting vector is affected by more than 100-fold, more preferably only 10-fold, and most preferably unaffected by such helper modifications.
[0207]
FIG. 7 shows that the presence of one ribozyme (pVirPac1.2Rz), or a ribozyme designed to affect helper RNA cell trafficking and an intron (pVirPac1.2RzIn), had no significant effect on vector titers. Indicates that FIG. 12 shows that PCR analysis of titer samples for co-packaged helper constructs showed high co-packaging in the absence of ribozyme versus reduced co-packaging in the presence of ribozyme. Is shown. The presence of the two ribozymes completely prevented simultaneous packaging of the helper vector into the vector virion. Thus, the present invention provides a two-plasmid packaging system to improve the integrity of such vectors by generating high vector titers and reducing or eliminating co-packaging of helper vectors. Effective means of using
[0208]
The data suggest that helper packaging into virions is not random but vector dependent. Another preferred embodiment that prevents co-packaging of the helper nucleic acid with the vector nucleic acid is to degenerate the helper construct in regions that are important for helper and vector sequence association. This approach can be further modified by completely denaturing the helper sequence to prevent simultaneous packaging of the helper and vector sequences.
[0209]
For example, the nucleotide sequence of the helper vector may be partially or completely modified. While such degeneracy significantly reduces or eliminates the tendency for homologous pairing and recombination of helper vectors and conditionally replicating vectors, it reduces the protein products required for viral replication and packaging. Does not affect the ability of the helper vector to code. This degeneracy can be directed to any possible gene or open reading frame (ORF) that is homologous between the helper and the conditionally replicating vector, or to a particular gene or ORF within the helper vector.
[0210]
It should be noted that full degeneracy is not necessary to reduce the tendency for homologous recombination, as deletion of sequence homology for nucleotides greater than 18 nucleotides is sufficient. Thus, degeneracy to the level where only 18, 17, 16, 15, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 nucleotides are identical between sequences controls recombination. Or will prevent. The sequences can be degenerate, if desired, using codons that have selective expression in humans or particular cell types.
[0211]
By way of example, the nucleotide sequence encoding gag, rev and / or tat may be completely or partially degenerate. Other examples include degeneracy of the first 42 nucleotides of the gag sequence, the first 208 nucleotides of the rev sequence, the last 183 nucleotides of the tat sequence, and the last 545 nucleotides of the Pol sequence. I do. Examples of helper vectors having such degeneracy are pVirPac1.2, pVirPac1.2Rz, pVirPac1.2Rz2 and pVirPac1.2RzIn or fusions thereof. Of course, nucleotide sequences encoding any other viral gene product may also be degenerate according to the present invention, thereby reducing, minimizing or eliminating the tendency for homologous recombination.
[0212]
Another aspect of the invention is to target and denature a conditionally replicating vector when the two vectors are co-localized, co-packaged, or otherwise paired together. Incorporation of one element into the helper vector. Ribozymes that selectively target conditionally replicating vectors are contemplated by the present invention. Multiple ribozymes, eg, double or triple ribozyme cassettes as described herein, may also be used. For example, a ribozyme can target the U5 region of a conditionally replicating vector (eg, HIV-1, HIV-2, or another retrovirus used in a conditionally replicating vector). Degradation of the conditionally replicating vector prevents recombination by the conditionally replicating vector from producing RCV, which is present when two vectors are co-packaged. Examples of ribozyme-containing helper vectors are pVIRPAC-1.1Rz and pVIRPAC-1.2Rz2, as shown in FIG.
[0213]
Yet another aspect of the invention is the replacement of a heterologous RRE, including any retroviral RRE, but preferably another lentiviral RRE, in a helper vector, eg, HIV-2 by HIV-1 RRE, CTB or PRE. This is a replacement for RRE. This approach contributes to reducing, minimizing or eliminating the possibility of homologous recombination based on different RREs with different sequences. A further advantage of such substitutions of the present invention is a surprising and unexpected increase in the production of conditionally replicating vectors, by a factor of about five.
[0214]
Without being bound by theory, the presence of different RREs between the helper vector and the conditionally replicating vector can be bound by one or more different cellular factors, which can be quantitatively limited. Thus, the use of different RREs can reduce competition between the two vectors for restricted cellular factors. Examples of helper vectors containing HIV-2 RRE elements for packaging HIV-1 based on retroviral vectors are pVIRPAC-1.2 and pVIRPA-1.2Rz2 as shown in FIG.
[0215]
Yet another embodiment is a heterologous env protein, such as VSV-G from vesicular stomatitis virus, Ravies G protein, GaLV, alphavirus 1/2 glycoprotein or RD114, a nucleotide encoding an env protein from a cat endogenous virus. Integration of the helper vector of the sequence. This allows packaging of the vector into a particle that conditionally replicates with the heterologous env protein. In addition, a ligand may optionally be inserted into the envelope protein to facilitate stimulation of target cells during transduction. Since modification of the envelope protein destroys its binding ability, a preferred embodiment is to express the modified ligand, including the chimeric envelope protein, along with the unemploymented pseudotyped envelope protein during production. is there.
[0216]
Thus, both types of envelope proteins are present on the surface of the cell, one stimulating the target cell and the other mediating binding and invasion. Preferred pseudotyped lentiviral vectors that also express a chimeric receptor for cell stimulation are the VSV-G envelope protein and the chimeric VSV-G envelope protein. A more preferred chimeric envelope protein is the VSV-G / RD114 chimeric envelope protein (see FIGS. 14A and 14B). Other preferred proteins (or fragments thereof) that create chimeras with VSV-G include notches, delta, FLT-3 ligand, TPO, Kit ligand, ligands that bind CD3, ligands that bind CD28, and GM-CSF. And ligands that bind
[0217]
The sequence encoding the heterologous env protein can be operably linked with a second inducible promoter to ensure more supply of the env protein for viral packaging. This approach can enhance virus production, especially under conditions where env protein production is the rate limiting factor. The heterologous env protein coding sequence can be contained on the same helper vector as the other complementary viral protein coding sequence, or on a separate vector. Optionally, it can also be incorporated into a production host cell or cell line.
[0218]
A further aspect of the present invention is that the alternative splicing from certain viral components acts on a helper vector such that it acts to minimize or eliminate the possibility of homologous recombination with conditional replication. Figure 4 is an alternative localization of splice donor and acceptor sites. For example, a splice site may be located to allow for the deletion of the RRE and / or the packaging or dimerization signal as a result of a splice event. Thus, the context of whether a nucleic acid is expressed can be regulated by the placement of the splice site in the vector. For example, a splice site can be located distal to a ribozyme or antisense sequence, such that the ribozyme or antisense is incorporated into unspliced RNA, but not into spliced RNA. On the other hand, a ribozyme or antisense can be placed downstream of a splice site if the objective is to express the ribozyme or antisense molecule from all mRNA molecules and not a particular subtype.
[0219]
If the vector is used according to the above method as a prophylactic treatment against viral infection, the vector may encode a protein that is not encoded by the wild-type virus, such as a mutant viral protein or a non-viral protein antigen. Thus, the antigen encoded by the vector can be of bacterial or cancer cell origin. In addition, a conditionally replicating viral vector, or preferably a lentiviral vector, can also encode an MHC gene for correct presentation of the antigen to the human immune system. Thus, such vectors provide a durable immunological response to various potential pathogens and / or endogenous proteins (eg, tumor-specific antigens) that are selectively expressed in abnormal cells. Can be used to facilitate.
[0220]
The above does not limit the use of the present invention to supply antigens to dendritic cells for immunotherapy. In contrast, the present invention provides means and methods by which any gene of interest can be supplied and expressed in any desired cell. Furthermore, it allows for the supply and expression of multiple genes through a single vector of the invention. Multiple genes can be expressed using any suitable means for gene expression. A non-limiting example is the use of an internal ribosome entry site (IRES) that can be located distally from the first gene that needs to be expressed.
[0221]
A preferred example is to express a gene of interest in the IRES that is distal to the mutant MGMT gene. A more preferred vector embodiment is to express the gene of interest and the IRES-linked gene of interest from the spliced message, unmodified or modified from the HIV-LTR. Further preferred vectors, when modified, express two genes of interest from unmodified or modified HIV-LTR promoters that are modified in sequences that bind transcription factors. Of course, any unmodified or modified lentiviral LTR can be used in addition to the HIV LTR.
[0222]
Another non-limiting example is expressing a gene of interest from the HIV-LTR using a splicing site derived from HIV. For example, one gene of interest is expressed from a Nef splice acceptor site, and a second gene of interest is expressed from a Tat splice acceptor site. In this case, two genes can be expressed without the need for an IRES element. Any splice site may be used, such as those sites of the Tat, Rev, Vif, Vpr, Vpu, Nef and Env genes. The Env protein is expressed from a single spliced message, so that expression of the gene of interest through this splice site requires Rev if significant expression of the gene of interest is required. Furthermore, expression from the HIV-LTR can be made Tat and Rev dependent, if desired.
[0223]
In addition, "helper-virus" (also referred to herein as "helper") expression vectors allow for cell-specific expression replication and spread. With the addition or exclusion of certain genetic elements / factors (in vectors or helper-virus expression constructs), they can be constructed to be expressed only in certain cell types (eg, stem cells, certain antigen presenting cells and tumor cells). Thus, the vector still expands due to complementation with the helper-virus construct, but this extension adds certain genetic elements / factors or is eliminated from the vector or helper-virus expression construct. Depending on, it is cell-specific. This can be used alone or in combination with the other of the means mentioned above.
[0224]
For example, a conditionally replicating HIV vector can be designed to replicate specifically in macrophages in macrophages, rather than in T-cells. Vectors that make up Tat-deficient HIV (the vector encodes other HIV proteins, but it is not expressed due to the absence of Tat transcriptional transactivity) degrade wild-type HIV, but conditionally. It can encode a ribozyme that does not degrade replicating HIV RNA. The helper expression vector for this vector can encode a tat gene expressed from a macrophage specific promoter. Thus, crHIV conditionally replicates only in macrophage cells, but cannot replicate in T-cells or other cells.
[0225]
On the other hand, the tat gene can be operably linked to a tumor-specific promoter; thus, crHIV replicates only in tumor CD4 cells and does not replicate in normal CD4 cells. The genetic element / factor may also be a modification of the promoter sequence of the vector, so that it is expressed only in certain cell types and in other cell types in cooperation with the "helper-virus" expression construct Not done.
[0226]
In another embodiment, the helper-expression construct or the vector construct envelope protein (if such a construct is constructed to include the envelope protein) is modified so that the vector-virion is of a certain cell type. It specifically infects (eg, tumor cells) but cannot infect other cell types (eg, normal cells). In yet another embodiment, adenoviruses lacking one or several key factors for replication are supplemented by using a helper construct that provides such factors linked to a tumor-specific promoter. obtain. Thus, factors that complement adenovirus replication are simply expressed in tumor cells, thereby allowing virus replication in tumor cells (with the expression of proteins required for cell killing), but It is not possible in normal cells.
[0227]
In a further aspect, the vector comprises O⁶Expressing a negative selection gene for killing cells by using a guanine alkylating agent, such as, but not limited to, BCNU (or a functional analog of BCNU) as a negative selection agent I was able to. Lentiviral vectors expressing antisense or ribozymes targeted to the MGMT gene can be supplied to normal cells. At a later point in time, the cells can be treated with BCNU or an analog thereof, ex vivo or in vivo, to kill the vector-transduced cells.
[0228]
In a preferred embodiment, normal cells are transduced with MGMT antisense, which expresses the vector prophylactically, and later killed if the cells become abnormal. An example of this latter approach is when the transduced cells become cancerous cells that can be killed by treatment of the cells with BCNU or a functional analog thereof. Another preferred embodiment is to express an anti-MGMT antisense or ribozyme molecule from the U1 promoter and contained within the U1 snRNA. An even more preferred embodiment is to transduce the anti-MGMT lentiviral vector into a population of hematopoietic cells, such as lymphocyte stem cells and dendritic cells.
[0229]
In another preferred embodiment, the normal cells are hematopoietic cells, preferably T cells, transduced by the vector prior to transplantation into an allogeneic host. Such cells can be killed by treatment with BCNU or a functional analog thereof if the cells become harmful to the host (eg, where a graft-versus-host reaction occurs). In yet another embodiment, the same anti-MGMT antisense or ribozyme-expressing lentiviral vector can be used to purge unwanted cells from a mixed cell population. A non-limiting example is the case of bone marrow contaminated with cancer cells, which is easy for transplantation.
[0230]
Tumor cells have been observed to be very efficiently transduced with a relatively low MOI in a single transduction (see FIG. 13A). In contrast, normal cells, especially CD34⁺Hematopoietic stem cells, but not limited to, are difficult to transduce effectively and require multiple transductions for high efficiency (see FIG. 13B).
[0231]
Thus, the preferred method of purging is through transduction of contaminated bone marrow with an anti-MGMT antisense-containing vector using an MOI that effectively transduces contaminating tumor cells, but not normal cells. This example does not limit the scope of the invention to ex vivo use, or simply to cancer related applications. One use is for in vivo selection gene delivery, especially for brain cancer and any other disease where there is a suggested efficiency of transduction between diseased and normal cells that can be developed for targeting according to the invention. Includes, but is not limited to, processing.
[0232]
Accordingly, the present invention also provides a method for treating cancer, and particularly for treating T-cell leukemia. “Treatment of cancer” of the present invention comprises administering to a host an additional modified vector as set forth herein to produce a therapeutic response. Such a response can be assessed, for example, by monitoring tumor growth weakening and / or tumor regression. "Tumor growth" includes an increase in tumor size and / or number of tumors. “Tumor regression” includes a reduction in tumor mass.
[0233]
"Cancer" of the present invention includes cancers characterized by abnormal cell growth and absence of contact inhibition, which may be evident by tumorigenesis. The term encompasses cancers that are localized to tumors and cancers that are not localized to tumors, for example those cancer cells that expand locally by invasion or systemically by metastasis. In theory, any type of cancer can be targeted for treatment according to the present invention. However, preferably, the cancer is of viral origin.
[0234]
Finally, the vectors can be used directly for in vivo gene therapy. Current methods for gene therapy have drawbacks because they cannot mediate a high rate of gene supply to cells; only a certain percentage of cells are infected. This is particularly important in anti-tumor methods where gene transduction of the entire tumor population is critical. By adding the "vector" together with the "helper", the transduced cells can immediately infect neighboring cells, and thus provide viral particles that enable high and possible complete transduction efficiencies. Will generate.
[0235]
In one aspect of the invention, a human retrovirus (which may be an HIV or retrotransposon element) or a "helper" construct may be provided in a tissue (or cell in vitro). Cells containing the vector and helper will soon produce the virus and conditionally package the vector in virions. These virions could mediate high efficiency transduction of neighboring cells (since cell-cell contact is the most effective means for transducing cells). The transduced cells die or do not die, depending on whether the vector / helper combination results in a cytolytic infection.
[0236]
In the case of a retrotransposon, the helper need not include structural proteins, as normal or tumor cells can contain the proteins / factors required for inclusion in virions. In this case, the helper can be, but is not limited to, simply a transactivator protein that activates the transcription of a factor required for retrotransposon inclusion. In the case of HIV, other factors are required, but not necessary, for the inclusion of the HIV genome in progeny virions for infection / transduction of cells.
[0237]
The vectors can also be used in anti-biological and anti-chemotherapy methods. For example, a conditionally replicating vector can be provided in an individual who has been infected with a highly pathogenic virus or bacterium or a bacterial agent (eg, a toxin). The vector will prevent replication of the pathogenic virus, as described previously. However, conditionally replicating vectors can also be used for antimicrobial or anti-chemical methods in cooperation with helper expression vectors ("helpers").
[0238]
For example, a conditionally replicating vector can secrete antimicrobial or anti-toxin antibodies after the "helper" has enabled its expression and propagation. “Helpers” are fungi, cytokines (in response to antimicrobial infections), antibiotics (for tetracycline-inducible promoter systems (Paulus et al., J. Virol., 70, 62-67 (1996)), or chemicals ( For example, based on activation by a toxin, itself), it can, but need not, be driven from an inducible promoter that allows its expression. Not only selectively grow with the help of "helpers" in response to (as a result of the activation of helpers), but also inhibit the pathological effects of tumor antigens, pathogens or chemicals (eg toxins) In addition, it can secrete anti-pathogen or anti-toxin antibodies.
Thus, any protein, factor or genetic element that can be transcribed into either mRNA and / or protein inhibits the pathogen response in cooperation with "helpers" that promote its selective growth and expression. Can be inserted into a conditionally replicating vector (the product of the helper is conditionally expressed and thus selective, for example,
[0239]
(A) an inducible promoter system, ie, a factor in a tumor cell, activates the production of a helper factor, a toxin responsive element expresses a helper factor, or a cytokine responsive promoter triggers the production of a helper factor;
(B) helper RNA / protein / factor is selectively stabilized in certain cells and not in other cells, and
(C) Indirect induction of a third party gene affects, but is not limited to, helper virus protein production, concomitant, targeting, structure or another bifunctionality. Such methods can be used on transgenic plants and animals to protect them from pathogens. Similarly, such methods can be used in transgenic systems to produce valuable heterologous proteins / factors.
[0240]
In another embodiment of the method of the present invention, one cell is developed for drug / factor screening to determine that a portion of the protein / factor is important for a particular function. Can be done. Vectors can be created to express the mutated protein of interest in a given cell line. However, the RNA encoding the mutagenized protein is rendered resistant to the ribozyme by insertion of a silent point mutation. However, wild-type protein expression is inhibited in cell lines.
[0241]
Vectors expressing a ribozyme for the protein of interest can also be configured to express the mutated test protein. When the vector is transduced into cells, most of the native RNA encoding the normal protein is degraded, but the mutated test protein is expressed. This method is a recently developed supply and selection technique that is a fast and powerful technique for determining how a given protein functions and how a given factor / drug interacts with the protein. It can be used with techniques.
[0242]
In yet another embodiment for applying the vector to treat a blood-borne disease, the patient undergoes leukocyte export to extract a sufficient amount of white blood cells from the blood. After leukocyte export, the desired cells are isolated, transduced with the vector, and then expanded in culture to the desired cell number. During ex vivo expansion of the desired cell type, antibodies targeted to cell surface proteins on the desired cell type are infused into the patient, and a sufficient amount of the desired cell type is expanded, and for a period of time, Destroy cells. Next, the transduced cells are re-injected into the patient to replace the cellular in vivo antibody-mediated loss.
[0243]
A preferred means of performing the above is to use CD4 from leukocyte-exported material.⁺ It involves the isolation of T cells and then transduction of said cells with an HIV vector containing anti-HIV antisense or ribozyme sequences for ex vivo expansion. During ex vivo expansion of cells, the patient's endogenous CD4⁺ T cells may be supplied, for example, by anti-thymocyte globulin (ATG) (supplied by Pasteur Merieux Serums et Vaccins, Lyon, France, SangStat Medical Corporation, Menlo Park, CA, USA), or Atgamba, Comm. UAS) is destroyed, but any cytoreductive antibody can be used after appropriate screening as will be apparent to those skilled in the art. After expansion, the transduced CD4 + T cells are returned to the patient. In a preferred embodiment, CD4⁺CD4-negative cells obtained during cell isolation are retained, frozen and transduced CD4⁺The cells are thawed for injection at the time they are returned to the patient.
[0244]
The above example shows that the scope of the present invention can be applied to both ATG and⁺ It is not restricted to T cells. Any cell type can be targeted by the use of antibodies that bind cell surface proteins on the cell and are cytocidal. The antibody can be introduced exogenously into the patient, or a second vector secreting the antibody can be introduced into the body. The vectors may be used to confer antibodies either constitutively (for cell life), transiently (eg, but not limited to integrase-negative vectors), or inducibly (eg, but not limited to tetracycline-inducible promoter systems). Not limited to), could be generated.
[0245]
There are also many uses of the methods and vectors of the present invention in vitro. For example, vectors can be used to ascertain certain nuances of viral replication and ribozyme function. Similarly, ribozyme-containing vectors are used as a diagnostic tool to assess mutations present in diseased cells or to test for genetic drift. Vectors can also be used to determine or confirm the function of a gene, regardless of whether the function is previously known, unknown, or simply proposed or planned. This preceding discussion is by no means comprehensive with respect to the use of the present invention.
[0246]
Advantages of the invention:
The advantages of using crHIV methods for gene therapy treatment of AIDS and other diseases can be considered. For example, the problem of targeting the vector to cells infected by HIV has been solved. Infected CD4⁺After in vivo transfection of crHIV into cells, crHIV becomes packaged in progeny virions using endogenous infectious HIV envelope proteins. Thus, crHIV RNA labels along internal progeny virions and infects cell types normally infected by certain strains of HIV, producing non-pathogenic virions. This involves the difficulty of targeting cells that are major reservoirs for HIV infection of the central nervous system, such as microglia. Uninfected CD4 because the viral proteins are not encoded by the crHIV vector⁺There is probably little toxicity associated with the crHIV vector infecting the cells.
[0247]
In addition, the consequence of crHIV vector competition with wild-type HIV results in the generation of non-pathogenic particles resulting in reduced viral load. Decreasing pathogenic HIV-1 loading not only can increase the survival time of infected individuals, since crHIV particles can also be systemically expanded (ie, like infectious HIV). And the speed of transmission to uninfected individuals can be reduced. Reduced pathogenic HIV-1 load in the blood is a problem in pregnant HIV-infected individuals because the production of crHIV can also reduce the transmission of HIV from infected mothers to the fetus in their uterus. May be particularly important in
[0248]
Plasmid DNA / lipid mixtures that can be used to introduce crHIV vectors into host cells should be stable and inexpensive to produce, avoiding expensive ex vivo methods. Of course, the method of the invention is inherently flexible as long as it is also used for ex vivo gene delivery, which should be desired. Nevertheless, the availability of a liposome-mediated approach opens up the possibility for treatment of the general population (ie, which cannot be performed with current gene therapy). crHIV vectors can also be constructed to contain several ribozymes that can be produced to different targets on the HIV genome. This reduces the likelihood that infectious HIV will mutate and escape the effects of anti-HIV ribozymes. In addition, conditional replication competent virus methods can be applied to treat other viral infections, especially those with high virus turnover.
[0249]
A particular feature of crHIV vectors is that they can be used to post-transcribedly express genetic antiviral agents, such as ribozymes. Thus, infection of uninfected cells with the crHIV vector results in low toxicity, as most expression results from the long terminal reaction (LTR) promoter of HIV in the absence of the Tat protein. High levels of crHIV expression and the resulting antiviral activity are only present when the Tat protein is provided by complementation with wild-type HIV. Thus, crHIV vectors are not designed to protect cells from HIV infection, but are designed to reduce the overall wild-type HIV viral load through selective accumulation of non-pathogenic crHIV particles.
[0250]
While not being bound by any particular theory regarding the operation or function of the present invention, ribozymes are identified in the following examples to provide selective advantages to the crHIV genome for two useful properties: Could be used to: (1) they have a high degree of specificity, and (2) they depend on their ability to co-localize with the target RNA to increase relative efficiency. (Cech, Science, 236, 1532-1539 (1987)). Ribozyme specificity is conferred by their specific hybridization to a complementary target sequence containing an XUY site. Ribozymes are relatively effective only when they effectively co-localize with the target RNA because they degrade target RNA with high potency.
[0251]
In mixed HIV / crHIV infections, co-localization of wild-type HIV RNA with ribozyme-containing crHIV should occur because the HIV RNA genome dimerizes before packaging into progeny virions. is there. Degradation of genomic species of wild-type HIV RNA, required for the production of viral proteins, is likely to be less effective than degradation of genomic wild-type HIV RNA unless non-genomic HIV RNA dimerizes . The selective advantage conferred on crHIV RNA was due to the selective packaging of crHIV into viral virions. The results suggest that the most effective degradation occurs intracellularly during dimerization, resulting in the selective destruction of wild-type HIV RNA by host nucleases. This allows for selective packaging of crHIV RNA into virus particles.
[0252]
The application of crHIV vectors for HIV therapy is not limited to genomic selection of crHIV, but can also include cell selection of cells that produce crHIV particles. On the other hand, cells that produce wild-type HIV will produce wild-type HIV particles with selective advantages over cells that produce crHIV particles, and will rapidly outperform. The selective advantage has a survival advantage over cells expressing wild-type HIV. CrHIV expressing cells (in the presence of a drug) can be conferred on crHIV expressing cells by inserting one gene into a crHIV genome (eg, a multi-drug gene). Under these conditions, wild-type HIV expressing cells die progressively, but there are still crHIV-expressing cells that produce some wild-type HIV, but preferentially produce crHIV.
[0253]
Infection of crHIV-containing cells with remaining wild-type HIV will result in further production of crHIV, including viral particles. Thus, the viral genome shift is due to the CD4 by crHIV genome.⁺Due to the cumulative infection of the cells, thereby altering the viral balance in the host to pathogenic wild-type HIV non-pathogenic crHIV genome. Such methods can result in the clearance of wild-type HIV when the balance of the HIV genome is selectively shifted from wild-type HIV to crHIV. Viral replication eventually stops because crHIV can replicate in the presence of the wild-type HIV helper genome. Thus, it is possible not only to reduce wild-type HIV under such mutually restrictive conditions, but also to clear the virus from the HIV-infected host.
[0254]
Means of generation:
The vector may be produced by a complementation step by using transient transfection of the vector and helper genome into a cell line, preferably the well-known 293T cell, or by using a packaging cell line. Preferably, the cells are transiently transfected with a transfection reagent, preferably calcium phosphate, electroporation or lipid transfection reagent, with high transfection efficiency. As soon as transfection occurs, the vector is harvested from the supernatant 12 hours to 7 days after transfection. The vector can optionally be concentrated by high speed centrifugation, if desired, without precipitation or ultracentrifugation. Preferred centrifugation conditions are about 5000-12,000 xg, more preferably about 10,000 xg. More preferred conditions are centrifugation at 4 ° C. overnight.
[0255]
Methods for vector purification:
Method 1:
1. Clarification of virus supernatant;
2. Concentration by ultrafiltration;
3. Diafiltration with or without benzonase treatment;
4. Ion-exchange chromatography;
5. Size exclusion chromatography or diafiltration;
6. Optionally, concentration by ultrafiltration.
[0256]
Method 2:
1. Clarification of the virus supernatant;
2. Ion exchange chromatography;
3. Diafiltration or size exclusion chromatography;
4. Optional benzonase treatment;
5. Size exclusion chromatography or diafiltration;
6. Optionally, concentration by ultrafiltration.
Different resins can be used for the above method, for example, Poros 50HQ from Perseptive Biosystems. Hollow fiber cartridges can be used for ultrafiltration or diafiltration, such as cartridges from A / G Technologies (UFP-750 series).
[0257]
Means of administration:
According to the present invention, the vector is introduced into a host cell in need of gene therapy for viral infection, as previously described. The means of introduction, according to the invention, comprises contacting the virus with a host that can be infected. Preferably, such contacting comprises any means by which the vector is introduced into the host cell; the method is not dependent on any particular means of introduction and is to be construed as such. is not. Means of introduction are well known to those skilled in the art and are also exemplified herein.
[0258]
Thus, introduction can be effected, for example, in vitro (eg, in an ex vivo method of gene therapy) or in vivo, and can be performed by electroporation, transformation, transduction, conjugation or triple parenting, transfection, infection, Includes membrane fusion with cationic lipids, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitates, direct microinjection into single cells, and the like. Other methods are also available and are known to those skilled in the art.
[0259]
However, preferably the vector or ribozyme is introduced by a cationic lipid, such as a ribosome. Such ribosomes are commercially available (eg, Lipofectin supplied by Life Technologies, Gibco BRL, Gaithersburg, Md.)^TM, Lipofectamine^TMAnd similar). In addition, liposomes with increased transfer capacity and / or reduced toxicity in vivo (as reconsidered in PCT Patent Application No. WO 95/21259) can be used in the present invention. For liposome administration, the recommendations identified in PCT Patent Application No. WO 93/23569 are followed. In general, for such administration, the formulation will be taken up by most lymphocytes within 8 hours at 37 ° C. and more than 50% of the injected dose will be in the spleen 1 hour after intravenous administration. Is detected. Similarly, other delivery vehicles include hydrogels and controlled release polymers.
[0260]
The form of the vector introduced into the host cell can vary, depending in part on whether the vector is introduced in vitro or in vivo. For example, the nucleic acid can be circularly closed, nicked, or linearized, depending on whether the vector is maintained extragenomically (ie, in the case of an autonomously replicating vector), a provirus or a provirus. It is integrated as a phage, transiently transfected, transfected by the use of replication-defective or conditionally replicating viruses, or stably integrated into the host genome through double or single cross-over recombination events. Can be introduced.
[0261]
Prior to introduction into a host, the vectors of the invention can be formulated into various compositions for use in therapeutic and prophylactic treatment methods. In particular, the vectors can be made into pharmaceutical compositions by combining with a suitable pharmaceutically acceptable carrier or diluent, and formulated as appropriate for human or veterinary application.
[0262]
Thus, a composition for use in the method of the invention may comprise one or more of the above vectors, preferably in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art when appropriate methods of administration. The choice of carrier will be determined in part by the particular vector, and the particular method used to administer the composition. One of skill in the art will also appreciate that various routes of administration of the composition are available, and that more than one route may be used for administration, but that certain routes will provide more immediate efficacy than another route. You will understand that you can. Accordingly, there is a wide variety of suitable formulations of the compositions of the present invention.
[0263]
Compositions comprising the vectors of the present invention, alone or in combination with other antiviral compounds, can be prepared in formulations suitable for parenteral administration, preferably for intraperitoneal administration. Such formulations can include antioxidants, buffers, bacteriostats, and solutes that render the intended recipient blood isotonic, aqueous and non-aqueous, isotonic, sterile injectables. Solutions and sterile aqueous and non-aqueous suspensions which can include suspending agents, solubilizing agents, thickening agents, stabilizers and preservatives can be included. The formulations are supplied in unit-dose or multi-dose sealed containers, such as ampules and vials, and stored under lyophilization conditions, and are used immediately before use by addition of a sterile liquid carrier, such as water. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets as described herein.
[0264]
Vectors can be stored in any suitable solution, buffer or lyophilized form, if desired. Preferred storage buffers are Dulbecco's phosphate buffer; Dulbecco's phosphate buffer mixed with a 1-50% solution of trehalose (1: 1) in water, preferably a 10% solution of trehalose (1: 1) in water. Solution (final concentration is 5% trehalose); Dulbecco's phosphate buffered solution mixed with a 1-50% solution of glucose (1: 1) in water, preferably a 10% solution of glucose (1: 1) in water (Final glucose concentration is 5%); 1-50% solution of trehalose (1: 1) in water, preferably 20 mM HEPES-buffer solution mixed with 10% solution of trehalose (1: 1) in water (Final trehalose concentration is 5%); or a 1-50% solution of mannitol (1: 1) in water, preferably a 5% solution of mannitol (1: 1) in water. Dulbecco's phosphate buffered solution is mixed with (final mannitol concentration is 2.5%) is.
[0265]
Formulations suitable for oral administration include an effective amount of the compound dissolved in a liquid solution, eg, a diluent such as water, saline, or fruit juice; a predetermined amount of the active ingredient as a solid or granule, respectively. Capsules, sachets or tablets; solutions or suspensions in aqueous solutions; and oil-in-water or oil-in-water emulsions. Tablet form may contain one or more lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other additives. Excipients, colorants, diluents, humectants, preservatives, flavoring agents and pharmacologically compatible carriers can be included.
[0266]
Aerosol formulations suitable for administration via inhalation may also be prepared. Aerosol formulations can be placed in pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen and the like.
Similarly, formulations suitable for oral administration include a lozenge form comprising the active ingredient in a flavoring agent, usually sucrose and acacia or togacanth; an inert substrate such as gelatin and glycerin, or active in sucrose and acacia. Lozenges comprising the ingredients; and a mouthwash comprising the active ingredients in a suitable liquid carrier; and creams, emulsions, glues and the like, which contain, in addition to the active ingredients, carriers known in the art. Is included.
[0267]
Formulations suitable for topical administration may be in the form of chrome, ointment or lotion.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, carriers known in the art. Similarly, the active ingredient may be combined with a lubricant as a coating on a condom.
[0268]
In the present invention, the dose administered to an animal, eg, a human, should be sufficient to effect a therapeutic response in the infected individual over an appropriate period of time. The dose will be determined by the ability of the particular vector to be used for treatment, the severity of the disease state, and the weight and age of the infected individual. The size of the dose will be determined by the presence of any side effects that accompany the use of the particular vector used. It is always desirable whenever possible to keep side effects to a minimum.
[0269]
The dose can be in unit dosage form, for example, in tablet or capsule form. The term “unit dose form” as used herein refers to physically discrete units suitable as unit volumes for human and animal subjects, where each unit is a predetermined amount. Of the present invention, alone or together with a pharmaceutically acceptable diluent, carrier or vehicle, in combination with other antiviral agents calculated in an amount sufficient to produce the desired effect. The specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, as well as the pharmacokinetics associated with the particular compound in the host. The dose administered should be an "antivirally effective amount", or the amount necessary to achieve an "effective level" in an individual patient.
[0270]
As "effective level" is used as the preferred endpoint for administration, the actual dosage and schedule may vary depending on individual differences in pharmacokinetics, drug distribution and metabolism. An "effective level" refers to the desired blood or blood concentration in a patient, e.g., corresponding to the concentration of one or more vectors of the invention that inhibit a virus, e. Can be defined as organization level. "Effective levels" for the compounds of the present invention can vary when the compositions of the present invention are used with zidovudine or other known antiviral compounds or combinations thereof.
[0271]
One of skill in the art can readily determine the appropriate dose, schedule and method of administration for the exact formulation of the composition to be used, in order to achieve the desired "effective level" in the individual patient. Those skilled in the art will also appreciate that suitable (eg, blood and / or tissue) patient samples (eg, analytical chemistry analysis) or indirect (eg, surrogate indicators of viral infection, eg, for the treatment of AIDS or AIDS-like diseases). (By p24 or reverse transcriptase) can easily determine and use a suitable indicator of the "effective level" of the compound of the present invention.
[0272]
Furthermore, with regard to determining the effective level in patients for the treatment of AIDS or AIDS-like diseases, particularly suitable animal models are available and widely practiced to evaluate the in vivo efficacy of various gene therapy protocols against HIV. (Sarver et al. (1993b), supra). Those models include mice, monkeys and cats. A chimeric mouse model (eg, SCID, reconstituted by human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver / thymus or other tissues, even if those animals are not naturally susceptible to HIV disease) bg / nu / xid, NOD / SCID, SCID-hu, immunocompetent SCID-hu, bone marrow-depleted BALB / c) can be infected by vectors or HIV, and models for HIV pathogenesis and gene therapy Can be used as Similarly, the simian immunodeficiency virus (SIV) / monkey model can be used in the same way as the feline immunodeficiency virus (FIV) / cat model.
[0273]
These models can also be used to determine vector safety for evaluation of vector systems for clinical trials. An important application is the use of those animal models for biodistribution studies. Transduced cells, preferably but not limited to human cells containing the vector, are injected into a non-human animal model, and the stability of the vector is determined by the absence of vector genetic material in animal tissue. You. The absence of vector genetic material in animal tissues does not allow the vector to replicate autonomously without the presence of helper or wild-type virus, and therefore appears to be safe for clinical use in humans.
[0274]
The presence of a helper vector or a vector in the absence of a helper virus is considered a safety hazard if the vector is not expected to replicate autonomously. However, if the vector is expected to replicate autonomously, other criteria for safety need to be established, such as a lack of replication in certain tissues or the level of replication in animals. The presence or absence of the vector can be determined by PCR, or by FACS analysis if the vector being tested expresses a marker gene that can be visualized by FACS, but is not limited to such means of detection.
[0275]
Generally, sufficient vector to achieve a tissue concentration of ribozyme (or vector) to be administered of from about 5 μg / kg to about 300 mg / kg body weight / day, especially from about 10 μg / kg to about 200 mg / kg body weight / day. Is preferred. For certain applications, for example topical, ocular or vaginal applications, multiple daily doses are preferred. In addition, the number of doses will vary depending on the means of delivery and the particular vector being administered.
[0276]
In individual treatments of some of the virus infections, it is desirable to use a "mega-dose" regime, where a large dose of the vector is administered, allowing the compound to work, and then using the appropriate reagents. Is administered to an individual to inactivate the active compound. In the method of the invention, processing (ie, replication of the vector in competition with the virus to be processed) is necessarily restricted. In other words, for example, as the level of HIV decreases, the level of the vector will also depend, depending on the HIV for the production of virions.
[0277]
Pharmaceutical compositions, when used to treat AIDS therapeutically, can include other medicaments along with the vectors of the present invention. These other medicaments may be used in their conventional manner (ie, as agents for treating HIV infection), and more particularly, in methods of selecting the crHIV virus in vivo. Such selection as described herein promotes conditionally replicating HIV spread and conditionally replicates with wild-type HIV, which will necessarily limit wild-type HIV pathogenicity Enable more effective competition for HIV.
[0278]
In particular, it is envisaged that an antiretroviral agent, such as preferably zidovudine, is used. Further representative examples of those additional medicaments that may be used in addition to those described so far include antiviral compounds, immune modulators, immunostimulants, antibiotics and other agents, and for treating AIDS. Includes processing regimes that can be used, including those recognized as other drugs.
[0279]
Antiviral compounds include ddI, ddC, ganciclovir, fluorinated dideoxynucleotides, non-nucleoside analog compounds such as nevirapine (Shih et al., PNAS, 88, 9878-9882 (1991)), TIBO derivatives such as R82913 (White) Inc., Antibiotic Research, 16, 257-266 (1991)), and BI-RJ-70 (Shih et al., Am. J. Med., 90 (Suppl. 4A), 8S-17S (1991)). I do. Immunomodulators and immunostimulants include, but are not limited to, various interleukins, CD4, cytokines, antibody preparations, blood transfusions and cell transfer. Antibiotics include, but are not limited to, antifungals, antibacterials and anti-pneumocystis carinii agents.
[0280]
Administration of a virus inhibitory compound together with other anti-retroviral agents and especially known RT inhibitors such as ddC, zidovudine, ddI, ddA, or other inhibitors acting on other HIV proteins, such as anti-TAT agents. Will generally inhibit most or all replication steps of the virus life cycle. Dosing of ddC and zidovudine for AIDS or ARC patients is publicly available. The static virus range for ddC is generally between 0.05 μM and 1.0 μM. The range of about 0.005 to 0.25 mg / kg body weight is viral in most patients.
[0281]
The dose range for oral administration is somewhat broader; for example, 0.001 to 0.25 mg / kg is given in one or more doses at time intervals of 2, 4, 6, 8, and 12, etc. . Preferably, 0.01 mg / kg body weight ddC is given every 8 hours. When given in a combined therapy, the other antiviral compounds will be given at the same time as the vector of the invention, or the dose may be varied as desired. Vectors can also be combined into compositions. Individual doses are lower when used in combination than when used alone.
[0282]
Example
The compounds and methods of the present invention are further described in the following examples. These examples further illustrate the invention and do not limit the scope of the invention.
Example 1.
This example describes the construction of a conditionally replicating competent vector of the invention. In particular, this example describes the construction of HIV, a conditionally replicating vector based on the crHIV vector.
[0283]
One of the most prominent aspects of the HIV-1 pathogen is the generation of genetic variants of the virus. The rapid generation of HIV mutants in vivo indicates that the virus appears to be within the skeleton of the Darwin genetic model (see, eg, Coffin, Curr. Top. Microbiol. Immunol., 176, 143-164 (1992). And Coffin, Science, 267, 483-489 (1995)). Mutants are the result of non-replication of the HIV-1 reverse transcriptase molecule, which creates mutations in newly transcribed provirus from viral genomic RNA.
[0284]
Thus, without significant interference, and under in vivo conditions of many replication cycles, substantial variations can result in the production of many viral variants. Furthermore, wild-type HIV is still outstanding under such non-restrictive conditions, as it has the highest selective advantage. However, in the presence of inhibitors, such as zidovudine, conferred higher selectivity advantages than the wild-type strain, and would therefore select for prominent viral variants (Coffin (1992) and (1995), Above). On this basis, the invention provides selective advantages over pathogenic wild-type HIV-1 in a viral vector method that conditionally replicates against the non-pathogenic HIV-1 genome.
[0285]
Those non-pathogenic, conditionally replicating HIV (crHIV) vectors are defective HIV that undergo replication and packaging only in cells infected by wild-type HIV. The crHIV genome competes with and reduces the pathogenic wild-type HIV viral load. The effect of reducing wild-type HIV viral load in the infected host should lead to enhanced life expectancy. It should also reduce the ability of the infected host to transmit wild-type HIV to uninfected individuals. For favorable competition between wild-type HIV-1 and crHIV, two factors appear to be important: (1) the selective advantage of the crHIV genome over the wild-type HIV genome, and (2) ) Selective advantage of crHIV-expressing cells over wild-type HIV-expressing cells (ie, selective advantage for crHIV virion production from crHIV expression over wild-type HIV-expressing cells).
[0286]
crHIV vectors contain the sequences required for RNA expression, dimerization and packaging, but are conditioned by the fact that they do not express functional (ie, wild-type) HIV-1 protein. Duplicate with date. A selective advantage is conferred on crHIV vectors by inserting a ribozyme cassette that degrades in the U5 region of the wild-type HIV genome, but does not degrade in crHIV U5 RNA.
[0287]
The ribozymes present in the vector have been modified by conserved base substitutions (base substitutions present in other HIV strains) so that the U5 region of crHIV RNA prevents the ribozyme from effectively binding and degrading those sites. Therefore, it does not degrade crHIV RNA. In addition, crHIV shows that they have CD4⁺It is non-pathogenic because it does not code for a protein believed to be responsible for cell death. When an HIV-infected cell (transfected with a crHIV vector) is activated, it results in the production of crHIV progeny virions, which are not able to recruit crHIV genomic death.
[0288]
In general, the crHIV genome was composed of a full length infectious HIV clone, pNL4-3 (Adachi et al. (1986), supra). All cloning reactions and DNA, RNA and protein manipulations are well known to those skilled in the art and are described, for example, in Maniatis et al. , Molecular Cloning: A Laboratory Manual. 2^nd ed. (Card Spring Harbor Laboratory, NY (1982)). Enzymes and reagents used in those reactions are obtained from commercial suppliers (eg, New England Biolabs, Inc., Beverly, Mass .; Clontech, Palo Alto, Calif .; ) And used according to the manufacturer's recommendations. In addition, vector maintenance and propagation is accomplished using techniques commonly known and described previously (eg, Dropulic 'et al. (1992), supra; and Dropulic' et al. (1993), supra). went.
[0289]
pNL4-3 is degraded by the enzymes PstI (degraded in gag at +1000 from the start of transcription) and XhoI (degraded in nef at +8400 from the start of transcription) and a convenient restriction site A polylinker containing was inserted. A 0.86 kb Bgl II to BamHI fragment containing the Rev responsive element (RRE) was cloned into the BamHI site present in the polylinker. These manipulations resulted in the deletion of the HIV wild-type genome within the gag coding region to the U3 coding region (ie, the deletion of the nef gene). Although vectors are capable of producing truncated gag transcripts, functional Gag proteins of sufficient length are not produced by some vectors. However, as long as wild-type Gag function is not required by the present invention, gag sequences can be mutagenized to prevent translation of the Gag protein.
[0290]
A ribozyme cassette containing single or multiple ribozymes as described in the present invention was inserted into the SalI site downstream from the BamHI site. To accomplish this, a complementary deoxyoligonucleotide encoding the ribozyme sequence was synthesized, annealed, and then cloned into the Sal I site. The ribozyme used for the construction of the crHIV vector was a hammerhead ribozyme. The ribozymes contained a catalytic domain of 22 base pairs, and two hybridization domains of 9 base pairs each. Ribozymes were targeted to either the +115 or +133 sites of the U5 RNA sequence (ie, corresponding to base pair numbers downstream from the start of transcription).
[0291]
The hybridization and catalytic domains (underlined) of the ribozymes targeted at +115 and +133 are as follows:
CACACAACACTGATGAGGGCCGAAAGGCCGAAACGGGCACA ("+115 ribozyme") SEQ ID NO: 3; and
ATCTCTAGTCTGATGAGGCCGAAAGGCCGAAACCAGAGTC ("+133 ribozyme") SEQ ID NO: 4.
[0292]
Ribozyme cassettes consisted of either single, double or triple ribozymes arranged in tandem. Vectors containing single or triple ribozymes can be readily constructed and targeted to the same site of U5 HIV RNA at position +115. Vectors containing dual ribozymes can be readily constructed and targeted to either the same site at position +115 or different sites at positions +115 and +133 of U5 HIV RNA.
[0293]
To complete the construction of the vector, crHIV vectors are ribozyme-degraded (ie, in their representation as RNA) by mutagenizing a site recognized by the hammerhead ribozyme present in the U5 region of crHIV. Resistant. To achieve this, a double-stranded oligonucleotide containing the base substitutions shown in SEQ ID NO: 2 of Figure 2 (ie, AAGCTTGCCTTGAGTGCTCAAAGTAGTGTGTGCCCCACCTGTTGTGTGACTCTGGCAGCTAGAGATCCCACAGACCCTTTTAGTCAGTGTCGGATAGCTCT was added to the sequence in the vector GAGATACT). In particular, base substitutions were constructed in ribozyme hybridization and degradation sites at base pairs 115 and 133.
[0294]
In particular, mutations were introduced at base pairs 133, 114, 132, 134 and 142, as shown in FIG. The sites can be modified to include any mutation (ie, GTGTGCCCNNCTGTTGTGTGACTCTGGNANCTAGAGANC, where N is any mutated nucleotide), SEQ ID NO: 14. However, preferably, the sequence is replaced, for example, by a G to A substitution at site +113 (ie, the sequence includes GTGTGCCCCATCTGTTGTGTGACTCTGGTAACTAGAGATC SEQ ID NO: 15); U at site +114 SEQ ID NO: 5 (ie, according to the DNA SEQ ID NO: Substitution at position +132 of U (ie, T according to the DNA sequence) to C; substitution of A at position +134 for G (ie, the sequence GTGTGCCCGTCTGTTGTGTGACTCTGGTAGCTAGAGATCC SEQ ID NO: 16); and a mutation such that there is a U to A (ie, T according to the DNA sequence) to A substitution at the site +142 (mutations can be performed alone or in combination). Trigger.
[0295]
In particular, in the absence of other U5 mutations, the substitution of U (ie, T according to the DNA sequence) from C at site +114 SEQ ID NO: 5 and / or site +132 SEQ ID NO: 6 in crHIV US RNA is a (Uhlenbeck (1987), supra). Inserted base-substitutions are present in a variety of other strains of HIV (Myers et al., HIV Sequence Database, Los Alamos Nat. Lab. (1994)), which indicates that these substitutions replicate the HIV genome. Indicates that the function will not be reduced.
[0296]
The method as set forth in the present invention may be used to construct other conditionally replicating vectors, eg, consisting of different viral genomes (eg, different RNA viruses) or consisting of different genetic antiviral agents. Can be used for In addition, conditionally replicating vectors can be further modified to confer selective advantages on the host cell into which the vector is introduced, over host cells containing the wild-type virus. For example, such vectors can be modified to further encode multidrug resistance, or a mutagenized protease or reverse transcriptase.
Those methods can, of course, be used to construct the various helper vector constructs described herein.
[0297]
Example 2.
This example describes the resistance of conditionally replicating vectors and especially crHIV vectors to ribozyme degradation.
To confirm the resistance of the crHIV vector to ribozyme degradation, in vitro transcription was performed. To achieve this, the ribozyme sequence was cloned into the XhoI site of pBluescript KSII (Stratagene, La Jolla, Calif.). A 0.21 kilobase pair (kb) BglII fragment containing the mutated crHIV U5 region was similarly excised from the crHIV vector and inserted into the BamHI site of pBluescript KSII. The resulting modified pBluescript KSII vector was linearized with BassHII prior to in vitro transcription. A similar plasmid expressing wild-type HIV U5 RNA (Myers et al. (1994), supra) was used as a control. It was linearized with EcoRI before in vitro transcription.
[0298]
Radiolabeled U5 HIV RNA and ribozyme RNA were generated by in vitro transcription of the vector as previously described (Dropulic 'et al. (1992), supra). The radiolabeled transcript was prepared using 40 mM Tris-HCl, pH 7.5, 6 mM MgCl₂, And incubated together (target: ribozyme at a 1: 2 molar ratio) in 1 × transfer buffer containing 2 mM spermidine and 10 mM NaCl. The sample was heated to 65 ° C and then brought to 37 ° C before the addition of a stop buffer solution containing 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol FF. Cool for 5 minutes. Next, the products were resolved by denaturing polyacrylamide gel electrophoresis (PAGE) and detected by autoradiography.
[0299]
When wild-type U5-HIV RNA was incubated with a transcript containing a single ribozyme at site +115, degradation was readily observed. Such a decomposition results in products P1 and P2. Degradation can also be found when wild-type HIV RVA is incubated with RNA containing double ribozymes at the same or different sites. When ribozyme-containing transcripts, which are directed to two different sites, were incubated with wild-type HIV RNA, products P1, P2 and P3 were produced. P3 results from degradation at the +133 site.
[0300]
By comparison, when the modified U5-containing crHIV RNA is incubated with a single ribozyme directed to the +115 site or a dual ribozyme directed to either the +115 or +133 site, the degradation products are: Could not be detected. Thus, the results confirm that erHIV U5 RNA is resistant to ribozymes, whereas wild-type HIV-U5 RNA is degraded by anti-US ribozymes. Furthermore, the results show that the selective advantage for replication when the approach of the present invention is introduced into host cells compared to wild-type virus strains is a conditionally replicating vector (other than a crHIV vector). (Including vectors).
[0301]
Example 3.
This example briefly illustrates the key steps in generating the helper vector construct of the present invention. Unless otherwise stated, nucleic acid sequences encoding various retroviral elements were obtained from public sources. All described steps required detection of errors, if necessary, and sequencing of the complete coding region to dictate correction.
[0302]
To degenerate the tat sequence and allow its targeting by the anti-tat ribozyme, the plasmid pTAT was cloned using the QuickChange kit from Stratagene to include a targeting site for the anti-tat ribozyme cassette. Mutagenesis. Subsequently, the mutagenized tat sequence was cloned into an expression vector based on the pMG plasmid from InvitroGen. The tat sequence was fused to the IRES at the first ATG using an NcoI site. The vector was further modified to include the ampicillin resistance gene and the SV40 origin of replication.
[0303]
The sequence containing the HIV-1 rev and RRE elements was obtained by excising the third exon of rev together with the RRE from pNL4-3 and cloned into the pLITMUS38 plasmid. The rev sequence up to nucleotide 208, including the second exon and part of the third exon up to the BamHI site, was assembled from three synthetic nucleotides using ETR and PCR. The PCR product was cloned into pUC18 and then subcloned into pLIT / RRrev3. The two fragments of rev were fused using the QuickChange kit. The RRE from HIV-2 was replaced by the HIV-1 RRE, and both constructs were cloned into the gag / pol / Zz vector. The degenerated rev sequence was also used directly for subcloning into a vector, such as a packaging construct for pVIRPac-2.
[0304]
To degenerate the first 42 nucleotides of gag, which is required for packaging and should be part of the vector plasmid, the BssHII / SpeI fragment from pNL4-3 was inserted into pLITMUS38. Subcloned. The gag sequence was degenerate using the Quick Change kit, thus eliminating all components of the packaging signal and reinserting the HIV major splice donor before the first ATG codon of gag. The degenerate gag is replaced with Dr. in place of the 5 'LTR to form a continuous gag sequence. Subcloned into the pNgp plasmid from Conde.
[0305]
The resulting construct contains the splice acceptor (SA) and splice donor (SD) sequences used to express the vif protein. This splicing results in the undesired degradation of the spliced retroviral (conditionally replicating) vector RNA and the reduction of the ribozyme cassette in the spliced mRNA, which is undesirable due to the reduced titer of the packaged transduction vector. Can bring about existence. Overlapping PCR was used to mutagenize the SA and SD sequences. The PCR product was inserted into the gag / pol construct and cloned, followed by insertion of the anti-U5 ribozyme cassette and rev and RRE elements.
[0306]
Vesicular stomatitis protein G (VSV-G) env protein was cloned into the first MCS of the pMG-tat vector to generate an envelope expression cassette. To further simplify the cloning step, VSV-G was cloned as a blunt-ended fragment into Bgl II filled in with Klenow polymerase. Constructs with the SV40 origin of replication (ori) were generated. The rev sequence was subcloned into a second MCS to make the packaging plasmid critical as a helper vector. The resulting plasmid was named pVIRPAC-2 (see FIG. 6).
[0307]
The gag / pol / Rz / RRE / rev cassette was subcloned into the pMG-tat-G plasmid (without SV40 ori) to generate a Herber vector from several packaging plasmids. Constructs with RRE from either HIV-1 or HIV-2 were produced (pVIRPac-1 and 1.2Rz in FIG. 6). As a control to test ribozyme function, the ribozyme cassette was deleted in the final HIV-2 RRE containing construct, generating pVIRPac-1.2. Finally, in order to test the hypothesis that forcing gag / pol RNA into the splicing machinery can prevent degradation of retroviral (conditionally replicating) vector genomic RNA, introns from β-globin were replaced with Inserted before the SV40 poly A signal (pVIRPac-1.2RzIn). Of course, in other aspects of the invention, the inserted intron is derived from HIV, preferably a complete HIV intron that ensures the directing of the RNA into the cell splicing machinery (eg, splicesomes).
[0308]
Example 4.
This example describes the efficacy of various helper vector constructs. A standardized protocol for retroviral vector production by transient transfection was used for those experiments. Large quantities of plasmid DNA were produced using known techniques. To generate the virus, 293T cells were co-transfected with a retroviral vector and a helper vector plasmid using the calcium phosphate method. Cell supernatants were collected about 40 hours after transfection, filtered through a 0.45 μm syringe filter, and dropped on HT1080 cells. Vector titers were calculated about 72 hours post-infection based on their transduction efficiencies as determined by flow cytometry. Arbitrary expression N^*400,000^*(Where N is the transduced cell fraction; 400,000 is the approximate number of cells at the time of infection; and F is the dilution factor) was used for titer calculations. .
[0309]
To enhance the safety of helper vectors by reducing the likelihood of producing RCV based on retroviral vector-based recombination with HIV-1, an anti-U5 ribozyme cassette is used in certain helper vector constructs. Was inserted. It cleaves ribozymes and destroys retroviral vector RNA when co-packaged in virions. However, ribozymes can also degrade vector RNA in producer cells, thus preventing virus production. To further increase stability, the helper construct included an RRE-2 element to further reduce the tendency for helpers and vectors to be co-packaged into viral particles. These helpers were used to package the pN1 (cPT) GFP vector.
[0310]
As shown in FIG. 7, the presence of the ribozyme on the helper construct had little effect on vector titers. Importantly, apart from vector RNA, helper constructs containing introns that affect cell trafficking of helper RNA (pVirPac1.2RzIn) also had no significant effect on vector titers. Vector titers produced this 2-plasmid system comparable to that obtained with the conventional 3-plasmid transfection system. A similar experiment with the helper construct, RRE-1, containing pVirPac1.1 showed a 5-fold lower packaging efficiency. Thus, incorporation of RRE-2 into the helper vector construct not only increased its stability by reducing the likelihood of homologous recombination, but also unexpectedly increased the efficiency of packaging.
[0311]
Example 5.
This example tests whether the expression of the MGMT gene from HIV-LTR spliced mRNA is sufficient for efficient selection of MGMT transduced cells by BG and BCNU. FIG. 10A shows the vector used. FIG. 10B shows effective cell viability and expansion in the presence of cell killing concentrations of BG / BCNU. The latter names are as follows: M, MGMT; I, IRES; G, GFP; W, Woodchuck-transcriptional element; and C, CMV promoter. All of the above elements are located downstream of the RRE element so that any genetic death 3 ′ to the RRE element is expressed from the spliced mRNA since the splice receptor site is located proximal to the gene insertion site. In the pN1 vector. The figure shows that control cells that were not transduced by the lentiviral vector ("CGFP") did not expand or survive, whereas all vectors expressing MGMT survived and expanded.
[0312]
Cells transduced by pN1CGIG died after 14 weeks, whereas cells transduced by pN1MCG died after 21 weeks. Importantly, cells transduced by pN1MIG and pN1MIG-W survive after 29 weeks, indicating that the cells transduced by the vector without the inserted internal polomotor had a prolonged period. Shows viability.
[0313]
Surprisingly, the vector expressing MGMT from the HIV-LTR spliced mRNA was very effectively selected. MGMT expression does not limit the range of loading genes to either MGMT or selection genes. The results indicate that either gene can be expressed from the HIV-LTR promoter in T cells. Similar data was obtained in non-T cells, indicating that expression of the gene from the HIV-LTR spliced mRNA could be spliced for gene expression in many cell types.
Furthermore, the bcistronic nature of the "MIG" construct indicates that multiple genes can be expressed when bound by an IRES. Other examples include MGMT or other selections with chemokines, interferons or other genetic antiviral agents.
[0314]
Example 6.
Human CD4⁺ T cells were tested for their sensitivity to BC or BCNU. CD14 wasted CD4⁺Cells were purified from peripheral blood with or without cytokine transfer. Cells were cultured in 3 μg / ml IL-2 for 4-7 days before BC and BCNU treatment. Under appropriate conditions in the absence of BFG and BCNU, CD4 + cells were expanded 100-fold in culture for 10 days. The doses tested were 0, 0.2, 0.5, 2, 5, 10, 20, 30, and 40 μM BC and 0, 2, 5, 10, 20, 30, 30 and 40 μM BCNU. A series of three experiments were performed.
[0315]
FIG. 11 shows representative results from the above. Expansion was reduced 20-fold after treatment with 10 μM BC and μM BCNU, and about 10-fold or less after treatment with 20 μM or more BCNU. CD4 by BCNU⁺Treatment of the cells did not result in growth retardation. Thus, unlike the Sup T1 cells used in the above example, CD4⁺Cells are not sensitive to low doses of BCNU alone. This indicates high MGMT-expressing primary CD4 + cells, although variations in MGMT expression are expected based on variations between T cell donors.
[0316]
The presence of both BG and BCNU at 10 μM or higher was best for sensitization of CD4 + cells, and BG at 0.5 μM and BCNU at 15 μM or higher sensitized the cells as well. Increasing BG dose did not increase cell killing, except that toxicity to CD4 cells increased with dose of BCNU.
As shown in FIG. 11, the level of selection generally increased 5-fold from baseline 20% to 100%. In one experiment, the selectivity was between 3% and 90%. A 10-100 fold expansion was found after selection and one week; a 20-400 fold expansion was found at a 2.5 fold selection level, with an average expansion of 80 fold.
[0317]
Example 7.
Previous studies suggest an interrelation between purging bone marrow autografts with improved disease-free viability after autologous transplantation. Purging of normal T cells from autologous grafts has also been used to reduce the risk of graft versus host disease. FIG. 13A shows the efficiency of transduction of tumor cells by a single transduction with pN1 (cPT) ASEnvGFP. FIG. 13B shows that the tumor cells were CD34.⁺The CD34 as stem cells do not show significant transduction after one transduction.⁺4 shows that they can be killed more selectively and effectively than stem cells.
[0318]
The above application for purging tumor cells from autologous bone marrow begins with harvesting bone marrow or collecting peripheral blood stem cells. CD34 + cells are purified through a CD34 antibody column and then transduced with a conditionally replicating vector at a MOI of 2-400 for 2-16 hours. The cells are optionally incubated in the presence of one or more cell activators, eg, cytokines, to help transduce or maintain the cells. Next, the cells are transferred to the subject to be treated.
[0319]
The ability to similarly target T cells in autologous transplants is the ability of Selective Transduction of T Cells to be simultaneously transduced by the US Attorney Docket No. 397272000400 filed August 31, 2000. Use a conditionally replicating vector in combination with the high efficiency stable transduction protocol in the patent application (not yet assigned).
[0320]
Example 8.
FIG. 14 (panel B) shows the effective pseudotyping of the HIV-1 vectors of the present invention by various envelope proteins.
Initial titers more similar to lentiviral vectors pseudotyped with the VSV-G envelope protein can be effectively increased through purification using either high speed centrifugation or diafiltration. The vector pN1 (cPT2) ASenvGFP was packaged with pVirPac1.2Rz2. The table below shows (a) total protein in the sample, (b) vector titer in the sample, and (c) purification of the vector from exogenous material (magnification) in the sample.
[0321]
[Table 1]

[0322]
As can be seen from the table, after clarification of cell debris, the total protein in the vector supernatant sample was 3027 mg, and the vector titer at that stage was 5.9 × 10⁶It is. Next, the sample underwent concentration by ultrafiltration, by which time the synthetic protein in this sample was 722 mg and the vector titer was 8.2 × 10⁷With a 61.7-fold purification. After concentration by ultrafiltration, the sample was diafiltered, by which time the total protein in this sample was further reduced to 224 mg and the vector titer was 6.8 x 10⁸/ Ml with 1650-fold purification. This indicates that non-vector exogenous proteins have been removed and the lentiviral vector pseudotyped with VSV-G can be purified using the above protocol.
[0323]
The above, as shown in FIG.¹⁰Can be used in combination with a vector produced in a Hela-tat cell line that produces a titer of
[0324]
Example 9.
Other helper constructs for pseudotyping a conditionally replicating vector have been developed by placing VSV-G expression under Rev-dependent control. Since RRE-containing mRNAs were not expressed in the absence of Rev, they were presumed to be defective due to the presence of the cis repressor sequence (CRS) present in the gag / pol, pol and env genes. CRS has been reported to entrap mRNA in the nucleus, destabilize the mRNA, or prevent binding of the ribosome to the mRNA. Thus, the presence of trans-supplied CRS and Rev, combined with mRNA splicing, can be used to regulate gene expression.
[0325]
The Rev / RRE / CRS system was used to control VSV-G expression in a Rev-dependent manner. Helper constructs for this approach include 5 'splice donor sites; HIV-2 RRE and 3' splice acceptor sites (preferably for tat / rev); propensity for recombination with HIV-1 based vectors. And optionally a non-inducible promoter in place of any inducible promoter, such as the immediate CMV (IE) promoter. VSV-G encoding RNA is produced without induction, but is regulated by the presence of Rev, as only unspliced RNA can be transported to the cytoplasm without Rev and expressed. . VSV-G expression remains Rev-mediated because Rev prevents mRNA splicing and nuclear retention of transcripts controlled by CRS.
[0326]
Preferably, the splice donor site is not a synthetic 5 '[beta] -globin donor site, but is a native HIV, or HIV analog, splice donor site. HIV-2 RRE is a 280 bp fragment containing both the HIV-2 RRE, and a splice acceptor site for Tat / Rev; and the CRS is a 550 bp internal fragment from the HIV-2 pol sequence. CRS and RRE elements from HIV-2 can be prepared by PCR. Non-limiting examples of such helper constructs include pCGCRSRRF, pCGCRSRzRRE, and others shown in FIGS. 15A-15E. The final plasmid construct was confirmed by multiple restriction enzyme digests. pEH-GP and pCMV-GP are constructs that constitutively express the gag-pol polyprotein, but other promoters, including lentivirus or HIV promoters, can be used if desired.
[0327]
FIG. 17 shows the results from the use of many different splice donor combinations for Rev-dependent expression of VSV-G. As shown, removal of tetracycline results in Rev-dependent expression.
Additional examples of splice-donor site combinations and consensus sequences are provided below. All can be used, but HIV major, HIV-1 env, HIV-2 major, and analog splice-donor combinations are preferred.
[0328]

[0329]
An example 10.
Prior to use, various storage buffers and conditions were tested to identify appropriate conditions for storage of the vector. The result is shown in FIG. The ability to store the vector at -20 ° C and in a pharmaceutically acceptable buffer without significant loss of vector recovery would be of enormous advantage in maintaining the vector prior to use. Of course, storage of the vector at about -80 ° C, about -20 ° C, and about 4 ° C in other pharmaceutically acceptable compositions can also be easily performed.
[0330]
An example 11.
This example describes the amino acid sequence of a chimeric HIV CTL epitope for use in practicing the present invention. The sequence comprises the first methionine (M) to initiate translation, followed by various contiguous subsequences corresponding to p17, p24, p15, pol, Rev, gp120env, gp41env and nef, respectively.
[0331]
[Table 2]

[0332]
An example 12.
This example provides elements of aspects of the invention as described above. The invention includes a conditionally replicating viral vector comprising one gene (or other nucleic acid) to be expressed, and one or more first nucleotide sequences, wherein the vector comprises (a) B.) Replicating in the host cell only on the basis of complementation with a wild-type virus strain, a helper virus or a helper vector, each of which is sensitive to the presence of said one or more first nucleotide sequences; C.) Comprising one or more substitutions, insertions or deletions that are resistant to said one or more first nucleotide sequences and (c) reduce the tendency to generate replication competent vectors.
[0333]
To reduce the tendency to generate replication competent vectors, the present invention provides vectors comprising degenerate sequences with respect to one or more sequences of helper constructs, host cell nucleic acid content, or other nucleic acids. I do. In addition, the helper construct can also include degenerate sequences for one or more sequences of vectors, host cell nucleic acid content or other nucleic acids.
[0334]
The vectors of the present invention can also include one or more sequences that target a helper construct. Such sequences include a genetic antiviral agent and are preferably ribozymes or antisense nucleic acids (or encode). Preferably, the ribozyme and antisense sequences ensure their base pair complementarity with their respective targets through GU base pairing in addition to standard AT, AU and GC base pairing. Can receive. Similarly, helper constructs of the present invention can also include the same type of sequence to target a vector.
[0335]
A helper vector of the invention can also include one or more sequences that affect the localization of RNA expressed by the helper relative to the vector, or cell trafficking. For example, a helper vector can include an intron, a heterologous RRE, as compared to a vector, or a heterologous gag sequence, as compared to a vector. The helper vector can also include a degenerated gag and / or pol sequence relative to the vector, such that the helper and vector RNA are probably co-localized. Helper vectors can also include a splice-donor site so that the packaging and RRE signals (sequences) are removed from the RNA to prevent their co-localization with the vector RNA. Such a splice-donor site can also be present in the helper vector to provide differential tracking of the vector as compared to the helper vector or wild-type virus.
[0336]
The helper vector of the invention can also pseudotype the vector of the invention. Typical examples include VSV-G, RD114, rabies virus, GALV and chimeric envelope proteins. The helper vector of the invention also preferably relates to the vector, from a heterologous virus. An example is an HIV-2 derived helper vector. The method of the invention involves the use of such a helper vector to replicate the vector of the invention.
[0337]
Exemplary vectors of the invention include those in the series of pN1 and pN2 shown in FIGS. 1A-1K, with the removal or replacement of the GFP sequence. Preferably, the vector carries at least an antisense sequence and a cPT inclusion sequence. Exemplary helper constructs of the invention include those of the series of pVirPac as shown in FIGS. 6A-6G, with and without ribozymes.
[0338]
All vectors and helpers of the present invention may be degenerate as described above or, if necessary, in other parts of the construct. Preferred degeneracy is for codons used selectively in human cells. Vectors and helpers can also be chimeric in nature, such that they are derived from different naturally occurring and synthetic nucleic acids. However, particularly preferred vectors are derived from HIV-1 or encode the HIV-1 Tat protein. On the other hand, the vector does not encode a tat protein supplied by a helper vector, wild-type virus or host cell.
[0339]
The vectors of the present invention also preferably do not encode or express viral accessory proteins. Vectors can also comprise nucleic acids that, when present in a host cell, reduce, minimize or prevent viral infection, or render the cell resistant to toxic agents. Examples of such nucleic acids include cell surface proteins or truncated CCR5 proteins, preferably CCR5Δ32T proteins. Other examples include nucleic acids encoding resistance to drugs or DNA damaging agents, preferably multiple drug resistance, or MGMT variants, particularly G156A variants. Additional examples include nucleic acids encoding a transdominant phenotype. A typical example is the transdominant HIV-1 gag sequence.
[0340]
The vectors of the present invention can also include sequences that assist in expressing the heterologous sequence. A typical example is an insulator sequence. Vectors can also include sequences that improve the transduction of the vector into cells. A typical example is a 545 base pair sequence comprising the cPT described herein, although smaller or larger fragments containing the cPT may be used. Vectors can also include LTRs for expressing genes and nucleic acids, including LTRs modified at the transcription binding site located thereon. Preferably, such a modified LTR is included in a lentiviral vector. The method of the invention involves the use of such vectors.
[0341]
The vectors of the present invention can also express at least two genes using appropriately located splice sites or IRES elements. Vectors can also be packaged in viral particles with one or more chimeric envelope proteins that stimulate the target or host cell for transduction. In addition, the vector may be packaged in a cell, wherein the helper comprises a protein or the helper comprises a Rev / RRE / CRS system to regulate gene expression. The method of the invention involves the use of such vectors.
[0342]
The vectors of the present invention can also be used to selectively kill transduced cells, in combination with the administered agents that are not normally toxic to non-transduced cells. Preferably, such vectors work by producing nucleic acid molecules rather than proteins. A typical example is the production of anti-MGMT antisense or ribozyme nucleic acid molecules. The method of the invention involves the use of such vectors.
[0343]
The present invention also provides a method for storing the vector of the present invention.
All references cited herein, such as patents, patent applications, and publications, are incorporated herein by reference. As used herein, all terms used in the singular include both the singular and the plural. Although the present invention has been described by emphasis on the preferred embodiments, variations in the preferred embodiments may be prepared and used, and the invention may be practiced otherwise than as specifically described herein. It will be apparent to those skilled in the art that The present invention is intended to cover such variations and other implementations. Accordingly, the present invention includes all modifications that fall within the scope of the present invention.
[Brief description of the drawings]
FIG.
Figures 1A-1K show specific improved, conditionally replicating vectors encompassed by the present invention: pN1 (cPT) ASenvGFP (452), pN1 (cPT2) ASenvGFP, pN1 (cPT) cGFP, pN1GFP (cPT). ) T, pN1 (cPT) GFPTAR, pN1GFP (cPT) VT, pN2GFP, pN2ASenvGFP (418) and pN2 (spe) ASenvGFP. Of course, the marker gene for green fluorescent protein (GFP) may be removed prior to using the vector for the uses described herein. Indication: N1, the packaging sequence from gag without the gag / pol sequence but shown as gag ′ or gag ″, and the termination codon located about 40 base pairs from the ATG of the gag sequence. A minimal HIV-1 derived vector; N2, a vector derived from HIV-1 capable of expressing gag / pol sequence; AS, antisense; ASenv, env sequence present in antisense orientation; gag, pol, and env, respectively Coding sequences for viral core, reverse transcriptase and envelope-forming proteins; tat, rev, rre and nef; additional viral genes; cPTc, minimal central polypyrimidine-based sequence; cPT, large insertion of approximately 548 base pairs. Central polypyrimidine-based body; cPT2, about 438 Central polypyrimidine system comprising the sequence of base pairs (not encompass the same gag sequences cPT); spe, gag / pol sequences are not translated; GFP, encoded edge color fluorescent proteins.
FIG. 2
FIG. 2 shows the DNA sequences of wild-type HIV U5 RNA SEQ ID NO: 1 (A) and the modified crHIV U5 RNA SEQ ID NO: (B). The numbers refer to the number of bases downstream from transcription.
FIG. 3
Figures 3A-3E show the effect of the ratio of helper vector: conditionally replicating (cr) vector on the titer of the resulting cr vector. FIG. 3A shows a molar ratio of 1: 0.5 to produce the highest titer for pN1 (cPTc) GFP; FIGS. 3B and 3C show the highest titers for pN1 (cPT) GFP and pN1 (cPT2) ASenvGFP, respectively. 3D and 3E show a molar ratio of 1: 1.5 to provide the highest titers for pN1cGFP and pN2cGFP, respectively. The vectors are shown in FIG. 1, except that pN1 (cPT) GFP lacking the antisense env sequence, and pN1cGFP or pN2cGFP with the inserted cytomegalovirus (CMV) promoter to express GFP. except. The figure is at least 1.5 × 10⁷Shows that the conditions for the production of a titer of 5 transduction units / ml were achieved with the two plasmid systems.
FIG. 4
4A and 4B show maps of two conditionally replicating vectors based on HIV-2; pS1cGFP and pS2cGFP. The designations pS1 and pS2 refer to the absence or presence of the gag / pol sequence as described above for pN1 and pN2. Display c shows the presence of the CMV promoter to direct GFP expression.
FIG. 5
5A and 5B show the effect of different helper vector constructs on pS1cGFP and pS2cGFP packaging. The effect of different molar ratios between the helper vector and the conditionally replicating vector was tested, along with the effect of different RREs on the helper vector. As shown, the use of a 1: 1 ratio of helper vector: conditionally replicating vector was more effective at generating functional vector particles than the other ratios tested.
FIG. 6
FIGS. 6A-6G show the following various helper vector constructs: pVIRPAC-1, pVIRPAC-2, pVIRPAC-1.1Rz, pVIRPAC-1.2, VirPac1.2Rz, pVIRPAC-1 as encompassed by the present invention. 2 shows the structures of 2Rz2 and pVIRPAC1.2RzIn. Rz refers to the presence of the anti-U5 ribozyme, and 1.1 and 1.2 refer to helpers with RREs from HIV-1 and HIV-2, respectively.
FIG. 7
FIG. 7 shows the effect of one or more ribozymes on viral vector titers. pN1 (cPT) GFP is composed of pVirPac1.2 (without ribozyme), pVirPac1.2RzIn (including one ribozyme and an intron), pVP1.2Rz (including one ribozyme) or pVP1.2Rz2 (containing two ribozymes). ) In the presence of HeLa-tat cells. As shown by the graph, the presence of one ribozyme (pVirPac1.2Rz), or an intron designed to affect cell trafficking of ribozymes and helper RNA (pVirPac1.2RzIn), was significant relative to vector titer. Had no significant effect. PCR analysis of titer samples for co-packaged helper constructs showed low co-packaging in the presence of ribozyme versus high co-packaging in the absence of ribozyme. The indicator "VirPac" may also be indicated as "VIRPAC" or "VP".
FIG. 8
FIG. 8 shows the inhibitory effect of vectors from a series of pN1 and pN2 on wild-type HIV replication in T cells. T cells were first transduced with the vector and then challenged with wild-type virus at a multiple infection of 0.1 for 100% of the transduced cells. Virus replication was assayed by the p24 ELISA antigen capture assay. pN2ASenvGFP showed sufficient inhibition of wild-type virus replication.
FIG. 9
Figures 9A and 9B show potent inhibition of wild-type HIV replication by pNl and pN2 based vectors. FIG. 9A shows the potent inhibition of wild-type HIV in human T cells by pN1GFP (cPT) VT and pN2ASEnvGFP as compared to control tumor cells based on infection with wild-type HIV. FIG. 9B shows similar results in T cells for pN1 (cPT) ASEnvGFP and pN2ASenvGFP.
FIG. 10
10A and 10B show vectors and their use for selecting transduced cells. FIG. 10A shows the mechanism of the vector used, where pN1CMIG and pN1MCG contain an internal CMV promoter, and pN1MIG and pN1MIG-W express the MGMT gene through the HIV-LTR promoter. FIG. 10B shows a graph of the expansion of SupT1 cells transduced with the above vector and selected by BC and BCNU, as described in Example 5 below.
FIG. 11
FIG. 11 (panels AF) shows selection of transduced primary CD4 + cells by BG and BCNU. CD4 transduced by pN2MIG⁺Cells were cultured in 0, 0.5, 2, 5, 10, and 10 μM BG (shown in panels AF, respectively) with the indicated concentrations of BCNU. Panel F further shows the results of a 1: 5 fold dilution of the transduced cells, followed by treatment with 10 μM BG and the indicated concentrations of BCNU. The starting level of 3% GFP + cells in Panel F increased at least 32 fold to 97% (so far not found in vitro).
FIG.
FIG. 12 shows the effect of ribozymes on preventing co-packaging of helper RNA into vector preparations. The virus-containing supernatant was extracted by "boom extraction" and DNase I digestion followed by qualitative reverse transcriptase-PCR (RT-PCR) detection of the HIV-1 gag-pol region found in the helper construct. Entrusted to you. As shown, the presence of one or two ribozymes (pVP1.2Rz or pVP1.2Rz2) in the helper construct used is⁻The amount of co-packaged helper vector was significantly lower than the helper (pVP1.2).
FIG. 13
13A and 13B show CD34.⁺5 shows the ability to purge tumor cells from stem cells. FIG. 13A shows that at an MOI of 10, pN1 (cPT) ASenvGFP transduced 98.52% of SupT1 tumor cells after one transduction. FIG. 13B shows CD34⁺Indicates that no significant transduction was found in the transduction of the cells after one transduction. Only after 3 rounds was significant transduction observed. Indication: 1/2 / A refers to one transduction, two MOIs and the presence of viral accessory protein in the transduced vector, and 3/50 three transductions and 50 MOIs. To mention.
FIG. 14
FIG. 14A shows the structures of VSV-G wild type, RD114 wild type and chimeric envelope proteins with the indicated extracellular, transmembrane and cellular domains. FIG. 14B shows the power of the HIV-1 vector pseudotyped in HT1080 by different envelope proteins (VSV-G wild type, rabies virus G, RD114 wild type and RD114E, chimeric VSV-G and RD114 construct). Indicates the value.
FIG.
FIGS. 15A-15E are diagrams of specific packaging-based constructs encompassed by the present invention: p (CGCRSRRE), p (TRETTApuro), p (BI-RevTat), p (EH-GP) and p (EH-GP). CMV-GP). FIG. 15F shows the configuration of the rev-dependent VSV-G construct.
FIG.
FIG. 16 shows the yield of pN1 (cPT) GFP vector per cell construct before and after enrichment in Hela-tat cells. As shown, the yield was approximately 10% under either set of conditions.¹⁰To survive.
FIG.
FIG. 17 shows the results from using the Rev / RRE / CRS system to control VSV-G expression.
FIG.
FIG. 18 shows the effect of storage buffer on vector recovery after 3-5 weeks of storage at different temperatures. As shown, the presence of 10% trehalose or 10% glucose in either D-PBS or HBS provided good recovery of the vector after storage at -20 or -80 <0> C.

Claims (22)

A conditionally replicating viral vector comprising the expressed gene and one or more first nucleotide sequences,
(A) replicating in a host cell only upon complementation by a wild type strain of a virus, helper virus or helper vector, each of which is susceptible to the presence of said one or more first nucleotide sequences;
(B) resistant to the presence of said first nucleotide sequence; and (c) comprising one or more substitutions, insertions or deletions, which reduce the likelihood of generating a replication competent vector;
A vector, characterized in that: 2. The vector of claim 1, wherein said one or more substitutions is a degeneracy to a codon selected in a human cell. The vector according to claim 1, wherein the vector is a chimeric vector comprising sequences from HIV-1 and HIV-2. 2. The vector of claim 1, wherein said vector does not encode or express a viral accessory protein. The vector according to claim 1, wherein the vector is derived from HIV-1 or encodes an HIV-1 Tat protein. The vector according to claim 1, wherein the one or more first nucleotide sequences are one or more ribozyme or antisense sequences. 2. The vector of claim 1, wherein said vector does not encode or does not express a Tat protein. The vector of claim 1, further comprising a nucleic acid sequence that reduces, minimizes, or prevents viral infection when present in a host cell, or a nucleic acid sequence that renders the cell resistant to a toxic agent. 2. The vector of claim 1, wherein said first nucleic acid sequence encodes a transdominant phenotype. 2. The vector of claim 1, wherein said vector further comprises an insulator sequence. The vector according to claim 1, further comprising an LTR or a modified LTR to regulate the expression of a nucleic acid sequence present in the vector. 2. The vector of claim 1, further comprising an HIV splice site or an IRES. A helper vector capable of complementing the vector according to any one of claims 1 to 12. 14. The helper vector according to claim 13, wherein the vector is capable of further pseudo-classifying a vector that can be replicated under the condition. 14. The helper vector of claim 13, further comprising one or more ribozyme or antisense sequences targeting the conditionally replicating vector. 14. The helper vector according to claim 13, wherein the vector is derived from HIV-2. 13. The vector according to any one of claims 1 to 12, packaged in a viral particle comprising one or more chimeric envelope proteins. A helper that is a protein that can complement the vector according to any one of claims 1 to 12. A method for expressing one or more nucleic acids of interest using the vector according to any one of claims 1 to 12 or 17. 18. A method for producing a vector according to any one of claims 1 to 12 or 17, comprising high speed centrifugation rather than ultracentrifugation. A cell killing method comprising transducing a cell with the vector according to any one of claims 1 to 12 or 17. A method for selecting hematopoietic stem cells (HSCs), comprising contacting a cell population containing HSCs with a cytotoxic agent after the cells are contacted with the vector according to any one of claims 1 to 12 or 17. A method comprising:

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