JP2023106545A - ACE-tRNAを用いた遺伝的再帰属を介して終止コドンレスキューする方法 - Google Patents
ACE-tRNAを用いた遺伝的再帰属を介して終止コドンレスキューする方法 Download PDFInfo
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Abstract
Description
連邦により資金援助された研究に関する記述
ナンセンス変異
expression)に適している。簡潔に述べれば、ヒト細胞中で機能するtRNA遺伝子の構造成分を含むオリゴヌクレオチドが合成される。このオリゴヌクレオチドの配列は、特定のtRNAにナンセンスまたはその他の特異的な変異を認識させるtRNAのアンチコドン領域中になされる置換を有する既知の配列に基づいて設計される。
アンチコドン編集tRNA(ACE-tRNA)
発現カセットおよびベクター
レトロウイルス;レトロウイルスベクター
al.,J.Virol.1999 73:4991-5000)。複製能のないウイルスが首尾よく産生された。
レトロウイルスベクター系
パッケージングシグナル
パッケージング系;パッケージングベクター;パッケージング細胞株
導入遺伝子ベクター
発現カセットおよびベクター
アデノ随伴ウイルス(AAV)
ITR」という用語内にあると考えられるためには、ヌクレオチド配列は、AAV2 ITRをAAV1 ITRと区別する、ここに記載されている一方または両方の特徴を保持しなければならない。(1)(AAV1中での4つではなく)3つの「GAGC」リピートおよび(2)AAV2 ITR Rep結合部位において、最初の2つの「GAGC」リピート中の4番目のヌクレオチドがTではなくCである。
AAVベクター
DTT、33μg/mL BSA、10mM~50mM NaClおよび40μM ATP、0.01~0.02(Weiss)単位のT4 DNAリガーゼ、0℃(「粘着末端」ライゲーションのため)または1mM ATP、0.3~0.6(Weiss)単位のT4 DNAリガーゼ、14℃(「平滑末端」ライゲーションのため)のいずれかの中で達成することができる。分子間「粘着末端」ライゲーションは、30~100μg/mLの全DNA濃度(5~100nMの総最終濃度)で通常行われる。ITRを含有するAAVベクター。
治療剤をコードする核酸
遺伝子材料を細胞中に導入するための方法
疾患状態および処置の方法
本発明の薬剤の投与量、製剤および投与の経路
定義
遺伝コードは、順次トリプレットコドンを形成する4つのヌクレオチドを使用し、これが、DNAのタンパク質への翻訳の基礎を成す。合計64個のコドンが存在し、そのうち61個がアミノ酸をコードするために使用され、そのうち3つ(TAG、TGAおよびTAA)がタンパク質終結「終止」または「ナンセンス」コドンをコードする
結果
方法
[実施例2]
[実施例3]
[実施例4]
[実施例5]
未成熟終結コドンの抑制のための操作された転移RNA
要約
序論
cellular machinery)によって、なお認識されるべきである、図21A。このようなサプレッサーtRNAは、限定的な様式で、β-サラセミア25、色素性乾皮症26およびトランスジェニックPTCレポーター遺伝子27と関連するインフレーム終止コドンをレスキューことが示されている。
結果
考察
材料および方法
ナンセンスレポーターHTCプラスミド
ACE-tRNAライブラリーのHTC
ATP、10mM DTT、400単位のT4 DNAリガーゼおよび10単位のBbsI-HFを含有し、ddH2Oで10μLになるように列に並べた(queued)。96ウェルプレートは、サーモサイクラー中で、以下のようにサイクルを行い、([37℃で5分、20℃で5分]×30サイクル、37℃で10分、80℃で10分、4℃まで冷却した。ディープウェルの96ウェルプレート中で、10μLの化学的に形質転換受容性のあるDH5α細胞(ThermoFisher、アメリカ)に、1μLのGolden Gate反応物を添加し、42℃で30秒間、熱ショックを加え、100μLのSuper Optimal Broth(S.O.C.;Thermofisher,アメリカ)中に再懸濁した。37℃、1時間、250rpmで、形質転換体を増殖させ、次いで、100μg/mLカルベニシリンが補充された2mLのLuria-Bertani液体培地(LB)に添加し、被覆されたディープ48ウェルプレート中において、37℃で20時間、300rpmで成長させた。大腸菌の増殖は、ディープウェルプレートおよびEnzyscreen(http://www.enzyscreen.com)のクランプ中で行った。大腸菌懸濁培養物を沈降させ(室温で10分、4,000g)、プラスミドDNAを調製し、125ng/μLまで希釈した(IBI scientific,アメリカ)。全てのクローンは、配列を確認した。この方法を用いて、100%のクローニング効率が達成された。
ACE-tRNAライブラリーのHTS
試薬対緩衝液;Promega、アメリカ)。回転振盪後に、プレートを2分間インキュベーションし、SpectraMax i3プレートリーダーを用いて読み取った(Molecular Devices、アメリカ;積分時間、200m秒;全ての波長は、エンドポイントモードで収集した)。各実験に対して、3つのウェルにわたって、発光を平均し、この様式で、全てのACE-tRNAを3回より多く繰り返した。各プレートは、形質移入効率およびベースラインPTCリードスルーに対する対照としての役割を果たすために、ACE-tRNAなしのオールインワンナンセンスレポーターで形質移入されたウェルも3つ組で含有した。全ての値は、ベースラインPTCリードスルー発光を上回るACE-tRNA発光の比±SEMとして報告されている。一元配置ANOVAは、所定のアミノ酸ファミリー中の全てのACE-tRNAにわたって、チューキーの事後分析を用いて行った。
CFTR、HDH-his-strepおよび4×ACE-tRNA発現プラスミド
細胞培養、タンパク質発現およびウェスタンブロット
PVDF(Bio-Rad、アメリカ)に移入した。2%脱脂乳中の抗CFTR抗体M3A7(1:1000;Millipore、アメリカ)を用いて、PVDFをイムノブロットし、LI-COR Odyssey Imaging System(LI-COR、アメリカ)上で画像化した。HDH-His-Strep発現細胞については、100mMスクロース、1mM DTT、1mM EDTA、20mM tris-HCl pH8.0、0.5μg/mLぺプスタチン、2.5μg/mLアプロチニン、2.5μg/mLロイペプチン、0.1mM PMSFおよび0.75mMベンズアミジン中で細胞ペレットを激しくダウンスホモジナイズした。100,000gで30分間、4℃で、溶解物を遠心した。1%の2-メルカプトエタノールの存在下での4~12%Bis-Tris SDS-pageアクリルアミドゲル(ThermoFisher、アメリカ)上で、上清(可溶性細胞タンパク質)を分離し、0.22μM LF PVDF(Bio-Rad、アメリカ)に移入し、2%脱脂乳中で抗Strep抗体(1:5000;iba、ドイツ)を用いてイムノブロットし、LI-COR Odyssey Imaging System(LI-COR、アメリカ)上で画像化した。
質量分析
RNアーゼ阻害剤(Thermo Scientific)を添加する前に、穏やかに撹拌しながら、室温で45分間、A260溶解物当たり100UのRNアーゼI(Ambion、アメリカ)を用いて、溶解物を消化した。次いで、改変されたポリソーム緩衝液(20mM Tris-HCl/pH7.4、150mM NaCl、8.5mM MgCl2、0.5mM DTT、20 U ml-1RiboLock RNアーゼ阻害剤)中に調製された1Mスクロースクッション上に溶解物を載せることによって、リボソーム保護されたmRNA断片を単離し、Beckmen TLA-110ローターを用いて、4℃で2時間、70,000rpmで遠心した。TRIzolを用いてmRNAフットプリントを含有するリボソームペレットを抽出し、8M尿素を含有する変性12%ポリアクリルアミドゲル上で分離した。SYBR Gold(Invitrogen)で染色されたゲルから、26~34ntの範囲のサイズを有するRNA断片を手作業で切り出し、リボソーム保護された断片のライブラリーを作製するために単離した。Ribo-Zeroキット(Illumina)を用いて、夾雑するrRNA断片を枯渇させた。3’オリゴヌクレオチドアダプターライゲーション、逆転写、環状化およびビオチン化されたrRNA枯渇オリゴ(表9)を用いた二度目のrRNA枯渇は、記載されているとおりに行った55。PCR増幅の間に、各試料に対してインデックスプライマーを使用して、ライブラリーにバーコードを付けた。次いで、3%PhiX(Illumina)とともに、バーコードが付けられたライブラリーをプールし、典型的には、試料当たり1800万~2700万の読み取り情報を生成するために、製造業者のプロトコールのとおりに、Illumina NextSeq500中で配列を決定した。
Laboratories、アメリカ)を用いて、PhoenixGP細胞(PMID:7690960)を、pNLuc-STOP-pQCXIPおよびcmv-VSV-G(VSV-G外被シュードタイピング)プラスミドで共形質移入し、33℃のCO2調節された(5%)細胞恒温器中に、48時間置いた。レトロウイルスの粒子を含有する培地(20mls)を4℃に冷却し、細胞細片を除去するために10,000gで遠心し、0.45μmMCE膜シリンジフィルター(Millipore、アメリカ)を通して、30%の集密度で、低継代HEK293細胞が播種された2つの10cm皿上にろ過した。細胞培養皿をParafilmで密封し、90分間、3,500gで、24℃で遠心し、37℃のCO2調節された(5%)細胞培養恒温器中に置いた。対照皿(感染なし)が完全な細胞死を示すまで、ピューロマイシン(1μg/mL)で、24時間後に細胞を選択した。FACSを用いて、96ウェルプレート中に細胞を単分散し、続いて、クローン集団を単分散した。実験操作の間、選択されたクローンを維持するために、ピューロマイシンは使用せず、全ての研究において、標準的なDMEM培地(DMEM-ダルベッコ変法イーグル培地-10%FBS、1%Pen/Stepおよび2mM L-グルタミンが補充された、L-グルタミンを加えた高グルコース;Thermofisher、アメリカ)を使用した。
Software(Perkin Elmer)を用いて分析した。
参考文献
本発明は、例えば以下の項目を提供する。
(項目1)
Tアームと、Dアームと、アンチコドンアームと、アクセプタアームとを含む修飾された転移RNA(tRNA)であって、前記Tアームが伸長因子1-α1(EF1α)と相互作用するヌクレオチドを有するTステムを含む、修飾された転移RNA(tRNA)。
(項目2)
配列番号1~538のいずれか一つからなる修飾された転移RNA(tRNA)であって、チミジンがウラシルで置換されている、修飾された転移RNA(tRNA)。
(項目3)
前記tRNAが配列番号4からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目4)
前記tRNAが配列番号16からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目5)
前記tRNAが配列番号24からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目6)
前記tRNAが配列番号33からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目7)
前記tRNAが配列番号38からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目8)
前記tRNAが配列番号44からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目9)
前記tRNAが配列番号48からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目10)
前記tRNAが配列番号53からなり、チミジンがウラシルで置換されている、項目2に記載の修飾された転移RNA(tRNA)。
(項目11)
配列番号56~60、62~66、84~86、90~111、113、128~143、147~149、153~156、161~174、176、178、181、184~186、192、196~197、199~201、205、213~240、246、255~256、258~285、299、305~312、314、318~332、335~344、346、350~354、357~360、362、365~370、372~383、388~390、392、394~401、403~407、414~416、418、422、425、428~433、437、444~445、452、455、459~463、470、472~474、476、487~492、525、530~539、545~550、553~555、561~563および567~579のいずれか1つからなる修飾された転移RNA(tRNA)であって、チミジンがウラシルで置換されている、修飾された転移RNA(tRNA)。
(項目12)
Tアームと、Dアームと、アンチコドンアームと、アクセプタアームとを含む修飾された転移RNA(tRNA)であって、
(a)前記アンチコドンアームがトリヌクレオチドアンチコドンを含み、前記アンチコドンが5’-UCA-3’であり、かつTGA終止コドンを認識し、前記アクセプタアームがアルギニン、トリプトファン若しくはグリシンに作動可能に連結されており;
(b)前記アンチコドンアームがトリヌクレオチドアンチコドンを含み、前記アンチコドンが5’-UUA-3’であり、かつTAA終止コドンを認識し、前記アクセプタアームがグルタミン若しくはグルタミン酸に作動可能に連結されており;または
(c)前記アンチコドンアームがトリヌクレオチドアンチコドンを含み、前記アンチコドンが5’-CUA-3’であり、かつTAG終止コドンを認識し、前記アクセプタアームがトリプトファン、グルタミン酸若しくはグルタミンに作動可能に連結されている;
修飾された転移RNA(tRNA)。
(項目13)
前記Tアームが伸長因子1-α1(EF1α)との相互作用を増強しまたは調整する合理的なヌクレオチド置換を含む、項目12に記載の修飾されたtRNA。
(項目14)
オリゴヌクレオチドが150ヌクレオチド未満の全長を有する、項目1~13のいずれか一項に記載の修飾されたtRNAをコードするオリゴヌクレオチド配列。
(項目15)
第一のオリゴヌクレオチド配列と第二のオリゴヌクレオチド配列とを含むオリゴヌクレオチドであって、前記第一および第二のオリゴヌクレオチド配列が、項目1~13のいずれか一項に記載の修飾されたtRNAを独立にコードし、前記第一および第二のオリゴヌクレオチドが150ヌクレオチド未満の全長を独立に有し、前記2つの配列がタンデムである、オリゴヌクレオチド。
(項目16)
前記オリゴヌクレオチドがDNAである、項目15に記載のオリゴヌクレオチド。
(項目17)
プロモーターと、項目1~13のいずれか一項に記載の修飾されたtRNAをコードする核酸または項目14~16のいずれか一項に記載のオリゴヌクレオチド配列とを含む発現カセット。
(項目18)
項目14~16のいずれか一項に記載のオリゴヌクレオチドまたは項目17に記載の発現カセットを含むベクター。
(項目19)
前記ベクターがウイルスベクターまたはプラスミドベクターである、項目18に記載のベクター。
(項目20)
項目1~13のいずれか一項に記載の修飾されたtRNAと、項目14~16のいずれか一項に記載のオリゴヌクレオチドと、または項目18若しくは19に記載のベクターと、
薬学的に許容され得る担体と、
を含む、組成物。
(項目21)
前記担体がリポソームである、項目20に記載の組成物。
(項目22)
項目18または19に記載のベクターを含む細胞。
(項目23)
終止コドン関連遺伝子疾患を処置する方法であって、終止コドン関連遺伝子疾患を処置することを必要とする患者に、項目20または21に記載の組成物を投与することを含む、方法。
(項目24)
未成熟終止コドンと関連する前記遺伝子疾患が、嚢胞性線維症、筋ジストロフィー、β-サラセミアまたはリドル症候群である、項目23に記載の方法。
(項目25)
細胞中のナンセンス変異を含むヌクレオチド配列への翻訳を回復させる方法であって、項目20または21に記載の組成物を前記細胞に導入することを含み、修飾されたtRNAが、ナンセンス変異を含む前記ヌクレオチド配列への翻訳を回復させる、方法。
Claims (1)
- 明細書に記載の発明。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762580887P | 2017-11-02 | 2017-11-02 | |
US62/580,887 | 2017-11-02 | ||
US201862687015P | 2018-06-19 | 2018-06-19 | |
US62/687,015 | 2018-06-19 | ||
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BR112020008733A2 (pt) | 2017-11-02 | 2020-11-03 | University Of Iowa Research Foundation | método de resgate de códons de parada através de reatribuição genética com ace-trna |
WO2021087401A1 (en) * | 2019-11-01 | 2021-05-06 | Tevard Bio, Inc. | Methods and compositions for treating a premature termination codon-mediated disorder |
EP4069841A1 (en) * | 2019-12-02 | 2022-10-12 | Shape Therapeutics Inc. | Targeted transfer rnas for treatment of diseases |
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CA3181680A1 (en) | 2020-06-12 | 2021-12-16 | University Of Rochester | Encoding and expression of ace-trnas |
KR20230127269A (ko) * | 2020-12-31 | 2023-08-31 | 유니버시티 오브 아이오와 리써치 파운데이션 | 센스, 억제자 트랜스퍼 rna 조성물 및 관련된 용도와기능 |
JP2024509123A (ja) * | 2021-02-25 | 2024-02-29 | リボズ エルエルシー | 合成リボ核酸(rna)からのタンパク質発現を向上させる組換え発現構築物 |
BR112023022805A2 (pt) * | 2021-05-05 | 2024-01-16 | Tevard Biosciences Inc | Métodos e composições para tratamento de um distúrbio mediado por códon de terminação prematura |
CN113308463A (zh) * | 2021-05-12 | 2021-08-27 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 经过反密码子编辑的tRNA作为分子开关在精准控制蛋白表达及病毒复制中的应用 |
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