JP2019198329A - HIV−1プロウイルスDNAのin vivo切除のための組成物及び方法 - Google Patents
HIV−1プロウイルスDNAのin vivo切除のための組成物及び方法 Download PDFInfo
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- 241001529453 unidentified herpesvirus Species 0.000 description 1
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Abstract
Description
EVDGVDEVAKKKSKK (配列番号26) (Schreiberら(1992) EMBO J. 11:3263-3269)又はステロイドホルモン受容体(ヒト)グルココルチコイドの配列RKCLQAGMNLEARKTKK (配列番号27) (Cadepondら(1992) Exp. Cell Res. 201:99-108)等の二分核局在配列を用いることも可能である。ある実施態様において、NLSはcasタンパク質のN末端に局在する。他の実施態様において、NLSはcasタンパク質のC末端に局在する。
HIV-1プロウイルスの長鎖末端反復領域内に位置する5種類の特定の領域の一つに特異的に結合するガイドRNA分子をコードするプラスミドDNAを作製する(図1)。同様に、cas9ヌクレアーゼをコードするプラスミドを作製する。両方のプラスミドをヒト細胞に送達し、そこでcas9及びガイドRNA遺伝子座が転写され、cas9転写産物は翻訳される。続いて、ガイドRNA部分がcas9ヌクレアーゼに結合し、ハイブリッド複合体を形成する。核局在配列を加えているので、このハイブリッド複合体は核内に入り、核においてガイドRNA部分がHIV-1プロウイルスDNA内の相補配列に結合する。ヌクレアーゼがプロウイルスDNAを切り、通常修復不可能な二重らせんDNA切片をもたらす。図2を参照。この方法を用いることにより、HIV-1プロウイルスDNAを感染細胞のゲノムから切除することができる。
J-Lat細胞株(ヒトT細胞がん株)を用いて、ガイドRNA:hCas9複合体によるHIV-1長鎖末端反復(LTR)の切断がこれらの細胞の転写活性を変化させるか否かを決定した。フローサイトメトリーを用いてトランスフェクション22時間後にLTRプロモーター由来の緑色蛍光タンパク質(GFP)を生産する細胞を定量した。フローサイトメトリー解析により、GFPを発現する細胞の割合が得られ、またGFP+細胞の平均蛍光強度(MFI)を定量することにより細胞群内の発現の強度を定量することもできる。フローサイトメトリーにより、転写を減少させるLTR領域内の変異と、転写を完全に消失させるLTR領域内の変異とを識別することができる。
CRISPR/Cas法のHIV-1 LTR内での切断及び転写の改変の有効性を決定するために、Jurkat細胞を3種類の異なるプラスミドでトランスフェクションした。これらのプラスミドには、様々なガイドRNA、ヒト化Cas9プラスミド、及び無傷のHIV-1 LTR及び5'配列がGFPの発現を促進する第3の(レポーター)プラスミドが含まれる。
Claims (11)
- 真核細胞における標的のヒト免疫不全ウイルス1(HIV-1)のDNA配列の機能又は存在を阻害する方法であって、標的HIV-1 DNA配列を保有する真核細胞を、
(a) 一種以上のガイドRNA、又は前記一種以上のガイドRNAをコードする核酸、及び
(b) 規則的な間隔をもってクラスター化された短鎖反復回文配列関連(Clustered Regularly Interspaced Short Palindromic Repeats-Associated:cas)タンパク質、又は前記casタンパク質をコードする核酸
と接触させる工程を含み、
前記ガイドRNAが前記標的HIV-1 DNA配列とハイブリダイズし、これにより前記標的HIV-1 DNA配列の機能又は存在を阻害するものである、方法。 - 前記標的HIV-1 DNA配列が配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9及び配列番号10から成る群から選択される、請求項1に記載の方法。
- 前記casタンパク質がcas9である、請求項1に記載の方法。
- 前記casタンパク質がヒト細胞での発現用にコドン最適化されている、請求項1に記載の方法。
- 前記casタンパク質が核局在配列をさらに含む、請求項1に記載の方法。
- (a) 一種以上のガイドRNA、又は前記一種以上のガイドRNAをコードする核酸であって、前記ガイドRNAが標的のヒト免疫不全ウイルス1 (HIV-1)のDNA配列とハイブリダイズするもの;及び
(b) 規則的な間隔をもってクラスター化された短鎖反復回文配列関連(Clustered Regularly Interspaced Short Palindromic Repeats-Associated:cas)タンパク質;又は前記タンパク質をコードする核酸
を含むキット。 - 標的HIV-1 DNA配列が、配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9及び配列番号10から成る群から選択される、請求項6に記載のキット。
- 前記casタンパク質がcas9である、請求項6に記載のキット。
- 前記casタンパク質がヒト細胞での発現用にコドン最適化されている、請求項6に記載のキット。
- 前記casタンパク質が核局在配列をさらに含む、請求項6に記載のキット。
- 一種以上のガイドRNAをコードする核酸を含み、前記ガイドRNAが配列番号2、配列番号3、配列番号4、配列番号5、配列番号6、配列番号7、配列番号8、配列番号9及び配列番号10から成る群から選択される標的のヒト免疫不全ウイルス1のDNA配列とハイブリダイズする、ベクター。
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