JP4646625B2 - Ras媒介性腫瘍形成の治療のためのRas不活性化キナーゼサプレッサ - Google Patents
Ras媒介性腫瘍形成の治療のためのRas不活性化キナーゼサプレッサ Download PDFInfo
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- JP4646625B2 JP4646625B2 JP2004508744A JP2004508744A JP4646625B2 JP 4646625 B2 JP4646625 B2 JP 4646625B2 JP 2004508744 A JP2004508744 A JP 2004508744A JP 2004508744 A JP2004508744 A JP 2004508744A JP 4646625 B2 JP4646625 B2 JP 4646625B2
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Description
本発明に至った研究は、少なくともその一部は、米国国立衛生研究所の研究助成金グラント番号第CA42385およびCA52462号によって支援された。従って、政府も本発明にはいくつかの権利を保有する可能性がある。
本発明は、Rasのキナーゼサプレッサー(KSR)の特異的抑制のための方法および組成物に関する。特に、本発明は、KSRの特異的抑制、特に、KSR発現の特異的抑制のための遺伝学的方法および核酸を提供する。本発明は、KSRを特異的に抑制し、かつ、gf Ras介在性腫瘍形成を阻止する、KSR RNAに対して相補的なアンチセンス・オリゴヌクレオチド、および核酸の発現に関する。
Rasは、発癌遺伝子による形質変換および発癌において不可欠な役割を果たす。発癌遺伝子H−、K−およびN−Rasは、少数の部位(アミノ酸の12、13、59および61部位)に限局した点突然変異から生ずる。正常のRasと違って、発癌遺伝子rasの蛋白は、内在性のGTP活性を欠くので、体質的に活性化されたままである(Trahey,M.,and McCormick,F.(1987)Science 238:542−5;Tabin,C.J.et al.(1982)Nature,300:143−9;Taparowsky,E.et al.(1982)Nature,300:762−5)。ヒト癌における発癌遺伝子rasの関与は30%と推定されている(Almoguera,C.et al.(1988)Cell,53:549−54)。
本発明は、Rasのキナーゼサプレッサー(KSR)を特異的に抑制するための方法および組成物に関する。本発明の組成物および方法は、KSRの発現および/または活性を抑制する。特に、本発明は、KSRを特異的に抑制するための遺伝学的方法および核酸を提供する。KSRの特異的抑制においては、Ras経路が破壊され、また、Ras介在性腫瘍および腫瘍形成が特異的に抑制または阻止され、既存の腫瘍は後退し、転移は抑制され、かつ、腫瘍すなわち癌細胞の増殖は抑制される。
(a)KSRを発現する細胞を、候補化合物または薬剤の存在下、または、不在下にインキュベートする過程、および、
(b)候補化合物または薬剤の存在下、または、不在下においてKSRの発現を検出または測定する過程、
を含む方法であって、前記候補化合物または薬剤の存在下におけるKSR発現が、前記候補化合物または薬剤の不在下の場合に対して低下していることから、前記化合物または薬剤がKSRの発現を抑制することが示される方法が提供される。
本発明においては、従来技術の範囲内の、通例の分子生物学、微生物学および組み替えDNA技術の使用が可能である。このような技術は文献の中に十分に記載されている。例えば、Sambrook et al.,「Molecular Cloning:A Laboratory Manual」(1989);「Current Protocols in Molecular Biology」 Volumes I−III[Ausubel,R.M.,ed.(1994)];「Cell Biology:A Laboratory Handbook」 Volumes I−III[J.E.Celis,ed.(1994)];「Current Protocols in Immunology」 Volume I−III[Coligan,J.E.,ed.(1994)];「Oligonucleotid Synthesis」(M.J.Gait ed.1984);「Nucleic Acid Hybridization」[B.D.Hames & S.J.Higgins eds.(1985)];「Transcription and Translation」[B.D.Hames & S.J.Higgins,eds.(1984)];「Animal Cell Culture」[R.I.Freshney,ed.(1986)];「Immobilized Cells and Enzymes」[IRL Press(1986)];B.Perbal,「A Practical Guide to Molecular Cloning」(1984)を参照されたい。
(非極性R基を持つアミノ酸)
アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、トリプトファン、メチオニン
(非荷電・極性R基を持つアミノ酸)
グリシン、セリン、トレオニン、システイン、チロシン、アスパラギン、グルタミン
(荷電・極性R基を持つアミノ酸(pH6.0で負に荷電))
アスパラギン酸、グルタミン酸
(塩基性アミノ酸(pH6.0で正に荷電))
リシン、アルギニン、ヒスチジン(pH6.0において)
別のグループ分けとして、フェニル基を持つアミノ酸であってもよい。
フェニルアラニン、トリプトファン、チロシン
別のグループ分けとして分子量(すなわち、R基のサイズ)によるものであってもよい。
グリシン 75
アラニン 89
セリン 105
プロリン 115
バリン 117
トレオニン 119
システイン 121
ロイシン 131
イソロイシン 131
アスパラギン 132
アスパラギン酸 133
グルタミン 146
リシン 146
グルタミン酸 147
メチオニン 149
ヒスチジン(pH6.0で) 155
フェニルアラニン 165
アルギニン 174
チロシン 181
トリプトファン 204
特に好ましい置換は、
−Argの代わりにLys、および、その逆で、正電荷を維持する。
−Aspの代わりにGlu、および、その逆で、負電荷を維持する。
−Thrの代わりにSerとし、遊離の−OHが維持されるようにする。および、
−Asnの代わりにGlnとし、遊離NH2が維持されるようにする。
(a)KSRを発現する細胞を、候補化合物または薬剤の存在下、または、不在下にインキュベートする過程、および、
(b)候補化合物または薬剤の存在下、または、不在下においてKSRの発現を検出または測定する過程、
を含む方法であって、前記候補化合物または薬剤の存在下におけるKSR発現が、前記候補化合物または薬剤の不在下の場合に対して低下していることから、前記化合物または薬剤がKSRの発現を抑制することが示される方法が提供される。
(a)KSRを発現する細胞を、候補化合物または薬剤の存在下、または、不在下にインキュベートする過程、および、
(b)候補化合物または薬剤の存在下、または、不在下においてKSRの活性を検出または測定する過程、
を含む方法であって、前記候補化合物または薬剤の存在下におけるKSR発現が、前記候補化合物または薬剤の不在下の場合に対して低下していることから、前記化合物または薬剤がKSRの発現を抑制することが示される方法が提供される。本発明の一つの局面では、KSRの活性および/または発現は、KSRキナーゼ標的の、または、キナーゼ標的配列ドメインを含むペプチドのリン酸化状態を定量することによって評価または監視してもよい。例えば、RafまたはRaf−由来ペプチドのリン酸化状態をそのような評価に利用してもよい。KSRの活性または発現はまた、インビトロの腫瘍細胞において、または、インビボの腫瘍動物モデルにおいて評価し、それによって、本出願の実施例中に記述されるように、Ras媒介性腫瘍形成、細胞増殖、または、転移を監視するようにしてもよい。
(要約)
Drosophila melanogaster(ショウジョウバエ)およびCaenorhabditis elegans(線虫)では、Rasのキナーゼサプレッサー(KSR)は、Rafの上流において、または、平行してRas/マイトジェン活性化プロテインキナーゼ(MAPK)のシグナル伝達を積極的に修飾する1−3。しかしながら、哺乳類KSRの正確なシグナル伝達機構、および、Ras−介在性形質変換におけるその役割は未だに不明のままである。組み換えKSRを著明に過剰発現する細胞を利用して、ある研究グループは、KSRが、MAPK活性化およびRas−介在性形質変換を抑制すると報告したが、4−6、一方別のグループは強調作用を観察した。7−10証拠から、この不一致は、遺伝子用量効果を反映することが示唆された。11インビボにおけるKSR機能に関してさらに深い理解を得るために、我々は、ホモのKSR欠損マウスを作成した。ksr−/−マウスは生存可能で、大きな発達障害を持たない。しかしながら、マウス新生児は、EGFR−欠損マウスで以前観察された、独特の毛嚢表現型を呈する。これは、EGFR、RasおよびKSRは、哺乳類において同じシグナル伝達経路に乗るという説に対して遺伝学的支持を与える。ksr−/−動物胎児の線維芽細胞は、MAPK経路のEGF活性化において欠陥があり、増殖能の低下およびRas−依存性形質変換能の欠損を示した。皮膚特異的ν−Ha−ras発現によってもたらされるTg.ACマウスの腫瘍形成は、ksr−/−背景において打ち消された。従って、本出願に提示される証拠は、KSRは、EGFR−/Ras−介在性新生物形成を誘導することであって、抗KSR治療方策が潜在的に標的とするのはこのKSRの新生物形成の誘導であることを示唆する。
序論
Rasのキナーゼサプレッサー(KSR)は、Drosophila melanogasterおよびCaenorhabditis elegansでは、Rafの上流において、または、平行してRas/マイトジェン活性化プロテインキナーゼ(MAPK)のシグナル伝達を積極的に修飾する因子として特定された(1−3)。哺乳類KSRの生化学的性質の解明に向けて集中的努力が注がれてきたけれども、その正確なシグナル伝達機構は不明のままである。特に、Ras−介在性形質変換におけるKSRの役割については納得できる取り組みがなされていない。KSRはMAPK活性化およびRas−介在性形質変換を抑制するというグループもあれば(4−6)、一方、強化作用を観察したグループもある(8−10)。これらの実験では、内因性KSRをはるかに越える濃度で組み換えKSRを過剰発現する細胞システムが用いられており、証拠から、上記不一致は遺伝子用量を反映する可能性のあることが示唆されている(11)。我々および他の研究者は、Raf−MAPKカスケードの最適活性化には、キナーゼとKSRの支柱機能の両方が必要であることを論じているのであるが(26−30)、別の人々は、KSRは、支柱機能を通してのみシグナル伝達すると考えている(9,12,31)。
結果および討論
哺乳類におけるKSRのインビボ機能を調べるために、我々は、マウスのksr座位に標的を定めた。これは、KSR発現に欠損のあるマウスを得るためである。ksr−/−マウスは、図1aに示すpF9ターゲッテイングベクターを用いて胚性幹細胞(ES)中にホモ組み換えを行って生成した。標的とされた領域は、KSR独特のCA1ドメインの85%をカバーする、開始メチオニン(ksr cDNAにおけるnt 83のATGコドン)とそれに続く74個のアミノ酸を含んでいた。2種類の標的ESクローン(図1b)を、C57BL/6杯盤胞にマイクロインジェクションしたところ、両方ともキメラマウスをもたらし、これらは、突然変異ksr対立遺伝子を後代に伝えて生殖系列を確立した。ksr+/−マウスを交雑したところ、期待されたメンデル頻度の遺伝型を持つ子孫が得られた。マウスゲノムDNAから、野生型(wt)および突然変異型の両方の対立遺伝子を検出するためのPCRによるスクリーニング法を開発した(図1c)。
方法
(遺伝子ターゲティング)マウスksrゲノムDNAクローンを、マウス129/sv株(Stratagene、ラホヤ、カリフォルニア州)から、マウスksr cDNA(ジーンバンク、アクセス#U43585)の5’コード領域(nt1−786)を用いて調製したλFixIIファージライブラリーをスクリーニングして単離した。このマウスksr cDNA配列を図12Aに示す。標的発射ベクターpF9を、マウスksrゲノムクローンの5’末端から調製した2.5−kb SpeI−SmaI充填フラグメントを、pPGK−NTKベクター(Frank Sirotnak博士の供与による)のNotI充填部位に挿入することによって構築した。マウスksrゲノムクローンの3’下流領域から調製した6.3−kb SpeI−HindIII充填フラグメントを、このベクターのClaI充填部位に挿入した。得られたプラスミドをKpnIにより直線化し、129/Sv−由来W9.5 ES細胞(Chrysalis DNX Transgenic Sciences、プリンストン、ニュージャージー州)中に電気穿孔により挿入した。200個のG418/ガンシクロビア耐性ES細胞クローンを、pF9中に存在するもののすぐ外側(5’)のゲノム配列から得られた0.6 kb BglII−SpeIプローブを用いてサザンブロットによって分析した。このプローブは、野生型ksr対立遺伝子では5.7−kb DNAフラグメントと、脱失された対立遺伝子では3.1−kbフラグメントとハイブリダイズする。ヘテロ接合体ES細胞を、スローンケッタリング研究所のトランスジェニック中心施設において、胚盤胞段階のC57BL/6マウス胚にマイクロインジェクトした。次に、注入を受けた胚盤胞を、擬似妊娠C57BL/6マウスの子宮に移植した。キメラ雄をC57BL/6雌と交雑した。生殖系統の伝達を、アグーチのF1子孫におけるサザンブロットにて監視した。マウスの遺伝型分析のために、マウスの尾部からゲノムDNAをDNeasy kit(Qiagen社、バレンシア、カリフォルニア州)を用いて単離し、これを、BglIIおよびXhoIにて消化し、ES細胞の場合と同様サザンブロットで調べるか、または、2組のプライマーを用いてPCR増幅によって分析した。野生型対立遺伝子用プライマーは、マウスksr CA1ドメインのcDNA配列から得られたもので、上流プライマー5’−TATCTCCATCGGCAGTCT−3’(配列特定番号20)、下流プライマー5’−TCGACGCTCACACTTCAA−3’(配列特定番号21)である。突然変異対立遺伝子用のプライマーは、ネオマイシン・フォスフォトランスフェラーゼ遺伝子の配列から得られたもので、上流プライマーは5’−CTGACCGCTTCCTCGTG−3’(配列特定番号22)、下流プライマーは5’−ATAGAGCCCACCGCATCC−3’(配列特定番号23)である。予想される産物のサイズは、野生型対立遺伝子では493−bpで、欠失対立遺伝子では312−bpである。標準的なPCR条件を用いた、すなわち、94℃で5分間の初期変性の後、56℃でアニーリング、72℃で伸長、および、94℃で変性、全体30秒から成るサイクルを30サイクルである。
(参考文献)
(要約)
ヒト癌における発癌遺伝子ras突然変異の高頻度が知られている今、機能獲得(gf)Rasシグナル伝達の選択的不活性化は、これまで臨床的には実現されたことがないが、極めて魅力的な治療法と映る。これに鑑みて、gfRasシグナル伝達を、gfRasに対して選択的な直接下流のエフェクターであるRas1のキナーゼサプレッサー(KSR1)を遺伝学的にまたは薬理学的に不活性化することによって間接的に標的した。KSR1不活性化は、いくつかのヒト腫瘍細胞系統およびヌードマウス異種移植組織において、内部的に活性化された上皮成長因子または発癌性K−Ras突然変異によって誘発されたgfRas介在性腫瘍形成を解消した。KSRアンチセンス・オリゴヌクレオチド(AS−ODN)連続注入によるksr1の抑制は、ヌードマウスにおける、発癌性K−ras−依存性ヒトPANC−1膵臓異種移植組織、および、A549非小細胞型肺癌(NSCLC)異種移植組織の成長を阻止し、確立されたPANC−1腫瘍の寛解をもたらし、A549肺癌転移を抑制した。しかも、これらを外見上毒性を示すことなく実現した。これらの研究は、KSR AS−ODNは、gfRas−依存性ヒト悪性腫瘍の治療剤、特に、現在のところ有効な治癒法が存在しない膵臓癌の治療剤として使えることを示唆する。
(ksr1遺伝子発現の抑制は、A431細胞に形態変化を誘発する)線虫では、KSR1は、LET−23−EGFR相同体である−を経由して起動される経路である、陰門発達のgfRasシグナル伝達を制御する(10,11)。EGFR−介在性腫瘍形成における哺乳類KSR1の役割を探るために、我々は、A431類表皮癌腫瘍系統を用いた。この系統では、腫瘍成長が、野生型Rasにより、活性化EGFR/HER1(107受容体/細胞)の100倍駆動される(14)。我々は、Retro−Tet−Offシステムを用いて、野生型KSR1(KSR−S)、アンチセンスKSR1(KSR−AS)、および、優勢陰性KSR1(DN−KSR)の誘発可能形態を安定に発現するA431細胞系統を創成した。Flag標識KSR−SとDN−KSRが同レベルで発現されたのに対して(図5A)、KSR−ASの安定的発現は、内因性KSR発現の60%低下をもたらした(図5B)。さらに、ドキシサイクリン(Dox)は、KSR−S発現の用量依存性抑制を引き起こし(図5C)、しかも、Dox添加後にそれを取り去ると、KSR−R発現は効果的に復帰した(図示せず)。同様の結果が、DN−KSRおよびKSR−AS細胞でも観察された(図示せず)。これらの所見は、KSR−Tet−OffシステムはDoxによって緊密に制御されることを示す。
A431細胞の悪性、および、インビトロにおけるA431細胞のEGFに対する反応に及ぼすKSR1抑制結果を評価するために、細胞増殖、侵入および形質変換のアッセイを実行した。KSR−SがA431細胞によって過剰に発現された場合、基礎増殖とEGF刺激増殖(図6A)、侵入(図6C)、および、形質変換(図6D)はいずれも著明に強化された。それと対照的に、KSR−AS細胞におけるksr1発現の消失は、基礎増殖、侵入および形質変換に対する有意な抑制をもたらし(図6、それぞれp<0.005)、かつ、EGF反応を遮断した(図6)。DN−KSR作用は、KSR−ASで観察されたものと同様であった(図6)。
討論
本研究は、KSR1が、組織レベルにおけるgfRasシグナル伝達に結合すること、および、ksr1発現の抑制はgfRas依存性腫瘍の選択的寛解をもたらすことを示す証拠を与える。Ras/Raf−1/MAPKシグナル伝達カスケードの要素を抑制することによってgfRas依存性腫瘍を治療しようと設計された従来の臨床実験は、これまでのところほとんど不成功であった(9)。多くの薬剤において毒性は受容可能ではあるものの、治療効果が、最近のデータでは別々の生物機能を持つことが示唆されている(24−26)、様々なRas異性形を抑制する点で特異性が欠けていること、生理的Rasシグナル伝達に対してgfに対する選択性の欠けるために(8,9)限られていた。同様の問題が、Raf−1/MEK1/MAPKカスケードの要素を抑制するように設計された実験薬についても存在する(9)。KSR AS−ODNの作用に関する本研究は、Ras依存性ヒト腫瘍の治療においてgfRasシグナル伝達を特異的に減衰させる方法を提供する。
方法
(Retro−Tet−OffA431細胞系統の培養および作製)ヒト上皮癌細胞系統A431、肺癌細胞系統A549、および、膵臓癌細胞系統PANC−1、Capan−2、PL−45、HPAF−II、AsPc−1、および、MiapaPa−2を、ATCC(マナサス、バージニア州)から入手した。完全長の、マウスの野生型ksr1 cDNA−これは、ヒトksr1と90%以上同じ(12)−を、pPetro−Tet−Off(Clontech、パロアルト、カリフォルニア州)においてドキシサイクリン誘発性プロモーターの下で、pRetor−TRE中において、センス(KSR−S)、アンチセンス(KSR−AS)両方の向きでクローンした。DN−KSR(D683A/D700A/R589M)を同様にサブクローンした。KSR−S、KSR−AS、DN−KSR、または、空のベクターによってトランスフェクトされたPT67パッケージング細胞から採取した培養液でA431細胞を感染させ、二重選択(0.1mg/mlネオマイシン、および、0.1mg/mlヒグロマイシン)の下に維持した。
(文献)
さらに別のアンチセンス・オリゴヌクレオチドを合成し、表1に示すようにA431細胞における増殖アッセイによって試験した。アンチセンス・ヌクレオチドおよびその配列は、安定なホモの二量体形成の無いこと、ヘアピンループ形成の無いこと、自己相補性配列を持たないこと、および、安定な2本鎖形成の無いこと(<−6 kcal/mol)という基準(ハイブリダイゼーション条件に基づく)に基づいて選ばれた。配列は、Oligo 4プログラム(Molecular Biology Insights,Inc.、カスケード、コロラド州)を用いて選択し、次に、Oligo Techプログラム(Oligo Therapeutics Inc.、ウィルソンビル、オレゴン州)を用いてその有効性を確認した。アンチセンス・オリゴヌクレオチドは、KSR CA1ドメインの、ヌクレオチド151から179に対して(ASオリゴ配列5’−CAGCCCGCGCAGACTGCCG−3’)(配列特定番号6)、および、ヌクレオチド181から198に対して(ASオリゴ配列5’−GAGGTCGTTAGACACTGA−3’)(配列特定番号7)(両方共P−Sオリゴヌクレオチド)生成された。これらのオリゴヌクレオチドを、実施例2で記述した、ヌクレオチド214から231に対するAS−ODNオリゴヌクレオチド(ASオリゴヌクレオチド配列特定番号8)と共に試験した。これらのアンチセンス・オリゴヌクレオチドは、それぞれ、ヒトKSR CA1ドメインのアミノ酸配列GSLRGI(配列特定番号17)、AVSNDL(配列特定番号18)および、RTLEAK(配列特定番号19)をコードする核酸の逆相補体を表す。A431細胞を、表示量のKSR AS−ODNにてトランスフェクトし、この処置の72時間後に細胞増殖を評価した。AS−ODNのA431細胞増殖に及ぼす作用は、ベヒクル処理(オリゴフェクタミンのみ)コントロールに対する抑制パーセントで表した。これは、4回の同様実験の内の一つを表す。
gfRasシグナル伝達仲介におけるKSR1の特異性を確定するために、よく特徴が明らかにされているヒト慢性骨髄性白血病細胞系統K562を用いた。K562をBcr−abl駆動して、gfRasシグナル伝達とは無縁とした。AS−ODN処置による、PANC−1細胞増殖の特異的、用量依存的抑制を図13に示す。PANC−1およびK562細胞を、表示の用量のコントロールまたはAS−ODNで処理し、細胞増殖アッセイを実行した(図13A)。AS−ODN−1およびAS−ODN−2は、それぞれ、ksr1 cDNAのヌクレオチド214−231および181−198に対応する。PANC−1細胞増殖は、予想通り抑制されたが、K562細胞増殖はODN処理による影響を受けなかった。K562細胞を5μM KSR AS−ODN−1で処理したところ、ウェスタンブロット分析で調べると、内因性KSR1遺伝子の発現が、PANC−1細胞で観察されたものと同程度に抑制(80%を越える)された(図13B)。にも拘わらず、増殖の抑制はPANC−1細胞のみに観察された(図13A)。ゲル負荷の等しいことは、全体P44/42 MAPKを用いて確認した。ウェスタンブロットの陽性コントロールとなる精製Flag−KSRが、Flagタグのせいで内因性KSRよりもややゆっくりと移動することに注意(図13B)。これらの結果は、gfRasシグナル伝達がKSR1と特異的に結合することを示す証拠を与える。
Claims (36)
- ストリンジェントな条件下で5′−GGCAGTCTGCGCGGGCTGC−3′、5′−TCAGTGTCTAACGACCTC−3′、または5′−CGGACCCTAGAGGCAAAG−3′にハイブリダイズ可能な配列を含む、アンチセンス・オリゴヌクレオチド。
- 5′−GGCAGTCTGCGCGGGCTGC−3′、5′−TCAGTGTCTAACGACCTC−3′、または5′−CGGACCCTAGAGGCAAAG−3′に100%相補的である配列を含む、アンチセンス・オリゴヌクレオチド。
- 5′−CGGACCCTAGAGGCAAAG−3′に100%相補的である配列を含む、アンチセンス・オリゴヌクレオチド。
- 5′−CAGCCCGCGCAGACTGCCG−3′、5′−GAGGTCGTTAGACACTGA−3′、または5′−CTTTGCCTCTAGGGTCCG−3′の配列を含む、アンチセンス・オリゴヌクレオチド。
- 5′−CTTTGCCTCTAGGGTCCG−3′の配列を含む、請求項4に記載のアンチセンス・オリゴヌクレオチド。
- 請求項1〜5のいずれか一項に記載のオリゴヌクレオチドであって、該オリゴヌクレオチドが18〜30ヌクレオチドの長さであるオリゴヌクレオチド。
- 請求項1〜5のいずれか一項に記載のオリゴヌクレオチドであって、該オリゴヌクレオチドが25ヌクレオチドまでの長さであるオリゴヌクレオチド。
- 請求項1〜5のいずれか一項に記載のオリゴヌクレオチドであって、該オリゴヌクレオチドが18ヌクレオチドの長さであるオリゴヌクレオチド。
- 検出可能な標識によって標識された請求項1〜5および7〜8のいずれか一項に記載のオリゴヌクレオチド。
- 前記標識は、酵素、リガンド、蛍光を発する化学薬品または放射性元素である、請求項9に記載のオリゴヌクレオチド。
- 前記オリゴヌクレオチドは、修飾されたバックボーンを含む、請求項1〜5および7〜10のいずれか一項に記載のオリゴヌクレオチド。
- 請求項11に記載のオリゴヌクレオチドであって、前記修飾されたバックボーンが、フォスフォロチオエート、フォスフォトリエステル、メチルフォスフォネート、ポリアミド、およびモルフォリノバックボーンから選択される、オリゴヌクレオチド。
- 請求項1〜5および7〜12のいずれか一項に記載のオリゴヌクレオチドであって、該オリゴヌクレオチドが少なくとも一つのフォスフォロチオエート結合を含有する、オリゴヌクレオチド。
- 前記オリゴヌクレオチドがオリゴデオキシヌクレオチドである、請求項1〜5および7〜13のいずれか一項に記載のオリゴヌクレオチド。
- 前記修飾されたバックボーンがモルフォリノバックボーンである、請求項1〜5および7〜12のいずれか一項に記載のオリゴヌクレオチド。
- 請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドを転写時にコードする核酸配列を含む組み換えDNA分子。
- 請求項16に記載の組み換えDNA分子であって、ここで前記核酸配列が転写制御配列に作動可能に連結される、組み換えDNA分子。
- 請求項16または17に記載の組み換えDNA分子によりトランスフェクトされた細胞系統。
- 請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドを発現させる発現ベクター。
- 治療有効量の請求項1〜5および7〜15のいずれか一項に記載のアンチセンス・オリゴヌクレオチド、および、薬学的に受容可能なキャリアまたは希釈剤を含む製薬組成物。
- 請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドおよび薬学的に受容可能なキャリアまたは希釈剤を含む組成物。
- 1種以上の化学療法剤または放射性治療剤、および請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドを含む組成物。
- 請求項1〜5および7〜15のいずれか一項に記載の、哺乳動物KSRの発現を抑制するための有効量のオリゴヌクレオチド。
- 請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドであって、哺乳動物における、gf−Ras発現、または、Rasの発現上昇と関連する癌の治療における使用のためのオリゴヌクレオチド。
- 請求項24に記載のオリゴヌクレオチドであって、ここで治療有効量の該オリゴヌクレオチドが前記哺乳動物に投与される、オリゴヌクレオチド。
- 請求項24に記載のオリゴヌクレオチドであって、ここで治療有効量の該オリゴヌクレオチドが前記哺乳動物において発現される、オリゴヌクレオチド。
- 請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドであって、哺乳動物における癌の進行の治療または抑制における使用のためのオリゴヌクレオチド。
- 請求項27に記載のオリゴヌクレオチドであって、ここで治療有効量の該オリゴヌクレオチドが前記哺乳動物に投与される、オリゴヌクレオチド。
- 請求項27または28に記載のオリゴヌクレオチドであって、ここで前記癌は、膵臓癌、肺癌、皮膚癌、尿路癌、膀胱癌、肝臓癌、甲状腺癌、結腸癌、小腸癌、白血病、リンパ腫、神経芽細胞腫、頭部・頚部癌、乳癌、卵巣癌、胃癌、食道癌、および前立腺癌からなる群より選ばれる、オリゴヌクレオチド。
- 前記癌が膵臓癌である、請求項29に記載のオリゴヌクレオチド。
- 前記癌が肺癌である、請求項29に記載のオリゴヌクレオチド。
- 哺乳動物における、gf−Ras発現、または、Rasの発現上昇と関連する癌を治療するための医薬の製造のための、請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドの使用。
- 哺乳動物において癌の進行を治療または抑制するための医薬の製造のための、請求項1〜5および7〜15のいずれか一項に記載のオリゴヌクレオチドの使用。
- 前記癌は、膵臓癌、肺癌、皮膚癌、尿路癌、膀胱癌、肝臓癌、甲状腺癌、結腸癌、小腸癌、白血病、リンパ腫、神経芽細胞腫、頭部・頚部癌、乳癌、卵巣癌、胃癌、食道癌、および前立腺癌からなる群より選ばれる、請求項33に記載の使用。
- 前記癌が膵臓癌である、請求項33または34に記載の使用。
- 前記癌が肺癌である、請求項33または34に記載の使用。
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US8895701B2 (en) | 2008-01-05 | 2014-11-25 | Sloan-Kettering Institute For Cancer Research | Peptide-conjugated oligonucleotide therapeutic and method of making and using same |
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