high-throughput-quantitative-real-time-rt-pcr-assay-for-determining

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

US Food and Drug Administration

Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 10(12) viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIalpha and XIIIalpha in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 x 10(11) viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity.

Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector.

&#x6811;&#x7A81;&#x72B6;&#x7EC6;&#x80DE; (Dc) &#x53D1;&#x8D77;&#x548C;&#x6307;&#x5BFC;&#x6297;&#x539F;&#x7279;&#x5F02;&#x6027; T &#x7EC6;&#x80DE;&#x53CD;&#x5E94;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;&#x57FA;&#x56E0;&#x6539;&#x9020;&#x7684;&#x76F4;&#x6D41;&#x76EE;&#x524D;&#x6B63;&#x5728;&#x7814;&#x7A76;&#x7684;&#x80BF;&#x7624;&#x514D;&#x75AB;&#x6CBB;&#x7597;&#x3001; &#x75AB;&#x82D7;&#x5F00;&#x53D1;&#x548C;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x6297;&#x539F;&#x514D;&#x75AB;&#x6291;&#x5236;&#x3002;&#x578B;&#x817A;&#x75C5;&#x6BD2; (Ad) 5 (Ad5)-&#x4ECB;&#x5BFC;&#x7684;&#x57FA;&#x56E0;&#x8F6C;&#x79FB;&#x9F20;&#x6807;&#x9AA8;&#x9AD3; DCs &#x548C;&#x4EBA;&#x7C7B;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x6E90;&#x6027; DCs &#x662F;&#x4F4E;&#x6548;&#x7684;&#x56E0;&#x4E3A;&#x65E2;&#x4E0D;&#x8868;&#x793A;&#x540C;&#x6E90;&#x7684;&#x9AD8;&#x4EB2;&#x548C;&#x6027;&#x5E7F;&#x544A;&#x53D7;&#x4F53;&#x3002;&#x6211;&#x4EEC;&#x8868;&#x660E; co-precipitating &#x4E0E;&#x9499;&#x78F7;&#x9178;&#x817A;&#x75C5;&#x6BD2;&#x8F7D;&#x4F53; (CaPi) &#x589E;&#x52A0;&#x57FA;&#x56E0;&#x8868;&#x8FBE; &#xFF08;2000-fold&#xFF09; &#x548C;&#x8F6C;&#x5BFC;&#x6548;&#x7387; &#xFF08;50-fold&#xFF09; &#x5728;&#x9F20;&#x6807;&#x76F4;&#x6D41;&#xFF0C;&#x4E3B;&#x8981;&#x662F;&#x56E0;&#x4E3A;&#x53D7;&#x4F53;&#x72EC;&#x7ACB;&#x75C5;&#x6BD2;&#x6027;&#x5438;&#x6536;&#x3002;&#x6B64;&#x5916;&#xFF0C;Ad5:CaPi-&#x5904;&#x7406;&#x96C6;&#x6563;&#x63A7;&#x5236;&#x7CFB;&#x7EDF;&#x88AB;&#x6FC0;&#x6D3B;&#x8868;&#x793A;&#x6210;&#x719F;&#x8868;&#x9762;&#x5206;&#x5B50; CD40 &#x548C; CD86&#xFF0C;&#x5E76;&#x5206;&#x6CCC;&#x708E;&#x6027;&#x7EC6;&#x80DE;&#x56E0;&#x5B50;&#x80BF;&#x7624;&#x574F;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x548C;&#x767D;&#x7EC6;&#x80DE;&#x4ECB;&#x7D20; 6&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x76F4;&#x6D41;&#x8F6C;&#x5BFC;&#x65E2;&#x4E0D;&#x6210;&#x719F;&#x662F;&#x4F9D;&#x8D56;&#x4E8E;&#x75C5;&#x6BD2;&#x86CB;&#x767D;&#x8D28;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x4E0E;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x7684;&#x6574;&#x5408;&#x3002;Ad5:CaPi &#x8FD8;&#x6BD4; Ad5 &#x72EC;&#x81EA;&#xFF0C;&#x7C7B;&#x4F3C;&#x4E8E;&#x9488;&#x5BF9; CD46 &#x53D7;&#x4F53;&#x7684;&#x8F6C;&#x57FA;&#x56E0;&#x8F7D;&#x4F53; (Ad5f35) &#x66F4;&#x6709;&#x6548;&#x8F6C;&#x5BFC;&#x4EBA;&#x76F4;&#x6D41;&#x3002;&#x4E3A;&#x6B64;&#xFF0C;&#x8FD9;&#x79CD;&#x65B9;&#x6CD5;&#x5C06;&#x6838;&#x53D7;&#x4F53;&#x72EC;&#x7ACB;&#x975E;&#x75C5;&#x6BD2;&#x57FA;&#x56E0;&#x8F7D;&#x4F53;&#x7684;&#x8D29;&#x8FD0;&#x548C;&#x817A;&#x75C5;&#x6BD2;&#x4ECB;&#x5BFC;&#x7684; endosomal &#x8F6C;&#x4E49;&#x7684;&#x6548;&#x7387;&#x7ED3;&#x5408;&#x8D77;&#x6765;&#x3002;&#x91CD;&#x8981;&#x7684;&#x662F;&#xFF0C;CaPi &#x6C89;&#x6DC0;&#x53EF;&#x7528;&#x4E8E;&#x529F;&#x80FD;&#x4FEE;&#x6539;&#x76F4;&#x6D41;&#x6FC0;&#x6D3B;&#x548C;&#x6269;&#x5927;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x7279;&#x5B9A;&#x8BB0;&#x5FC6;&#x7EC6;&#x80DE;&#x6BD2; T &#x6DCB;&#x5DF4;&#x7EC6;&#x80DE;&#x3002;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x6807;&#x8BC6;&#x4E00;&#x4E2A;&#x7B80;&#x5355;&#x7684;&#x65B9;&#x6CD5;&#x6765;&#x6539;&#x5584;&#x5F53;&#x524D; Ad5 &#x57FA;&#x56E0;&#x8F6C;&#x79FB;&#xFF0C;&#x652F;&#x6301;&#x4E34;&#x5E8A;&#x8FC7;&#x7EE7;&#x514D;&#x75AB;&#x6CBB;&#x7597;&#x7684;&#x529F;&#x6548;&#x3002;

&#x6811;&#x7A81;&#x72B6;&#x7EC6;&#x80DE;&#x540E;&#x4E0E;&#x817A;&#x75C5;&#x6BD2;&#x9499;&#x78F7; co-precipitates &#x57FA;&#x56E0;&#x8F6C;&#x79FB;&#x7684;&#x529F;&#x80FD;&#x3002;

Dendritic cells (DCs) are essential for initiating and directing antigen-specific T-cell responses. Genetic modification of DC is under study for cancer immunotherapy, vaccine development, and antigen-targeted immunosuppression. Adenovirus (Ad) type 5 (Ad5)-mediated gene transfer to mouse bone marrow DCs and human monocyte-derived DCs is inefficient because neither express the cognate high-affinity Ads receptor. We show that co-precipitating adenoviral vectors with calcium phosphate (CaPi) increased gene expression (2000-fold) and transduction efficiency (50-fold) in mouse DC, primarily owing to receptor-independent viral uptake. Moreover, Ad5:CaPi-treated DCs were activated to express the maturation surface molecules CD40 and CD86, and to secrete proinflammatory cytokines tumor necrosis factor-alpha and interleukin 6. However, neither DC transduction nor maturation was dependent on viral protein interactions with cell surface integrin. Ad5:CaPi also transduced human DC more efficiently than Ad5 alone, similar to a genetically modified vector (Ad5f35) targeted to the CD46 receptor. As such, this approach combines the efficiency of adenoviral-mediated endosomal escape and nuclear trafficking with the receptor independence of nonviral gene delivery. Importantly, CaPi co-precipitation could be used to functionally modify DC to activate and expand cytomegalovirus-specific memory cytotoxic T lymphocytes. This study identifies a simple technique to improve the efficacy of current Ad5 gene transfer, in support of clinical adoptive immunotherapy.

Dendritic cell function after gene transfer with adenovirus-calcium phosphate co-precipitates.

Les cellules dendritiques (CD) sont essentiels pour initier et diriger les r&eacute;ponses de cellules T sp&eacute;cifiques de l&#39;antig&egrave;ne. La modification g&eacute;n&eacute;tique de DC est &agrave; l&#39;&eacute;tude pour l&#39;immunoth&eacute;rapie contre le cancer, le d&eacute;veloppement de vaccins et immunosuppression ciblant l&#39;antig&egrave;ne. Ad&eacute;novirus (Ad) de type 5 (Ad5)-transfert de g&egrave;ne m&eacute;diatis&eacute;e &agrave; souris DCs de la moelle osseuse et DCs de d&eacute;riv&eacute;s de monocytes humains est inefficace car ni expriment le r&eacute;cepteur d&#39;annonces de haute affinit&eacute; apparent&eacute;. Nous montrons que co-precipitating des vecteurs ad&eacute;noviraux avec le phosphate de calcium (CaPi) augmente l&#39;expression des g&egrave;nes (soit) et l&#39;efficacit&eacute; de la transduction (50 fois) chez la souris DC, principalement en raison de l&#39;absorption virale r&eacute;cepteurs ind&eacute;pendants. En outre, Ad5:CaPi-DCs trait&eacute;es ont &eacute;t&eacute; activ&eacute;s pour exprimer les mol&eacute;cules de surface de maturation CD40 et CD86 et s&eacute;cr&egrave;tent des cytokines pro-inflammatoires facteur de n&eacute;crose tumorale alpha et interleukine 6. Cependant, transduction DC ni maturation d&eacute;pendait des interactions de prot&eacute;ine virale avec cell surface integrin. AD5:CaPi transduites aussi humaine DC plus efficacement que Ad5 seul, semblable &agrave; un vecteur d&#39;organismes g&eacute;n&eacute;tiquement modifi&eacute; (Ad5f35) cibl&eacute; le r&eacute;cepteur CD46. Par cons&eacute;quent, cette approche combine l&#39;efficacit&eacute; d&#39;&eacute;vasion endosomal ad&eacute;noviraux m&eacute;diation et nucl&eacute;aire traite avec l&#39;ind&eacute;pendance du r&eacute;cepteur de la livraison de g&egrave;ne nonviral. Ce qui est important, CaPi copr&eacute;cipitation pourrait servir &agrave; modifier fonctionnellement DC pour activer et d&eacute;velopper les lymphocytes T cytotoxiques sp&eacute;cifiques &agrave; cytom&eacute;galovirus m&eacute;moire. Cette &eacute;tude identifie une technique simple pour am&eacute;liorer l&#39;efficacit&eacute; du transfert de g&egrave;nes Ad5 actuel, &agrave; l&#39;appui de la clinique immunoth&eacute;rapie adoptive.

Fonction des cellules dendritiques apr&egrave;s transfert de g&egrave;nes avec les ad&eacute;novirus-calcium phosphate copr&eacute;cipit&eacute;s.

Dendritische Zellen (DCs) sind essentiell f&uuml;r die Initiierung und Leitung von Antigen-spezifischen T-Zell-Antworten. Genetische Ver&auml;nderung von DC ist unter Studie f&uuml;r Krebs-Immuntherapie, Entwicklung von Impfstoffen und Antigen-bezogene Immunsuppression. Adenoviren (Ad) Typ 5 (Ad5)-vermittelten Gentransfers auf Knochenmark DCs und menschlichen Monozyte abgeleitete DCs Maus ist ineffizient, weil keiner den verwandten hoher Affinit&auml;t-Ads-Rezeptor Ausdr&uuml;cken. Wir zeigen, dass co-precipitating adenoviral Vektoren mit Kalziumphosphat (CaPi) Genexpression (2000-fold) erh&ouml;ht und Transduktion Effizienz (50-fold) Maus DC, haupts&auml;chlich aufgrund der Rezeptor-unabh&auml;ngige virale Verbreitung. Dar&uuml;ber hinaus Ad5:CaPi-behandelter DCs wurden aktiviert, die Reifung Oberfl&auml;che Molek&uuml;le CD40 und CD86 auszudr&uuml;cken, und sezernieren Proinflammatory Cytokines Tumor-Nekrose-Faktor-Alpha und Interleukin 6. Jedoch war die DC-Transduktion weder Reifung viral Protein-Interaktionen mit Zelle Oberfl&auml;che Integrin abh&auml;ngig. Ad5:CaPi transduzierten auch menschliche DC effizienter als Ad5 allein, &auml;hnlich wie ein genetisch ver&auml;nderter Vektor (Ad5f35) gezielt an den CD46-Rezeptor. Dieser Ansatz kombiniert als solche die Wirksamkeit adenoviral-vermittelte Endosomen Fluchtwege und nuklearen Handel mit der Rezeptor-Unabh&auml;ngigkeit der nonviral gen Lieferung. Wichtig ist, k&ouml;nnte CaPi Temperaturen verwendet werden, um funktionell &auml;ndern DC zu aktivieren und Zytomegalie-Virus-spezifische Speicher cytotoxische T-Lymphozyten zu erweitern. Diese Studie zeigt eine einfache Technik, um die Wirksamkeit der aktuellen Ad5-Gentransfer zur Unterst&uuml;tzung klinischer Adoptive Immuntherapie zu verbessern.

Dendritische Zelle-Funktion nach Gentransfer mit Adenovirus-Kalzium-Phosphat-Co-precipitates.

Le cellule dendritiche (DCs) sono essenziali per avviare e dirigere le risposte delle cellule T antigene-specifiche. Modificazione genetica della DC &egrave; sotto studio per immunoterapia del cancro, sviluppo di un vaccino e immunosoppressione antigene mirati. Tipo adenovirus (annuncio) 5 (Ad5)-trasferimento genico mediato per mouse midollo DCs e DCs umano monocyte-derivati &egrave; inefficiente perch&eacute; n&eacute; esprimere il recettore di annunci affine ad alta affinit&agrave;. Mostriamo che co-precipitating vettori adenovirali con fosfato di calcio (CaPi) aumentata espressione del gene (2000-fold) e l&#39;efficienza di trasduzione (50 volte) in mouse DC, principalmente a causa della diffusione virale recettore-indipendente. Inoltre, Ad5:CaPi-DCs trattati sono stati attivati per esprimere le molecole superficiali di maturazione CD40 e CD86 e secernono citochine proinfiammatorie fattore di necrosi tumorale-alfa e interleuchina 6. Trasduzione DC n&eacute; maturazione per&ograve; dipendono le interazioni della proteina virale con cellule superficiali integrina. Ad5:CaPi anche transduced DC umana in modo pi&ugrave; efficiente rispetto a Ad5 da sola, simile a un vettore geneticamente modificato (Ad5f35) mirato al recettore CD46. Come tale, questo approccio combina l&#39;efficienza di fuga endosomal adenovirali-mediata e trafficare con indipendenza della consegna virali del gene del recettore nucleare. Importante, CaPi coprecipitazione potrebbe essere utilizzato per modificare funzionalmente DC per attivare ed espandere i linfociti T citotossici specifici del citomegalovirus memoria. Questo studio identifica una tecnica semplice per migliorare l&#39;efficacia dell&#39;attuale trasferimento genico Ad5, a sostegno della clinica adoptive immunoterapia.

Funzione delle cellule dendritiche dopo il trasferimento del gene con adenovirus-calcio fosfato co-precipitates.

&#x6A39;&#x72B6;&#x7D30;&#x80DE; (Dc) &#x306F;&#x3001;&#x958B;&#x59CB;&#x3057;&#x3066;&#x6297;&#x539F;&#x7279;&#x7570;&#x7684; T &#x7D30;&#x80DE;&#x5FDC;&#x7B54;&#x3092;&#x76E3;&#x7763;&#x306B;&#x3068;&#x3063;&#x3066;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3059;&#x3002;DC &#x306E;&#x907A;&#x4F1D;&#x7684;&#x6539;&#x5909;&#x7814;&#x7A76;&#x764C;&#x514D;&#x75AB;&#x7642;&#x6CD5;&#x3001;&#x30EF;&#x30AF;&#x30C1;&#x30F3;&#x958B;&#x767A;&#x3001;&#x6297;&#x539F;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3068;&#x3057;&#x305F;&#x514D;&#x75AB;&#x6291;&#x5236;&#x4E0B;&#x3067;&#x3059;&#x3002;&#x578B;&#x30A2;&#x30C7;&#x30CE; &#x30A6;&#x30A4;&#x30EB;&#x30B9; (Ad) 5 (Ad5)-&#x30DE;&#x30A6;&#x30B9;&#x9AA8;&#x9AC4; DCs &#x3068;&#x3072;&#x3068;&#x5358;&#x7403;&#x7531;&#x6765;&#x306E; DCs &#x3092;&#x4ECB;&#x3057;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x4F1D;&#x9054;&#x3069;&#x3061;&#x3089;&#x3082;&#x540C;&#x7A2E;&#x306E;&#x9AD8;&#x89AA;&#x548C;&#x6027;&#x5E83;&#x544A;&#x53D7;&#x5BB9;&#x4F53;&#x30A8;&#x30AF;&#x30B9;&#x30D7;&#x30EC;&#x30B9;&#x306E;&#x3067;&#x52B9;&#x7387;&#x7684;&#x3067;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3002;&#x6211;&#x3005; &#x30A2;&#x30C7;&#x30CE; &#x30EA;&#x30F3;&#x9178;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x3068; co-precipitating (CaPi) &#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE; (2000-fold) &#x5897;&#x52A0;&#x3092;&#x793A;&#x3059;&#x3068;&#x4F1D;&#x9054;&#x52B9;&#x7387; (50-fold) &#x53D7;&#x5BB9;&#x4F53;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x306A;&#x3044;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x53D6;&#x308A;&#x8FBC;&#x307F;&#x306B;&#x304A;&#x304D;&#x307E;&#x3057;&#x3066; DC&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x3067;&#x3002;&#x307E;&#x305F;&#x3001;Ad5:CaPi-&#x6271;&#x308F;&#x308C;&#x305F; DCs &#x304C;&#x6D3B;&#x6027;&#x5316;&#x3057;&#x305F; CD40 &#x3068; CD86, &#x6210;&#x719F;&#x8868;&#x9762;&#x5206;&#x5B50;&#x3092;&#x8868;&#x73FE;&#x3057;&#x3066;&#x708E;&#x75C7;&#x6027;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30A4;&#x30F3;&#x816B;&#x760D;&#x58CA;&#x6B7B;&#x56E0;&#x5B50;-&alpha; &#x304A;&#x3088;&#x3073;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30ED;&#x30A4;&#x30AD;&#x30F3; 6 &#x3092;&#x5206;&#x6CCC;&#x3057;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;DC &#x4F1D;&#x9054;&#x3082;&#x6210;&#x719F;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30EA;&#x30F3;&#x3068;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x305F;&#x3002;Ad5:CaPi &#x3082;&#x4EBA;&#x9593; DC Ad5 CD46 &#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x6A19;&#x7684;&#x907A;&#x4F1D;&#x5B50;&#x7D44;&#x307F;&#x63DB;&#x3048;&#x30D9;&#x30AF;&#x30BF;&#x30FC; (Ad5f35) &#x306E;&#x3088;&#x3046;&#x306A;&#x5358;&#x72EC;&#x3067;&#x3088;&#x308A;&#x52B9;&#x7387;&#x7684;&#x306B;&#x5C0E;&#x5165;&#x3002;&#x305D;&#x306E;&#x305F;&#x3081;&#x3001;&#x3053;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x52B9;&#x7387;&#x30A2;&#x30C7;&#x30CE; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x5A92;&#x4ECB;&#x30A8;&#x30F3;&#x30C9;&#x30BD;&#x30FC;&#x30E0;&#x8131;&#x51FA;&#x3068;&#x6838; nonviral &#x907A;&#x4F1D;&#x5B50;&#x30C7;&#x30EA;&#x30D0;&#x30EA;&#x30FC;&#x306E;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x72EC;&#x7ACB;&#x3068;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3092;&#x517C;&#x306D;&#x5099;&#x3048;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x91CD;&#x8981;&#x306A;&#x306F;&#x3001;CaPi zno-al_2o_3 &#x50BE;&#x659C;&#x6A5F;&#x80FD;&#x3092;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x3057;&#x3001;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30AC;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x56FA;&#x6709;&#x306E;&#x30E1;&#x30E2;&#x30EA;&#x7D30;&#x80DE;&#x50B7;&#x5BB3;&#x6027; T &#x30EA;&#x30F3;&#x30D1;&#x7403;&#x3092;&#x5C55;&#x958B;&#x3059;&#x308B; DC &#x304C;&#x5909;&#x66F4;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x81E8;&#x5E8A;&#x306E;&#x990A;&#x5B50;&#x514D;&#x75AB;&#x7642;&#x6CD5;&#x306E;&#x652F;&#x63F4;&#x306E;&#x73FE;&#x5728; Ad5 &#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x306E;&#x6709;&#x52B9;&#x6027;&#x3092;&#x6539;&#x5584;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x7C21;&#x5358;&#x306A;&#x30C6;&#x30AF;&#x30CB;&#x30C3;&#x30AF;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30A2;&#x30C7;&#x30CE; &#x30A6;&#x30A4;&#x30EB;&#x30B9; &#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5869;&#x306E;&#x6C88;&#x3068;&#x907A;&#x4F1D;&#x5B50;&#x5C0E;&#x5165;&#x5F8C;&#x306E;&#x6A39;&#x72B6;&#x7D30;&#x80DE;&#x6A5F;&#x80FD;&#x3002;

&#xC218;&#xC9C0;&#xC0C1; &#xC138;&#xD3EC; (DCs) &#xC2DC;&#xC791; &#xD558; &#xACE0; antigen &#xAD00;&#xB828; T&#xC758; &#xC138;&#xD3EC; &#xC751;&#xB2F5;&#xC744; &#xAC10;&#xB3C5;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;. DC&#xC758; &#xC720;&#xC804;&#xC790; &#xC870;&#xC791; &#xC554; immunotherapy, &#xBC31;&#xC2E0; &#xAC1C;&#xBC1C;&#xACFC; &#xD56D; &#xC6D0;&#xC744; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xBA74;&#xC5ED; &#xC5B5;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xC5F0;&#xAD6C; &#xC911; &#xC774;&#xB2E4;. &#xC544; &#xB370; &#xB178; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; (Ad) &#xC720;&#xD615; 5 (Ad5)-&#xB458; &#xB2E4; &#xB3D9;&#xC871; &#xC120;&#xD638;&#xB3C4; &#xB192;&#xC740; &#xAD11;&#xACE0; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xD45C;&#xD604; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xC911;&#xC7AC; &#xC720;&#xC804;&#xC790; &#xC804;&#xC1A1;&#xC744; &#xB9C8;&#xC6B0;&#xC2A4; &#xACE8; &#xC218; Dc&#xC640; &#xC778;&#xAC04;&#xC758; monocyte &#xD30C;&#xC0DD; &#xB41C; DCs &#xBE44;&#xD6A8;&#xC728;&#xC801;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xCE7C;&#xC298; &#xC778;&#xC0B0; &#xC5FC;&#xC73C;&#xB85C; adenoviral &#xBCA1;&#xD130; co-precipitating (CaPi) &#xC99D;&#xAC00; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604; (2000-fold)&#xC640; &#xBCC0;&#xD658; &#xD6A8;&#xC728; (50-fold) &#xC218;&#xC6A9; &#xCCB4;&#xC5D0; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xAE00;&#xADC0; &#xB54C;&#xBB38;&#xC5D0; &#xC8FC;&#xB85C; DC, &#xB9C8;&#xC6B0;&#xC2A4;&#xC5D0;. &#xB610;&#xD55C;, Ad5:CaPi-&#xB300;&#xC6B0; DCs cd40&#xACFC; CD86, &#xC131;&#xC219; &#xD45C;&#xBA74; &#xBD84;&#xC790;&#xB97C; &#xD45C;&#xD604; &#xD558; &#xACE0; proinflammatory cytokines &#xC885;&#xC591; &#xAD34; &#xC0AC; &#xC778;&#xC790;-&#xC54C;&#xD30C;&#xC640; &#xC778;&#xD130; &#xB8E8; &#xD0A8; 6 &#xBD84; &#xBE44;&#xB97C; &#xD65C;&#xC131;&#xD654; &#xD588;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; DC &#xBCC0;&#xD658;&#xB3C4; &#xC131;&#xC219; &#xC138;&#xD3EC; &#xD45C;&#xBA74; integrin&#xC640; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0; &#xC758;&#xC874; &#xD588;&#xB2E4;. Ad5:CaPi &#xB610;&#xD55C; &#xC778;&#xAC04;&#xC758; DC Ad5 &#xD63C;&#xC790;, &#xBE44;&#xC2B7;&#xD55C; CD46 &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615;&#xB41C; &#xBCA1;&#xD130; (Ad5f35) &#xBCF4;&#xB2E4; &#xB354; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xBD88;&#xB9AC;&#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;,&#xC774; &#xC774;&#xB807;&#xAC8C; adenoviral &#xC911;&#xC7AC; endosomal &#xD0C8;&#xCD9C; &#xD575; &#xC218;&#xC6A9; &#xCCB4; &#xB3C5;&#xB9BD; nonviral &#xC720;&#xC804;&#xC790; &#xC804;&#xB2EC;&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4; &#xD6A8;&#xC728;&#xC131;&#xC744; &#xACB0;&#xD569; &#xD569;&#xB2C8;&#xB2E4;. &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC740;, CaPi co-precipitation &#xAE30;&#xB2A5;&#xC801;&#xC73C;&#xB85C; DC&#xB97C; &#xD65C;&#xC131;&#xD654; &#xD558; &#xACE0; &#xC138;&#xD3EC; &#xD2B9;&#xC815; &#xBA54;&#xBAA8;&#xB9AC; cytotoxic T &#xC138;&#xD3EC; &#xD655;&#xC7A5;&#xC744; &#xC218;&#xC815; &#xD558;&#xB824;&#xBA74; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xB294; &#xC784;&#xC0C1; &#xC785;&#xC591; immunotherapy &#xC9C0;&#xC6D0; &#xD604;&#xC7AC; Ad5 &#xC720;&#xC804;&#xC790; &#xC774;&#xB3D9;&#xC758; &#xD6A8;&#xB2A5;&#xC744; &#xAC1C;&#xC120; &#xD558;&#xAE30; &#xC704;&#xD574; &#xAC04;&#xB2E8;&#xD55C; &#xAE30;&#xC220;&#xC744; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

&#xC544; &#xB370; &#xB178; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xCE7C;&#xC298; &#xC778;&#xC0B0; &#xC5FC; co-precipitates&#xC640; &#xC720;&#xC804;&#xC790; &#xC774;&#xB3D9; &#xD6C4; &#xC218;&#xC9C0;&#xC0C1; &#xC138;&#xD3EC; &#xAE30;&#xB2A5;.

Las c&eacute;lulas dendr&iacute;ticas (DCs) son esenciales para iniciar y dirigir las respuestas de T-cell antigen-specific. La modificaci&oacute;n gen&eacute;tica de DC es bajo estudio para inmunoterapia del c&aacute;ncer, desarrollo de vacunas y orientada por el ant&iacute;geno de la inmunosupresi&oacute;n. Tipo de adenovirus (Ad) 5 (Ad5)-transferencia de genes mediada Mouse DCs de la m&eacute;dula &oacute;sea y DCs derivados de monocitos humanos es ineficiente porque ni expresar el receptor cognado de anuncios de alta afinidad. Mostramos que co-precipitating vectores adenoviral con fosfato de calcio (CaPi) mayor expresi&oacute;n g&eacute;nica (2000-fold) y la eficacia de transducci&oacute;n (ha) en rat&oacute;n DC, principalmente debido a la absorci&oacute;n viral independiente del receptor. Por otra parte, Ad5:CaPi-DCs tratados fueron activados para expresar las mol&eacute;culas superficiales de maduraci&oacute;n CD40 y CD86 y a secretar citoquinas proinflamatorias factor de necrosis tumoral alfa e interleucina 6. Sin embargo, transducci&oacute;n DC ni maduraci&oacute;n era dependiente en las interacciones de la prote&iacute;na viral con la integrina superficial de la c&eacute;lula. Ad5:CaPi transduced tambi&eacute;n DC humano m&aacute;s eficientemente que Ad5 solo, similar a un vector gen&eacute;ticamente modificado (Ad5f35) dirigido al receptor CD46. Por lo tanto, este enfoque combina la eficacia de escape endosomal adenoviral-mediada y trata con la independencia del receptor de nonviral genes nucleares. Lo importante, CaPi co-precipitaci&oacute;n podr&iacute;a usarse para modificar funcionalmente DC para activar y ampliar los linfocitos T citot&oacute;xicos memoria espec&iacute;fica del citomegalovirus. Este estudio identifica una t&eacute;cnica sencilla para mejorar la eficacia de la actual transferencia g&eacute;nica Ad5, en apoyo de la cl&iacute;nica de inmunoterapia adoptiva.

Funci&oacute;n de las c&eacute;lulas dendr&iacute;ticas despu&eacute;s de la transferencia de genes con adenovirus-calcio fosfato co-precipitates.

Helper-dependent adenoviral vectors (HDAds) are attractive for liver-directed gene therapy because they can mediate long-term, high-level transgene expression without chronic toxicity. However, systemic delivery requires high vector doses for efficient hepatic transduction, resulting in dose-dependent acute toxicity. Clearly, strategies to improve hepatic transduction with low vector doses are needed. In this regard, we have previously shown that hydrodynamic injection of helper-dependent adenoviral vectors into mice results in increased hepatic transduction, reduced systemic vector dissemination, and reduced pro-inflammatory cytokines compared with conventional injection and thus has the potential to improve dramatically the therapeutic index of helper-dependent adenoviral vectors. Unfortunately, the rapid, large-volume injection used in this method cannot be applied to larger animals. Therefore, we have developed a novel balloon occlusion catheter-based method to mimic hydrodynamic injection of helper-dependent adenoviral vectors into non-human primates that does not require rapid, large-volume injection. Using a low, clinically relevant vector dose, this minimally invasive method results in high-efficiency hepatic transduction with minimal toxicity and stable long-term transgene expression for at least 413 days.

Pseudo-hydrodynamic delivery of helper-dependent adenoviral vectors into non-human primates for liver-directed gene therapy.

To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors. Genes that maintain cellular structure or homeostasis, referred to as housekeeping genes, or 18S ribosomal RNA are often used for this purpose. Although unstable or inconsistent housekeeping gene expression will misrepresent experimental effects on target gene expression, housekeeping genes are often chosen arbitrarily rather than systematically. We designed a simple and systematic approach towards selection of housekeeping genes based on Ct variance (as reflected by the standard deviation) and normality of distribution. We validated this approach by comparing stability and consistency of expression of 11 housekeeping genes across different types of cells, experimental treatments, and human donors. Finally, we demonstrated the consequences of inconsistent housekeeping gene expression on the calculation of target gene expression, and conclude that validation of stability of housekeeping gene expression by considering both distribution normality and standard deviation is straightforward and critical for proper experimental design.

Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.

&#x6700;&#x8FD1;&#x7684;&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x5173;&#x8054;&#x7814;&#x7A76;&#x8868;&#x660E;&#x4E0D;&#x540C;&#x7684;&#x89D2;&#x8272;&#xFF0C;&#x4E3A; 12 &#x4EBA;&#x5E72;&#x6270;&#x7D20; (&#x5E72;&#x6270;&#x7D20;-&alpha;) &#x548C; 3 &#x5E72;&#x6270;&#x7D20; &lambda; &#x4E9A;&#x578B;&#xFF0C;&#x53EF;&#x901A;&#x8FC7;&#x5B9A;&#x4E49;&#x8FD9;&#x4E9B;&#x7EC4;&#x7684;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x6765;&#x9610;&#x660E;&#x3002;&#x4E3A;&#x4E86;&#x514B;&#x670D;&#x4E4B;&#x95F4;&#x6BCF;&#x96C6;&#x9AD8;&#x540C;&#x6E90;&#x6027;&#x7684;&#x969C;&#x788D;&#xFF0C;&#x6211;&#x4EEC;&#x8BBE;&#x8BA1;&#x91C7;&#x7528;&#x5206;&#x5B50;&#x4FE1;&#x6807;&#x548C;&#x9501;&#x7684;&#x6838;&#x9178; (&#x4F4E;&#x566A;&#x58F0;&#x653E;&#x5927;&#x5668;) &#x63A2;&#x6D4B;&#x5668;&#xFF0C;&#x4EE5;&#x53CA;&#x5728;&#x67D0;&#x4E9B;&#x60C5;&#x51B5;&#x4E0B;&#xFF0C;&#x4F4E;&#x566A;&#x58F0;&#x653E;&#x5927;&#x5668;&#x5BE1;&#x6838;&#x82F7;&#x9178;&#x6291;&#x5236;&#x5242;&#x7684;&#x5B9A;&#x91CF;&#x5B9E;&#x65F6; pcr&#x3002;&#x7136;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x6D4B;&#x91CF;&#x5E72;&#x6270;&#x7D20;&#x5B50;&#x7C7B;&#x578B;&#x8868;&#x8FBE;&#x4EBA;&#x7C7B;&#x5916;&#x5468;&#x8840;&#x5355;&#x4E2A;&#x6838;&#x7EC6;&#x80DE;&#x548C;&#x7EAF;&#x51C0;&#x7684;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x3001; &#x6811;&#x7A81;&#x72B6;&#x7EC6;&#x80DE; (mDC)&#x3001; &#x6D46;&#x7EC6;&#x80DE;&#x6837;&#x6811;&#x7A81;&#x72B6;&#x7EC6;&#x80DE; (pDC) &#x548C;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x6E90;&#x6027;&#x5DE8;&#x566C;&#x7EC6;&#x80DE; (MDM) &#x548C;-&#x6811;&#x7A81;&#x72B6;&#x7EC6;&#x80DE; &#xFF08;&#x6811;&#x7A81;) &#x4FDD;&#x5229;&#x56DE;&#x5E94;&#x6211;&#xFF1A; C&#x3001; &#x8102;&#x591A;&#x7CD6; (LPS)&#x3001; &#x54AA;&#x55B9;&#x83AB;&#x7279;&#x548C; CpG &#x5BE1;&#x6838;&#x82F7;&#x9178;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#xFF0C;&#x9488;&#x5BF9;&#x4FDD;&#x5229;&#x6211;&#xFF1A; &#x5B50;&#x7C7B;&#x578B;&#x6A21;&#x5F0F;&#x4E3B;&#x8981;&#x9650;&#x4E8E;&#x5E72;&#x6270;&#x7D20; &beta; &#x548C;&#x5E72;&#x6270;&#x7D20; &lambda;1 C &#x548C;&#x8102;&#x591A;&#x7CD6;&#xFF0C;&#x5355;&#x6838;&#x7EC6;&#x80DE;&#x7684; MDM &#x548C;&#x6811;&#x7A81;&#x5FEB;&#x9012;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x540C;&#x65F6;&#x5F15;&#x8D77;&#x4E86;&#x4E2D;&#x592E;&#x4EBA;&#x6C11;&#x653F;&#x5E9C;&#x8868;&#x8FBE;&#x6240;&#x6709;&#x7684; I &#x578B;&#x5E72;&#x6270;&#x7D20;&#x5B50;&#x7C7B;&#x578B;&#x7531; pDC&#x3001; &#x54AA;&#x55B9;&#x83AB;&#x7279;&#x5374;&#x6CA1;&#x6709;&#x8FD9;&#x6837;&#x505A;&#x3002;&#x6B64;&#x5916;&#xFF0C;MDM &#x548C; mDC &#x7684;&#x9AD8;&#x6548;&#x8868;&#x8FBE;&#x5E72;&#x6270;&#x7D20;-&lambda; &#x548C;&#x4E9A;&#x578B;&#x7684;&#x5E72;&#x6270;&#x7D20; &lambda; &#x5E72;&#x6270;&#x7D20;-&lambda;2&#xFF0C;&#x7136;&#x540E;&#x662F;&#x5E72;&#x6270;&#x7D20; &lambda;3 &#x6309;&#x5C42;&#x6B21;&#x7ED3;&#x6784;&#x8868;&#x793A;&#x5E72;&#x6270;&#x7D20; &lambda;1 &#x9075;&#x5FAA;&#x7684;&#x987A;&#x5E8F;&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x652F;&#x6301;&#x534F;&#x8C03;&#x4E00;&#x81F4;&#x7684;&#x5355;&#x5143;&#x683C;&#x548C;&#x914D;&#x4F53;&#x7279;&#x5B9A;&#x8868;&#x8FBE;&#x5F0F;&#x7684;&#x7C7B;&#x578B;&#x7684;&#x6A21;&#x578B;&#xFF0C;&#x6211;&#x548C;&#x4E09;&#x5E72;&#x6270;&#x7D20;&#x3002;&#x5728;&#x5404;&#x79CD;&#x4E0A;&#x4E0B;&#x6587;&#x4E2D;&#x5B9A;&#x4E49;&#x5E72;&#x6270;&#x7D20;&#x5B50;&#x7C7B;&#x578B;&#x8868;&#x8FBE;&#x8C31;&#x53EF;&#x6F84;&#x6E05;&#x4E9A;&#x578B;&#x5E72;&#x6270;&#x7D20;&#x4F5C;&#x4E3A;&#x9632;&#x62A4;&#x3001; &#x6CBB;&#x7597;&#x6216;&#x81F4;&#x75C5;&#x7684;&#x8C03;&#x89E3;&#x5458;&#x7684;&#x7279;&#x6B8A;&#x4F5C;&#x7528;&#x3002;

&#x4EBA;&#x4F53;&#x5E72;&#x6270;&#x7D20; &alpha; &#x5E72;&#x6270;&#x7D20; lambda &#x4E9A;&#x578B;&#x7684;&#x8868;&#x8FBE;&#x8C31;&#x662F;&#x914D;&#x4F53;&#x548C;&#x4F9D;&#x8D56;&#x4E8E;&#x5355;&#x5143;&#x683C;&#x7684;&#x3002;

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-Î±) and 3 IFN-Î» subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-Î² and IFN-Î»1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-Î», and the subtypes of IFN-Î» are expressed hierarchically in the order IFN-Î»1 followed by IFN-Î»2, and then IFN-Î»3. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent.

Des &eacute;tudes r&eacute;centes de genome-wide association sugg&egrave;rent des r&ocirc;les distincts pour 12 humain interf&eacute;ron-alpha (IFN-&alpha;) et IFN-&lambda; 3 sous-types qui peut &ecirc;tre &eacute;lucid&eacute; en d&eacute;finissant les patrons d&#39;expression de ces g&egrave;nes. Pour surmonter l&#39;obstacle de la forte homologie entre chacun des ensembles, nous avons con&ccedil;u une quantitative PCR en temps r&eacute;el qui incorpore l&#39;utilisation des phares mol&eacute;culaires et des sondes d&#39;acide nucl&eacute;ique verrouill&eacute; (LNA) et dans certains cas, les inhibiteurs d&#39;oligonucl&eacute;otide de LNA. Nous avons ensuite mesur&eacute; IFN sous-type expression par les cellules mononucl&eacute;aires de sang p&eacute;riph&eacute;rique humain purifi&eacute;s des monocytes, cellules dendritiques my&eacute;lo&iuml;des (mDC), plasmacytoides (pDC), les cellules dendritiques et macrophages d&eacute;riv&eacute;s de monocytes (MDM) et - les cellules dendritiques (CDMD) en r&eacute;ponse &agrave; poly je: C, lipopolysaccharide (LPS), imiquimod et oligonucl&eacute;otides CpG. Nous avons constat&eacute; qu&#39;en r&eacute;ponse &agrave; poly je: C et LPS, monocytes, MDM et CDMD express un motif de sous-type limit&eacute; principalement &agrave; l&#39;IFN-&beta; et IFN-&lambda;1. En outre, tandis que l&#39;expression de CpG a suscit&eacute; de tout type je sous-types IFN de pDC, imiquimod n&#39;a pas. En outre, MDM et mDC hautement expriment IFN-&lambda; et les sous-types d&#39;IFN-&lambda; sont exprim&eacute;s hi&eacute;rarchiquement dans l&#39;ordre IFN-&lambda;1 suivie par IFN-&lambda;2, puis IFN-&lambda;3. Ces donn&eacute;es prennent en charge un mod&egrave;le d&#39;expression coordonn&eacute;e et ligand-sp&eacute;cifiques des cellules de types I et III IFN. D&eacute;finition des profils d&#39;expression IFN sous-type dans une vari&eacute;t&eacute; de contextes peut &eacute;lucider des r&ocirc;les sp&eacute;cifiques pour les sous-types de IFN comme m&eacute;diateurs protectrices, th&eacute;rapeutiques ou pathog&egrave;nes.

Profils d&#39;expression de l&#39;interf&eacute;ron-alpha humain et sous-types de l&#39;interf&eacute;ron-lambda sont ligand et d&eacute;pendant des lymphocytes.

Neuere genomweiten Verband-Studien legen nahe, unterschiedliche Rollen f&uuml;r 12 menschlichen Interferon-Alpha (IFN-&alpha;) und 3 IFN-&lambda; Subtypen, die durch die Definition der Ausdrucksmuster dieser Gruppen von Genen aufgekl&auml;rt werden kann. Um die Behinderung durch hohe Homologie zwischen den einzelnen Sets zu &uuml;berwinden, haben wir eine quantitative Echtzeit-PCR-Assay, das beinhaltet die Verwendung von molekularen Leuchtfeuer und verschlossene Nukleins&auml;ure (LNA) Sonden, und in einigen F&auml;llen LNA-Oligonukleotid-Hemmer entwickelt. Wir messen dann IFN Untertyp Ausdruck von Menschenblut peripheren mononukle&auml;ren Zellen und gereinigten Monozyten, myeloide dendritische Zellen (mDC), Plasmacytoid von dendritischen Zellen (pDC), und Monozyte abgeleiteten Makrophagen (MDM) und -dendritische Zellen (MDDC) als Reaktion auf Poly ich: C, Lipopolysaccharid (LPS), Imiquimod und CpG-Oligonukleotiden. Wir fanden, dass als Reaktion auf Poly ich: C und LPS, Monozyten, MDM und MDDC Express eine Subtyp-Muster in erster Linie auf IFN-&beta; und IFN-&lambda;1 beschr&auml;nkt. Dar&uuml;ber hinaus w&auml;hrend der CpG entlockt Ausdruck aller Typ I IFN-Subtypen von pDC, Imiquimod nicht. Dar&uuml;ber hinaus MDM und mDC sehr schnelles IFN-&lambda; und die Untertypen von IFN-&lambda; werden in der Reihenfolge, die IFN-&lambda;1 gefolgt von IFN-&lambda;2 und IFN-&lambda;3 hierarchisch ausgedr&uuml;ckt. Diese Daten unterst&uuml;tzen ein Modell f&uuml;r die koordinierte und Ligand-zellspezifische Ausdruck der Typen I und III IFN. Definieren von IFN Untertyp Ausdruck Profilen in einer Vielzahl von Kontexten kann bestimmte Rollen f&uuml;r IFN-Subtypen als Schutz-, therapeutische oder pathogenen Vermittler erhellen.

Ausdruck profile menschlichen Interferon-Alpha und Interferon-Lambda-Untertypen sind Ligand und Zelle-abh&auml;ngige.

Recenti studi di associazione genome-wide suggeriscono ruoli distinti per 12 umana interferone-alfa (IFN-&alpha;) e 3 IFN-&lambda; subtypes che pu&ograve; essere chiarita definendo il pattern di espressione di questi insiemi di geni. Per superare l&#39;impedimento di elevata omologia tra ciascuno dei set, abbiamo progettato un dosaggio quantitativo PCR in tempo reale che incorpora l&#39;utilizzo di sonde di acido nucleico locked (LNA) e molecular beacon ed in alcuni casi, gli inibitori del oligonucleotide LNA. Abbiamo quindi misurato IFN sottotipo espressione di cellule mononucleari di sangue periferico umano purificati monociti, cellule dendritiche mieloidi (mDC), plasmacytoid (pDC), le cellule dendritiche e macrofagi monocyte-derivati (MDM) e - cellule dendritiche (MDDC) in risposta a poly sono: C, lipopolisaccaride (LPS), imiquimod e oligonucleotidi CpG. Abbiamo trovato che in risposta a poly sono: C e LPS, monociti, MDM e MDDC express un pattern di sottotipo limitato principalmente a IFN-&beta; e IFN-&lambda;1. Inoltre, mentre CpG manifesta espressione di tutti i tipo I sottotipi IFN da pDC, imiquimod non ha fatto. Inoltre, MDM e mDC altamente esprimono IFN-&lambda; e i sottotipi di IFN-&lambda; sono espressi in ordine gerarchico nell&#39;ordine di IFN-&lambda;1 seguita da IFN-&lambda;2 e &lambda;3 IFN. Questi dati supportano un modello di espressione di specifiche cellule e ligando coordinato dei tipi I e III IFN. Definizione di profili di espressione del sottotipo IFN in una variet&agrave; di contesti pu&ograve; chiarire ruoli specifici per i sottotipi di IFN come mediatori protettivi, terapeutici o patogeni.

Profili di espressione di interferone-alfa e l&#39;interferone-lambda sottotipi sono ligando e cellula-dipendente.

&#x6700;&#x8FD1;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x30EF;&#x30A4;&#x30C9;&#x95A2;&#x9023;&#x7814;&#x7A76;&#x304A;&#x52E7;&#x3081; 12 &#x4EBA;&#x9593;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30ED;&#x30F3;-&alpha; (IFN-&alpha;) &#x306E;&#x7570;&#x306A;&#x308B;&#x5F79;&#x5272;&#x3068; 3 IFN &lambda; &#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7;&#x306F;&#x3053;&#x308C;&#x3089;&#x306E;&#x30BB;&#x30C3;&#x30C8;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x89E3;&#x660E;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x5404;&#x30BB;&#x30C3;&#x30C8;&#x306E;&#x9593;&#x3067;&#x9AD8;&#x3044;&#x76F8;&#x540C;&#x6027;&#x306E;&#x969C;&#x5BB3;&#x3092;&#x514B;&#x670D;&#x3059;&#x308B;&#x3068;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30A4;&#x30F3;&#x30B9;&#x30BF;&#x30F3;&#x30B9;&#x306F;&#x3001;LNA &#x306E;&#x30AA;&#x30EA;&#x30B4;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x306E;&#x6291;&#x5236;&#x5264;&#x5206;&#x5B50;&#x306E;&#x30D3;&#x30FC;&#x30B3;&#x30F3;&#x3068;&#x30ED;&#x30C3;&#x30AF;&#x3055;&#x308C;&#x305F;&#x6838;&#x9178; (LNA) &#x306E;&#x30D7;&#x30ED;&#x30FC;&#x30D6;&#x306E;&#x4F7F;&#x7528;&#x304C;&#x7D44;&#x307F;&#x8FBC;&#x307E;&#x308C;&#x3066;&#x3001;&#x5B9A;&#x91CF;&#x7684;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0; PCR &#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3092;&#x8A2D;&#x8A08;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x305D;&#x306E;&#x5F8C; IFN &#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7;&#x767A;&#x73FE;&#x7CBE;&#x88FD;&#x5358;&#x7403;, &#x9AA8;&#x9AC4;&#x7CFB;&#x6A39;&#x72B6;&#x7D30;&#x80DE; (mDC), &#x5F62;&#x8CEA;&#x7D30;&#x80DE;&#x69D8;&#x3067;&#x3072;&#x3068;&#x672B;&#x68A2;&#x8840;&#x5358;&#x6838;&#x7D30;&#x80DE;&#x306B;&#x3088;&#x308A;&#x6E2C;&#x5B9A;&#x3057;&#x305F;&#x6A39;&#x72B6;&#x7D30;&#x80DE; (pDC) &#x3068;&#x5358;&#x7403;&#x7531;&#x6765;&#x30DE;&#x30AF;&#x30ED;&#x30D5;&#x30A1;&#x30FC;&#x30B8; (MDM) &#x3068; - &#x6A39;&#x72B6;&#x7D30;&#x80DE; (MDDC) &#x30DD;&#x30EA;&#x306B;&#x5FDC;&#x3048;&#x3066;&#x79C1;&#xFF1A; C &#x30EA;&#x30DD;&#x30DD;&#x30EA;&#x30B5;&#x30C3;&#x30AB;&#x30E9;&#x30A4;&#x30C9; (LPS)&#x3001;imiquimod&#x3001;CpG &#x30AA;&#x30EA;&#x30B4;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x3002;&#x6211;&#x3005; &#x30DD;&#x30EA; &#x30EC;&#x30B9;&#x30DD;&#x30F3;&#x30B9;&#x305D;&#x306E;&#x3067;&#x304C;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x3057;&#x305F;&#x79C1;&#x306F;: &#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7; &#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x5236;&#x9650;&#x3042;&#x308A; IFN &beta; &#x304A;&#x3088;&#x3073; IFN &lambda;1 &#x3092;&#x4E3B;&#x306B; C &#x304A;&#x3088;&#x3073; LP&#x3001;&#x5358;&#x7403;&#x3001;MDM &#x3068; MDDC &#x30A8;&#x30AF;&#x30B9;&#x30D7;&#x30EC;&#x30B9;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x306A;&#x304C;&#x3089;&#x8A98;&#x767A; CpG &#x5F0F;&#x3059;&#x3079;&#x3066;&#x306E; I &#x578B; IFN &#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7; pDC &#x306B;&#x3088;&#x3063;&#x3066;&#x3001;imiquimod &#x3092;&#x3067;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;MDM &#x3068; mDC &#x9AD8; IFN &lambda; &#x3092;&#x8868;&#x73FE;&#x3057;&#x3001;IFN-&lambda;2 &#x304A;&#x3088;&#x3073; [IFN &lambda;3 &#x306B;&#x3088;&#x3063;&#x3066;&#x3001;IFN &lambda; &#x306E;&#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7;&#x306F;&#x3001;&#x968E;&#x5C64;&#x7684;&#x306B; IFN &lambda;1 &#x7D9A;&#x3044;&#x3066;&#x9806;&#x756A;&#x3092;&#x8868;&#x73FE;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x3057;&#x3001;III IFN &#x5354;&#x8ABF;&#x7D30;&#x80DE;&#x3068;&#x30EA;&#x30AC;&#x30F3;&#x30C9;&#x7279;&#x7570;&#x767A;&#x73FE;&#x578B;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;IFN &#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7;&#x767A;&#x73FE;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x306F;&#x3001;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x3067;&#x5B9A;&#x7FA9;&#x3059;&#x308B; IFN &#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7;&#x3092;&#x7279;&#x5B9A;&#x306E;&#x30ED;&#x30FC;&#x30EB;&#x4FDD;&#x8B77;&#x3001;&#x6CBB;&#x7642;&#x307E;&#x305F;&#x306F;&#x75C5;&#x539F;&#x6027;&#x30E1;&#x30C7;&#x30A3;&#x30A8;&#x30FC;&#x30BF;&#x30FC;&#x3068;&#x3057;&#x3066;&#x89E3;&#x660E;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x767A;&#x73FE;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x4EBA;&#x9593;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30ED;&#x30F3;-&alpha; &#x3068;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30ED;&#x30F3; &#x30E9;&#x30E0;&#x30C0; &#x30B5;&#x30D6;&#x30BF;&#x30A4;&#x30D7;&#x306E;&#x30EA;&#x30AC;&#x30F3;&#x30C9;&#x3068;&#x30BB;&#x30EB;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x307E;&#x3059;&#x3002;

&#xCD5C;&#xADFC; &#xAC8C;&#xB188; &#xB113;&#xC740; &#xD611;&#xD68C; &#xC5F0;&#xAD6C; 12 &#xC778;&#xAC04;&#xC758; &#xC778;&#xD130;&#xD398;&#xB860;-&#xC54C;&#xD30C; (IFN-&alpha;)&#xC5D0; &#xB300; &#xD55C; &#xB69C;&#xB837;&#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC548; &#xD558; &#xACE0; 3 IFN-&lambda; subtypes&#xC744; &#xC720;&#xC804;&#xC790;&#xC758; &#xC774;&#xB7EC;&#xD55C; &#xC9D1;&#xD569;&#xC758; &#xD45C;&#xD604; &#xD328;&#xD134;&#xC744; &#xC815;&#xC758; &#xD558; &#xC5EC; &#xD574;&#xBA85; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xAC01; &#xC9D1;&#xD569; &#xAC04;&#xC758; &#xB192;&#xC740; homology &#xC7A5;&#xC560;&#xB97C; &#xADF9;&#xBCF5; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC6B0;&#xB9AC;&#xB294; &#xBD84;&#xC790; &#xBE44;&#xCF58; &#xBC0F; &#xD504;&#xB85C;&#xBE0C; &#xC7A0;&#xAE34;&#xB41C; &#xD575; &#xC0B0; (LNA), &#xADF8;&#xB9AC;&#xACE0; &#xACBD;&#xC6B0;&#xC5D0; &#xB530;&#xB77C;&#xC11C;&#xB294; LNA oligonucleotide &#xC5B5;&#xC81C;&#xC81C; &#xC0AC;&#xC6A9; &#xD1B5;&#xD569; &#xC815;&#xB7C9; &#xC2E4;&#xC2DC;&#xAC04; PCR &#xC2DC;&#xD5D8;&#xC744; &#xC124;&#xACC4; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xB2E4;&#xC74C; IFN &#xD558;&#xC704; &#xC2DD;&#xC744; &#xC778;&#xAC04;&#xC758; &#xB9D0; &#xCD08; &#xD608;&#xC561; &#xB2E8; &#xC138;&#xD3EC; &#xACE0; &#xC815;&#xD654; monocytes, &#xACE8;&#xC218;&#xC131; &#xC218;&#xC9C0;&#xC0C1; &#xC138;&#xD3EC; (mDC), plasmacytoid&#xC5D0; &#xC758;&#xD574; &#xCE21;&#xC815; &#xB41C; &#xC218;&#xC9C0;&#xC0C1; &#xC138;&#xD3EC; (pDC) &#xBC0F; monocyte &#xD30C;&#xC0DD; &#xB41C; &#xB300; &#xC2DD; &#xC138;&#xD3EC; (MDM) &#xBC0F;-&#xD3F4; &#xB9AC;&#xC5D0; &#xB300; &#xD55C; &#xC751;&#xB2F5;&#xC5D0;&#xC11C; &#xC218;&#xC9C0;&#xC0C1; &#xC138;&#xD3EC; (MDDC) &#xB098;: C, lipopolysaccharide (LPS), imiquimod &#xBC0F; &#xD3EC;&#xC7A5; &#xC18C;&#xBE44;&#xC7AC; oligonucleotides. &#xC751;&#xB2F5; &#xD3F4; &#xB9AC;&#xC5D0; &#xADF8;&#xB97C; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB098;: C&#xC640; LPS, monocytes, MDM &#xBC0F; MDDC &#xC775;&#xC2A4;&#xD504;&#xB808;&#xC2A4; &#xD558;&#xC704; &#xD328;&#xD134; IFN-&beta;&#xC640; IFN-&lambda;1&#xC73C;&#xB85C; &#xC8FC;&#xB85C; &#xC81C;&#xD55C;. &#xB610;&#xD55C;, &#xBAA8;&#xB4E0; &#xD3EC;&#xC7A5; &#xC18C;&#xBE44;&#xC7AC; elicited &#xC2DD;&#xC740; &#xB3D9;&#xC548; &#xC720;&#xD615; I IFN &#xD558;&#xC704; Pdc&#xC5D0; &#xC758;&#xD574;, imiquimod &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4;. &#xB610;&#xD55C;, MDM &#xBC0F; mDC &#xB192;&#xC740; IFN-&lambda;, &#xC775;&#xC2A4;&#xD504;&#xB808;&#xC2A4;&#xC640; IFN-&lambda;&#xC758; &#xD558;&#xC704; IFN-&lambda;2, &#xADF8;&#xB9AC;&#xACE0; IFN-&lambda;3&#xC5D0; &#xC758;&#xD574; IFN-&lambda;1 &#xB2E4;&#xC74C; &#xC21C;&#xC11C;&#xC5D0; &#xB530;&#xB77C; &#xACC4;&#xCE35;&#xC801;&#xC73C;&#xB85C; &#xD45C;&#xD604; &#xB429;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; I &#xBC0F; III IFN &#xC870;&#xC815;&#xB41C; &#xC140; &#xBC0F; &#xB9AC;&#xAC04;&#xB4DC; &#xD2B9;&#xC815; &#xC2DD; &#xD615;&#xC2DD;&#xC758; &#xBAA8;&#xB378;&#xC744; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;. &#xB2E4;&#xC591; &#xD55C; &#xC0C1;&#xD669;&#xC5D0;&#xC11C;&#xC5D0;&#xC11C; IFN &#xD558;&#xC704; &#xC2DD; &#xD504;&#xB85C;&#xD544; &#xC815;&#xC758; &#xBCF4;&#xD638;, &#xCE58;&#xB8CC; &#xB610;&#xB294; &#xBCD1;&#xC6D0; &#xC131; &#xC911;&#xC7AC;&#xC790;&#xB85C; IFN &#xD558;&#xC704; &#xC720;&#xD615;&#xC5D0; &#xB300; &#xD55C; &#xD2B9;&#xC815; &#xC5ED;&#xD560;&#xC744; &#xBA85;&#xB8CC; &#xD558; &#xAC8C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC778;&#xAC04;&#xC758; &#xC778;&#xD130;&#xD398;&#xB860;-&#xC54C;&#xD30C; &#xBC0F; &#xC778;&#xD130;&#xD398;&#xB860; &#xB78C;&#xB2E4; &#xD558;&#xC704; &#xC2DD; &#xD504;&#xB85C;&#xD544;&#xC740; &#xB9AC;&#xAC04;&#xB4DC; &#xBC0F; &#xC138;&#xD3EC; &#xC885;&#xC18D;.

Estudios de Asociaci&oacute;n de genoma recientes sugieren diferentes roles para 12 humano interfer&oacute;n-alfa (IFN-&alpha;) y &lambda;-IFN 3 subtipos que puede ser aclarado definiendo los patrones de expresi&oacute;n de estos conjuntos de genes. Para superar el impedimento de alta homolog&iacute;a entre cada uno de los conjuntos, hemos dise&ntilde;ado un ensayo PCR cuantitativo en tiempo real que incorpora el uso de marcador molecular y sondas de &aacute;cido nucleico bloqueado (LNA) y en algunos casos, inhibidores de oligonucle&oacute;tidos LNA. Luego medimos IFN subtipo expresi&oacute;n por las c&eacute;lulas mononucleares de sangre perif&eacute;rica por purificada monocitos, c&eacute;lulas dendr&iacute;ticas mieloides (mDC), plasmacytoid (pDC), las c&eacute;lulas dendr&iacute;ticas y macr&oacute;fagos derivados de monocitos (MDM) y - las c&eacute;lulas dendr&iacute;ticas (MDDC) en respuesta a poly me: C, lipopolisac&aacute;rido (LPS), imiquimod y oligonucle&oacute;tidos CpG. Encontramos que en respuesta a poly me: C y LPS, monocitos, MDM y MDDC express un patr&oacute;n de subtipo limitado principalmente a IFN-&beta; y del IFN-&lambda;1. Adem&aacute;s, mientras que la expresi&oacute;n de CpG sacado de todo tipo I subtipos IFN por el pDC, imiquimod no. Adem&aacute;s, MDM y mDC altamente expresan IFN-&lambda;, y los subtipos de IFN-&lambda; expresan jer&aacute;rquico en el orden de IFN-&lambda;1 seguido por IFN-&lambda;2 y luego IFN-&lambda;3. Estos datos apoyan un modelo de expresi&oacute;n espec&iacute;ficos de la c&eacute;lula y ligando coordinada de tipos I y III IFN. Definici&oacute;n de perfiles de expresi&oacute;n de subtipo IFN en una variedad de contextos puede aclarar funciones espec&iacute;ficas para los subtipos IFN como mediadores de protecci&oacute;n, terap&eacute;uticas o pat&oacute;genos.

Perfiles de expresi&oacute;n de interfer&oacute;n-alfa humana y subtipos de interfer&oacute;n-lambda son dependientes de ligando y celular.

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.

Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors.

Thiolated polymers containing disulfide linkages are commonly researched in gene delivery with the assumption that the thiolated complexes form disulfide bonds. This study investigates the extent of disulfide linking in a thiol-containing polymer and determines the impact that free thiols have on the polymer's delivery potential. A fluorescent cationic polymer containing thiol pendant chains was prepared from poly(allylamine) and 2-iminothiolate (Traut's reagent). Polymer fluorescence was determined by UV plate readings and fluorescent microscopy. Transfection efficiency and cytotoxicity were assessed in MCF-7 breast cancer cells. Results show that thiolated polymers exhibited fluorescence at ex/em ∼595/620. Fluorescent measurements, microscopy imaging, and DNA electrophoresis show that thiolated polymers are not internalized by cells in a culture, yet, they bind to the cell surface, perhaps valuable for applications requiring cell adhesion.

Article	Year
Lifelong elimination of hyperbilirubinemia in the Gunn rat with a single injection of helper-dependent adenoviral vector. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15753292	2005
Dendritic cell function after gene transfer with adenovirus-calcium phosphate co-precipitates. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 17235318	2007
Pseudo-hydrodynamic delivery of helper-dependent adenoviral vectors into non-human primates for liver-directed gene therapy. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 17285138	2007
Systematic method for determining an ideal housekeeping gene for real-time PCR analysis. Journal of biomolecular techniques : JBT\| PubMed ID: 19183798	2008
Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent. Immunology and cell biology\| PubMed ID: 22249201	2012
Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors. Biomacromolecules\| PubMed ID: 22515194	2012
Investigating polymer thiolation in gene delivery. Journal of biomaterials science. Polymer edition\| PubMed ID: 23647248	2013

Viraj P. Mane

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