A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Oregon Health & Science University

&#x91CD;&#x7EC4;&#x817A;&#x76F8;&#x5173;&#x75C5;&#x6BD2; (rAAV) &#x8F7D;&#x4F53;&#x7684;&#x57FA;&#x56E0;&#x6CBB;&#x7597;&#x6709;&#x524D;&#x9014;&#x3002;&#x5C3D;&#x7BA1;&#x8F7D;&#x4F53;&#x57FA;&#x56E0;&#x7EC4;&#x67D3;&#x8272;&#x4F53;&#x878D;&#x5408;&#x4F4E;&#x9891;&#x7387;&#xFF0C;&#x6700;&#x8FD1;&#x7684;&#x7814;&#x7A76;&#x63D0;&#x51FA;&#x7684;&#x817A;&#x76F8;&#x5173;&#x75C5;&#x6BD2;&#x6574;&#x5408;&#x98CE;&#x9669;&#x62C5;&#x5FE7;&#x56E0;&#x4E3A;&#x4E00;&#x4F53;&#x5316;&#x4F18;&#x5148;&#x53D1;&#x751F;&#x5728;&#x57FA;&#x56E0;&#x548C;&#x4F34;&#x968F;&#x67D3;&#x8272;&#x4F53;&#x7F3A;&#x5931;&#xFF0C;&#x8FD9;&#x53EF;&#x80FD;&#x4F1A;&#x5BFC;&#x81F4;&#x635F;&#x5931;&#x7684;&#x51FD;&#x6570;&#x63D2;&#x5165;&#x7A81;&#x53D8;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x901A;&#x8FC7;&#x5206;&#x6790;&#x5C0F;&#x9F20; 347 rAAV &#x96C6;&#x6210;&#xFF0C;&#x6211;&#x4EEC;&#x6F84;&#x6E05; rAAV &#x4E00;&#x4F53;&#x5316;&#x7684;&#x65B0;&#x578B;&#x7279;&#x70B9;&#xFF1A; &#x96C6;&#x6210;&#x7684;&#x70ED;&#x70B9;&#x548C;&#x9644;&#x8FD1;&#x57FA;&#x56E0;&#x8C03;&#x63A7;&#x5E8F;&#x5217;&#x6574;&#x5408;&#x5F3A;&#x70C8;&#x504F;&#x597D;&#x7684;&#x5B58;&#x5728;&#x3002;&#x6700;&#x7A81;&#x51FA;&#x7684;&#x70ED;&#x70B9;&#x662F;&#x65E0;&#x5BB3;&#x7684; rRNA &#x57FA;&#x56E0;&#x67D3;&#x8272;&#x4F53;&#x9F9B;&#x91CD;&#x590D;&#xFF0C;&#x800C;&#x5C06;&#x8FD1;&#x4E00;&#x534A;&#x7684;&#x8F6C;&#x5F55;&#x9644;&#x8FD1;&#x964D;&#x843D;&#x7684;&#x96C6;&#x6210;&#x542F;&#x52A8;&#x7AD9;&#x70B9;&#x6216; CpG &#x5C9B;&#xFF0C;&#x6697;&#x793A;&#x7684;&#x6FC0;&#x6D3B;&#x7EC6;&#x80DE;&#x75BE;&#x75C5;&#x57FA;&#x56E0;&#x4FA7;&#x7FFC;&#x77E2;&#x91CF;&#x4E00;&#x4F53;&#x5316;&#xFF0C;&#x7C7B;&#x4F3C;&#x4E8E;&#x53CD;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x7684;&#x589E;&#x76CA;&#x7684;&#x51FD;&#x6570;&#x63D2;&#x5165;&#x7A81;&#x53D8;&#x7684;&#x53EF;&#x80FD;&#x6027;&#x3002;&#x53EF;&#x80FD;&#x4E0E;&#x764C;&#x75C7;&#x6709;&#x5173;&#x7684;&#x57FA;&#x56E0;&#x88AB;&#x51FB;&#x4E2D;&#x7684;&#x817A;&#x76F8;&#x5173;&#x75C5;&#x6BD2;&#x96C6;&#x6210;&#x5728; 3.5%&#x7684;&#x9891;&#x7387;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x5728; 218 &#x96C6;&#x6210;&#x7AD9;&#x70B9;&#x548C; 602 &#x65AD;&#x70B9;&#x7684;&#x8F7D;&#x4F53;&#x57FA;&#x56E0;&#x7EC4;&#x67D3;&#x8272;&#x4F53;&#x53D8;&#x5316;&#x6709;&#x5173;&#x7684;&#x4FE1;&#x606F;&#x63D0;&#x4F9B;&#x4E86;&#x77E2;&#x91CF;&#x7EC8;&#x7AEF;&#x5982;&#x4F55;&#x91CD;&#x590D;&#x7684;&#x7EBF;&#x7D22;&#x548C;&#x5728;&#x878D;&#x5408;&#x8FC7;&#x7A0B;&#x4E2D;&#x52A0;&#x5165;&#x4E86;&#x5BBF;&#x4E3B;&#x67D3;&#x8272;&#x4F53; DNA&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x672C;&#x7814;&#x7A76;&#x62A5;&#x544A;&#x63D0;&#x4F9B;&#x4E86;&#x65B0;&#x7684;&#x89C1;&#x89E3;&#x7684;&#x817A;&#x76F8;&#x5173;&#x75C5;&#x6BD2;&#x878D;&#x5408;&#x673A;&#x5236;&#x548C;&#x817A;&#x76F8;&#x5173;&#x75C5;&#x6BD2;&#x4ECB;&#x5BFC;&#x7684;&#x63D2;&#x5165;&#x7A81;&#x53D8;&#x7684;&#x98CE;&#x9669;&#x3002;

&#x5927;&#x89C4;&#x6A21;&#x7684;&#x5206;&#x5B50;&#x751F;&#x7269;&#x5B66;&#x7814;&#x7A76;&#x5C0F;&#x9F20;&#x809D;&#x817A;&#x76F8;&#x5173;&#x75C5;&#x6BD2;&#x8F7D;&#x4F53;&#x4E00;&#x4F53;&#x5316;&#x3002;

Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.

Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver.

Recombinant virus ad&#xE9;no-associ&#xE9; (rAAV) vecteur prometteur pour la th&#xE9;rapie g&#xE9;nique. Malgr&#xE9; une faible fr&#xE9;quence de l&#x26;#39;int&#xE9;gration chromosomique des g&#xE9;nomes de vecteurs, des &#xE9;tudes r&#xE9;centes ont soulev&#xE9; des pr&#xE9;occupations au sujet du risque de l&#x26;#39;int&#xE9;gration rAAV parce que l&#x26;#39;int&#xE9;gration se fait pr&#xE9;f&#xE9;rentiellement dans les g&#xE8;nes et accompagne des d&#xE9;l&#xE9;tions chromosomiques, ce qui peut conduire &#xE0; la perte de fonction de mutagen&#xE8;se insertionnelle. Ici, en analysant 347 int&#xE9;grations rAAV chez la souris, nous avons &#xE9;labor&#xE9; de nouvelles caract&#xE9;ristiques de l&#x26;#39;int&#xE9;gration rAAV: la pr&#xE9;sence de points chauds pour l&#x26;#39;int&#xE9;gration et une forte pr&#xE9;f&#xE9;rence pour l&#x26;#39;int&#xE9;gration de pr&#xE8;s de s&#xE9;quences r&#xE9;gulatrices de g&#xE8;nes. L&#x26;#39;endroit le plus &#xE9;minent &#xE9;tait une niche chaude inoffensifs chromosomique dans les r&#xE9;p&#xE9;titions de g&#xE8;nes ARNr, alors que pr&#xE8;s de la moiti&#xE9; des int&#xE9;grations atterri &#xE0; proximit&#xE9; des sites de d&#xE9;but de transcription ou &#xEE;lots CpG, sugg&#xE9;rant la possibilit&#xE9; d&#x26;#39;activer des g&#xE8;nes de maladies d&#x26;#39;accompagnement cellulaires par l&#x26;#39;int&#xE9;gration du vecteur, semblable &#xE0; r&#xE9;troviral gain de mutagen&#xE8;se insertionnelle fonction-. Possibles li&#xE9;s au cancer g&#xE8;nes ont &#xE9;t&#xE9; touch&#xE9;s par l&#x26;#39;int&#xE9;gration rAAV &#xE0; une fr&#xE9;quence de 3,5%. En outre, l&#x26;#39;information sur les changements chromosomiques &#xE0; 218 sites d&#x26;#39;int&#xE9;gration et 602 points d&#x26;#39;arr&#xEA;t de g&#xE9;nomes vecteurs ont fourni un indice &#xE0; la fa&#xE7;on dont r&#xE9;p&#xE9;titions terminales vecteur et l&#x26;#39;ADN chromosomique de l&#x26;#39;h&#xF4;te sont r&#xE9;unies dans le processus d&#x26;#39;int&#xE9;gration. Ainsi, la pr&#xE9;sente &#xE9;tude fournit de nouvelles connaissances sur le risque de rAAV &#xE0; m&#xE9;diation mutagen&#xE8;se insertionnelle et des m&#xE9;canismes d&#x26;#39;int&#xE9;gration rAAV.

&#xC0; grande &#xE9;chelle Caract&#xE9;risation mol&#xE9;culaire des virus ad&#xE9;no-associ&#xE9; vecteur d&#x26;#39;int&#xE9;gration du virus dans le foie de souris

Gro&#xDF;fl&#xE4;chige molekulare Charakterisierung von Adeno-assoziierten Virus-Vektor-Integration in M&#xE4;useleber.

Vettore ricombinante virus adeno-associati (rAAV) mantiene la promessa per la terapia genica. Nonostante una bassa frequenza di integrazione cromosomica di genomi di vettore, recenti studi hanno sollevato preoccupazioni circa il rischio di rAAV integrazione perch&eacute; l&#39;integrazione si verifica preferenzialmente nei geni e accompagna delezioni cromosomiche, che possono portare alla perdita di funzione mutagenesi inserzionale. Qui, analizzando 347 rAAV integrazioni nei topi, abbiamo delucidare romanzo caratteristiche di integrazione rAAV: la presenza di hot spots per l&#39;integrazione e una forte preferenza per l&#39;integrazione nei pressi di sequenze regolatrici del gene. L&#39;hot spot pi&ugrave; prominente era un&#39;innocua nicchia cromosomica del gene del rRNA ripete, considerando che quasi la met&agrave; delle integrazioni atterrate vicino trascrizione avviare siti o isole CpG, suggerendo la possibilit&agrave; di attivare geni malattia cellulare di accompagnamento tramite integrazione vettoriale, simile a retrovirale mutagenesi inserzionale guadagno di funzione. Possibili geni legati al cancro sono stati colpiti da rAAV integrazione a una frequenza di 3.5%. Inoltre, le informazioni sulle modifiche cromosomiche a 218 siti di integrazione e 602 punti di interruzione dei genomi di vettore hanno fornito un indizio di come terminale di vettore si ripete e DNA cromosomico host sono unite nel processo di integrazione. Pertanto, il presente studio fornisce nuove intuizioni il rischio di mutagenesi inserzionale rAAV-mediata e i meccanismi di integrazione rAAV.

Caratterizzazione molecolare su larga scala di integrazione vettoriale virus adeno-associati in fegato del mouse.

&#x7D44;&#x63DB;&#x3048;&#x30A2;&#x30C7;&#x30CE;&#x968F;&#x4F34;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#xFF08;rAAV&#xFF09;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x306F;&#x907A;&#x4F1D;&#x5B50;&#x6CBB;&#x7642;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x79D8;&#x3081;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x7D71;&#x5408;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x306B;&#x512A;&#x5148;&#x7684;&#x306B;&#x767A;&#x751F;&#x3057;&#x3001;&#x6A5F;&#x80FD;&#x55AA;&#x5931;&#x633F;&#x5165;&#x7A81;&#x7136;&#x5909;&#x7570;&#x8A98;&#x767A;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x67D3;&#x8272;&#x4F53;&#x6B20;&#x5931;&#x3092;&#x3001;&#x4ED8;&#x5C5E;&#x3059;&#x308B;&#x306E;&#x3067;&#x3001;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x67D3;&#x8272;&#x4F53;&#x7D44;&#x8FBC;&#x307F;&#x306E;&#x4F4E;&#x5468;&#x6CE2;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x3001;&#x6700;&#x8FD1;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;rAAV&#x306E;&#x7D71;&#x5408;&#x306E;&#x30EA;&#x30B9;&#x30AF;&#x306B;&#x3064;&#x3044;&#x3066;&#x61F8;&#x5FF5;&#x3092;&#x8868;&#x660E;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x7D71;&#x5408;&#x3068;&#x907A;&#x4F1D;&#x5B50;&#x8ABF;&#x7BC0;&#x914D;&#x5217;&#x306E;&#x8FD1;&#x304F;&#x306B;&#x7D71;&#x5408;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5F37;&#x529B;&#x306A;&#x597D;&#x307F;&#x306E;&#x30DB;&#x30C3;&#x30C8;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x304C;&#x5B58;&#x5728;&#x3059;&#x308B;&#xFF1A;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x3067;347 rAAV&#x306E;&#x7D71;&#x5408;&#x3092;&#x5206;&#x6790;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;rAAV&#x306E;&#x7D71;&#x5408;&#x306E;&#x65B0;&#x898F;&#x306A;&#x7279;&#x5FB4;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3059;&#x308B;&#x3002;&#x7D71;&#x5408;&#x306E;&#x307B;&#x307C;&#x534A;&#x5206;&#x306F;&#x30EC;&#x30C8;&#x30ED;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x5229;&#x5F97;&#x306E;&#x3068;&#x540C;&#x69D8;&#x306B;&#x3001;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x306E;&#x7D71;&#x5408;&#x306B;&#x3088;&#x308A;&#x96A3;&#x63A5;&#x3059;&#x308B;&#x7D30;&#x80DE;&#x306E;&#x75BE;&#x60A3;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x305B;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x793A;&#x5506;&#x3057;&#x3001;&#x8EE2;&#x5199;&#x958B;&#x59CB;&#x90E8;&#x4F4D;&#x307E;&#x305F;&#x306F;CpG&#x30A2;&#x30A4;&#x30E9;&#x30F3;&#x30C9;&#x306E;&#x8FD1;&#x304F;&#x306B;&#x4E0A;&#x9678;&#x3057;&#x305F;&#x306E;&#x306B;&#x5BFE;&#x3057;&#x3001;&#x6700;&#x3082;&#x9855;&#x8457;&#x306A;&#x30DB;&#x30C3;&#x30C8;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x306F;&#x3001;rRNA&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x7E70;&#x308A;&#x8FD4;&#x3057;&#x3067;&#x7121;&#x5BB3;&#x306A;&#x67D3;&#x8272;&#x4F53;&#x306E;&#x30CB;&#x30C3;&#x30C1;&#x3067;&#x3042;&#x3063;&#x305F;&#x95A2;&#x6570;&#x633F;&#x5165;&#x7A81;&#x7136;&#x5909;&#x7570;&#x3002;&#x53EF;&#x80FD;&#x306A;&#x764C;&#x95A2;&#x9023;&#x907A;&#x4F1D;&#x5B50;&#x306F;3.5&#xFF05;&#x306E;&#x983B;&#x5EA6;&#x3067;rAAV&#x306E;&#x7D71;&#x5408;&#x306B;&#x3088;&#x3063;&#x3066;&#x6253;&#x6483;&#x3092;&#x53D7;&#x3051;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x67D3;&#x8272;&#x4F53;&#x306E;218&#x7D71;&#x5408;&#x30B5;&#x30A4;&#x30C8;&#x3067;&#x306E;&#x5909;&#x66F4;&#x70B9;&#x3068;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306E;602&#x30D6;&#x30EC;&#x30FC;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x306B;&#x95A2;&#x3059;&#x308B;&#x60C5;&#x5831;&#x306F;&#x3001;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x306E;&#x672B;&#x7AEF;&#x53CD;&#x5FA9;&#x914D;&#x5217;&#x3068;&#x5BBF;&#x4E3B;&#x67D3;&#x8272;&#x4F53;DNA&#x306F;&#x3001;&#x7D71;&#x5408;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306B;&#x53C2;&#x52A0;&#x3055;&#x308C;&#x308B;&#x65B9;&#x6CD5;&#x3078;&#x306E;&#x624B;&#x304C;&#x304B;&#x308A;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x304D;&#x307E;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;rAAV&#x306E;&#x5A92;&#x4ECB;&#x633F;&#x5165;&#x5909;&#x7570;&#x3068;rAAV&#x306E;&#x7D71;&#x5408;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306E;&#x30EA;&#x30B9;&#x30AF;&#x65B0;&#x305F;&#x306A;&#x6D1E;&#x5BDF;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30DE;&#x30A6;&#x30B9;&#x809D;&#x81D3;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30C7;&#x30CE;&#x968F;&#x4F34;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x306E;&#x7D71;&#x5408;&#x306E;&#x5927;&#x898F;&#x6A21;&#x5206;&#x5B50;&#x306E;&#x30AD;&#x30E3;&#x30E9;&#x30AF;&#x30BF;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;

&#xC7AC;&#xC870;&#xD569; adeno &#xC81C;&#xD734;&#xD574; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; (rAAV) &#xBCA1;&#xD130;&#xB294; &#xC720;&#xC804;&#xC790; &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; &#xC57D;&#xC18D;&#xC744; &#xBCF4;&#xC720;. &#xBCA1;&#xD130; &#xC720;&#xC804;&#xC790;&#xC758; &#xC5FC;&#xC0C9;&#xCCB4; &#xD1B5;&#xD569;&#xC758; &#xC800;&#xC8FC;&#xD30C;&#xC5D0;&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0; &#xCD5C;&#xADFC; &#xC5F0;&#xAD6C; &#xD1B5;&#xD569; &#xC720;&#xC804;&#xC790;&#xC5D0; &#xC6B0;&#xC120;&#xC801;&#xC73C;&#xB85C; &#xBC1C;&#xC0DD; &#xD558; &#xACE0; &#xD568;&#xAED8; &#xC5FC;&#xC0C9;&#xCCB4; &#xC0AD;&#xC81C; &#xAE30;&#xB2A5; &#xC190;&#xC2E4; insertional mutagenesis &#xC774;&#xC5B4;&#xC9C8; &#xC218; &#xC788;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; rAAV &#xD1B5;&#xD569;&#xC758; &#xC704;&#xD5D8;&#xC5D0; &#xB300; &#xD55C; &#xC6B0;&#xB824;&#xB97C; &#xC81C;&#xAE30; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;&#xC5D0;, &#xC950;&#xC5D0;&#xC11C; 347 rAAV &#xD1B5;&#xD569; &#xBD84;&#xC11D; &#xD558; &#xC5EC; &#xC6B0;&#xB9AC; &#xBA85;&#xB8CC; rAAV &#xD1B5;&#xD569;&#xC758; &#xC0C8;&#xB85C;&#xC6B4; &#xAE30;&#xB2A5;: &#xD1B5;&#xD569;&#xC5D0; &#xB300; &#xD55C; hot spots &#xBC0F; &#xC720;&#xC804;&#xC790; &#xADDC;&#xC81C; &#xC2DC;&#xD000;&#xC2A4; &#xADFC;&#xCC98; &#xD1B5;&#xD569;&#xC5D0; &#xB300; &#xD55C; &#xAC15;&#xD55C; &#xC120;&#xD638;&#xC758; &#xC874;&#xC7AC;. &#xAC00;&#xC7A5; &#xB208;&#xC5D0; &#xB744;&#xB294; &#xD56B; &#xC2A4;&#xD31F; &#xD544; &#xC0AC; &#xADFC;&#xCC98;&#xC5D0; &#xCC29;&#xB959; &#xD558;&#xB294; &#xD1B5;&#xD569;&#xC758; &#xAC70;&#xC758; &#xC808;&#xBC18; &#xC2DC;&#xC791; &#xC0AC;&#xC774;&#xD2B8; &#xB610;&#xB294; CpG &#xC12C;, &#xD65C;&#xC131;&#xD654; retroviral &#xD568;&#xC218; &#xC774;&#xB4DD; insertional mutagenesis &#xC720;&#xC0AC;&#xD55C; &#xBCA1;&#xD130; &#xD1B5;&#xD569; &#xD558; &#xC5EC; &#xC140;&#xB8F0;&#xB7EC; &#xC9C8;&#xBCD1; &#xC720;&#xC804;&#xC790; &#xCE21;&#xBA74;&#xC758; &#xAC00;&#xB2A5;&#xC131;&#xC744; &#xC81C;&#xC548; &#xD558;&#xB294; &#xBC18;&#xBA74; rRNA &#xC720;&#xC804;&#xC790;&#xC5D0; &#xBB34;&#xD574; &#xD55C; &#xC5FC;&#xC0C9;&#xCCB4; &#xD2C8;&#xC0C8; &#xBC18;&#xBCF5; &#xD588;&#xB2E4;. &#xAC00;&#xB2A5;&#xD55C; &#xC554; &#xAD00;&#xB828; &#xC720;&#xC804;&#xC790;&#xB294; 3.5%&#xC758; &#xBE48;&#xB3C4;&#xB85C; rAAV &#xD1B5;&#xD569;&#xC5D0; &#xC758;&#xD574; &#xB9DE; &#xC558;. &#xB610;&#xD55C;, 218 &#xD1B5;&#xD569; &#xC0AC;&#xC774;&#xD2B8; &#xBC0F; &#xBCA1;&#xD130; &#xAC8C;&#xB188;&#xC758; 602 &#xC911;&#xB2E8;&#xC810;&#xC5D0;&#xC11C; &#xC5FC;&#xC0C9;&#xCCB4; &#xBCC0;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xB0B4;&#xC6A9;&#xC740; &#xBCA1;&#xD130; &#xD130;&#xBBF8;&#xB110; &#xBC18;&#xBCF5; &#xD558;&#xB294; &#xBC29;&#xBC95; &#xB2E8;&#xC11C;&#xB97C; &#xC81C;&#xACF5; &#xD558; &#xACE0; &#xD1B5;&#xD569; &#xACFC;&#xC815;&#xC5D0;&#xC11C; &#xC870;&#xC778; &#xB418;&#xB294; &#xD638;&#xC2A4;&#xD2B8; &#xC5FC;&#xC0C9;&#xCCB4; DNA. &#xB530;&#xB77C;&#xC11C;, &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C; insertional mutagenesis rAAV &#xC911;&#xC7AC;&#xC758; &#xC704;&#xD5D8; &#xBC0F; rAAV &#xD1B5;&#xD569; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xC81C;&#xACF5; &#xD55C;&#xB2E4;.

&#xB9C8;&#xC6B0;&#xC2A4; &#xAC04; adeno &#xC81C;&#xD734;&#xD574; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBCA1;&#xD130; &#xD1B5;&#xD569;&#xC758; &#xB300;&#xADDC;&#xBAA8; &#xBD84;&#xC790; &#xD2B9;&#xC131;.

Recombinante virus adeno-asociado (rAAV) vectores es una promesa para la terapia g&#xE9;nica. A pesar de la baja frecuencia de la integraci&#xF3;n cromos&#xF3;mica de los genomas de vectores, estudios recientes han surgido preocupaciones sobre el riesgo de la integraci&#xF3;n rAAV porque la integraci&#xF3;n se produce preferentemente en los genes y acompa&#xF1;a a deleciones cromos&#xF3;micas, lo que puede conducir a la p&#xE9;rdida de mutag&#xE9;nesis de la funci&#xF3;n de inserci&#xF3;n. En este caso, mediante el an&#xE1;lisis de 347 integraciones rAAV en ratones, que dilucidar nuevas caracter&#xED;sticas de integraci&#xF3;n rAAV: la presencia de puntos calientes para la integraci&#xF3;n y una fuerte preferencia por la integraci&#xF3;n de cerca de secuencias reguladoras de genes. El punto caliente m&#xE1;s prominente era un nicho inofensiva cromos&#xF3;mica en las repeticiones de genes rRNA, mientras que casi la mitad de las integraciones aterriz&#xF3; cerca de los sitios de inicio de transcripci&#xF3;n o islas CpG, lo que sugiere la posibilidad de activar los genes que flanquean la enfermedad de celulares por la integraci&#xF3;n de vectores, similar a retroviral ganancia de -funci&#xF3;n de inserci&#xF3;n mutag&#xE9;nesis. Posibles genes relacionados con el c&#xE1;ncer se vieron afectados por la integraci&#xF3;n rAAV a una frecuencia de 3,5%. Adem&#xE1;s, la informaci&#xF3;n sobre los cambios cromos&#xF3;micos en 218 sitios de integraci&#xF3;n y 602 puntos de ruptura de los genomas de vectores han proporcionado una pista de c&#xF3;mo se repite de vectores de terminales y el ADN cromos&#xF3;mico del hu&#xE9;sped se unen en el proceso de integraci&#xF3;n. Por lo tanto, el presente estudio proporciona nuevos conocimientos sobre el riesgo de rAAV mediada por mutag&#xE9;nesis de inserci&#xF3;n y de los mecanismos de integraci&#xF3;n rAAV.

A gran escala Caracterizaci&#xF3;n molecular de adeno-asociado vector de integraci&#xF3;n del virus en el h&#xED;gado del rat&#xF3;n

Although recombinant adeno-associated viral (rAAV) vectors are promising tools for gene therapy of genetic disorders, they remain mostly episomal and hence are lost during cell replication. For this reason, rAAV vectors capable of chromosomal integration would be desirable. Ribosomal DNA (rDNA) repeat sequences are overrepresented during random integration of rAAV. We therefore sought to enhance AAV integration frequency by including 28S rDNA homology arms into our vector design. A vector containing ~1 kb of homology on each side of a cDNA expression cassette for human fumarylacetoacetate hydrolase (FAH) was constructed. rAAV of serotypes 2 and 8 were injected into Fah-deficient mice, a model for human tyrosinemia type 1. Integrated FAH transgenes are positively selected in this model and rDNA-containing AAV vectors had a ~30× higher integration frequency than controls. Integration by homologous recombination (HR) into the 28S rDNA locus was seen in multiple tissues. Furthermore, rDNA-containing AAV vectors for human factor IX (hFIX) demonstrated increased transgene persistence after liver regeneration. We conclude that rDNA containing AAV vectors may be superior to conventional vector design for the treatment of genetic diseases, especially those associated with increased hepatocyte replication.

AAV vectors containing rDNA homology display increased chromosomal integration and transgene persistence.

Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by the absence of a functional dystrophin protein. Lack of dystrophin protein disrupts the dystrophin-glycoprotein complex causing muscle membrane instability and degeneration. One of the secondary manifestations resulting from lack of functional dystrophin in muscle tissue is an increased level of cytokines that recruit inflammatory cells, leading to chronic upregulation of the nuclear factor (NF)-κB. Negative regulators of the classical NF-κB pathway improve muscle health in the mdx mouse model for DMD. We have previously shown in vitro that a negative regulator of the NF-κB pathway, A20, plays a role in muscle regeneration. Here, we show that overexpression of A20 by using a muscle-specific promoter delivered with an adeno-associated virus serotype 8 (AAV8) vector to the mdx mouse decreases activation of the NF-κB pathway in skeletal muscle. Recombinant A20 expression resulted in a reduction in number of fibers with centrally placed nuclei and a reduction in the number of T cells infiltrating muscle transduced with the AAV8-A20 vector. Taken together, we conclude that overexpression of A20 in mdx skeletal muscle provides improved muscle health by reduction of chronic inflammation and muscle degeneration. These results suggest A20 is a potential therapeutic target to ameliorate symptoms of DMD.

Adeno-associated virus serotype 8 (AAV8) delivery of recombinant A20 to skeletal muscle reduces pathological activation of nuclear factor (NF)-κB in muscle of mdx mice.

We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10(9) viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.

Intraganglionic AAV6 results in efficient and long-term gene transfer to peripheral sensory nervous system in adult rats.

Overlapping open reading frames (ORFs) in viral genomes undergo co-evolution; however, how individual amino acids coded by overlapping ORFs are structurally, functionally, and co-evolutionarily constrained remains difficult to address by conventional homologous sequence alignment approaches. We report here a new experimental and computational evolution-based methodology to address this question and report its preliminary application to elucidating a mode of co-evolution of the frame-shifted overlapping ORFs in the adeno-associated virus (AAV) serotype 2 viral genome. These ORFs encode both capsid VP protein and non-structural assembly-activating protein (AAP). To show proof of principle of the new method, we focused on the evolutionarily conserved QVKEVTQ and KSKRSRR motifs, a pair of overlapping heptapeptides in VP and AAP, respectively. In the new method, we first identified a large number of capsid-forming VP3 mutants and functionally competent AAP mutants of these motifs from mutant libraries by experimental directed evolution under no co-evolutionary constraints. We used Illumina sequencing to obtain a large dataset and then statistically assessed the viability of VP and AAP heptapeptide mutants. The obtained heptapeptide information was then integrated into an evolutionary algorithm, with which VP and AAP were co-evolved from random or native nucleotide sequences in silico. As a result, we demonstrate that these two heptapeptide motifs could exhibit high degeneracy if coded by separate nucleotide sequences, and elucidate how overlap-evoked co-evolutionary constraints play a role in making the VP and AAP heptapeptide sequences into the present shape. Specifically, we demonstrate that two valine (V) residues and β-strand propensity in QVKEVTQ are structurally important, the strongly negative and hydrophilic nature of KSKRSRR is functionally important, and overlap-evoked co-evolution imposes strong constraints on serine (S) residues in KSKRSRR, despite high degeneracy of the motifs in the absence of co-evolutionary constraints. 

An experimental and computational evolution-based method to study a mode of co-evolution of overlapping open reading frames in the AAV2 viral genome.

Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research. 

Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.

Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2(144-184)). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase-AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2(144-184) has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly.

Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein.

Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.

Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11.

The intent of this contribution is to provide an update of the progress we have made towards developing a method/treatment to permanently sterilize cats. Our approach employs two complementary methodologies: RNA interference (RNAi) to silence genes involved in the central control of reproduction and a virus-based gene therapy system intended to deliver RNAi selectively to the hypothalamus (where these genes are expressed) via the systemic administration of modified viruses. We selected the hypothalamus because it contains neurons expressing Kiss1 and Tac3, two genes essential for reproduction and fertility. We chose the non-pathogenic adeno-associated virus (AAV) as a vector whose tropism could be modified to target the hypothalamus. The issues that must be overcome to utilize this vector as a delivery vehicle to induce sterility include modification of the wild-type AAV to target the hypothalamic region of the brain with a simultaneous reduction in targeting of peripheral tissues and non-hypothalamic brain regions, identification of RNAi targets that will effectively reduce the expression of Kiss1 and Tac3 without off-target effects, and determination if neutralizing antibodies to the AAV serotype of choice are present in cats. Successful resolution of these issues will pave the way for the development of a powerful tool to induce the permanent sterility in cats.

Engineering a gene silencing viral construct that targets the cat hypothalamus to induce permanent sterility: An update.

Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from  gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of  mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.

Hiroyuki Nakai

Department of Molecular and Medical Genetics,
Department of Molecular Microbiology and Immunology

Oregon Health & Science University

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

Hiroyuki Nakai

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular and Medical Genetics,Department of Molecular Microbiology and Immunology

Oregon Health & Science University

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

Department of Molecular and Medical Genetics,
Department of Molecular Microbiology and Immunology