JP7402479B2 - 干渉rna分子を送達する飼料としてのトランスジェニック微細藻類およびその使用 - Google Patents
干渉rna分子を送達する飼料としてのトランスジェニック微細藻類およびその使用 Download PDFInfo
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Description
本発明は、標的生物体にRNAi分子を効率的で、安定であり、かつ環境に安全な方法で経口送達する組成物および方法を提供する。特に、本発明は、RNAiを仲介するポリヌクレオチド分子を含む非増殖性トランスジェニック微細藻類およびそれを含む可食性組成物を提供する。
「微細藻類」という用語は、本明細書ではその最も広い範囲で使用され、通常淡水系および海水系にみられる顕微レベルの単細胞真核藻類を指す。微細藻類の大きさは、種に応じて数マイクロメートル(μm)から数百マイクロメートルの範囲であり得る。本発明の特定の実施形態では、この用語は海産真核微細藻類を指す。
藻の培養および回収
藻の培養および回収を本発明の出願者に対する米国特許出願公開第2011/0081706号に記載されている通りに実施した。簡潔に述べれば、珪藻生育用のF/2栄養素(Andersen Rら,2005.Recipes for freshwater and seawater media.In:Algal Culturing Techniques(R.A.Andersen編),pp.429-538.Elsevier,Amsterdamのものを改変)を多量に含み、ろ過された海水で、藻(フェオダクチラム・トリコルヌタム(Phaeodactylum tricornutum))を培養した。F/2を最終培養体積に対して1:1000の量で72時間毎に加えた。21℃の一定温度条件を維持した。1m2s1当たり100μmol光子の光強度で明:暗を16時間:8時間に設定した。CO2と空気を混合し、比を制御した状態でエアレーションシステムから培養物に供給した。実験には、藻をその最大培養密度付近で回収した。藻を凝集させるため、水酸化カルシウムを0.15g/mlのCa(OH)2を含有する微細な粒子懸濁水の形で培養物に添加し、次いで、培養物をろ過または遠心分離した。得られた藻の沈殿物を凍結乾燥させて藻粉末にした。藻粉末を真空バッグ中、2~8℃の温度で保管した。
I.粒子ボンバードメントによる形質転換
新鮮な藻培養物を上記の人工海水(ASW)F/2培地で対数中期(2~5×106細胞/ml)まで増殖させた。ボンバードメントの24時間前に細胞を回収し、新鮮なASW+F/2で2回洗浄し、元の細胞体積の10分の1をASW+F/2に再懸濁させた。凝固したASW+F/2培地の入った55mmのペトリ皿の中央に細胞懸濁液0.5mlをスポットした。プレートを通常の増殖条件下で乾燥させた。PDS 1000/He微粒子銃形質転換システム(BioRad Laboratories社、ハーキュリーズ、カリフォルニア州、米国)を製造業者の指示通りに用い、直径2ミクロン超の細胞にはM17タングステン末(BioRad Laboratories社)を、これより小さい細胞には0.6ミクロン未満の粒子からなるタングステン末(FW06、Canada Fujian Jinxin Powder Metallurgy社、マーカム、オンタリオ州、カナダ)を使用して、ボンバードメントを実施した。タングステンには直線状DNAをコーティングした。1100psi(約7580kPa)または1350psi(約9300kPa)の破裂板を使用した。消耗品はいずれもBioRad Laboratories社から購入した。ボンバードメント後、プレートを通常の増殖条件下で24時間インキュベートした後、細胞を選択固形培地に播き、単一コロニーが出現するまで通常の増殖条件下でインキュベートした。
新鮮な藻培養物を上記のASW+F/2培地で対数中期まで増殖させた。培養物の試料10mlを回収し、ダルベッコのリン酸緩衝生理食塩水(DPBS、Gibco、Invitrogen社、カースルバッド、カリフォルニア州、米国)で2回洗浄し、緩衝液R(マイクロポレーション装置およびキットの製造者であるDigital Bio、NanoEnTek社(ソウル、韓国)により供給されたもの)250μlに再懸濁させた。細胞100μl毎に直線状DNAを8μg加えた後、細胞にパルスを加えた。通常、細胞の種類に応じて700~1700ボルト、パルス長10~40ミリ秒の範囲の様々なパルスが必要であり、各試料にパルスを1~5回加えた。パルスを加えた直後、細胞を200μlの新鮮な培地(非選択培地)に移した。25℃、微光下で24時間インキュベートした後、細胞を選択固形培地上に播き、単一コロニーが出現するまで通常の増殖条件下でインキュベートした。
動物(本明細書でのちに記載する種のもの)を水で洗浄し、8~10個体のプールの血リンパをエッペンドルフチューブに採取した。試料を14000g、4℃で5分間遠心分離し、Trizol(Ambion life technologies社;カタログ番号15596026)の入った新たなエッペンドルフチューブに移した。試料を液体窒素の中に入れ、使用するまで凍結保存した。
Master Pure Plant RNAキット(Epicentre社;カタログ番号MPR09100)を製造業者の指示通りに用いて、藻からのRNA抽出を実施した。試料を無RNアーゼ水に再懸濁させ、TURBO DNAフリーキット(Ambion社、カタログ番号AM1907)を製造業者の指示通りに用いてインキュベートした。
ランダムヘキサマープライマー(Fermentas社;カタログ番号S0142)を添加したSuperScriptII Reverse Transcriptase(Invitrogen社;カタログ番号100004925)またはiScript(商標)cDNA Synthesis Kit(Bio-Rad社;カタログ番号170-8890)を製造業者の指示通りに用いたcDNA合成には、全RNA1μgを鋳型として用いた。
Biorad CFX96にPlatinum SYBRグリーン(Invitrogen社;カタログ番号11744500)を製造業者の指示通りに用いたqRT-PCRによる相対的定量化によってcDNAの解析を実施した。プライマーds-GFP Q2 For juncおよびds-GFP Q2 Rev linker(表1)を用いて、mRNAをコードし緑色蛍光タンパク質(GFP)に標的化されたdsRNAを増幅した。TBP(TATAボックス結合タンパク質をコードする)を参照遺伝子として使用し、Q-TBP-fwおよびQ-TBP-rv(表1)を用いて増幅した。ds-mCherry Q1 For 5’およびds-mCherry Q1 Rev junc(表1)を用いて、赤色蛍光タンパク質であるmCherryに標的化されたdsRNAを増幅した。内部対照(Primerdesign社、イギリス;カタログ番号INT-RNA-FAM)を参照に用いた。ΔΔCt法(Livakら,2001.Methods 25:402-408)に従ってデータ解析を実施した。
サバクトビバッタ(Schistocerca gregaria)のclk mRNAに標的化されたdsRNAの構築
clk遺伝子のセンスフラグメント(配列番号2)およびアンチセンスフラグメント(配列番号3)をBiomatik USA社にて合成した。BamHI、SacIIによってセンスフラグメントをクローン化し、SacI、KpnIによってアンチセンスフラグメントをクローン化した。clkフラグメントをpPhaTカセットに含まれるイントロンにセンス/アンチセンスの方向でクローン化した(図1、配列番号37に記載される核酸配列を有するpPhatカセット)。
以下のオリゴヌクレオチドを用いたPCRによりGFPセンス鎖(配列番号4)を増幅した:BamHIセンスGFP F 5’-GGGATCCATGGTGAGCAAGGGCGAGG-3′(配列番号25)。SacIIセンスGFP R 5’-GCCGCGGGGCGAGCTGCACGCTGCC-3’(配列番号26)。以下のオリゴヌクレオチドを用いたPCRによりGFPアンチセンス鎖(配列番号5)を増幅した:SacIアンチセンスGFP F 5’-GGAGCTCATGGTGAGCAAGGGCGAGG-3’(配列番号27)。KpnIアンチセンスGFP R 5’-GGGTACCGGCGAGCTGCACGCTGCC-3’(配列番号28)。
チャイロコメノゴミムシダマシ(Tenebrio molitor)の幼虫を一定温度24℃および相対湿度50%、12時間/12時間の明/暗照明条件の栽培チャンバに入れた。キャベツの葉に、mCherryに標的化されたdsRNA(dsRNA-mCherryまたはdsmCherry)を発現するトランスジェニック藻の粉末または野生型藻の粉末を、いずれの葉も藻粉末で均一に覆われるよう塗布した。藻粉末を植物原料に良好に塗布できるように、(野生型および形質転換藻の)藻粉末と、人工海水(ASW)にゼラチンを5%溶かし再蒸留水(DDW)で1:3に希釈したものとを混合した。チャイロコメノゴミムシダマシ(Tenebrio molitor)の幼虫には16時間餌を与えなかった。実験当日、藻を塗布したキャベツの葉またはゼラチンのみで覆われた葉(mock)を幼虫に3~5時間給餌した。そののち、血リンパを採取し、試料を急速冷凍し、RNA抽出まで-80℃で保管した。
エジプトヨトウ(Spodoptera(Prodenia)littoralis)の5齢段階の幼虫を一定温度24℃および相対湿度50%、12時間/12時間の明/暗照明条件の栽培チャンバに入れた。トウゴマ(Ricinus communis)(トウゴマ)の葉に、GFPに標的化されたdsRNA(dsRNA-GFP)を発現するトランスジェニック藻の粉末または野生型藻の粉末を、いずれの葉も藻粉末(上記のようにゼラチンと混合したもの)で均一に覆われるよう塗布した。藻粉末を塗布した葉をエジプトヨトウ(Spodoptera littoralis)に給餌した。各処理には、それぞれ10個体の幼虫からなる5つの独立した反復を用いた。5齢幼虫から血リンパを採取し、RNAを調製した後、dsRNA-GFPの特異的プライマー(配列番号9~10)および負荷対照のTATAボックス結合タンパク質(TBP)mRNA(配列番号7~8)(表4)を用いたqRT-PCR解析に供した。RT-PCR産物をアガロースゲルに負荷した。血リンパ試料全8例(野生型藻の粉末およびトランスジェニック藻の粉末で覆われた葉を給餌した幼虫から採取したもの)にTBPセグメントの175bpの完全長アンプリコン産物が検出された。83bpのdsRNA-GFPの完全長アンプリコン産物は、dsRNA-GFP発現藻を給餌した個体の血リンパ試料4例にのみ検出され、WT藻を給餌した個体の血リンパには検出されなかった(図5)。以上の結果は、藻に発現したdsRNA分子を、この実施例ではエジプトヨトウ(Spodoptera littoralis)の幼虫に給餌することによって送達することが可能であることをさらに示すものである。
サバクトビバッタ(Schistocerca gregaria)のtimeless遺伝子およびperiod遺伝子は、発現がclockタンパク質によって直接調節される2つの遺伝子である。これらの遺伝子の発現レベルは、clockの発現および活性のダウンレギュレーションを明らかにするのによく用いられる(Tobback Jら,2011,同上)。
dsRNA-clockを発現する藻の粉末を塗布したコムギ新芽またはWT藻の粉末を塗布したコムギ新芽を上記のようにサバクトビバッタ(Schistocerca gregaria)の齢幼虫(4~5齢)に継続的に給餌した。バッタが性的に成熟した第17日、砂を満たした鉢を各ケージの中に置き、雌に産卵させた。48時間毎に鉢を回収し、37℃で10日間、恒温放置して幼虫を孵化させた。卵莢および孵化した幼虫をカウントし、各処理の卵莢数に対する孵化した幼虫の割合を算出した。dsRNA-clockを発現するトランスジェニック藻を給餌した雌バッタは卵莢を31個産卵し、そのうち128個が孵化したが、WT藻を給餌した雌バッタは卵莢を28個産卵し、そのうち262個が孵化した。卵莢数に対する孵化した幼虫の割合(全反復の平均値)は、dsRNA-clock発現藻を給餌したバッタの方がWT藻を給餌したバッタよりも2.3倍低い値であった(図7A)。
dsRNA-clockを発現する藻の粉末またはWT藻の粉末(上記のようにゼラチンと混合した藻粉末)を塗布したコムギ新芽をサバクトビバッタ(Schistocerca gregaria)に継続的に給餌した。各処理のケージの1つをLifeCam HD-3000カメラで常時モニターし、ActiveWebCamプログラムで記録した。3日間の給餌の間のバッタの運動量の測定から、dsRNA-clock発現藻を給餌したバッタの総運動量は、WT藻を給餌したバッタと比較よりも約1.7~2.5倍少ないことが明らかになった(図8;A~C)。dsRNA-clockを発現する藻を経口給餌することにより、バッタの活性がWT藻を給餌した対照バッタよりも低下した。
レッドクロウ(Cherax quadricarinatus)個体(30±10g)をケージに入れ各処理に供した。レッドクロウにdsRNA-GFP発現藻またはWT藻を含有する飼料ペレットを28日間、毎日給餌した(7.5g藻/g個体)。第0日および第28日、給餌から72時間後に各個体から血リンパ100μlを採取した。dsRNA-GFP発現藻では、6つの独立した反復を用い、WT藻では、3つの独立した反復を用いた。採取した血リンパをRNA調製に供し、試料をRNAドットブロットハイブリダイゼーションにより解析した。ドットブロットハイブリダイゼーション法を製造業者の指示に従って実施した(Roche Applied Science社、カタログ番号11 603 558 001、カタログ番号11 585 762 001、カタログ番号11 093 274 910、カタログ番号11 685 627 001)。製造業者の指示に従って、直線化した鋳型DNAからジゴキシゲニン-11-dUTPで標識したdsRNA-GFPの一本鎖RNAプローブを合成した(Roche Applied Science社、カタログ番号11175025910)。
サバクトビバッタ(Schistocerca gregaria)の3齢幼虫を温度勾配30~37℃、14時間/10時間の明/暗照明条件に制御された45×45×50cmのチャンバに100個体の密度で入れた。各処理には、それぞれ100個体のバッタからなる4つの独立した反復を用いた。鉢で生育させたコムギ新芽に、clkに標的化されたdsRNA(dsRNA-clock)またはGFPに標的化されたdsRNA(dsGFP)を発現するトランスジェニック藻の粉末を、いずれの葉も藻粉末(上記のようにゼラチンと混合した藻粉末)で均一に覆われるまで塗布した。次いで、藻を塗布したコムギ新芽をバッタに4週間給餌し、その間、処理が継続的に実施されるように、藻を塗布した新しいコムギ新芽に毎日与えた。次いで各処理について、個体数をカウントし、雌に対する雄の比を算出した。dsRNA-clock発現藻を塗布した葉を給餌したバッタの比は、dsGFP発現藻を塗布した葉を給餌したバッタよりも有意に(p値=0.01)小さい値であった(48%の減少、図9)。以上の結果から、サバクトビバッタ(Schistocerca gregaria)のclock遺伝子に標的化されたdsRNAを発現する藻の経口送達によりバッタ集団内の性分化が変化したことがわかる。これまでに、概日時計と性的発育および性行動との間に何らかの関係があることが示唆されている。例えば、takeout遺伝子は強力な概日調節遺伝子であることが明らかにされている。Dauwalderらは、ショウジョウバエのtakeout遺伝子ファミリーが性特異的機能を有する複数の因子をコードすることを示唆しており、同遺伝子ファミリーが生物体の性別、栄養状態および概日周期に関する情報の統合に何らかの役割を果たして成体雄の行動に影響を及ぼすと提唱している(Dauwalderら,2002.Genes & Development 16:2879-2892)。clock遺伝子に標的化されたdsRNAを発現する藻で覆った葉をサバクトビバッタ(S.gregaria)に給餌したときに観察された雌の数の増加から、dsRNAによるclock遺伝子のダウンレギュレーションが示唆される。
GEA Westfalia Separator(モデルSSD18-06-007、回転数9790rpm)またはAlfa Laval SeparatorモデルIFB 303X-73(7500rpm)を用いて藻の液体培養物を回収した。藻ペーストを1cm×14cm×14cmのトレーまたは2cm×25cm×52cmのプレートに置き、-80℃で24時間保管した。翌日、VirTis凍結乾燥機(商品番号270389)を用いて-50℃、2×10-1Torr(約27Pa)でトレーを24~48時間、凍結乾燥させた。藻粉末を生存能アッセイに用いた。
サケジラミ(Lepeophtheirus salmonis)COPB2遺伝子のセンスフラグメント(配列番号35)およびアンチセンスフラグメント(配列番号36)をBiomatik USA社にて合成する。BamHI、SacIIによってセンスフラグメントをクローン化し、EcoRI、KpnIによってアンチセンスフラグメントをクローン化する。COPB2のフラグメントをpPhaTカセットに含まれるイントロンにセンス/アンチセンスの方向でクローン化する。
平均体重80グラムのタイセイヨウサケ(Salmo salar)を0.75m3のプラスチック水槽に入れ、温度10℃の海水(塩分濃度34.5ppt)で維持する。魚に1日2回、15%のCOPB2 dsRNA-発現藻の粉末(魚の飼料の総重量に対するw/w)で均一に覆った市販の魚の飼料またはGFP dsRNAを発現する藻の粉末(15%(w/w))で均一に覆った市販の魚の飼料を魚の体重の1%給餌する。各処理には、25個体の魚からなる3つの独立した反復を用いる。
Claims (7)
- 少なくとも1つの異種dsRNA分子を含む非増殖性トランスジェニック フェオダクチラム・トリコルヌタム(Phaeodactylum tricornutum)微細藻類であって、
前記dsRNA分子が、異種生物体内に存在する内在性ポリヌクレオチドに標的化されており、
前記トランスジェニック フェオダクチラム・トリコルヌタム微細藻類が前記異種生物体によって摂取されると、前記dsRNA分子が、前記異種生物体内に存在する前記内在性ポリヌクレオチドの発現をサイレンシングし、
前記非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類が、乾燥形態であり、
乾燥形態である前記トランスジェニック フェオダクチラム・トリコルヌタム微細藻類を含む調製物が、前記調製物の総質量のうちの15%以下の水分を含み、
前記異種生物体が、直翅目、鱗翅目および鞘翅目からなる群より選択される目に属する、植物に対する有害生物である、
非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類。 - 前記植物に対する有害生物が、サバクトビバッタ(Schistocerca gregaria)およびトノサマバッタ(Locusta migratoria)からなる群より選択されるバッタである、請求項1に記載の非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類。
- 前記植物に対する有害生物がサバクトビバッタ(Schistocerca gregaria)であり、前記dsRNA分子が、配列番号1に記載される核酸配列を含むclk遺伝子に標的化されている、請求項2に記載の非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類。
- 前記dsRNA分子が、配列番号2に記載される核酸配列またはそのフラグメントを含むセンス鎖と、前記配列番号2またはそのフラグメントに相補的なアンチセンス鎖と、を含む、請求項3に記載の非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類。
- 前記dsRNA分子が、配列番号2に記載される核酸配列を含むセンス鎖と、配列番号6に記載される核酸配列を含むリンカー配列と、配列番号3に記載される核酸配列を含むアンチセンス鎖と、を含む、請求項4に記載の非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類。
- 請求項1~5のいずれか1項に記載の非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類と、可食性の希釈剤、補形剤または担体からなる群より選択される物質と、を含む、可食性組成物あるいは植物、動物またはその一部に適用される製剤。
- 生物体内でのポリヌクレオチドの発現を阻害するための方法であって、可食性の請求項1~5のいずれか1項に記載の非増殖性トランスジェニック フェオダクチラム・トリコルヌタム微細藻類を前記生物体に与えることを含む、方法。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007530414A (ja) | 2003-07-02 | 2007-11-01 | エムユーエスシー ファウンデイション フォー リサーチ デべロップメント | 甲殻類動物、及び他の無脊椎動物における、dsRNAで誘導された特異的、及び非特異的免疫、及びそこで使用する生物送達媒体 |
WO2012054919A2 (en) | 2010-10-22 | 2012-04-26 | Donald Danforth Plant Science Center | Control of pathogens and parasites |
WO2012023960A3 (en) | 2010-08-17 | 2014-03-27 | Transalgae (Israel) Ltd | Transgenically mitigating the establishment and spread of transgenic algae in natural ecosystems |
US20140271559A1 (en) | 2013-03-15 | 2014-09-18 | Monsanto Technology Llc | Compositions and methods for the production and delivery of double stranded rna |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6027900A (en) | 1996-04-12 | 2000-02-22 | Carnegie Institution Of Washington | Methods and tools for transformation of eukaryotic algae |
WO2001098335A2 (en) | 2000-06-20 | 2001-12-27 | Phycotransgenics, Llc | Transgenic algae for delivering antigens to an animal |
CN1860220A (zh) * | 2003-07-02 | 2006-11-08 | 南卡罗来纳医科大学研究发展基金会 | dsRNA 诱导的在甲壳动物和其它无脊椎动物中的特异性和非特异性免疫及其中所用的生物送递载体 |
CN1946733A (zh) * | 2004-02-06 | 2007-04-11 | 高级生物营养公司 | 以rna介导的干扰来控制陆生和水产养殖动物疾病 |
GB0608838D0 (en) | 2006-05-04 | 2006-06-14 | Novartis Ag | Organic compounds |
WO2008027235A1 (en) | 2006-08-25 | 2008-03-06 | University Of New Mexico | Methods and compositions for control of disease in aquaculture |
EP2094086B1 (en) | 2006-11-08 | 2013-08-28 | Veritas Bio, LLC | In vivo delivery of double stranded rna to a target cell |
US20110081706A1 (en) | 2009-10-02 | 2011-04-07 | TransAlgae Ltd | Method and system for efficient harvesting of microalgae and cyanobacteria |
TWI407964B (zh) | 2011-05-10 | 2013-09-11 | Univ Fu Jen Catholic | 干擾性核糖核酸用於治療或減緩疼痛之用途 |
US10017747B2 (en) * | 2011-09-21 | 2018-07-10 | University Of Florida Research Foundation, Inc. | Citrus tristeza virus based vectors for foreign gene/s expression |
CN102550864B (zh) * | 2012-01-04 | 2013-11-06 | 河南科技大学 | 一种抗禽流感的功能性饲料添加剂的制备方法 |
CN104754954B (zh) * | 2012-08-23 | 2018-08-28 | 特郎萨格以色列有限公司 | 转基因微藻和其用于口服递送蛋白质的用途 |
AR095275A1 (es) * | 2013-03-13 | 2015-09-30 | E I Dupont De Nemours & Company | Composiciones y métodos para el control insecticida de chinches |
AU2016291813B2 (en) * | 2015-07-14 | 2022-09-01 | Transalgae Israel Ltd. | Transgenic microalgae and use thereof as a feed for delivery of interfering RNA molecules |
-
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007530414A (ja) | 2003-07-02 | 2007-11-01 | エムユーエスシー ファウンデイション フォー リサーチ デべロップメント | 甲殻類動物、及び他の無脊椎動物における、dsRNAで誘導された特異的、及び非特異的免疫、及びそこで使用する生物送達媒体 |
WO2012023960A3 (en) | 2010-08-17 | 2014-03-27 | Transalgae (Israel) Ltd | Transgenically mitigating the establishment and spread of transgenic algae in natural ecosystems |
WO2012054919A2 (en) | 2010-10-22 | 2012-04-26 | Donald Danforth Plant Science Center | Control of pathogens and parasites |
US20140271559A1 (en) | 2013-03-15 | 2014-09-18 | Monsanto Technology Llc | Compositions and methods for the production and delivery of double stranded rna |
Non-Patent Citations (6)
Title |
---|
British Journal of Nutrition,2014年,vol.112,p.1055-1064 |
Insect Biochemistry and Molecular Biology,2011年,vol.41,p.313-321 |
Insect Molecular Biology,2012年,vol.21(3),p.369-381 |
Journal of Antivirals & Antiretrovirals,2011年,S9,p.1-12 |
Malaria World Journal,2013年,vol.4(6),p.1-7 |
水産増殖,2013年,vol.61(4),p.389-394 |
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