JP5409636B2 - 抗c型肝炎ウイルス組成物 - Google Patents
抗c型肝炎ウイルス組成物 Download PDFInfo
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- JP5409636B2 JP5409636B2 JP2010526785A JP2010526785A JP5409636B2 JP 5409636 B2 JP5409636 B2 JP 5409636B2 JP 2010526785 A JP2010526785 A JP 2010526785A JP 2010526785 A JP2010526785 A JP 2010526785A JP 5409636 B2 JP5409636 B2 JP 5409636B2
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- hepatitis
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
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Description
(a)PA28γ遺伝子の発現又は機能を抑制する核酸
(b)(a)に記載の核酸を修飾してなる修飾核酸
(c)(a)に記載の核酸を発現することのできる発現ベクター。
試験物質をPA28γ遺伝子の発現細胞又は発現能を有する細胞に接触させる工程、
試験物質を接触させた細胞と対照細胞におけるPA28γ遺伝子の発現レベルを比較する工程、及び、前記比較によりPA28γ遺伝子の発現を阻害すると評価された試験物質を選択する工程を備えたスクリーニング方法。
試験物質をPA28γ遺伝子の発現細胞又は発現能を有する細胞に接触させる工程、
試験物質を接触させた細胞と対照細胞におけるPA28γ遺伝子の発現レベルを比較する工程、及び、前記比較において対照よりPA28γ遺伝子の発現レベルを低下させる試験物質を選択する工程を備えたスクリーニング方法。
PA28γ、トリプシン様基質及び20Sプロテアソームを含む反応系に、試験物質を投与する工程、
試験物質を投与した系と対照におけるプロテアソームのトリプシン様活性を比較する工程、及び
前記比較において対照よりトリプシン様活性を抑制させる試験物質を選択する工程
を備えたスクリーニング方法。
本発明の抗HCV組成物は、PA28γ遺伝子の発現又は機能を抑制する物質を有効成分として含む。
本発明の抗HCV組成物又はそれを製剤化したものを、被験体に投与することにより、HCV感染予防又は増殖抑制を行うことができる。
本発明の抗C型肝炎ウイルス組成物の有効成分は、PA28γ遺伝子の発現又は機能の抑制を指標とするスクリーニング方法によって選択することができる。
試験物質をPA28γ遺伝子の発現細胞又は発現能を有する細胞に接触させる工程、
試験物質を接触させた細胞と対照細胞におけるPA28γ遺伝子の発現レベルを比較する工程、及び、
前記比較によりPA28γ遺伝子の発現を阻害すると評価された試験物質を選択する工程
を備えた方法が挙げられる。
PA28γ、トリプシン様基質及び20Sプロテアソームを含む反応系に試験物質を投与する工程、
試験物質を投与した系と対照におけるプロテアソームのトリプシン様活性を比較する工程、及び
前記比較において対照よりトリプシン様活性を抑制させる試験物質を選択する工程
を備えたスクリーニング方法が挙げられる。
ヒト PA28γ 遺伝子をRNA 干渉によりノックダウンするためのshort hairpinRNA をコードするDNA フラグメントを以下の手順により設計した。
配列番号1:AAGTGAAGCTCAAGGTTGATT
配列番号2:AAGGTTGGATGAGTGTGAAGA
配列番号3:AAGTGAGGCAGAAGACTTGGT
配列番号4:AAGGTGGATCAGGAAGTGAAG
このうち、発現抑制効果が顕著な配列番号4の配列を選択した。配列番号4の配列は、配列番号5に示すPA28γコード領域の16-36番目に相当する。
Insert Design Tool for the pSilencer(登録商標)Vectors(Ambion 社、http://www.ambion.com/jp/techlib/misc/psilencer_converter.html)を使用して設計した。設計したフラグメントの配列を、配列表の配列番号6に示す。
上記1.のshRNAのPA28γ標的領域であるPA28γコード領域の16-36番目の内、18番目のGをA、24 番目T をC、30 番目のA をG に変異させたPA28γ遺伝子を、Huang et al. Oncogene 14:405-414, 1997に基づいて作製したプラスミドpEFFLAGGspGBKに組み込み、shRNA に対して耐性をもつPA28γを発現するためのプラスミド(pEFFLAGGsPA28gammaSI)を作製した。
コントロール細胞(Control shRNA)、PA28γノックダウン細胞C5 およびC7 にpEFFLAGGspGBKあるいはpEFFLAGGsPA28gammaSI プラスミドをトランスフェクションし、翌日、3μg/mLピューロマイシンを含む10%牛胎児血清ダルベッコ変法イーグル培地に置換し、更に24時間培養し、生き残った細胞を12 well plate に2.5 x 104 Cell/well で撒き直した。M.O.Iが0.024 の割合でC型肝炎ウイルスJFH1を感染させた。
PA28 γ 遺伝子コード領域の162-180位を標的配列とするsiRNA( Ambion、 Cat No. 138669)と0.1mL のOpti-MEM(Invitrogen)を混和し、1μL のLipofectamineTM RNAiMax(Invitrogen)を加えさらによく混和し、室温で20 分間静置した。コンフルエントになったHuh7 OK1 細胞をトリプシンによって剥がし、10%牛胎児血清ダルベッコ変法イーグル培地に入れ、105個/mL の濃度になるように混和した。先に調製したsiRNA 混液0.1mL を24 ウェルプレートの1 wellに移し、さらに0.5mL の細胞混液を加えて、よく混ぜ合わせた。
Krieger らの方法(Krieger et al., J. Virol., 75:4614-4624,2001)に従い、上記1で作製したコントロール細胞及びノックダウン細胞を使用する以外は同様にして、レプリコンによるウイルス複製を次のように評価した。
上記1.に記載のHuh7 Ok1細胞を0.25 x 105/wellで24 well-plateに播いた。
配列番号2:PA28γ遺伝子RNA干渉標的配列の1例
配列番号3:PA28γ遺伝子RNA干渉標的配列の1例
配列番号4:PA28γ遺伝子RNA干渉標的配列の1例
配列番号5:PA28γコード領域
配列番号6:配列番号4の配列に対するshort hairpinRNA をコードするDNA フラグメント
Claims (4)
- 以下の(a)〜(c)のいずれかの、PA28γによる20Sプロテアソームのトリプシン様活性の活性化を阻害する化合物を有効成分として含む抗C型肝炎ウイルス組成物:
(a)PA28γの20Sプロテアソームのトリプシン様活性の活性化を抑制するPA28γに対するRNA、
(b)(a)に記載のRNAを修飾してなる修飾RNA、
(c)(a)に記載のRNAを発現することのできる発現ベクター、
ここで、該RNAはsiRNA(small interfering RNA)又はshRNA(short hairpin RNA)である。 - C型肝炎ウイルス感染予防用又は増殖抑制用である請求項1に記載の組成物。
- 請求項1または2に記載の組成物を動物に投与する工程を含む、C型肝炎ウイルス感染予防又は増殖抑制方法(但し、ヒトに対する医療行為を除く)。
- 請求項1または2に記載の抗C型肝炎ウイルス組成物の有効成分のスクリーニング方法であって、
PA28γ、トリプシン様基質及び20Sプロテアソームを含む反応系に、試験物質を投与する工程、
試験物質を投与した系と対照におけるプロテアソームのトリプシン様活性を比較する工程、及び
前記比較において対照よりトリプシン様活性を抑制させる試験物質を選択する工程
を備えたスクリーニング方法。
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GB2506086A (en) | 2011-10-21 | 2014-03-19 | Abbvie Inc | Methods for treating HCV comprising at least two direct acting antiviral agent, ribavirin but not interferon |
CA2811250C (en) | 2011-10-21 | 2015-08-11 | Abbvie Inc. | Methods for treating hcv |
US8466159B2 (en) | 2011-10-21 | 2013-06-18 | Abbvie Inc. | Methods for treating HCV |
US8492386B2 (en) | 2011-10-21 | 2013-07-23 | Abbvie Inc. | Methods for treating HCV |
CN102899329B (zh) * | 2012-11-07 | 2015-04-15 | 南开大学 | 一种更加稳定的人源蛋白酶体激活因子突变体的构建方法 |
US11192914B2 (en) | 2016-04-28 | 2021-12-07 | Emory University | Alkyne containing nucleotide and nucleoside therapeutic compositions and uses related thereto |
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