Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Children's Hospital of Eastern Ontario (CHEO) Research Institute

University of Ottawa

&#x82E5;&#x8981;&#x5C55;&#x5F00;&#x6211;&#x4EEC;&#x7684;&#x5B89;&#x5168;&#x548C;&#x70C8;&#x6027;&#x6EB6;&#x7624;&#x75C5;&#x6BD2;&#x7684;&#x5F53;&#x524D;&#x6570;&#x7EC4;&#xFF0C;&#x6211;&#x4EEC;&#x7504;&#x522B;&#x5404;&#x79CD;&#x5404;&#x6837;&#x7684;&#x91CE;&#x751F;&#x578B; (WT) rhabdoviruses &#x5BF9;&#x80BF;&#x7624;&#x7EC6;&#x80DE;&#x7CFB;&#x7684;&#x9762;&#x677F;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x5C4F;&#x5E55;&#x786E;&#x5B9A;&#x75C5;&#x6BD2;&#x7684;&#x6570;&#x91CF;&#x6709;&#x4E0D;&#x540C;&#x7A0B;&#x5EA6;&#x7684;&#x6740;&#x4F24;&#x6D3B;&#x6027;&#x3002;&#x822A;&#x6D4B;&#x75C5;&#x6BD2;&#x662F;&#x6700;&#x6709;&#x529B;&#x7684;&#x8FD9;&#x4E9B;&#x83CC;&#x682A;&#x3002;&#x6211;&#x4EEC;&#x6784;&#x5EFA;&#x4E86;&#x4E00;&#x4E2A;&#x91CD;&#x7EC4;&#x7CFB;&#x7EDF;&#x822A;&#x6D4B;&#x75C5;&#x6BD2;&#x5E73;&#x53F0;&#xFF0C;&#x7B56;&#x5212;&#x4E00;&#x7CFB;&#x5217;&#x7684;&#x8870;&#x51CF;&#x7A81;&#x53D8;&#xFF0C;&#x6269;&#x5927;&#x5176;&#x6CBB;&#x7597;&#x6307;&#x6570;&#x548C;&#x6D4B;&#x8BD5;&#x5176;&#x6548;&#x4EF7;&#x4F53;&#x5916;&#x548C;&#x4F53;&#x5185;&#x3002;&#x5305;&#x542B; G &#x86CB;&#x767D; (Q242R) &#x548C; M &#x86CB;&#x767D; (L123W) &#x53CC;&#x7A81;&#x53D8;&#x4F53; (MG1) &#x5E94;&#x53D8;&#x7A81;&#x53D8;&#x51CF;&#x6BD2;&#x822A;&#x6D4B;&#x75C5;&#x6BD2;&#x5728;&#x6B63;&#x5E38;&#x4E8C;&#x500D;&#x4F53;&#x7EC6;&#x80DE;&#x884C;&#x4E2D;&#xFF0C;&#x4F46;&#x4F3C;&#x4E4E;&#x662F;&#x5728;&#x80BF;&#x7624;&#x7EC6;&#x80DE;&#x4E2D;&#x7684; hypervirulent&#x3002;&#x8FD9;&#x79CD;&#x9009;&#x62E9;&#x6027;&#x7684;&#x8870;&#x51CF;&#x4ECB;&#x5BFC;&#x5E72;&#x6270;&#x7D20; &#xFF08;&#x5E72;&#x6270;&#x7D20;&#xFF09; &#x901A;&#x8FC7;-&#x4ECE;&#x5C5E;&#x548C;&#x72EC;&#x7ACB;&#x7684;&#x673A;&#x5236;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x822A;&#x6D4B; MG1 &#x5E94;&#x53D8;&#x4E86; 100-fold &#x66F4;&#x5927;&#x6700;&#x5927;&#x5BB9;&#x8BB8;&#x5242;&#x91CF; &#xFF08;MTD&#xFF09; &#x6BD4; WT &#x822A;&#x6D4B;&#x4F53;&#x5185;&#xFF0C;&#x5E76;&#x5E26;&#x6765;&#x4E86;&#x6301;&#x4E45;&#x7684;&#x6CBB;&#x6108;&#x65F6;&#x5177;&#x6709;&#x7CFB;&#x7EDF;&#x91CD;&#x8981;&#x6027;&#x7684;&#x7BA1;&#x7406;&#x81EA;&#x4F53;&#x548C;&#x5F02;&#x79CD;&#x79FB;&#x690D;&#x6A21;&#x578B;&#x3002;&#x603B;&#x4E4B;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x5177;&#x6709;&#x72EC;&#x4E00;&#x65E0;&#x4E8C;&#x7684;&#x80BF;&#x7624;&#x9009;&#x62E9;&#x6027;&#x8870;&#x51CF;&#x7A81;&#x53D8;&#x70C8;&#x6027;&#x7684;&#x65B0;&#x6EB6;&#x7624;&#x75C5;&#x6BD2;&#x5E73;&#x53F0;&#x3002;

&#x4F5C;&#x4E3A;&#x6EB6;&#x7624;&#x75C5;&#x6BD2;&#x8F6C;&#x57FA;&#x56E0;&#x822A;&#x6D4B;&#x75C5;&#x6BD2;&#x9274;&#x5B9A;&#x3002;

To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations.

Identification of genetically modified Maraba virus as an oncolytic rhabdovirus.

Afin d&#x26;#39;&#xE9;largir notre &#xE9;ventail actuel de la s&#xE9;curit&#xE9; et puissant virus oncolytiques, nous avons projet&#xE9; une vari&#xE9;t&#xE9; de rhabdovirus de type sauvage (WT) contre un panel de lign&#xE9;es cellulaires tumorales. Notre &#xE9;cran identifi&#xE9; un certain nombre de virus avec des degr&#xE9;s de tuer l&#x26;#39;activit&#xE9;. Maraba virus &#xE9;tait le plus puissant de ces souches. Nous avons construit un syst&#xE8;me de recombinaison du virus pour la plate-forme de Maraba, a con&#xE7;u une s&#xE9;rie de mutations d&#x26;#39;att&#xE9;nuation d&#x26;#39;&#xE9;largir son index th&#xE9;rapeutique, et test&#xE9; leur efficacit&#xE9; in vitro et in vivo. Un double mutant (MG1) souche contenant &#xE0; la fois la prot&#xE9;ine G (Q242R) et la prot&#xE9;ine M (L123W) mutations att&#xE9;nu&#xE9; Maraba virus dans des lign&#xE9;es cellulaires normales diplo&#xEF;des, mais qui semblait &#xEA;tre hypervirulente dans les cellules canc&#xE9;reuses. Cette att&#xE9;nuation s&#xE9;lective a &#xE9;t&#xE9; v&#xE9;hicul&#xE9;e par l&#x26;#39;interf&#xE9;ron (IFN)-d&#xE9;pendante et des m&#xE9;canismes ind&#xE9;pendants. Enfin, la souche Maraba MG1 eu une 100 fois plus la dose maximale tol&#xE9;rable (DMT) de WT Maraba in vivo et a abouti dans les cures durables lorsque administr&#xE9; par voie syst&#xE9;mique dans les mod&#xE8;les syng&#xE9;niques et x&#xE9;nogreffe. En r&#xE9;sum&#xE9;, nous pr&#xE9;sentons une nouvelle plate-forme puissante rhabdovirus oncolytique avec uniques antitumorales s&#xE9;lectives mutations att&#xE9;nuantes.

Identification des aliments g&#xE9;n&#xE9;tiquement modifi&#xE9;s Maraba virus comme un rhabdovirus oncolytique

Um unsere aktuelle Array von sicheren und starken Onkolytische Viren zu erweitern, sehen wir eine Vielzahl von Wildtyp (WT) Rhabdoviruses gegen ein Gremium von Tumor-Zelllinien. Unser Bildschirm ermittelt eine Reihe von Viren mit unterschiedlichem T&ouml;tung Aktivit&auml;t. Maraba-Virus war der m&auml;chtigste dieser St&auml;mme. Wir ein rekombinantes System f&uuml;r die Maraba-Virus-Plattform, entwickelt eine Reihe von Mutationen um seinen therapeutischen Index erweitern abgeschw&auml;cht und ihre Wirksamkeit in vitro und in vivo getestet. Eine doppelte Mutant (MG1) Belastung mit G-Protein (Q242R) und M-Protein (L123W) Mutationen Maraba-Virus in normalen diploiden Zelllinien abgeschw&auml;cht, doch Hypervirulent in Krebszellen zu sein schien. Diese selektive D&auml;mpfung wurde vermittelt durch Interferon (IFN)-abh&auml;ngige und -unabh&auml;ngige Mechanismen. Schlie&szlig;lich hatte die Maraba MG1-Belastung eine verhundertfacht gr&ouml;&szlig;ere maximale tolerierbare Dosis (MTD) als WT Maraba in vivo und f&uuml;hrte dauerhaft Kuren wenn systemisch syngenen verabreicht und Xenograft-Modelle. Zusammenfassend l&auml;sst sich sagen berichten wir eine starke neue Onkolytische Rhabdovirus Plattform mit einzigartigen Tumor-selektive schalld&auml;mpfend Mutationen.

Nachweis von gentechnisch ver&auml;nderten Maraba-Virus als ein Rhabdovirus Onkolytische.

Per espandere la nostra matrice corrente dei virus oncolytic potente e sicuro, abbiamo proiettato una variet&agrave; di rhabdoviruses (WT) selvaggio-tipo contro un pannello di linee di cellule tumorali. Il nostro schermo identificato un certo numero di virus con diversi gradi di attivit&agrave; di uccisione. Maraba virus &egrave; stato il pi&ugrave; potente di questi ceppi. Abbiamo costruito un sistema per la piattaforma di Maraba virus ricombinante, progettato una serie di attenuanti mutazioni per espandere il suo indice terapeutico e testato la loro potenza in vitro e in vivo. Un ceppo mutante doppia (MG1) che contiene sia proteine G (Q242R) e la proteina M (L123W) mutazioni attenuato Maraba virus nella cellula diploide normale, eppure sembravano essere supervirulenti nelle cellule tumorali. Questa attenuazione selettiva &egrave; stato mediato attraverso l&#39;interferone (IFN)-dipendenti - indipendenti e meccanismi. Infine, il ceppo di Maraba MG1 aveva una 100 volte maggiore massima tollerabile dose (MTD) di Maraba WT in vivo e portato in cure durevoli quando somministrato sistematicamente in Carlo e xenoinnesto modelli. In sintesi, riportiamo una potente nuova piattaforma rhabdovirus oncolytic con uniche tumore-selettive mutazioni attenuante.

Identificazione del virus geneticamente modificato di Maraba come un rhabdovirus oncolytic.

&#x5B89;&#x5168;&#x304B;&#x3064;&#x5F37;&#x529B;&#x306A;&#x816B;&#x760D;&#x6EB6;&#x89E3;&#x6027;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x6211;&#x3005;&#x306E;&#x73FE;&#x5728;&#x306E;&#x30A2;&#x30EC;&#x30A4;&#x3092;&#x62E1;&#x5F35;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x816B;&#x760D;&#x7D30;&#x80DE;&#x682A;&#x306E;&#x30D1;&#x30CD;&#x30EB;&#x306B;&#x5BFE;&#x3057;&#x3066;&#x3001;&#x91CE;&#x751F;&#x578B;&#xFF08;WT&#xFF09;&#x30E9;&#x30D6;&#x30C9;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x69D8;&#x3005;&#x306A;&#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x3057;&#x305F;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306E;&#x753B;&#x9762;&#x306B;&#x306F;&#x3001;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D3;&#x30C6;&#x30A3;&#x3092;&#x6BBA;&#x3059;&#x306E;&#x5EA6;&#x5408;&#x3044;&#x3092;&#x5909;&#x5316;&#x3055;&#x305B;&#x305F;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x6570;&#x3092;&#x540C;&#x5B9A;&#x3057;&#x305F;&#x3002; Maraba&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x83CC;&#x682A;&#x306E;&#x4E2D;&#x3067;&#x6700;&#x3082;&#x5F37;&#x529B;&#x3067;&#x3042;&#x3063;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;Maraba&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30D7;&#x30E9;&#x30C3;&#x30C8;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x306E;&#x305F;&#x3081;&#x306E;&#x7D44;&#x63DB;&#x3048;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x69CB;&#x7BC9;&#x3057;&#x3001;&#x305D;&#x306E;&#x6CBB;&#x7642;&#x6307;&#x6570;&#x3092;&#x62E1;&#x5927;&#x3057;&#x3066;&#x5F31;&#x6BD2;&#x5316;&#x7A81;&#x7136;&#x5909;&#x7570;&#x306E;&#x30B7;&#x30EA;&#x30FC;&#x30BA;&#x3092;&#x8A2D;&#x8A08;&#x3057;&#x3001;in vitro&#x304A;&#x3088;&#x3073;in vivo&#x3067;&#x305D;&#x306E;&#x52B9;&#x529B;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x901A;&#x5E38;&#x306E;&#x4E8C;&#x500D;&#x4F53;&#x7D30;&#x80DE;&#x682A;&#x306B;&#x304A;&#x3051;&#x308B;G&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#xFF08;Q242R&#xFF09;&#x304A;&#x3088;&#x3073;M&#x86CB;&#x767D;&#x8CEA;&#xFF08;L123W&#xFF09;&#x5909;&#x7570;&#x5F31;&#x6BD2;Maraba&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x4E21;&#x65B9;&#x3092;&#x542B;&#x3080;&#x4E8C;&#x91CD;&#x5909;&#x7570;&#x682A;&#xFF08;MG1&#xFF09;&#x682A;&#x306F;&#x3001;&#x307E;&#x3060;&#x764C;&#x7D30;&#x80DE;&#x3067;hypervirulent&#x3088;&#x3046;&#x306B;&#x898B;&#x3048;&#x305F;&#x3002;&#x3053;&#x306E;&#x9078;&#x629E;&#x7684;&#x306A;&#x6E1B;&#x8870;&#x306F;&#x3001;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30ED;&#x30F3;&#xFF08;IFN&#xFF09;&#x4F9D;&#x5B58;&#x6027;&#x304A;&#x3088;&#x3073;&#x975E;&#x4F9D;&#x5B58;&#x6027;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x5A92;&#x4ECB;&#x3055;&#x308C;&#x305F;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;Maraba MG1&#x682A;&#x306F;in vivo&#x3067;&#x306E;WT Maraba&#x3088;&#x308A;100&#x500D;&#x4EE5;&#x4E0A;&#x306E;&#x6700;&#x5927;&#x8A31;&#x5BB9;&#x7528;&#x91CF;&#xFF08;MTD&#xFF09;&#x3092;&#x6301;&#x3063;&#x3066;&#x3001;&#x5168;&#x8EAB;&#x540C;&#x7CFB;&#x3068;&#x7570;&#x7A2E;&#x79FB;&#x690D;&#x30E2;&#x30C7;&#x30EB;&#x306B;&#x304A;&#x3044;&#x3066;&#x3001;&#x6295;&#x4E0E;&#x3055;&#x308C;&#x305F;&#x5834;&#x5408;&#x306B;&#x8010;&#x4E45;&#x6027;&#x306E;&#x3042;&#x308B;&#x6CBB;&#x7642;&#x6CD5;&#x3092;&#x3082;&#x305F;&#x3089;&#x3057;&#x305F;&#x3002;&#x8981;&#x7D04;&#x3059;&#x308B;&#x3068;&#x3001;&#x6211;&#x3005;&#x306F;&#x30E6;&#x30CB;&#x30FC;&#x30AF;&#x306A;&#x816B;&#x760D;&#x9078;&#x629E;&#x7684;&#x306A;&#x5F31;&#x6BD2;&#x5316;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3067;&#x5F37;&#x529B;&#x306A;&#x65B0;&#x3057;&#x3044;&#x816B;&#x760D;&#x6EB6;&#x89E3;&#x30E9;&#x30D6;&#x30D7;&#x30E9;&#x30C3;&#x30C8;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x3092;&#x5831;&#x544A;&#x3059;&#x308B;&#x3002;

&#x816B;&#x760D;&#x6EB6;&#x89E3;&#x6027;&#x30E9;&#x30D6;&#x3068;&#x3057;&#x3066;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x7D44;&#x63DB;&#x3048;Maraba&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x540C;&#x5B9A;

&#xC548;&#xC804; &#xD558; &#xACE0; &#xAC15;&#xB825;&#xD55C; oncolytic &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xC6B0;&#xB9AC;&#xC758; &#xD604;&#xC7AC; &#xBC30;&#xC5F4; &#xD655;&#xC7A5; &#xD558;&#xB824;&#xBA74; &#xC6B0;&#xB9AC;&#xB294; &#xB2E4;&#xC591; &#xD55C; &#xC885;&#xC591; &#xC138;&#xD3EC; &#xB77C;&#xC778;&#xC758; &#xD328;&#xB110;&#xC5D0; &#xB300; &#xD55C; &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; (WT) rhabdoviruses &#xC0C1;&#xC601;. &#xC6B0;&#xB9AC;&#xC758; &#xD654;&#xBA74; &#xC0B4;&#xC778; &#xD65C;&#xB3D9;&#xC758; &#xB2E4;&#xC591; &#xD55C; &#xD559;&#xC704;&#xC640; &#xB2E4;&#xC591; &#xD55C; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xB97C; &#xC2DD;&#xBCC4;&#xD569;&#xB2C8;&#xB2E4;. &#xB9C8; &#xB77C;&#xBC14; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xB294; &#xC774;&#xB7EC;&#xD55C; &#xAE34;&#xC7A5;&#xC758; &#xAC00;&#xC7A5; &#xAC15;&#xB825;&#xD55C;. &#xC6B0;&#xB9AC; &#xB9C8; &#xB77C;&#xBC14; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xD50C;&#xB7AB;&#xD3FC;&#xC744; &#xC704;&#xD55C; &#xC7AC;&#xC870;&#xD569; &#xD615; &#xC2DC;&#xC2A4;&#xD15C; &#xAD6C;&#xCD95; &#xACF5;&#xAC04;&#xC740; &#xCE58;&#xB8CC; &#xC778;&#xB371;&#xC2A4;&#xB85C; &#xD655;&#xC7A5; &#xD558;&#xB294; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC758; &#xC2DC;&#xB9AC;&#xC988;&#xB97C; &#xC124;&#xACC4; &#xD558; &#xACE0; &#xC0DD;&#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xADF8;&#xB9AC;&#xACE0; vivo&#xC5D0;&#xC11C; &#xADF8;&#xB4E4;&#xC758; &#xD6A8;&#xB2A5;&#xC744; &#xD14C;&#xC2A4;&#xD2B8;. G &#xB2E8;&#xBC31;&#xC9C8; (Q242R)&#xC640; M &#xB2E8;&#xBC31;&#xC9C8; (L123W)&#xC774; &#xD3EC;&#xD568; &#xB41C; &#xC774;&#xC911; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; (MG1) &#xC2A4;&#xD2B8;&#xB808;&#xC778; &#xBCC0;&#xC774; &#xC815;&#xC0C1; diploid &#xC138;&#xD3EC; &#xB77C;&#xC778;&#xC5D0; &#xB9C8; &#xB77C;&#xBC14; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xB97C; &#xAC10;&#xC1E0; &#xC544;&#xC9C1; &#xC554; &#xC138;&#xD3EC;&#xC5D0; hypervirulent &#xB420; &#xAC83;&#xC73C;&#xB85C; &#xBCF4;&#xC778;&#xB2E4;. &#xC774; &#xC120;&#xD0DD;&#xC801; &#xAC10;&#xC1C4;&#xB294; &#xC778;&#xD130;&#xD398;&#xB860; (IFN)&#xC744; &#xD1B5;&#xD574; &#xC911;&#xC7AC;-&#xC885;&#xC18D; &#xBC0F;-&#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xB9C8; &#xB77C;&#xBC14; MG1 &#xAE34;&#xC7A5; &#xD588;&#xB2E4; 100 &#xD070; &#xCD5C;&#xB300; &#xCF8C;&#xD65C; &#xBCF5;&#xC6A9;&#xB7C9; (MTD) WT &#xB9C8; &#xB77C;&#xBC14; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xBC0F; syngeneic&#xC5D0;&#xC11C; &#xCCB4;&#xACC4;&#xC801;&#xC73C;&#xB85C; &#xAD00;&#xB9AC; &#xD558;&#xB294; &#xACBD;&#xC6B0; &#xC9C0;&#xC18D;&#xC801;&#xC778; &#xCE58;&#xB8CC; &#xACB0;&#xACFC; &#xBCF4;&#xB2E4; &#xB1CC;&#xC885;&#xC591; &#xBAA8;&#xB378;. &#xC694;&#xC57D; &#xD558;&#xC790;&#xBA74;, &#xC6B0;&#xB9AC;&#xB294; &#xB3C5;&#xD2B9;&#xD55C; &#xC885;&#xC591; &#xC120;&#xD0DD;&#xC801; &#xC57D;&#xD558;&#xAC8C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC640; &#xAC15;&#xB825;&#xD55C; &#xC0C8;&#xB85C;&#xC6B4; oncolytic rhabdovirus &#xD50C;&#xB7AB;&#xD3FC;&#xC744; &#xBCF4;&#xACE0;&#xD569;&#xB2C8;&#xB2E4;.

Oncolytic rhabdovirus&#xB85C; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615;&#xB41C; &#xB9C8; &#xB77C;&#xBC14; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xC2DD;&#xBCC4;.

Para ampliar nuestra gama actual de virus oncol&#xED;ticos seguros y potentes, que exhibi&#xF3; una variedad de rabdovirus de tipo salvaje (WT) frente a un panel de l&#xED;neas celulares tumorales. Nuestra pantalla identificado una serie de virus con diferentes grados de matar a la actividad. Maraba virus era el m&#xE1;s potente de estas cepas. Se construy&#xF3; un sistema recombinante para el virus de la plataforma Maraba, dise&#xF1;ado una serie de mutaciones atenuantes para expandir su &#xED;ndice terap&#xE9;utico, y probado su potencia in vitro e in vivo. Un doble mutante (MG1) cepa que contiene tanto la prote&#xED;na G (Q242R) y prote&#xED;na M (L123W) mutaciones virus atenuado Maraba en l&#xED;neas de c&#xE9;lulas diploides normales, sin embargo, que parec&#xED;a ser hipervirulenta en las c&#xE9;lulas cancerosas. Esta atenuaci&#xF3;n selectiva se vio mediada por interfer&#xF3;n (IFN)-dependiente y de mecanismos independientes. Finalmente, la cepa Maraba MG1 ten&#xED;a un 100-veces mayor dosis m&#xE1;xima tolerada (MTD) que WT Maraba in vivo y result&#xF3; en curaciones durables cuando la administraci&#xF3;n sist&#xE9;mica en los modelos sing&#xE9;nicos y xenoinjerto. En resumen, presentamos una potente nueva plataforma rabdovirus oncol&#xED;tico con el &#xFA;nico tumor selectivos mutaciones atenuantes.

La identificaci&#xF3;n de virus gen&#xE9;ticamente modificados Maraba Como Rhabdovirus oncol&#xED;tica

This study characterizes the ability of novel oncolytic rhabdoviruses (Maraba MG1) to boost natural killer (NK) cell activity. Our results demonstrate that MG1 activates NK cells via direct infection and maturation of conventional dendritic cells. Using NK depletion and conventional dendritic cells ablation studies in vivo, we established that both are required for MG1 efficacy. We further explored the efficacy of attenuated MG1 (nonreplicating MG1-UV(2min) and single-cycle replicating MG1-Gless) and demonstrated that these viruses activate conventional dendritic cells, although to a lesser extent than live MG1. This translates to equivalent abilities to remove tumor metastases only at the highest viral doses of attenuated MG1. In tandem, we characterized the antitumor ability of NK cells following preoperative administration of live and attenuated MG1. Our results demonstrates that a similar level of NK activation and reduction in postoperative tumor metastases was achieved with equivalent high viral doses concluding that viral replication is important, but not necessary for NK activation. Biochemical characterization of a panel of UV-inactivated MG1 (2-120 minutes) revealed that intact viral particle and target cell recognition are essential for NK cell-mediated antitumor responses. These findings provide mechanistic insight and preclinical rationale for safe perioperative virotherapy to effectively reduce metastatic disease following cancer surgery.

Maraba MG1 virus enhances natural killer cell function via conventional dendritic cells to reduce postoperative metastatic disease.

Recent clinical data have emphatically shown the capacity of our immune systems to eradicate even advanced cancers. Although oncolytic viruses (OVs) were originally designed to function as tumour-lysing therapeutics, they have now been clinically shown to initiate systemic antitumour immune responses. Cell signalling pathways that are activated and promote the growth of tumour cells also favour the growth and replication of viruses within the cancer. The ability to engineer OVs that express immune-stimulating 'cargo', the induction of immunogenic tumour cell death by OVs and the selective targeting of OVs to tumour beds suggests that they are the ideal reagents to enhance antitumour immune responses. Coupling of OV therapy with tumour antigen vaccination, immune checkpoint inhibitors and adoptive cell therapy seems to be ready to converge towards a new generation of multimodal therapeutics to improve outcomes for cancer patients.

Going viral with cancer immunotherapy.

Large-scale assays, such as microarrays, next-generation sequencing and various "omics" technologies, have explored multiple aspects of the immune response following virus infection, often from a public health perspective. Yet a lack of similar data exists for monitoring immune engagement during oncolytic virus immunotherapy (OVIT) in the cancer setting. Tracking immune signatures at the tumour site can create a snapshot or longitudinally analyse immune cell activation, infiltration and functionality within global populations or individual cells. Mapping immune changes over the course of oncolytic biotherapy-from initial infection to tumour stabilisation/regression through to long-term cure or escape/relapse-has the potential to generate important therapeutic insights around virus-host interactions. Further, correlating such immune signatures with specific tumour outcomes has significant value for guiding the development of novel oncolytic virus immunotherapy strategies. Here, we provide insights for OVIT from large-scale analyses of immune populations in the infection, vaccination and immunotherapy setting. We analyse several approaches to manipulating immune engagement during OVIT. We further explore immunocentric changes in the tumour tissue following immunotherapy, and compile several immune signatures of therapeutic success. Ultimately, we highlight clinically relevant large-scale approaches with the potential to strengthen future oncolytic strategies to optimally engage the immune system.

Big Data Offers Novel Insights for Oncolytic Virus Immunotherapy.

High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms - including those based on herpes simplex virus, reovirus, and vaccinia virus - have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research.

High-throughput screening to enhance oncolytic virus immunotherapy.

Rhabdomyosarcoma (RMS), a neoplasm characterized by undifferentiated myoblasts, is the most common soft tissue tumour in children. Therapeutic resistance is common in RMS and is often caused by acquired defects in the cellular apoptotic program. Smac mimetic compounds (SMCs) are a novel class of inhibitor of apoptosis (IAP) antagonists that are currently under clinical development as cancer therapeutics. We previously reported that cIAP1 is overexpressed in human primary RMS tumours and in patient-derived RMS cell lines where it drives resistance to apoptosis. In this study, we investigated whether inflammatory cytokine production triggered by activators of innate immunity synergizes with LCL161 to induce bystander killing of RMS cells in vitro and in vivo. Indeed, we show that innate immune stimuli (oncolytic virus (VSVΔ51-GFP), interferon γ (IFNγ), and tumour necrosis factor-like weak inducer of apoptosis (TWEAK)) combine with SMCs in vitro to reduce cell viability in the Kym-1 RMS cancer cell line. Other human RMS cell lines (RH36, RH41, RD, RH18, RH28, and RH30) and the murine RMS cell line 76-9 are resistant to treatment with LCL161 alone or in combination with immune stimulants in in vitro cell viability assays. In contrast, we report that the combination of LCL161 and VSVΔ51-GFP reduces tumour volume and prolongs survival in a 76-9 syngeneic murine model. Our results support further exploration of the combined use of IAP antagonists and innate immune stimuli as a therapeutic approach for RMS cancers.

Oncolytic virus synergizes with Smac mimetic compounds to induce rhabdomyosarcoma cell death in a syngeneic murine model.

The viral-transforming proteins E6 and E7 make human papillomavirus-positive (HPV) malignancies an attractive target for cancer immunotherapy. However, therapeutic vaccination exerts limited efficacy in the setting of advanced disease. We designed a strategy to induce substantial specific immune responses against multiple epitopes of E6 and E7 proteins based on an attenuated transgene from HPV serotypes 16 and 18 that is incorporated into MG1-Maraba virotherapy (MG1-E6E7). Mutations introduced to the transgene abrogate the ability of E6 and E7 to perturb p53 and retinoblastoma, respectively, while maintaining the ability to invoke tumor-specific, multifunctional CD8T-cell responses. Boosting with MG1-E6E7 significantly increased the magnitude of T-cell responses compared with mice treated with a priming vaccine alone (greater than 50 × 10E7-specific CD8T cells per mouse was observed, representing a 39-fold mean increase in boosted animals). MG1-E6E7 vaccination in the HPVmurine model TC1 clears large tumors in a CD8-dependent manner and results in durable immunologic memory. MG1-Maraba can acutely alter the tumor microenvironmentand exploit molecular hallmarks of HPVcancer, as demonstrated by marked infection of HPVpatient tumor biopsies and is, therefore, ideally suited as an oncolytic treatment against clinical HPVcancer. This approach has the potential to be directly translatable to human clinical oncology to tackle a variety of HPV-associated neoplasms that cause significant morbidity and mortality globally..

David F. Stojdl

Children's Hospital of Eastern Ontario (CHEO) Research Institute

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy