JP2016505245A - 安全性及び抗がん活性が増加された組換えアデノウイルス及びこの用途 - Google Patents
安全性及び抗がん活性が増加された組換えアデノウイルス及びこの用途 Download PDFInfo
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Abstract
Description
1)本発明の組換えアデノウイルスをがん細胞に導入する段階;及び
2)前記段階1)のがん細胞でレポータータンパク質を検出する段階を含む肝がん診断のための情報提供方法を提供する。
組換えアデノウイルスの構築
本発明の組換えアデノウイルスを製作するために、リボザイムは公知の文献に記載の方法により製作した(Kwon et al.,Mol. Ther. 12:824-834, 2005)。また、PEPCK遺伝子のプロモーターは公知の文献に記載の方法により製作した(Kwon, B.S. et al, FEBS Lett 580,5033-5043、2006)。また、PEPCKプロモーター、ApoA1(Apolipoprotein A1)イントロン、リボザイム及びHSVtk遺伝子で構成されたPEPCK.Rz.HSVtkを含むpcDNA 3.1(Invitrogen)ベクター(pPEPCK.Rz.HSVtk)は公知の文献に記載の方法により製作した(Song MS, et. al. Cancer Gene Ther2009;16:113-125.)。それから、前記PEPCK.Rz.HSVtkを含むpcDNA3.1(Invitrogen))ベクター(pPEPCK.Rz.HSVtk)からPEPCK.HSVtkをSalIとSmaI制限酵素で切って、予めSalIとEcoRV制限酵素で切っておいたpZAP1.1ΔNotI/BglIIベクター(OD260、Boise、ID)と連結し、pZAP1.1(PEPCK.TK)を製作した。また、pZAP1.1ΔNotI/BglIIベクターのBglIIとNotIは、追ってribozymeを入れるべき制限酵素位の重複を避けようと予め除去した。その後、hTERTを認知するribozyme配列を下記プライマーを用いてCMV−Ribo−TK vector(檀国大学校のイ・ソンウック教授から収得)から増幅した後、前記製作したpZAP1.1(PEPCK−TK)vector(BglII NotI(treated by Klenow))を増幅したribozyme配列と(BamHI/HpaI処理)と共に結合してpZAP1.1(PEPCK.Rz/As.TK)を製作した。
細胞培養
本発明で使用した全てのヒトがん細胞及びヒト乳腺上皮細胞株(human mammary epithelial cell line;HMEC)は、ATCC(American Type Culture Collection)で購入した。
ApoEエンハンサーの存在によるPEPCKに誘導されたHSVtk活性を確認するために、前記<実施例1>と同じ方法で組織特異的なプロモーターPEPCKとHSVtkを含むpPEPCK−HSVtk(pPT−O)、及びPEPCK、HSVtk並びにApoEエンハンサーを含むpPEPCK−HSVtk−Enh(pPT−E)組換えアデノウイルスを製作した(図2)。それから、24ウェルプレートで前記それぞれの組換えアデノウイルス1 MOIをHep3B肝がん細胞株及びSK−OV3卵巣がん細胞株にそれぞれ感染させた後、48時間後、前記感染された細胞に1.0 mCi/ml[8−3H]penciclovir(PCV)処理及び非処理した後、37℃で2時間培養した後、前記細胞を0.1%SDS200mlに溶解させた後、放射能を測定した。
血清型35のファイバーノブと血清型5のシャフトを含む組換えアデノウイルスの感染性を確認するために、CMVプロモーター、緑色蛍光タンパク質(GFC)を含み、E1及びE3遺伝子が欠失した組換えアデノウイルス(Ad5CMB.GFP)を製作した。また、CMVプロモーター、緑色蛍光タンパク質(GFC)及び血清型35のファイバーノブと血清型5のシャフトを含み、E1及びE3遺伝子が欠失した組換えアデノウイルス(Ad5/35CMV.GFP)を製作した(図4)。それから、前記それぞれの組換えアデノウイルスをHep3B肝がん細胞株に感染させた後、フローサイトメトリー(flow cytometry)及び顕微鏡観察を通じてGFP−陽性の細胞数を確認した。さらに、前記Ad5CMV.GFP及びAd5/35CMV.GFPに感染されたHep3B肝がん細胞株で定量的−PCRを用いたアデノウイルスのゲノムの複製数を測定した。
がん組織特異的なPEPCKプロモーターとhTERT(human Telomerase reverse trasncriptase)を標的するリボザイム及び血清型(serotype)35のファイバーノブと血清型5のシャフトを含む組換えアデノウイルスの肝がん特異的な抗がん効果を確認するために、Ad−PRT、Ad−PT及びAd−Mockを多様な感染多重度(MOI:multiplicities of infection)でHep3B肝がん細胞株及びSK−OV3卵巣がん細胞株に感染させた後、100μmのガンシクロビル(GCV)を処置した。それから、前記処置後、5日目に生きている細胞の比率を確認するため、クリスタルバイオレット(crystal violet)分析を実施した。
前記<実施例1>で製作したAd−PRTのトランススプライシング作用を確認するために、Ad−PRT及びAd−Mockをそれぞれ0、1、5及び10MOIでHep3B肝がん細胞株及びSK−OV3卵巣がん細胞株に感染させた後、RNA分析を行った。
Ad−PRTの肝がん特異的な毒性の効果を確認するために、前記<実施例2>で培養したPEPCK−陽性及びhTERT−陽性の肝がん細胞株(HepG2、SNU398及びSNU739)、及びPEPCK−陰性並びにhTERT−陰性の非肝がん細胞株(ヒト上皮細胞(Human epithelial cell; HDF)、肺がん細胞株(SBC−5)及び大腸がん細胞株(HCT116))にAd−PRT 10MOIを処理した後、前記<実験例3>と同じ方法で細胞死の効果を確認した。
<6−1>実験動物
実験動物として4〜5週齢の雄Balb/c−ヌードマウス(オリエントバイオ、韓国)を使用した。実験動物は、無病菌の条件で飼育され、使用前に最小1週間実験室環境に馴化し、AAALACの国際動物管理規定(AAALAC International Animal Care policy)による国立がんセンターの動物管理センターで飼育された(accredited unitNational Cancer Center Research Institute: unit number−1392)。
生体内Ad−PRTの肝がん−特異的な標的化能力を確認するために、本発明の組換えウイルスをがんを有していない前記<6−1>マウスに前記Ad−PRTを全身に投与し、正常の肝への感染の可否を[18F]FHBG(9−(4−[18F]fluoro−3−hydroxymethylbutylguanine)造影剤を用いてmicro−PET/CTを通じて確認した。
がん誘発のマウスでAd−PRTの肝がん−特異的な標的化能力を確認するために、Ad−PRT及びAd−Mockのそれぞれをマウスに全身に投与し、がん細胞に感染可否を[18F]FHBG造影剤を用いたmicro−PET/MRを通じて確認した。
<8−1>生体内実験のためのマウスモデルの製造及びウイルスの投与
ヒト肝細胞がん腫(HCC)を模倣した多発性マウス肝がんモデルは、Balb/c−ヌードマウス(オリエントバイオ社、韓国)にHep3B 3×106の細胞を脾臓内に投与して製造した。前記動物は、AAALACの国際動物管理規定(AAALAC International Animal Care policy;公認規格(accredited unit)National Cancer Center Research Institute: ユニットナンバー(unit number)−1392)を遵守する国立がんセンターの動物管理センターで飼育された。殆ど全てのマウスはHep3B細胞投与の12または14日後に肝で、特に肝の境界部位で肉眼検査によって容易に検出される多発性小型腫瘍結節を確認した。さらに、マウスの殆どで脾臓腫瘍結節も示されたことを確認した。
本発明者は、Ad−PRTの安全性を評価するために、前記<8−1>で製造した腫瘍が形成されたマウス(n=10)に組換えアデノウイルス5×108pfuを静脈内に投与し、GCVを14日間処理した。
<9−1>腫瘍細胞の侵襲におけるhTERT除去の影響
本発明者は、腫瘍細胞の侵襲において、リボザイムによるhTERT除去の影響を確認するために、GCV処置されないアデノウイルス−処置細胞でマトリゲル侵襲検定法(matrigel invasion assay)を行った。
本発明者は、肝細胞がん腫マウスモデルでAd−PRTの抗−HCCの効果を分析するために、無作為にAd−PRT(20匹)群及びAd−Mock(10匹)群に分けて、30匹のヌードマウスにHep3B細胞株を投与した。Ad−PRTの静脈投与後、14日目に生体内の画像(n=10)及び生検(n=30)を用いてモニタリングした。
Claims (13)
- 肝組織特異的なPEPCK(phosphoenolpyruvate carboxykinase)遺伝子のプロモーター;
前記プロモーターと作動可能に連結されたがん特異的な遺伝子に作用するトランススプライシングリボザイム(trans−splicing ribozyme);
前記リボザイム3’エクソンに連結された治療遺伝子又はレポーター遺伝子;及び
血清型(serotype)35のファイバーノブ(fiber knob)と血清型5のシャフト(shaft)を含み、
アデノウイルスE1、E3及びE4 orf1乃至orf4遺伝子が欠損した組換えアデノウイルス。 - 前記肝組織特異的なPEPCK遺伝子のプロモーターは、配列番号1で記載される塩基配列を有することを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記プロモーターは、配列番号2で記載される塩基配列を有するApoA1のイントロンを更に含むことを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記がん特異的な遺伝子は、hTERT(human Telomerase Reverse Transcriptase)mRNA、AFP(alphafetoprotein)mRNA、CEA(carcinoembryonic antigen)mRNA、PSA(Prostatespecific antigen)mRNA、またはCKAP2(Cytoskeleton−associated protein 2)mRNAであることを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記リボザイムは、hTERT(human Telomerase Reverse Transcriptase)mRNAを特異的に標的するトランススプライシンググループIのリボザイムであることを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記治療遺伝子は、薬剤感受性遺伝子、細胞死遺伝子、細胞増殖抑制遺伝子、細胞毒性遺伝子、腫瘍抑制因子遺伝子、抗原性遺伝子、サイトカイン遺伝子及び抗新生血管生成遺伝子で構成された群から選択される何れか一つであることを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記レポーター遺伝子はLacZ、クロラムフェニコールアセチル転移酵素(CAT: chloramphenicol acetyl transferase)、レニラルシフェラーゼ(Renila luciferase)、ホタル(firefly)ルシフェラーゼ、赤色蛍光タンパク質(RFP)、緑色蛍光タンパク質(GFP)、分泌型胎盤アルカリホスファターゼ(secreted placental alkaline phosphatase、SEAP)またはHSV−tk(Herpes simplex virus−thymidine kinase)をコードする遺伝子であることを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記組換えアデノウイルスは、配列番号6で記載されるApoE(apolipoprotein E)遺伝子のエンハンサー、PEPCK(phosphoenolpyruvate carboxykinase)遺伝子のエンハンサー、血清アルブミン遺伝子のエンハンサー、AFP(alphafetoprotein)遺伝子のエンハンサー、CEA(carcinoembryonic antigen)遺伝子のエンハンサーまたはPSA(Prostate−specific antigen)遺伝子のエンハンサーをさらに含むことを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記トランススプライシングリボザイムは、配列番号3で表示される塩基配列を有することを特徴とする請求項1に記載の組換えアデノウイルス。
- 前記治療遺伝子は、配列番号4で表示される塩基配列を有するHSV−tk(Herpes simplex virus−thymidine kinase)遺伝子であることを特徴とする請求項1に記載の組換えアデノウイルス。
- 請求項1乃至10の何れかによる組換えアデノウイルスを有効成分として含む肝がん治療用薬学的組成物。
- 請求項1乃至10の何れかによる組換えアデノウイルスを有効成分として含む肝がん診断用組成物。
- 1)請求項1乃至10の何れかによる組換えアデノウイルスをがん細胞に導入する段階;及び
2)前記段階1)のがん細胞でレポータータンパク質を検出する段階を含む肝がん診断のための情報提供方法。
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