JP6131433B2 - 細胞rna発現のための方法 - Google Patents
細胞rna発現のための方法 Download PDFInfo
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Description
本発明は、RNAをトランスフェクトした細胞などの細胞におけるRNA発現を、RNA依存性プロテインキナーゼ(PKR)の活性を低減することによって強化することに関する。したがって本発明は、細胞中でRNAを発現させるための方法であって、その細胞におけるRNA依存性プロテインキナーゼ(PKR)の活性を低減するステップを含む方法を提供する。細胞におけるRNA依存性プロテインキナーゼ(PKR)の活性の低減は、その細胞におけるRNAの安定性を増加させ、かつ/またはRNAの発現を増加させる。
可逆的な遺伝子治療の一種としてRNAを使用することの利点には、一過性発現と非形質転換性とが含まれる。RNAは、核に進入しなくても発現することができ、そのうえ宿主ゲノムに組み込むこともできないので、腫瘍形成のリスクが排除される。RNAで達成できるトランスフェクション率は比較的高く、多くの細胞タイプで>90%にもなるため、トランスフェクト細胞を選択する必要がない。さらにまた、達成されるタンパク質の量も、生理的発現におけるそれに相当する。
以下に本発明を詳述するが、本発明は、本明細書に記載する特定の方法論、プロトコールおよび試薬類に限定されないと理解すべきである、というのも、これらはさまざまでありうるからである。本明細書において使用する術語が、特定の実施形態を説明するための術語に過ぎず、本願請求項によってのみ限定される本発明の範囲を限定しようとするものではないことも理解すべきである。別段の定義がある場合を除き、本明細書において使用する全ての技術用語および科学用語は、当業者に通常理解されている意味と同じ意味を有する。
実施例
初代ヒト新生児包皮線維芽細胞(CCD−1079Sk、CCD)、BJヒト新生児包皮線維芽細胞をATCC(米国バージニア州マナッサス)から入手し、10%熱非働化ウシ胎児血清Gold(PAA Labratories、オーストリア・パッシング)、1×非必須アミノ酸(Invitrogen)、1mMピルビン酸ナトリウム(Invitrogen)、2mMグルタミン(Invitrogen)、50U/mlペニシリン(Invitrogen)および50μg/mlストレプトマイシン(Invitrogen)を添加したMEM(Invitrogen,ドイツ・カールスルーエ)で培養した。もう一つのヒト新生児包皮線維芽細胞(HFF)をSBI(米国カリフォルニア州マウンテンビュー)から入手し、CCDおよびBJと同様に培養した。マウス胚性線維芽細胞(MEF)は、14日齢のC57BL/6マウス胚から単離し、15%熱非働化ウシ胎児血清、1×非必須アミノ酸、1mMピルビン酸ナトリウム、2mMグルタミン、50U/mlペニシリンおよび50μg/mlストレプトマイシンを添加したDMEM(Invitrogen)で培養した。ヒト表皮ケラチノサイトをPromoCell(ドイツ・ハイデルベルク)から入手し、同梱の添加剤を含むケラチノサイト基礎培地2(PromoCell,ドイツ・ハイデルベルク)で培養した。Lonza(米国メリーランド州ウォーカーズビル)からのヒト臍帯静脈内皮細胞(HUVEC)は、Clonetics(登録商標)Endothelial Cell System(Lonza)を使って培養した。
ルシフェラーゼ、eGFP、不安定化eGFP(d2eGFP)、OCT4、SOX2、KLF4、cMYC、LIN28、NANOG、SV40ラージT抗原、E6、p53−DD、E3およびK3をコードするRNAのインビトロ転写用のテンプレートとして使用するためのプラスミドコンストラクトは、pST1−2hBgUTR−A120(Holtkamp 2006)に基づいた。遺伝子のヌクレオチド配列をコドン最適化して、GC含量を増加し、ターゲット細胞における翻訳を強化した。インビトロ転写RNAの作製のために、クラスII制限エンドヌクレアーゼを使って、ポリ(A)テールの下流で、プラスミドを線状化した。線状化したプラスミドをフェノール−クロロホルム抽出によって精製し、分光光度法で定量し、MEGAscript T7キット(Ambion、米国テキサス州オースティン)を使って、T7 RNAポリメラーゼによるインビトロ転写に付した。キャップアナログβS−ARCA(D1)の組込みのために、6mMのアナログを反応に加える一方で、GTP濃度を1.5mMに低下させた。反応を37℃で3〜6時間インキュベートし、DNase(Ambion)処理し、MEGAclear Kit(Ambion)を製造者に従って使用することにより、精製した。
IVT−RNAの細胞への移入はエレクトロポレーション(EP)によって行った。この目的を達成するために、EP1回あたり1〜10×107細胞を回収し、2mM EDTAを添加したPBSおよびX−VIVO 15培地(Lonza)で逐次、洗浄し、125μlのX−VIVO 15培地に再懸濁し、2mmギャップ滅菌エレクトロポレーションキュベット(Bio−RAD,米国カリフォルニア州ハーキュリーズ)に移した。適当量のインビトロ転写RNAを加え、BTX ECM(登録商標)830エレクトロポレーションシステム(BTX、米国マサチューセッツ州ホリストン)と、予め最適化しておいたCCD用(110V、3×12msパルス、400msインターバル)、BJ用(110V、3×12msパルス、400msインターバル)、MEF用(125V、5×6msパルス、400msインターバル)、HFF用(125V、1×24msパルス)、ケラチノサイト用(125V、4×6msパルス、200msインターバル)およびHUVEC用(125V、1×20msパルス)のエレクトロポレーションパラメータとを使って、細胞をエレクトロポレーションに付した。ヒトPBMCおよびiDCは、4mmギャップ滅菌エレクトロポレーションキュベット(Bio−Rad)中、Gene−Pulser−II装置(Bio−Rad、ドイツ・ミュンヘン)を使って、450V/250μF(PBMC)、300V/150μF(iDC)の電圧およびキャパシタンス設定で、エレクトロポレーションした。エレクトロポレーション後、直ちに、細胞を培養培地で希釈した。
ルシフェラーゼアッセイのために、ルシフェラーゼをコードするインビトロ転写RNAをエレクトロポレーションした細胞を、96ウェル白色平底マイクロプレート(Nunc、ドイツ・ランゲンゼルボルト)にプレーティングし、37℃でインキュベートした。細胞の溶解は、「Bright−Glo Luciferase Assay System」(Promega、米国ウィスコンシン州マディソン)を使って行った。バイオルミネセンスフラックスを、Infinite M200マイクロプレートルミネセンスリーダー(Tecan、ドイツ・クライルスハイム)を使って1秒の積分時間で測定した。本明細書に提示するルシフェラーゼ活性のデータは単位がカウント毎秒である。
RNeasy Mini KitまたはRNeasy Micro Kit(Qiagen、ドイツ・ヒルデン)を使って、全細胞RNAを細胞から抽出し、Superscript II(Invitrogen、米国カリフォルニア州カールズバッド)を使ってオリゴdT18で逆転写し、QuantiTect SYBR Green PCR Kit(Qiagen)を使って、ABI PRISM 7700 Sequence Detection System機器およびソフトウェア(Applied Biosystems、米国カリフォルニア州フォスターシティ)でのリアルタイム定量分析に付した。反応は、トリプリケートで、コドン最適化OCT4(OCT4−s:5’−ACCTGGAAAACCTGTTCCTGC−3’(配列番号14);OCT4−as:5’−AGCTCGGATCCTCATCAGTTG−3’(配列番号15))、
SOX2(SOX2−s:5’−AACCAGCGGATGGACAGCTAC−3’(配列番号16);SOX2−as:5’−GCTTTTCACCACGCTGCCCAT−3’(配列番号17))、
KLF4(KLF4−s:5’−AAGACCTACACCAAGAGCAGC−3’(配列番号18);KLF4−as:5’−AGGTGGTCAGATCTGCTGAAG−3’(配列番号19))およびcMYC(cMYC−s:5’−CCCCTGAACGACAGCTCTAGC−3’(配列番号20);cMYC−as:5’−TTCTCCACGGACACCACGTCG−3’(配列番号21))、または
IFNα(IFNα−s:5’−AAATACAGCCCTTGTGCCTGG−3’(配列番号22);IFNα−as:5’−GGTGAGCTGGCATACGAATCA−3’(配列番号23))、
IFNβ(IFNβ−s:5’−AAGGCCAAGGAGTACAGTC−3’(配列番号24);IFNβ−as:5’−ATCTTCAGTTTCGGAGGTAA−3’(配列番号25))、
GDF3(GDF3−s:5’−TCCCAGACCAAGGTTTCTTTC−3’(配列番号26);GDF3−as:5’−TTACCTGGCTTAGGGGTGGTC−3’(配列番号27))および
TERT(TERT−s:5’−CCTGCTCAAGCTGACTCGACACCGTG−3’(配列番号28);TERT−as:5’−GGAAAAGCTGGCCCTGGGGTGGAGC−3’(配列番号29))の内在性転写産物の特異的領域を増幅するプライマー(各333nM)を使用し、95℃で15分間の初回変性/活性化、ならびに95℃で30秒、プライマー特異的なアニーリング温度(OCT4、cMYC、IFNα、IFNβ、GDF3、TERT:60℃;SOX2:64℃;KLF4:58℃)で30秒、および72℃で30秒の40サイクルで行った。内部標準としてのHPRTコードRNA(HPRT−s、5’−TGACACTGGCAAAACAATGCA−3’(配列番号30);HPRT−as:5’−GGTCCTTTTCACCAGCAAGCT−3’(配列番号31);上記のPCR条件で、アニーリング温度を62℃とする)と比較してΔΔCt計算を使って、転写産物の発現を定量し、RNAの量とインプット相補DNAの量の変動について標準化した。
OCT4、SOX2およびNANOGの細胞内FACS染色は、ヒト多能性幹細胞転写因子分析キットを製造者の説明(BD Bioscences)に従って使用することによって行った。簡単に述べると、3〜5×105個の細胞を回収し、PBSで2回洗浄し、250μlのBD Cytofixバッファー(BD Bioscieces)中、室温で20分間固定した。インキュベーション後に、細胞をPBSで洗浄し、1×Perm/Washバッファー(BD Biosciences)で2回洗浄することによって透過処理した後、50μlの1×Perm/Wash中、室温で10分間インキュベートした。アイソタイプ対照および特異的抗体による染色を、暗所、室温で30分間行った後、1×Perm/Washで2回洗浄した。細胞を、2%ホルムアルデヒドを添加したPBSに再懸濁し、上述のようにフローサイトメトリーで分析した。
Claims (49)
- インビトロで、細胞中でRNAを発現させるための方法であって、(i)RNAのトランスフェクションによって、細胞にRNAを導入するステップおよび(ii)細胞におけるRNA依存性プロテインキナーゼ(PKR)の活性を低減するステップを含み、
細胞におけるPKRの活性を低減するステップが、細胞を少なくとも1つのPKR阻害剤で処理することを含み、かつ
RNAがリプログラミング因子をコードする、
方法。 - RNAがエレクトロポレーションによって細胞中に導入されている、請求項1に記載の方法。
- RNAがインビトロ転写RNAである、請求項1または2に記載の方法。
- 細胞におけるPKRの活性を低減するステップが、細胞におけるRNAの安定性の強化および/または発現の強化をもたらす、請求項1〜3のいずれか一項に記載の方法。
- 細胞におけるRNAの発現の強化が、細胞におけるRNAの発現のレベルの増加および/または発現の持続時間の増加を含む、請求項4に記載の方法。
- 少なくとも1つのPKR阻害剤による細胞の処理が、細胞におけるRNAの安定性の強化および/または発現の強化をもたらすのに充分な時間にわたる、請求項1〜5のいずれか一項に記載の方法。
- PKR阻害剤がRNA誘導性PKR自己リン酸化を阻害する、請求項1〜6のいずれか一項に記載の方法。
- PKR阻害剤がPKRのATP結合部位指向性阻害剤である、請求項1〜7のいずれか一項に記載の方法。
- PKR阻害剤がイミダゾロ−オキシインドール化合物である、請求項1〜8のいずれか一項に記載の方法。
- PKR阻害剤が6,8−ジヒドロ−8−(1H−イミダゾール−5−イルメチレン)−7H−ピロロ[2,3−g]ベンゾチアゾール−7−オンである、請求項1〜9のいずれか一項に記載の方法。
- PKR阻害剤が2−アミノプリンである、請求項1〜8のいずれか一項に記載の方法。
- PKR阻害剤がPKRのウイルス由来阻害剤である、請求項1〜8のいずれか一項に記載の方法。
- PKRのウイルス由来阻害剤が、ワクシニアウイルスE3および/もしくはK3、またはそれらのRNAからなる群より選択される、請求項12に記載の方法。
- 細胞におけるPKRの活性を低減するステップが、PKR遺伝子の発現をサイレンシングすることを含む、請求項1〜13のいずれか一項に記載の方法。
- 細胞がバリア機能を有する細胞である、請求項1〜14のいずれか一項に記載の方法。
- 細胞が、線維芽細胞、ケラチノサイト、上皮細胞、または内皮細胞である、請求項1〜15のいずれか一項に記載の方法。
- 内皮細胞が、心臓の内皮細胞、肺の内皮細胞または臍帯静脈内皮細胞である、請求項16に記載の方法。
- 細胞がヒト細胞である、請求項1〜17のいずれか一項に記載の方法。
- 幹細胞特徴を有する細胞を提供するための方法であって、(i)体細胞を含む細胞集団を用意するステップ、(ii)体細胞におけるRNA依存性プロテインキナーゼ(PKR)の活性を低減するステップ、(iii)幹細胞特徴を有する細胞への体細胞のリプログラミングを可能にする1つ以上の因子を発現する能力を有するRNAを、RNAのトランスフェクションによって、体細胞の少なくとも一部に導入するステップ、および(iv)幹細胞特徴を有する細胞の発生を可能にするステップを含み、
細胞におけるPKRの活性を低減するステップが、少なくとも1つのPKR阻害剤で細胞を処理することを含む、
方法。 - RNAがエレクトロポレーションによって体細胞の少なくとも一部に導入される、請求項19に記載の方法。
- RNAがインビトロ転写RNAである、請求項19または20に記載の方法。
- 1つ以上の因子がOCT4およびSOX2を含む、請求項19〜21のいずれか一項に記載の方法。
- 1つ以上の因子が、さらにKLF4および/またはc−MYCを含む、請求項22に記載の方法。
- 1つ以上の因子がさらに、NANOGおよび/またはLIN28を含む、請求項22または23に記載の方法。
- 1つ以上の因子が、OCT4、SOX2、KLF4およびc−MYCを含む、請求項19〜21のいずれか一項に記載の方法。
- 1つ以上の因子がさらにLIN28を含む、請求項25に記載の方法。
- 1つ以上の因子が、OCT4、SOX2、NANOGおよびLIN28を含む、請求項19〜21のいずれか一項に記載の方法。
- 細胞におけるPKRの活性を低減するステップが、細胞におけるRNAの安定性の強化および/または発現の強化をもたらす、請求項19〜27のいずれか一項に記載の方法。
- 細胞におけるRNAの発現の強化が、細胞におけるRNAの発現のレベルの増加および/または発現の持続時間の増加を含む、請求項28に記載の方法。
- 少なくとも1つのPKR阻害剤による細胞の処理が、細胞におけるRNAの安定性の強化および/または発現の強化をもたらすのに充分な時間にわたる、請求項19〜29のいずれか一項に記載の方法。
- PKR阻害剤がRNA誘導性PKR自己リン酸化を阻害する、請求項19〜30のいずれか一項に記載の方法。
- PKR阻害剤がPKRのATP結合部位指向性阻害剤である、請求項19〜31のいずれか一項に記載の方法。
- PKR阻害剤がイミダゾロ−オキシインドール化合物である、請求項19〜32のいずれか一項に記載の方法。
- PKR阻害剤が6,8−ジヒドロ−8−(1H−イミダゾール−5−イルメチレン)−7H−ピロロ[2,3−g]ベンゾチアゾール−7−オンである、請求項19〜33のいずれか一項に記載の方法。
- PKR阻害剤が2−アミノプリンである、請求項19〜32のいずれか一項に記載の方法。
- PKR阻害剤がPKRのウイルス由来阻害剤である、請求項19〜32のいずれか一項に記載の方法。
- PKRのウイルス由来阻害剤が、ワクシニアウイルスE3および/もしくはK3、またはそれらのRNAからなる群より選択される、請求項36に記載の方法。
- 細胞におけるPKRの活性を低減するステップが、PKR遺伝子の発現をサイレンシングすることを含む、請求項19〜37のいずれか一項に記載の方法。
- 少なくとも1つのヒストンデアセチラーゼ阻害剤の存在下で体細胞を培養するステップをさらに含む、請求項19〜38のいずれか一項に記載の方法。
- 少なくとも1つのヒストンデアセチラーゼ阻害剤がバルプロ酸を含む、請求項39に記載の方法。
- ステップ(iv)が、胚性幹細胞培養条件下で体細胞を培養することを含む、請求項19〜40のいずれか一項に記載の方法。
- 幹細胞特徴が胚性幹細胞形態を含む、請求項19〜41のいずれか一項に記載の方法。
- 幹細胞特徴を有する細胞が正常核型を有し、テロメラーゼ活性を発現し、胚性幹細胞に特有の細胞表面マーカーを発現し、かつ/または胚性幹細胞に特有の遺伝子を発現する、請求項19〜42のいずれか一項に記載の方法。
- 幹細胞特徴を有する細胞が多能性状態を示す、請求項19〜43のいずれか一項に記載の方法。
- 幹細胞特徴を有する細胞が、3つ全ての一次胚葉の高度な派生物へと分化する発生能を有する、請求項19〜44のいずれか一項に記載の方法。
- 体細胞が線維芽細胞である、請求項19〜45のいずれか一項に記載の方法。
- 線維芽細胞が肺線維芽細胞、包皮線維芽細胞または真皮線維芽細胞である、請求項46に記載の方法。
- 体細胞がヒト細胞である、請求項19〜47のいずれか一項に記載の方法。
- 分化細胞タイプを提供するための方法であって、(i)請求項19〜48のいずれか一項に記載の方法を使って、幹細胞特徴を有する細胞を用意するステップ、および(ii)分化細胞タイプへの部分的なまたは完全な分化を誘導または指示する条件下で、幹細胞特徴を有する細胞を培養するステップを含む方法。
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IL126757A0 (en) * | 1998-09-07 | 1999-08-17 | Yissum Res Dev Co | Regulation of gene expression through manipulation of mRNA splicing and its uses |
ES2336887T5 (es) | 2000-03-30 | 2019-03-06 | Whitehead Inst Biomedical Res | Mediadores de interferencia por ARN específicos de secuencias de ARN |
EP2072618A1 (en) * | 2007-12-14 | 2009-06-24 | Johannes Gutenberg-Universität Mainz | Use of RNA for reprogramming somatic cells |
US20120115225A1 (en) * | 2009-04-23 | 2012-05-10 | Xu C W | Reprogramming of somatic cells with purified proteins |
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ES2640875T3 (es) | 2017-11-07 |
WO2012072096A8 (en) | 2012-07-26 |
PT2646557T (pt) | 2017-10-03 |
LT2646557T (lt) | 2017-10-10 |
WO2012072096A1 (en) | 2012-06-07 |
DK2646557T3 (en) | 2017-10-02 |
WO2012072269A1 (en) | 2012-06-07 |
US20140030808A1 (en) | 2014-01-30 |
CA2819522C (en) | 2019-07-16 |
AU2011335428A1 (en) | 2013-05-30 |
JP2017079749A (ja) | 2017-05-18 |
CA2819522A1 (en) | 2012-06-07 |
HUE034558T2 (en) | 2018-02-28 |
JP2013545469A (ja) | 2013-12-26 |
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