Wir danken Drs. R. Jude Samulski und Xiao Xiao für ihre Sachspenden von rekombinanten AAV3 und AAV-EGFP-Plasmiden, bzw., und Dr. Gail Tomlinson für die großzügige Bereitstellung von Hep293TT Zellen. Diese Arbeit wurde teilweise durch Public Health Service gewähren Zuschüsse R01 HL-076901, R01 HL-097088 und P01 DK-058327 (Projekt 1) von den National Institutes of Health (AS) unterstützt.

1. Verpackung von rekombinanten Adeno-assoziierten Virus Serotyp 3 (rAAV3) Vektoren <ol><li> Besuchen Sie die Website: &quot; <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> &quot;und berechnen Sie die DNA-Menge. </li><li> Pre-Mix Plasmide pHelper (Adenovirus Helfer), pACG2c3 (AAV-Helfer) und pdsAAV-CB-EGFP (rAAV-Vektor) in Medium ohne FBS und Antibiotika. Das Gesamtvolumen sollte 8.750 ul für zehn 15 cm 2 Platten sein. </li><li> Fügen Sie direkt 1.250 ul des PEI (0,1% m / v, Polysciences, Kat. Nr. 23966, pH 4,5) in die DNA-Lösung in Schritt 1.2. Vortex die DNA-PEI-Lösung für ein paar Sekunden. </li><li> Inkubieren DNA-PEI Lösung für ein komplexes für 5 min bei Raumtemperatur (RT) zu bilden. </li><li> 1 ml der DNA-PEI-Lösung pro 15 cm 2 Platten. </li><li> Entfernen Medium nach 4 Stunden und ersetzt mit frischen 25 ml Vollmedium +10% FBS (C-DMEM). </li><li> Zweiundsiebzig Stunden nach der Transfektion, kratzen Zellen und gießen in einen konischen 250 ml-Zentrifugenröhrchen. </li><li> Spin bei 3000 Umdrehungen pro Minute, bei 4 ° C für 10 min. Überstand abgießen. </li><li> Re-suspend das Zellpellet in 5 ml RB TMS-Puffer (50 mM Tris-HCl, 150 mM NaCl, pH 8,0). Transfer zu einem neuen 15 ml konischen Rohr. </li><li> Einfrieren in Trockeneis-Ethanol-Bad für 10 min und Auftauen bei 37 ° C für 10 min. Dreimal wiederholen. </li><li> Add 1 ul von 4,8 M MgCl 2 und 2 ul Benzonase (25 U / ul, Novagen, Kat. Nr. 70664-3). Vortex und inkubieren bei 37 ° C für 40 min. </li><li> Spin down bei 4.000 rpm, bei 4 ° C für 40 min und den Überstand (geklärte Lysat). </li><li> In einem 13 mL Quick-Seal-Zentrifugenröhrchen (Beckman, Kat. Nr. 342413), Last Iodixanol Gradienten (OptiPrep, Kat. Nr. 1114542) von oben nach unten: 2 ml 15%, 2 ml 25%, 1,5 ml 40% und 1,5 ml 60% Iodixanol. Legen Sie den Überstand (Schritt 1,12) auf der Oberseite des Iodixanol Gradienten. Füllen Sie das Röhrchen mit RB TMS Buffer. </li><li> Zentrifuge mit 75.000 UpM für 1 Stunde bei 16 ° C mit Beckman 90TI Rotor. </li><li> Sammeln Sie die 40% Iodixanol Fraktion durch Einlegen einer Spritze bei 40-60%-Grenze. </li><li> Übertragen Sie die 40% Iodixanol Bruchteil in einem 50 ml konischen Rohr und machen bis zu 40 ml mit Puffer A (20 mM Tris, 15 mM NaCl, pH 8,5). </li><li> Montieren Sie die Filtrationsvorrichtung mit HiTrap Q HP-Säule (GE Healthcare, Kat. Nr. 17-1154-01). Die Säule wird mit den folgenden Puffer: 25 ml Puffer A (20 mM Tris, 15 mM NaCl, pH 8,5), 25 ml Puffer B (10 mM Tris, 500 mM NaCl, pH 8,5); 50 ml Puffer A; 40 ml Virus-Probe in Puffer A, 50 ml Puffer A (20 mM Tris, 15 mM NaCl, pH 8,5); 20 ml Puffer C (20 mM Tris, 175 mM NaCl, pH 8,5); </li><li> Sammeln Buffer C in einem 20 ml Apollo-Konzentratoren (Orbital Biosciences). Zentrifuge bei 3.000 rpm bei 4 ° C für 10 min. </li><li> Verwerfen Sie den Durchfluss durch und fügen 20 mL gekühlter PBS und Zentrifuge bei 3.000 rpm bei 4 ° C für 10 min. </li><li> Verwerfen Sie den Durchfluss durch und stell 500 ul von gekühlten PBS. Waschen Sie die Vektoren aus der Membran und Transfer zum Silicon behandelten Eppendorf-Röhrchen und lagern in kleinen Aliquots bei -80 ° C eingefroren </li></ol> 2. Bestimmung der rAAV3 Vektortiter <ol><li> Tauwetter 10 ul des gereinigten Virus in ein Eppendorf-Röhrchen auf Eis. Add 40 ul ddH 2 O. </li><li> Add 0,2 ul der Benzonase (25 U / ul, Novagen, Kat. Nr. 70664) und bei 37 ° C für 1 Stunde. </li><li> Nehmen Sie 50 ul von Plasmid-Standard (0,2 ng / mL) in einem anderen Eppendorf-Röhrchen. </li><li> Add 50 ul 100 mM NaOH in jedes Röhrchen und inkubieren bei 65 ° C für 30 min. </li><li> Gefühlt auf Eis sofort für mindestens 5 min. </li><li> Cut Transfer-Membran (Millipore, Cat # INYC00010) nach Bio-Dot-Filterpapier (Bio-Rad, Kat. Nr. 1620161). Spülen Sie Membran in 10x SSC mit drei Filterpapiere für 20 min. </li><li> Set Slot-Blot-SF Apparatus (Bio-Dot, Kat. Nr. 170-6542). Schließen Sie eine Thermoskanne und 100 ul von 10x SSC auf die Slot-Blot-Apparatur. </li><li> Add 100 ul 20x SSC und 1 ul 6x Loading Dye zu jeder der Proben in Schritt 2.5. </li><li> Legen Sie 100 ul der einzelnen Proben auf die Membran in die Slot-Blot-Apparatur. </li><li> Add another 100 ul 10x SSC in jeder der Proben. </li><li> Wiederholen Sie die Schritte 2.9 und 2.10, bis alle die Probe geht durch die Membran. </li><li> Zerlegen Sie den Slot-Blot-Apparatur und entfernen Sie die Membran. Legen Sie es in Inspector Linker SL-1000 UV Crosslinker. Schalten Sie &quot;Energy&quot;, &quot;Optimal Crosslink&quot; (show 1200) und drücken Sie &quot;Start&quot;. </li><li> Boil 1 ml Lachssperma (SS) DNA (Fisher, Kat. Nr. NC9753983) für 5 min. Gefühlt auf Eis für mindestens 5 min. </li><li> Bereiten Sie die Pre-Hybridisierungslösung durch Mischen 27.5 mL ddH 2 O, 15 ml 20x SSC, 5 ml 50x Denhardt-Lösung, 1,25 ml 20% SDS, 0,25 ml PolyA und 1 ml SS DNA aus Schritt 2.13. </li><li> Suspend die Membran in 25 ml pre-Hybridisierungslösung in einem verschlossenen Glaszylinder und Inkubieren bei 65 ° C in einer Hybridisierung Kammer über Nacht. </li><li> Verdünnen Sie 50 ng DNA-Vorlagen in 10 ul ddH 2 O. Kochen bei 100 ° C für 5 min und Chill auf Eis sofort für mindestens 5 min. </li><li> Zentrifuge bei 3.000 rpm bei RT für 1 min. Add 2 &amp;mgr; l 10 mM 3 dNTPs (fehlt dCTP), 2 ul der Hexanukleotid Mix (Roche, Kat. Nr. 11277081001), 1 ul Klenow (Roche, Kat. Nr. 11008404001) und 5 ul α -32 P-dCTP. Bei 37 ° C für 10 min. </li><li> Zentrifuge G-50 Säulen (GE Healthcare, Kat. Nr. 27-5330-01) bei 3.000 rpm für 2 min. Laden Sie die Sonde in Step 2,17 in der Spalte. Zentrifuge bei 3.000 rpm für 2 min und sammeln Sie die Flow-Through. </li><li> Messen der Radioaktivität. Kochen Sie die Sonde bei 100 ° C für 5 min und Chill auf Eis sofort für mindestens 5 min. </li><li> Fügen Sie die Sonde (6x10 5 cpm / ml) in die Pre-Hybridisierungslösung mit Membran. </li><li> Inkubieren über Nacht bei 65 ° C in einer Hybridisierung Kammer. </li><li> Verwerfen der Hybridisierungslösung und spülen Sie die Membran in 2x SSC +0,1% SDS bei RT für 15 min. </li><li> Entsorgen Sie die Lösung und spülen Sie die Membran in 0,1 x SSC +0,1% SDS bei 65 ° C für 30 min. </li><li> Entsorgen Sie die Lösung und spülen Sie die Membran in 0,1 x SSC bei RT kurz. </li><li> Setzen Sie den Film bei -80 ° C für 6 Stunden und Entwicklung des Films. Ein typisches Ergebnis ist in Abbildung 1 dargestellt. </li></ol> 3. rAAV3 Vektor-vermittelte Transduktion und Transgenexpression in Human Liver Cancer Cells <ol><li> Seed 1x10 4 Zellen (entweder Huh7 oder Hep293TT) in jede Vertiefung der 96-Well-Platte. Inkubieren in einem befeuchteten CO 2-Inkubator für mindestens 18 Stunden. </li><li> Entfernen Sie Medium aus dem 96-Well-Platte. </li><li> Wash Zellen zweimal mit 50 ul Medium ohne FBS und Antibiotika und zu entfernen. </li><li> Add 50 ul Medium ohne FBS und Antibiotika und rAAV-Vektoren bei 5.000 vgs / Zelle. </li><li> Die Platten in der CO 2-Inkubator für 4 Stunden. </li><li> Entsorgen Sie das Medium. Wash Zellen zweimal mit 50 ul Vollmedium und zu entfernen. </li><li> Add 150 ul C-DMEM in jede Vertiefung und inkubieren Sie die Platte in CO 2-Inkubator für 72 Stunden, und visualisieren die EGFP-Expression mit einem Fluoreszenzmikroskop (DMI 4000B; Leica Microsystems). </li><li> Bilder aus drei Brunnen von Virus-infizierten Zellen quantitativ durch ImageJ-Analyse-Software (NIH, Bethesda, MD, USA) analysiert. </li><li> Transgenexpression als Gesamtfläche von grüner Fluoreszenz (Pixel 2) pro Gesichtsfeld beurteilt. Ein typisches Ergebnis ist in Abbildung 2 dargestellt. </li><li> Transgenexpression kann auch durch Durchflusszytometrie bestimmt werden. </li></ol> 4. Repräsentative Ergebnisse: Nach dem Protokoll oben genannten, kann man AAV-Serotyp Vektoren effizient zu generieren. Die typische Ausbeute ist ~ 500 ul mit ~ 10 11 vgs / ul des gereinigten Vektor Lager. Die Reinheit des Vektors Lager wird durch den Vergleich Hybridisierungssignale auf quantitative DNA-Slot-Blots, mit und ohne Benzonase Verdauung bestimmt. Gereinigtes rAAV3 Vektoren transduzieren menschlichen Leberkrebs - hepatoblastoma (HB) und des hepatozellulären Karzinoms (HCC) - Zelllinien effizient. <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Quantitative DNA-Slot-Blot-Analyse zur Bestimmung der Titer von rAAV3 Vektoren. Zweifache Verdünnungen des gereinigten viralen Aktien, mit Benzonase (obere Reihe) verdaut wurden quantitative DNA-Slot Blots mit 32 P-markierten EGFP-spezifischen DNA-Sonde analysiert. Der Titer AAV3 Vektor wurde durch Vergleich mit 1 ng (mittlere Reihe) oder 10 ng (untere Reihe) von AAV-EGFP-Plasmid-Standards auf der Membran geladen bestimmt. Die Zahlen zum DNA-Kopien entsprechen. <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Rekombinante AAV-Vektor-vermittelte Transgenexpression in der menschlichen Leber Krebszellen. Hep293TT Zellen, einer kürzlich etablierten humanen Zelllinie hepatoblastoma 4, wurden entweder mock-infiziert (linkes Bild), oder transduziert mit 5.000 vgs / Zelle entweder scAAV2-EGFP oder scAAV3-EGFP-Vektoren bei 37 ° C für 2 Stunden. Transgenexpression visualisiert wurde 72 Stunden nach der Transduktion mit einem Fluoreszenzmikroskop.

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A., Zolotukhin, S., Muzyczka, N., Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+of+adeno-associated+virus+2+vectors:+point+mutations+in+tyrosines+lead+to+high-efficiency+transduction+at+lower+doses.">Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.</a> Proc Natl Acad Sci USA. 105, 7827-7832 (2008).</li><li>Petrs-Silva, H., Dinculescu, A., Li, Q., Min, S. H., Chiodo, V., Pang, J. J., Zhong, L., Zolotukhin, S., Srivastava, A., Lewin, A. S., Hauswirth, W. 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W., Srivastava, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+adeno-associated+virus+(AAV)-protein+phosphatase-5+helper+viruses+for+efficient+liver+transduction+by+single-stranded+AAV+vectors:+therapeutic+expression+of+factor+IX+at+reduced+vector+doses.">Optimized adeno-associated virus (AAV)-protein phosphatase-5 helper viruses for efficient liver transduction by single-stranded AAV vectors: therapeutic expression of factor IX at reduced vector doses.</a> Hum. Gene Ther. 21, 271-283 (2010).</li><li>Li, M., Jayandharan, G. 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J. r., Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+long-term+transduction+and+multilineage+engraftment+of+human+hematopoietic+stem+cells+transduced+with+tyrosine-modified+recombinant+adeno-associated+virus+serotype+2.">Enhanced long-term transduction and multilineage engraftment of human hematopoietic stem cells transduced with tyrosine-modified recombinant adeno-associated virus serotype 2.</a> Hum Gene Ther. 21, 1129-1136 (2010).</li><li>Qiao, C., Zhang, W., Yuan, Z., Shin, J. H., Li, J., Jayandharan, G. R., Zhong, L., Srivastava, A., Xiao, X., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotype+6+capsid+tyrosine-to-phenylalanine+mutations+improve+gene+transfer+to+skeletal+muscle.">Adeno-associated virus serotype 6 capsid tyrosine-to-phenylalanine mutations improve gene transfer to skeletal muscle.</a> Hum Gene Ther. 21, 1343-1348 (2010).</li><li>Markusic, D. M., Herzog, R. W., Aslanidi, G. V., Hoffman, B. E., Li, B., Li, M., Jayandharan, G. R., Ling, C., Zolotukhin, I., Ma, W., Zolotukhin, S., Srivastava, A., Zhong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transduction+and+correction+of+murine+hemophilia+B+using+AAV2+vectors+devoid+of+multiple+surface-exposed+tyrosines.">High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.</a> Mol Ther. 18, 2048-2056 (2010).</li><li>Ojano-Dirain, C., Glushakova, L. G., Zhong, L., Zolotukhin, S., Muzyczka, N., Srivastava, A., Stacpoole, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+PDH+deficiency+using+AAV8-siRNA+vector-mediated+knockdown+of+pyruvate+dehydrogenase+E1&#945;.">An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1&#945;.</a> Mol Genet &#38; Metabol. 101, 183-191 (2010).</li><li>Petrs-Silva, H., Dinculescu, A., Li, Q., Deng, W. T., Pang, J. J., Min, S. H., Chiodo, V., Neeley, A. W., Govindasamy, L., Bennett, A., Agbandje-McKenna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+properties+of+tyrosine-mutant+AAV2+vectors+in+the+mouse+retina.">Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina.</a> Mol Ther. , (2011).</li></ol>

Rekombinante Vektoren auf einer nicht-pathogenen menschlichen Parvovirus, das Adeno-assoziierte Virus (AAV) wurden entwickelt und werden derzeit in einer Reihe von Gentherapie klinische Studien 5. Zuvor, von den 10 am häufigsten verwendete AAV-Serotypen, die Transduktionseffizienz von AAV3 Vektoren im Allgemeinen berichtet worden, um besonders niedrig sein, sowohl in vitro und in vivo 1. Aber unsere jüngsten Beobachtung, dass AAV3 Vektoren transduzieren menschlichen Leber Krebszelllinien außerordentlich gut, wie quantitativ mittels Durchflusszytometrie 2 bestimmt, und dass AAV3 nutzt die menschlichen Hepatozyten-Wachstumsfaktor-Rezeptor (hHGFR) als zelluläre Co-Rezeptor für die Bindung und die Eintragung in diese Zellen 3, empfehlen dringend eine selektive Gewebe-Tropismus AAV3 für den menschlichen Leberzellen im allgemeinen und der menschlichen Leber Krebszellen im Besonderen. Da jedoch AAV3 Vektoren auch transduzieren normalen menschlichen Hepatozyten effizient 2, würden ihre mögliche Verwendung in der Krebstherapie Anwendung in vivo werden negativ beeinflusst. Eine mögliche Strategie, um dieses mögliche Problem zu umgehen, besteht Transkriptions-Targeting von Krebszellen durch die Identifizierung eines Gens Produkt, das selektiv durch den Tumor produziert wird, aber nicht durch normale Hepatozyten. Zum Beispiel haben frühere Studien gezeigt, dass der Serumspiegel von α-Fetoprotein (AFP) als ein spezifischer Marker für die Verfolgung der Gegenwart, Progression und / oder Wiederauftreten bestimmter Arten von Leberkrebs eingesetzt werden, da normale Hepatozyten in der Regel sehr geringe Mengen produzieren dieses Proteins. In der Tat haben wir AAV3 Vektoren, die die AFP-Promotor Transgenexpression in Leberkrebszellen Ziel verwendet, aber nicht in normalen Hepatozyten 2 und Studien werden derzeit durchgeführt, um die Wirksamkeit dieses Ansatzes in einem murinen Xenograft-Modell von Leberkrebs zu testen. In unseren zusätzlichen Untersuchungen haben wir festgestellt, dass die Transduktion Effizienz verschiedener AAV-Serotyp Vektoren deutlich durch ortsspezifische Mutagenese von Oberflächen-exponierten Tyrosinreste in der viralen Kapside 6-14 erweitert werden. Seit 6 von 7 Oberflächen-exponierten Tyrosine auch in AAV3 konserviert sind, haben wir Mutagenese dieser Reste durchgeführt und festgestellt, dass die Transduktion von Tyrosin-Mutante AAV3 Vektoren deutlich wird in der menschlichen Leber Krebszellen (unveröffentlichte Daten) erweitert. Die Studien werden derzeit auch im Gange, um die Sicherheit und Wirksamkeit des Tyrosin-Mutante AAV3 Vektoren in Maus-Xenograft-Modellen für den menschlichen hepatoblastoma und des hepatozellulären Karzinoms zu bewerten und, sofern erfolgreich, die optimale Tyrosin-Mutante AAV3 Serotyp Vektoren können sich als nützlich erweisen für die Ausrichtung der menschlichen Leber Krebserkrankungen für die potenzielle Gentherapie.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-0170CM</td>
<td>&nbsp;</td>
<tr>
<td>PEI</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>23966</td>
<td>&nbsp;</td>
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<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70664-3</td>
<td>&nbsp;</td>
<tr>
<td>HiTrap Q HP column</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>17-1154-01</td>
<td>&nbsp;</td>
<tr>
<td>Salmon sperm DNA</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9753983</td>
<td>&nbsp;</td>
<tr>
<td>Iodixanol gradient</td>
<td>&nbsp;</td>
<td>OptiPrep</td>
<td>1114542</td>
<td>&nbsp;</td>
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<td>G-50 Columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>27-5330-01</td>
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high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

Rekombinante Vektoren auf einer nicht-pathogenen menschlichen Parvovirus basieren, haben die Adeno-assoziierten Virus 2 (AAV2) entwickelt worden und sind derzeit im Einsatz in einer Reihe von Gentherapie klinische Studien. In jüngerer Zeit haben eine Reihe von zusätzlichen AAV-Serotypen auch isoliert worden, von denen gezeigt wurde, um eine selektive Gewebe-Tropismus in verschiedenen kleinen und großen Tiermodellen 1 aufweisen. Von den 10 am häufigsten verwendete AAV-Serotypen ist AAV3 mit Abstand die am wenigsten effiziente Transduktion in Zellen und Geweben in vitro als auch in vivo. Doch in unserem kürzlich veröffentlichten Studien, haben wir dokumentiert, dass AAV3 Vektoren menschlichen Leberkrebs transduzieren - hepatoblastoma (HB) und des hepatozellulären Karzinoms (HCC) - Zelllinien äußerst effizient, weil AAV3 nutzt menschlichen Hepatozyten-Wachstumsfaktor-Rezeptor als zellulären Co-Rezeptor für die Bindung und die Eintragung in diese Zellen 2,3. In diesem Artikel beschreiben wir die notwendigen Schritte zur hocheffizienten Transduktion von humanen Leber-Krebszellen durch rekombinante AAV3 Vektoren, die ein Reporter-Gen zu erreichen. Die Verwendung von rekombinanten AAV3 Vektoren, die ein therapeutisches Gen kann schließlich das Potenzial der Gentherapie von Lebertumoren beim Menschen führen.

Watch this Scientific Journal Video about High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors at JoVE.com

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

In diesem Artikel beschreiben wir die Identifikation des Adeno-assoziierten Virus Serotyp 3 (AAV3) als die effizienteste Vektor für die Ausrichtung der menschlichen Leber Krebszellen.

High-Efficiency Transduktion von Liver Cancer Cells von rekombinanten Adeno-assoziierten Virus Serotyp 3 Vektoren

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

high-efficiency-transduction-liver-cancer-cells-recombinant-adeno

Research

JoVE Journal

Medicine

13.1K Aufrufe.  University of Florida. In diesem Artikel beschreiben wir die Identifikation des Adeno-assoziierten Virus Serotyp 3 (AAV3) als die effizienteste Vektor für die Ausrichtung der menschlichen Leber Krebszellen.

Serie: High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modeling and Imaging 3-Dimensional Collective Zellinvasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Forschung

Medizin

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Ein Multiplex-Luciferase-basierte Screening-Plattform zur Abfrage Krebs-assoziierte Signaltransduktion in kultivierten Zellen

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Molecular Profiling von der invasiven Tumor-Microenvironment in einem 3-Dimensional Model of Colorectal Cancer Cells und<em&gt; Ex-vivo-</em&gt; Fibroblasten

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Verwendung Adeno-assoziierte Virus als Werkzeug Retinal Barrieren in Disease Study

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studieren Bauchspeicheldrüsenkrebs Stammzelleigenschaften für die Entwicklung neuer Behandlungsstrategien

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Entwicklung eines Alpha-Synuklein Based Rattenmodell für Parkinson-Krankheit durch stereotaktische Injektion eines rekombinanten Adeno-assoziierten viralen Vektor,

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Eine schnelle und effiziente Erzeugung von rekombinanten humanen pluripotenten Stammzellen durch Rekombinase-vermittelten Kassettenaustausch in der<em&gt; AAVS1</em&gt; Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Adeno-assoziierten Virus-vermittelte Lieferung von CRISPR für kardiale gen Bearbeitung bei Mäusen

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Subkonjunktivale Verabreichung von Adeno-assoziierten Virusvektoren in Kleintiermodellen

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

AAV-Isolierung durch Doppel-Jodixanol-Dichtegradientenzentrifugation

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Autor im Rampenlicht: Effiziente Adeno-assoziierte Virusisolierung für präklinische Anwendungen

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

Herstellung und Aufreinigung von Suspensionskulturen von Adeno-assoziierten Viren durch Iodixanol-Dichtegradientenzentrifugation für In-vivo-Anwendungen