The authors do not have competing financial interests to disclose.

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Protocol development and studies presented in this paper were supported in part by American Heart Association Scientist Development grants (I.F. 0735110N and M.C. 10SDG4020003), U.S. National Heart, Lung, and Blood Institute grant HL 72108 (R.J.L.), and U.S. National Heart, Lung, and Blood Institute T32 007915 (S.P.F.). Angioplasty catheters were kindly donated by NuMED (Hopkinton, NY). The authors wish to acknowledge secretarial assistance of Ms. Susan Kerns.

<html> Das vorgestellte Protokoll beschreibt eine operative Methode zur Substrat vermittelt durch reversible Bindung von Adenovirus-Vektoren erreicht werden, um Oberflächen aus Edelstahl ohne Mantel Gen Lieferung. Während für den spezifischen Zweck der Stent-basierten Gentherapie von vaskulärer Restenose entwickelt hat diese Technik viel breitere Anwendungen auf den Gebieten der Biomaterialien, biomedizinische Implantate und Gentherapie. Obwohl vorgestellten Studien wurden nur verwendet E...

Die Wirksamkeit der Gentherapie als ein therapeutisches Mittel wird durch die schlechte Zielkapazität von Gentherapievektoren 1,2 behindert. Der Mangel an geeigneten Ziel Ergebnisse zu sub-therapeutischen Ebenen der Expression des Transgens am Zielort und führt zu einer weiten Verbreitung von Vektoren für Nichtzielorgane 3, auch die für die Montage Immunantwort sowohl gegen den Vektor und codiert therapeutisches Produkt verantwortlich 4, 5. Eine mögliche Mittel, um die Promiskuität der Transduktion Offset und zur Förderung der Ausrichtung ist es, Gen-Vektoren an der gewünschten Stelle in einer Form, die ihre freie Verbreitung über Blut und Lymphe schließt einzuführen. Typischerweise auf einem lokal injizierbares Abgabesysteme mit entweder viralen oder nicht-viralen Vektoren mit Fibrin, Kollagen oder Hyaluronsäure Hydrogelmatrices 6-10, die durch physikalisches Einschließen th fähig vorübergehend erhalt Genvektoren an der Injektionsstelle zugemischt verlassen diese Bemühungenem in einem polymeren Netzwerk. Ein weiteres allgemein akzeptierte Paradigma für lokalisierte Gentherapie verwendet Immobilisierung von Genvektoren auf der Oberfläche der implantierten Prothesen 11,12. Dauerhafte medizinische Implantate (endovaskuläre, Bronchial-, urologische und Magen-Darm-Stents, Herzschrittmacher, künstliche Gelenke, chirurgische und gynäkologische Maschen, etc.) Werden jährlich in Millionen von Patienten 13 verwendet. Während sie im Allgemeinen wirksam sind diese Geräte anfällig für Komplikationen, die unzureichend für die durch Strom Arztpraxen 14-17 gesteuert werden. Implantierbaren Prothesen bieten eine einzigartige Gelegenheit, als Proxy-Plattformen für die lokale Gentherapie Behandlung dienen. Von der pharmakokinetischen Sicht Oberflächenderivatisierung von medizinischen Implantaten mit relativ niedrigen Eingangs Dosen von Genvektoren Ergebnisse bei der Erreichung sowohl hohe lokale Konzentrationen von Genvektoren auf das Implantat / Gewebe-Grenzfläche und eine Verlangsamung der Kinetik der their Elimination aus diesem Ort. Als Folge längerer Verweilzeit und verbesserte Aufnahme durch die Zielzellpopulation, Immobilisierung Vektor minimiert Ausbreitung der Gen-Vektor. Damit die unbeabsichtigte Inokulation von Nicht-Zielgeweben verringert. Oberfläche Anbindehaltung von Genvektoren auf implantierbare Biomaterialien (auch als Substrat-vermittelte Gen Lieferung oder Festphasengentransfer bezeichnet) hat in der Zellkultur und Tierversuchen mit implementiert sowohl spezifische (Antigen-Antikörper 18-20, Avidin-Biotin 21,22) und nicht-spezifischen 23-26 (Ladung, van-der-Waals) Wechselwirkungen. Die kovalente Bindung von Vektoren an die Oberfläche der implantierten Vorrichtung zuvor als nicht-funktionell betrachtet worden, da zu starke Bindungen mit der Oberfläche entgegen Vektor Internalisierung durch die Zielzellen. Kürzlich wurde gezeigt, dass diese Begrenzung kann durch die Verwendung von spontan hydrolysierbaren Vernetzer als Tet verwendet überwindenihr zwischen dem modifizierten Metalloberfläche des Stents und Kapsidproteine ​​des adenoviralen Vektors 27,28. Darüber hinaus kann der Vektor Freisetzungsrate und Zeitverlauf der Expression des Transgens in vitro und in vivo unter Verwendung von hydrolysierbaren Vernetzern, die unterschiedliche Kinetik der Hydrolyse 28 moduliert werden. Die vorliegende Arbeit stellt ein detailliertes Protokoll für die reversible kovalente Bindung von adenoviralen Vektoren an aktivierte Metalloberfläche und stellt eine nützliche experimentellen Aufbau zur Untersuchung der folgenden Ereignisse Transduktion in vitro kultivierten glatten Muskelzellen und Endothelzellen und in vivo in der Rattenhalsschlagmodell Stentangioplastie .

<table border="0" cellpadding="0" cellspacing="0" width="625">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">316 stainless steel mesh disks</td>
			<td style="width:183px;">Electon Microscopy Sciences</td>
			<td style="width:104px;">E200-SS</td>
			<td style="width:75px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Generic 304-grade stainless steel stents</td>
			<td>Laserage</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AdeGFP</td>
			<td style="width:183px;">University of Pennsylvania Vector Core</td>
			<td>AD-5-PV0504</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AdLuc</td>
			<td style="width:183px;">University of Pennsylvania Vector Core</td>
			<td>AD-5-PV1028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">AdEMPTY</td>
			<td style="width:183px;">University of Pennsylvania Vector Core</td>
			<td style="width:104px;">A858</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:263px;">Cy3(NHS)2</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:104px;">PA23000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Sepharose 6B</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">6B100-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">UV 96-well plates</td>
			<td style="width:183px;">Costar</td>
			<td style="width:104px;">3635</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Fluorometry 96-well plates</td>
			<td style="width:183px;">Costar</td>
			<td style="width:104px;">3915</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Cell culture 96-well plates</td>
			<td style="width:183px;">Falcon</td>
			<td style="width:104px;">353072</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Tris(2-carboxyethyl)phosphine hydrochloride (TCEP )</td>
			<td style="width:183px;">Pierce Thermo Scientific</td>
			<td style="width:104px;">20490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">dithiothreitol (DTT)</td>
			<td style="width:183px;">Pierce Thermo Scientific</td>
			<td style="width:104px;">20290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">sulfo-LC-SPDP</td>
			<td style="width:183px;">Pierce Thermo Scientific</td>
			<td style="width:104px;">21650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Spectrophotometer</td>
			<td style="width:183px;">Molecular Devices&#160;</td>
			<td style="width:104px;">SpectraMax 190</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Spectrofluorometer</td>
			<td style="width:183px;">Molecular Devices</td>
			<td style="width:104px;">SpectraMax Gemini EM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Orbital shaker incubator</td>
			<td style="width:183px;">VWR</td>
			<td style="width:104px;">1575R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Horizontal airflow oven</td>
			<td style="width:183px;">Shel Lab</td>
			<td style="width:104px;">1350 FM</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">Centra-CL2 centrifuge&#160;</td>
			<td style="width:183px;">International Equipment Company</td>
			<td style="width:104px;">426</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Digital vortex mixerer</td>
			<td style="width:183px;">Fisher Thermo Scientific</td>
			<td style="width:104px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eclipse TE300 fluorescence microscope</td>
			<td>Nikon&#160;</td>
			<td>TE300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">DC 500 CCD camera</td>
			<td style="width:183px;">Leica</td>
			<td style="width:104px;">DC-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">7500 Real-Time PCR system</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IVIS Spectrum bioluminescence station</td>
			<td>Perkins-Elmer</td>
			<td>not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">EDTA dipotassium salt</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">ED2P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin fraction V (BSA)</td>
			<td>Fisher Thermo Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Tween-20</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">P1379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Dumont forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">11255-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">A10 cell line&#160;</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:104px;">CRL-1476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Bovine aortic endothelial cells</td>
			<td style="width:183px;">Lonza</td>
			<td style="width:104px;">BW-6002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Luciferin, potassium salt</td>
			<td style="width:183px;">Gold Biotechnology</td>
			<td style="width:104px;">LUCK-1Ge</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Pluronic F-127</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:104px;">P2443-250G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">PBS&#160; without calcium and magnesium</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:104px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Fetal bovine serum</td>
			<td style="width:183px;">Gemini Bio-Products</td>
			<td style="width:104px;">100-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Penicillin/Streptomycin solution</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:104px;">11540-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">DMEM, high glucose</td>
			<td style="width:183px;">Corning cellgro</td>
			<td style="width:104px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">0.25% Trypsin/EDTA</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:104px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">QIAamp DNA micro kit</td>
			<td style="width:183px;">Qiagen</td>
			<td style="width:104px;">56304</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Power Sybr Green PCR Master Mix</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:263px;">MicroAmp Optical 96-well Reaction Plate</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td style="width:104px;">N8010560</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">MicroAmp Optical Adhesive Film</td>
			<td style="width:183px;">Applied Biosystems</td>
			<td style="width:104px;">4360954</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Cephazolin&#160;</td>
			<td style="width:183px;">Apotex</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Loxicom (Meloxicam)</td>
			<td style="width:183px;">Norbrook</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Heparin sodium</td>
			<td style="width:183px;">APP Pharmaceuticals</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Ketavet (Ketamine)</td>
			<td style="width:183px;">VEDCO</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Anased (Xylazine)&#160;</td>
			<td style="width:183px;">Lloid</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Forane (Isoflurane)&#160;</td>
			<td style="width:183px;">Baxter</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Curved Moria iris forceps</td>
			<td style="width:183px;">Fine Science tools</td>
			<td style="width:104px;">11370-31</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">&#160;Curved extra-fine Graefe forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">11152-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Dumont #5 forceps</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">11252-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Vannas spring scissors</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">15018-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Fine scissors - ToughCut</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">14058-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Surgical scissors</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">14101-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Vicryl suture (5-0)</td>
			<td style="width:183px;">Ethicon</td>
			<td style="width:104px;">J385</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Suture thread (4/0 silk)&#160;</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">18020-40</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Michel suture clips</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">12040-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wound dilator (Lancaster eye specula)</td>
			<td>KLS Martin</td>
			<td>34-149-07</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Michel suture clip applicator</td>
			<td style="width:183px;">Fine Science Tools</td>
			<td style="width:104px;">112028-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Insyte Autoguard 24G IV catheter</td>
			<td style="width:183px;">Beckton-Dickinson</td>
			<td style="width:104px;">381412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">2F Fogarty catheter</td>
			<td style="width:183px;">Edwards Lifesciences</td>
			<td style="width:104px;">120602F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Teflon tubing</td>
			<td style="width:183px;">Vention</td>
			<td style="width:104px;">041100BST</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">PTA catheter</td>
			<td style="width:183px;">NuMed</td>
			<td style="width:104px;">custom order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Gauze pads</td>
			<td style="width:183px;">Kendall Healthcare</td>
			<td style="width:104px;">9024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Cotton applicators</td>
			<td style="width:183px;">Solon Manufacturing</td>
			<td style="width:104px;">WOD1003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Saline</td>
			<td style="width:183px;">Baxter</td>
			<td style="width:104px;">281321</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">10 ml syringe (Luer-Lok)</td>
			<td style="width:183px;">Beckton-Dickinson</td>
			<td style="width:104px;">309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">1 ml syringe (Luer-Lok)</td>
			<td style="width:183px;">Beckton-Dickinson</td>
			<td style="width:104px;">309628</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Clippers with #40 blade</td>
			<td style="width:183px;">Oster&#160;</td>
			<td style="width:104px;">78005-314</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Transpore surgical tape</td>
			<td style="width:183px;">3M</td>
			<td style="width:104px;">MM 15271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Puralube vet ointment</td>
			<td style="width:183px;">Pharmaderm</td>
			<td style="width:104px;">not available</td>
			<td></td>
		</tr>
	</tbody>
</table>

vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers

In-Stent-Restenose stellt eine wesentliche Komplikation der Stentbasis Revaskularisierungsverfahren weithin verwendet, um die Wiederherstellung des Blutflusses durch kritisch verengter Segmente der koronaren und peripheren Arterien. Endovaskulären Stents in der Lage, abstimmbaren Freisetzung von Genen, die mit Anti-Restenose-Aktivität kann eine alternative Strategie präsentieren, um derzeit verwendeten Medikamenten-freisetzenden Stents. Um klinische Umsetzung zu erreichen, müssen Gen-freisetzenden Stents vorhersehbar Kinetik der Stent-immobilisiert Genvektor Mitteilung und ortsspezifische Transduktion von Gefäßsystem aufweisen, während eine übermäßige Entzündungsreaktion in der Regel mit den Polymerbeschichtungen für physikalischen Einschluß des verwendeten Vektors verbunden. Dieser Beitrag beschreibt eine detaillierte Methode, ohne Mantel Tethering adenoviraler Genvektoren, Stents basierend auf einer reversiblen Bindung der adenoviralen Partikel Polyallylamin Bisphosphonat (PABT) modifizierte Edelstahl-Oberfläche über hydrolysierbarer Vernetzer (HC). Eine Familie vonbifunktionelle (Amin-und Thiol-reaktive) HC mit einer durchschnittlichen t 1/2 der in der Kette Esterhydrolyse im Bereich zwischen 5 und 50 Tage waren verwendet, um den Vektor mit dem Stent zu verbinden. Der Vektor Immobilisierungsverfahren wird typischerweise innerhalb von 9 h durchgeführt und besteht aus mehreren Schritten: 1) Inkubation der Metallproben in einer wässrigen Lösung von PABT (4 h); 2) Entschützen von Thiolgruppen in PABT mit Tris (2-carboxyethyl) phosphin (20 min) installiert ist; 3) Expansion von Thiol reaktiven Leistung von der Metalloberfläche durch Reaktion der Proben mit Polyethylenimin mit pyridyldithio (PDT) Gruppen (2 h) derivatisiert; 4) Umsetzung von PDT Gruppen Thiole mit Dithiothreitol (10 min); 5) Änderung von Adenoviren mit HC (1 Stunde); 6) Reinigung von modifizierten adenoviralen Partikel durch Größenausschluss-Säulenchromatographie (15 min) und 7) Immobilisierung von Thiol-reaktiven adenoviralen Partikel auf dem thiolierten Stahlfläche (1 h). Diese Technik hat breite potentielle Anwendbarkeit über Stents,durch die Erleichterung der Oberflächentechnik bioprothetischer Geräte ihrer Biokompatibilität durch das Substrat-vermittelte Gen-Lieferung an den Zellen Anbindung des implantierten Fremdmaterials zu verbessern.

Vektor-Freisetzungsversuche Anbinden von adenoviralen Vektoren in der Oberfläche von Implantaten, einschließlich interventionelle Vorrichtungen wie endovaskulären Stents, etwa der Vektor an die erkrankte Stelle, die teilweise Beseitigung der mangelnden physikalischen Zielvektoren. Jedoch um therapeutische Wirkungen über die Transduktion der Zielgewebe zu erreichen, muss der Vektor von der Oberfläche (2) freigegeben werden. Der Einsatz von hydrolysierbar...

Watch this Scientific Journal Video about Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers at JoVE.com

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

Gefäß Gentransfer von metallischen Stent Oberflächen mit Adenovirus-Vektoren durch Tethered Hydrolysierbare Vernetzer

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

vascular-gene-transfer-from-metallic-stent-surfaces-using-adenoviral

Research

JoVE Journal

Medicine

9.7K Aufrufe.  The Children's Hospital of Philadelphia, University of Pennsylvania. These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries. 

Serie: Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Gentransfer bei ischämischen Herzinsuffizienz in einem vorklinischen Modell

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Forschung

Medizin

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause destabilization of cell membrane, making it permeable to small molecules and allows transfer of large molecules such as DNA. It represents an alternative to viral vectors, due to its safety, efficacy and ease of application. For gene electrotransfer different electric pulse protocols are used in order to achieve maximum gene transfection, one of them is changing the electric field direction and orientation during the pulse delivery. Changing electric field direction and orientation increase the membrane area competent for DNA entry into the cell. In this video, we demonstrate the difference in gene electrotransfer efficacy when all pulses are delivered in the same direction and when pulses are delivered by changing alternatively the electric field direction and orientation. For this purpose tip with integrated electrodes and high-voltage prototype generator, which allows changing of electric field in different directions during electric pulse application, were used. Gene electrotransfer efficacy is determined 24h after pulse application as the number of cells expressing green fluorescent protein divided with the number of all cells. The results show that gene transfection is increased when the electric field orientation during electric pulse delivery is changed.

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Gene transfection by electroporation is improved approximately two times when orientation of electric field is changed during pulse application, while cell viability is not affected. The increase in gene transfection is caused by the increase of the membrane area which is made competent for DNA entry into the cell.

Ändern der Ausrichtung und Orientierung des elektrischen Feldes während elektrische Impulse Anwendung Verbessert Plasmid Gene Transfer In-vitro-</em

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

Subretinalen Injektion von Vektoren für die Gentherapie und Stammzellen in die Maus Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Neo-Islet Formation in Leber der diabetischen Mäuse von Helper-abhängigen Adenovirus-Vektor-vermittelten Gentransfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and perforations in vasculature. Such stent-grafts are manufactured by covering a stent with a polymer membrane. An ideal stent-graft should have a biocompatible stent covered by a porous, thromboresistant, and biocompatible polymer membrane which mimics the extracellular matrix thereby promoting injury site healing. The goal of this protocol is to manufacture a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. Electrospinning of polyurethane has been shown to assist in healing by mimicking native extracellular matrix, thereby promoting endothelialization. Electrospinning polyurethane nanofibers on a slowly rotating mandrel enabled us to precisely control the thickness of the nanofibrous membrane, which is essential to achieve a small caliber balloon expandable stent-graft. Mechanical validation by crimping and expansion of the stent-graft has shown that the nanofibrous polyurethane membrane is sufficiently flexible to crimp and expand while staying patent without showing any signs of tearing or delamination. Furthermore, stent-grafts fabricated using the methods described here are capable of being implanted using a coronary intervention procedure using standard size guide catheters.

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

Die Herstellung von Kleinkaliber Stentgrafts Mit Elektrospinnen und Balloon Expandable Bare Metal Stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Mit Hilfe der Sleeve-Technik in einem Mausmodell der Aorta Transplantation - ein Lehr-Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetes durch die Augen eines Fisches zu studieren: Mikrodissektion, Visualisierung und Analyse von den Erwachsenen tg(fli:EGFP) Zebrafisch Retinal Gefäßsystem

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

In Vivo Gentransfer in die Kaninchen gemeinsame Halsschlagader Endothel

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Handheld-Metalldetektor-Screening für metallische Fremdkörper Verschlucken bei Kindern

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

Murine Modell der zentralen venösen Stenose mit Aortocaval Fistel mit einem Abfluss Stenose

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...