JP6495653B2 - レンチウイルスパッケージ化のための非サブタイプbのgagタンパク質の使用 - Google Patents
レンチウイルスパッケージ化のための非サブタイプbのgagタンパク質の使用 Download PDFInfo
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- JP6495653B2 JP6495653B2 JP2014531334A JP2014531334A JP6495653B2 JP 6495653 B2 JP6495653 B2 JP 6495653B2 JP 2014531334 A JP2014531334 A JP 2014531334A JP 2014531334 A JP2014531334 A JP 2014531334A JP 6495653 B2 JP6495653 B2 JP 6495653B2
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Description
atgggtgcgagagcgtcagtattaagcgggggaaaattagatacatgggaaagaattcggttacggccaggaggaaagaaaaaatatgcactaaaacatttgatatgggcaagcagggagctagaacgatttacacttaatcctggccttttagagacatcagaaggctgtaaacaaataataggacagctacaaccatctattcaaacaggatcagaagaaattagatcattatataatacagtagcaaccctctattgtgtacatgaaaggatagaggtaaaagacaccaaagaagctgtagaaaagatggaggaagaacaaaacaaaagtaagaaaaagacacagcaagcagcagctgatagcagccaggtcagccaaaattaccctatagtgcagaacctacaggggcaaatggtacatcaggccatatcacctagaactttgaacgcatgggtaaaagtaatagaagaaaaggccttcagcccggaagtaatacccatgttttcagcattatcagaaggagccaccccacaagatttaaacaccatgctaaacacagtggggggacatcaagcagctatgcaaatgctaaaagagaccatcaatgacgaagctgcagaatgggacagattacatccagtgcatgcagggcctgttgcaccaggccaaatgagagaaccaaggggaagtgatatagcaggaactactagtacccttcaggaacaaatagcatggatgacaagcaacccacctatcccagtaggagaaatctataaaagatggataatcctgggattaaataaaatagtaagaatgtatagccctgtcagcattttggacataagacagggaccaaaggaaccttttagagactatgtagaccggttctataaaactctaagagccgagcaagcttcacaggatgtaaaaaactggatgacagaaaccttgttggtccaaaatgcaaacccagattgtaaaactatcttaaaagcattgggaccacaggctacactagaagaaatgatgacagcatgccagggagtgggggggcccggccataaagcaagagttttggctgaggcaatgagccaagtaacaggttcagctactgcagtaatgatgcagagaggcaattttaagggcccaagaaaaagtattaagtgtttcaactgtggcaaggaagggcacacagcaaaaaattgcagggcccctagaaaaaagggctgttggaaatgcggaagggaaggacaccaaatgaaagattgcactgaaagacaggctaattttttagggaagatttggccttcccacaagggaaggccggggaattttcttcagagcagaccagagccaacagccccaccagcagagagcttcgggtttggggaggagataaccccctctcagaaacaggagcagaaagacaaggaactgtatcctttagcttccctcaaatcactctttggcaacgacccctcgtcacaataaagatagggggacagctaaaggaagctctattagatacaggagcagatgatacagtattagaagaaataaatttgccaggaaaatggaagccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgatcaaatactcatagaaatctgtggatataaagctatgggtacagtattagtaggacctacacctgtcaacataattggaagaaatttgttgacccagattggctgcactttaaattttccaattagtcctattgaaactgtaccagtaaaattaaagccaggaatggatggcccaaaagttaaacaatggccattgacgaagaaaaaataaaagcattaacagaaatttgtacagaaatggaaaaggaaggaaaaatttcaagaattgggcctgaaaatccatataatactccaatatttgccataaagaaaaaagacagtaccaagtggagaaaattagtagatttcagagaacttaataagagaactcaagatttctgggaggttcaattaggaataccgcatcctgcagggctgaaaaagaaaaaatcagtaacagtactggatgtgggtgatgcatatttctcagttcccttagatgaagattttaggaaatataccgcatttaccatacctagtataaacaatgagacaccagggattagatatcagtacaatgtgctcccacagggatggaaaggatcaccggcaatattccaaagtagcatgacaaaaatcttagagccctttagaaaacaaaatccagaaatagttatctatcaatacatggatgatttgtatgtaggatctgacttagaaatagggcagcatagaacaaaaatagaggaattaagagaacatctattgaggtggggatttaccacaccagataaaaaacatcagaaagaacctccatttctttggatgggttatgaactccatcctgataaatggacagtacagcctataaacctgccagaaaaagaaagctggactgtcaatgatatacagaagttagtggggaaattaaactgggcaagccagatttatgcaggaattaaagtaaagcaattatgtaaactccttaggggaaccaaagcactaacagaagtagtaccactaacagaagaagcagaattagaactggcagaaaacagggaaattctaaaagaaccagtacatggagtgtattatgacccatcaaaagacttaatagcagaactacagaaacaaggggacggccaatggacataccaaatttatcaagaaccatttaaaaatctaaaaacaggaaagtatgcaagaacgaggggtgcccacactaatgatgtaaaacaattaacagaggcagtgcaaaaaatagccacagaaagcatagtgatatggggaaagactcctaaatttaaactacccatacaaaaggaaacatgggaaacatggtggatagagtattggcaagccacctggattcctgagtgggaatttgtcaatacccctcctttagtaaaattatggtaccagttagagaaggaacccataataggagcagaaactttctatgtagatggggcagctaatagagagactaaattaggaaaagcaggatatgttactgacagaggaagacagaaagttgtccctttcactgacacgacaaatcagaagactgagttacaagcaattaatctagctttacaggattcgggattagaagtaaacatagtaacagattcacaatatgcactaggaatcattcaagcacaaccagataagagtgaatcagagttagtcagtcaaataatagagcagctaataaaaaaggaaaaggtttacctggcatgggtaccagcacacaaaggaattggaggaaatgaacaagtagataaattagtcagtcagggaatcaggaaagtactatttttggatggaatagataaggctcaggaagaacatgagaaatatcacaacaattggagagcaatggctagtgattttaacctaccacctgtggtagcgaaagaaatagtagctagctgtgataaatgtcagctaaaaggagaagccatgcatggacaagtagactgtagtccaggaatatggcaattagattgtacacatctggaaggaaaagttatcctggtagcagttcatgtagccagtggctatatagaagcagaagttattccagcagaaacggggcaagaaacagcatactttctcttaaaattagcaggaagatggccagtaaaagtagtacatacagataatggcagcaatttcaccagtgctacagttaaggccgcctgttggtgggcagggatcaaacaggaatttggaattccctacaatccccaaagtcaaggagtagtagaatctatgaataaagaattaaagaaaattataggacaggtaagagatcaagctgaacatcttaagacagcagtacaaatggcagtatttatccacaattttaaaagaaaaggggggattgggggatacagtgcaggggaaagaataatagacataatagcaacagacatacaaactagagaattacaaaaacaaatcataaaaattcaaaattttcgggtttattacagggacagcagagatccaatttggaaaggaccagcaaagcttctctggaaaggtgaaggggcagtagtaatacaagacaatagtgacataaaggtagtaccaagaagaaaagtaaagatcattagggattatggaaaacagatggcaggtgatgattgtgtggcaagtagacaggatgaggattaac (配列番号:1)
MGARASVLSGGKLDAWERIRLRPGGKKKYALKHLIWASRELERIALNPGLLETSEGCKQIIGQLQPSIQTGSEELRSLYNTIATLYCVHERIEVKDTKEAVEKMEEEQNKSKKKTQQAAADSSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINDEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSTLQEQIAWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPQATLEEMMTACQGVGGPGHKARVLAEAMSQVTGSVTAVMMQRGNFKGPRKSIKCFNCGKEGHTAKNCRAPRKKGCWKCGREGHQMKDCSERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFGFGEEITPSQKQEQKDKELYPLASLKSLFGNDPSSQFFREDLAFPQGKAGEFSSEQTRANSPTSRELRVWGGDNPLSETGAEGQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGKWKPKMIGGIGGFIKVRQYDQILIEICGYKAMGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALTEICTEMEKEGKISRIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPEIVIYQYMDDLYVGSDLEIGQHRTKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIKLPEKESWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAELQKQGDGQWTYQIYQEPFKNLKTGKYARTRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWETWWIEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPFTDTTNQKTELQAINLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSQGIRKVLFLDGIDKAQEEHEKYHNNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKVVHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTRELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIRDYGKQMAGDDCVASRQDED (配列番号:2)
MGARASVLSGGKLDTWERIRLRPGGKKKYALKHLIWASRELERFTLNPGLLETSEGCKQIIGQLQPSIQTGSEEIRSLYNTVATLYCVHERIEVKDTKEAVEKMEEEQNKSKKKTQQAAADSSQVSQNY (配列番号:3)
サブタイプB HIV−1ウイルスは、サブタイプBウイルスが感染する効率が低く、異なる様式で感染する点で他のHIV−1サブタイプとは異なる。それにもかかわらず、サブタイプBウイルスは、HIV−1レンチウイルスベクターおよびレンチウイルスパッケージ化ベクターの製造に広く使用されてきた。非サブタイプBウイルスのGagおよびPolタンパク質がレンチウイルスパッケージ化ベクターの作製に使用することができるか否かを決定するために、レンチウイルスパッケージ化プラスミドのHIV−1サブタイプBのgag−pol遺伝子(コンストラクトp8.74)を、サブタイプDのHIV−1のgag−pol遺伝子に置き換え、コンストラクトpThV−GP−Nを生成した。レンチウイルスベクター製造にこのコンストラクトを用いた。コンストラクトp8.74と比較して、pThV−GP−Nプラスミドを用いた場合におよそ2倍高い力価が得られた(図3)。よって、パッケージ化ベクターにおけるサブタイプD HIV−1 Gag−Polは、サブタイプB HIV−1 Gag−Polによって観察された力価よりもレンチウイルスベクターの力価を増加させた。
本発明は、非サブタイプBのGagおよび/またはPolタンパク質をコードするパッケージ化ベクターを包含する。レンチウイルス「パッケージ化ベクター」は、ここでは、機能的なHIV−1 Envをコードせず、Ψ部位を欠いているが、パッケージ化のために好適なレンチウイルスのシス作用型シグナルを含むベクターと同時形質移入した場合にウイルス粒子内へ取り込まれうるレンチウイルスGagおよび/またはPolタンパク質を発現することができる核酸配列として定義される。本発明のレンチウイルスパッケージ化ベクターは、自身の配列をパッケージ化し、逆転写することによってそれ自身複製することができない。
アミノ酸12位のグルタミン酸の欠失;
アミノ酸15位のアルギニンの欠失;
アミノ酸46位のバリンの欠失;および
アミノ酸61位のロイシンの欠失。
12位のアミノ酸がリシンであり;
15位のアミノ酸がスレオニンであり;
15位のアミノ酸がアラニンであり;
46位のアミノ酸がロイシンであり;
61位のアミノ酸がイソロイシンであり;および/または
61位のアミノ酸がメチオニンである。
本発明は、非サブタイプBのGagおよび/またはPolタンパク質を発現する細胞を含むレンチウイルスパッケージ化システムを包含する。ここでは、レンチウイルスの「パッケージ化システム」は、Ψ部位の非存在下で少なくともレンチウイルスのGagおよびPolタンパク質を発現し、Ψ部位を含む外因性核酸をパッケージ化し、逆転写することができる細胞を含む、細胞ベースのシステムとして定義される。レンチウイルスパッケージ化システムの細胞はまた、他のウイルスタンパク質を発現することができる。好ましくは、レンチウイルスパッケージ化システムは、エンベロープタンパク質を発現する。エンベロープタンパク質は、レンチウイルス(例えばHIV−1 Env)または非レンチウイルス(例えばVSV、シンドビスウイルス、狂犬病ウイルス)のエンベロープタンパク質であってよい。様々な実施形態では、レンチウイルスパッケージ化システムは、HIV−1 Tatおよび/またはRevタンパク質を発現する。
本発明は、非サブタイプBのGagおよび/またはPolタンパク質をコードするパッケージ化ベクターの製造方法を包含する。パッケージ化ベクターは、本明細書で説明する特徴のいずれかを含みうる。
本発明はまた、HIV−1非サブタイプBのGagおよび/またはPolタンパク質をコードするパッケージ化ベクターを使用し、レンチウイルスベクターを作製するための方法を包含する。ある実施形態では、本発明は、レンチウイルスベクターを用いて、HIV−1非サブタイプBのGagまたはPolタンパク質をコードするパッケージ化ベクターを、細胞に投与することを包含する。パッケージ化ベクターは、本明細書で説明する特徴のいずれかを含みうる。
本発明はまた、HIV−1非サブタイプBのGagおよび/またはPolタンパク質を含むレンチウイルスベクター粒子を包含する。非サブタイプBのGagおよびPolタンパク質は、本明細書に記載の特徴のいずれかを含むことができる。
gag−pol遺伝子は、鋳型としてHIV−1 NDKのクローン、pNDK−Nおよび2つのプライマーを用いてPCRにより増幅した。pThV−GP−Nプラスミドを得るために、PCR産物をEagI /SalIにて消化し、EagI /SalIにて消化したパッケージ化コンストラクトp8.74に挿入した。
レンチウイルスベクター貯蔵液は、pFLAP CMV GFP bisおよびpTHV−VSV.G (INDI−CO)bisをp8.74またはpThV−GP−Nと組み合わせて用いて製造した。p8.74にて18回、pThV−GP−Nにて18回、合計36回の形質移入を行った。すべての上清物を−80℃に保存した。
ベクター力価は293T細胞におけるGFP発現の頻度によって決定した。細胞は、1ウェルあたり1×105細胞の密度に達するまで、10%FBSを含有するDMEM中で、24ウェルプレートにて培養した。次いで、細胞を300μLの終容量中で異なる容量のベクター上清を形質導入した。2時間後、10%FBSを含有する700μlの新鮮培地を各ウェルに添加した。形質導入の72時間後に、培地を除去し、細胞をダルベッコのリン酸緩衝生理食塩水(DPBS、Gibco)中で洗浄した。細胞を0.05%トリプシン−EDTA(Gibco)にて除去した。トリプシン処理は300μlの完全DMEMを添加して止め、細胞をFACSのためにチューブに移して、その後、GFP発現細胞の数を、509nmの励起波長を用いたFACSCalibur(BD Biosciences)にて計数した。30%未満のGFP陽性細胞の割合のみを考慮した。
HIV−1 BRUおよびHIV−1 NDKウイルスは、293T細胞上で作製し、形質導入されたP4 CCR5細胞に使用した。これらの細胞は、HIV LTRの制御下に安定したルシフェラーゼ遺伝子を包含する。これらがTATタンパク質を形質移入している場合(WT HIVに感染している場合)、LacZ遺伝子が発現され、ルシフェラーゼ発現を測定することができる。HIV−1のGag p24およびルシフェラーゼ発現を測定した。図4に結果を示し、野生型NDKウイルスが、野生型BRUウイルスよりも高い形質導入率を有することが確認される。
異なる比率のp8.74およびpSD GP NDKパッケージ化ベクターを用いて、レンチウイルスベクター粒子を製造した。それぞれの比率について力価およびp24レベルを測定した。結果を図5に示し、p24レベルおよび製造力価の亢進を担うNDKパッケージ化ベクターの存在が示された。
パッケージ化ベクターp8.74およびpTHV−GP−Nを用いて、レンチウイルスベクター粒子を含む上清を製造した。NIH抗P24 MAB(183−H12−5C)を用いて上清についてウエスタンブロットを行った。結果を図6に示し、BRUがウイルス上清中にp24のみを示す場合にNDKが高いp24合成を生成するようであるので(ウイルス上清にp24前駆体が存在)、NDKとBRUパッケージ化プラスミドとの差異は、p24タンパク質と前駆体との製造に因るものであることが確認される。
Claims (11)
- Ψ部位を欠き、サブタイプD HIV−1 Gag−Polタンパク質をコードする、複製欠損レンチウイルスパッケージ化ベクターであって、
当該HIV−1 Gag−Polタンパク質が、HIV−1 NDKのHIV−1 Gag−Polタンパク質であり、
当該HIV−1 Gag−Polタンパク質が、配列番号:2のアミノ酸配列、および配列番号:2と少なくとも95%同一でありかつサブタイプB HIV−1 Gag−Polタンパク質をコードするパッケージ化ベクターよりも高いベクター力価を提供するアミノ酸配列から選択されるアミノ酸配列からなる、複製欠損レンチウイルスパッケージ化ベクター。 - 前記ベクターが、配列番号:2のアミノ酸配列、および配列番号:2と少なくとも95%同一でありかつサブタイプB HIV−1 Gag−Polタンパク質をコードするパッケージ化ベクターよりも高いベクター力価を提供するアミノ酸配列から選択されるアミノ酸配列からなる、サブタイプD HIV−1 Gag−Polタンパク質をコードするヌクレオチド配列を含むプラスミドである、請求項1に記載のベクター。
- サブタイプD HIV−1 Gag−Polタンパク質をコードするヌクレオチド配列を、非HIVプロモーターの制御下でプラスミドに挿入し、パッケージ化ベクターを作製することを含む、パッケージ化ベクターの製造方法であって、
当該HIV−1 Gag−Polタンパク質が、HIV−1 NDKのHIV−1 Gag−Polタンパク質であり、
当該HIV−1 Gag−Polタンパク質が、配列番号:2のアミノ酸配列、および配列番号:2と少なくとも95%同一でありかつサブタイプB HIV−1 Gag−Polタンパク質をコードするパッケージ化ベクターよりも高いベクター力価を提供するアミノ酸配列から選択されるアミノ酸配列からなる、パッケージ化ベクターの製造方法。 - レンチウイルスベクターを用いて、サブタイプD HIV−1 Gag−Polタンパク質をコードするパッケージ化ベクターを、細胞に投与することを含む、レンチウイルスベクター粒子を作製するためのインビトロの方法であって、
当該HIV−1 Gag−Polタンパク質が、HIV−1 NDKのHIV−1 Gag−Polタンパク質であり、
当該HIV−1 Gag−Polタンパク質が、配列番号:2のアミノ酸配列、および配列番号:2と少なくとも95%同一でありかつサブタイプB HIV−1 Gag−Polタンパク質をコードするパッケージ化ベクターよりも高いベクター力価を提供するアミノ酸配列から選択されるアミノ酸配列からなる、レンチウイルスベクター粒子を作製するためのインビトロの方法。 - 前記パッケージ化ベクターおよびレンチウイルスベクターが、プラスミドである、請求項4に記載の方法。
- 前記細胞が、サブタイプD HIV−1 Gag−Polタンパク質を一時的に発現する、請求項4に記載の方法。
- 前記パッケージ化ベクターが、前記細胞のゲノムに組み込まれる、請求項4に記載の方法。
- さらに、サブタイプB HIV−1 Gag−Polタンパク質をコードするパッケージ化ベクターよりも高い力価のレンチウイルスベクターをパッケージ化するパッケージ化ベクターを選択することを含む、請求項4に記載の方法。
- 請求項1に記載のベクターを含む細胞。
- 前記ベクターが、前記細胞のゲノムに組み込まれる、請求項9に記載の細胞。
- サブタイプD HIV−1のGag−Polタンパク質とVSVのエンベロープ糖タンパク質を含むレンチウイルスベクター粒子であって、
当該HIV−1 Gag−Polタンパク質が、HIV−1 NDKのHIV−1 Gag−Polタンパク質であり、
当該HIV−1 Gag−Polタンパク質が、配列番号:2のアミノ酸配列、および配列番号:2と少なくとも95%同一でありかつサブタイプB HIV−1 Gag−Polタンパク質をコードするパッケージ化ベクターよりも高いベクター力価を提供するアミノ酸配列から選択されるアミノ酸配列をコードする核酸配列を発現させることによって製造されるGag−Polタンパク質である、レンチウイルスベクター粒子。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11306222 | 2011-09-26 | ||
EP11306222 | 2011-09-26 | ||
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EP3009144A1 (en) * | 2014-10-15 | 2016-04-20 | Theravectys | Lentiviral vectors for inducing CD4+ and CD8+ immune responses in vaccination of humans |
WO2015063707A1 (en) * | 2013-10-31 | 2015-05-07 | Theravectys | LENTIVIRAL VECTORS FOR INDUCING CD4+ and CD8+ IMMUNE RESPONSES IN VACCINATION OF HUMANS |
WO2016120489A2 (en) * | 2015-02-01 | 2016-08-04 | Theravectys | Lentiviral vectors for expression of mycobacterium tuberculosis antigens |
US20210102221A1 (en) * | 2019-10-07 | 2021-04-08 | University Of Utah Research Foundation | Methods and Compositions for A HIV Based Delivery System |
CN114107393A (zh) | 2021-04-07 | 2022-03-01 | 上海劲威生物科技有限公司 | 一种治疗乙型肝炎的慢病毒载体、慢病毒颗粒及其制备方法和应用 |
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UA68327C2 (en) * | 1995-07-04 | 2004-08-16 | Gsf Forschungszentrum Fur Unwe | A recombinant mva virus, an isolated eukaryotic cell, infected with recombinant mva virus, a method for production in vitro of polypeptides with use of said cell, a method for production in vitro of virus parts (variants), vaccine containing the recombinant mva virus, a method for immunization of animals |
US7262049B2 (en) * | 1999-03-16 | 2007-08-28 | Dana-Farber Cancer Institute, Inc. | Pseudotyped lentiviral vectors and uses thereof |
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US20070166784A1 (en) | 2003-09-15 | 2007-07-19 | Barnett Susan W | Combination approaches for generating immune responses |
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ZA201401780B (en) | 2015-09-30 |
KR20140116053A (ko) | 2014-10-01 |
ES2683820T3 (es) | 2018-09-28 |
US20140234908A1 (en) | 2014-08-21 |
CA2849652A1 (en) | 2013-04-04 |
KR101811736B1 (ko) | 2017-12-22 |
IL231666A0 (en) | 2014-05-28 |
CA2959867A1 (en) | 2013-04-04 |
CA2849652C (en) | 2017-11-21 |
CN104039968B (zh) | 2018-10-26 |
BR112014007027A2 (pt) | 2017-04-11 |
JP2018183165A (ja) | 2018-11-22 |
AU2012313960B2 (en) | 2016-05-12 |
US20170362607A1 (en) | 2017-12-21 |
HK1200870A1 (en) | 2015-08-14 |
JP2014530000A (ja) | 2014-11-17 |
CA2959867C (en) | 2020-04-28 |
CN104039968A (zh) | 2014-09-10 |
WO2013046034A3 (en) | 2013-05-30 |
US9752160B2 (en) | 2017-09-05 |
DK2761011T3 (en) | 2018-08-27 |
AU2012313960A1 (en) | 2014-04-10 |
EP2761011B1 (en) | 2018-05-23 |
NZ622860A (en) | 2016-05-27 |
IL231666B (en) | 2018-03-29 |
JP2017018132A (ja) | 2017-01-26 |
WO2013046034A2 (en) | 2013-04-04 |
SG11201400987QA (en) | 2014-07-30 |
EP2761011A2 (en) | 2014-08-06 |
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