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> O protocolo apresentado descreve um método operacional para a entrega de genes mediada substrato conseguida através de fixação reversível de vectores adenovirais sem casaco para as superfícies de aço inoxidável. Embora desenvolvido para a finalidade específica de terapia genética baseada em stent de reestenose vascular, esta técnica tem aplicações muito mais amplas nas áreas de biomateriais, implantes biomédicos e terapia gênica. Embora os estudos apresentados têm apen...

A eficácia da terapia do gene como uma modalidade terapêutica é dificultada pela capacidade de direccionamento pobre de vectores de terapia génica 1,2. A falta de segmentação resultados adequados nos níveis sub-terapêuticos de expressão do transgene no local de destino e leva a uma ampla disseminação de vetores de órgãos não-alvo 3, incluindo os responsáveis ​​pela montagem respostas imunes contra tanto o vetor e codificado produto terapêutico 4, 5. Um potencial significa para compensar a promiscuidade de transdução e promover a segmentação é introduzir vetores de genes no local desejado de uma forma que impede a sua disseminação através do sangue e da linfa. Tipicamente, esses esforços contar com um sistema de entrega localmente injectáveis ​​compreendendo quer de vectores virais ou não virais misturados com fibrina, colagénio ou matrizes de hidrogel de ácido hialurónico 6-10, que são capazes de sustentar transitoriamente vectores de genes no local de injecção por aprisionamento físico thin em uma rede polimérica. Outro paradigma geralmente aceite para terapia génica localizada utiliza imobilização de vectores de genes para a superfície de próteses implantadas 11,12. Implantes médicos permanentes (endovasculares, brônquios, os stents urológicas e gastrointestinais, pacemakers, juntas artificiais, malhas cirúrgicas e ginecológicas, etc.) São utilizados anualmente em dezenas de milhões de pacientes 13. Embora geralmente eficaz, estes dispositivos são propensas a complicações que são inadequadamente controlados pelo práticas médicas atuais 14-17. Próteses implantáveis ​​apresentam uma oportunidade única para servir de plataforma de proxy para tratamento de terapia genética localizada. Do ponto de vista farmacocinético, derivatização da superfície dos implantes médicos com doses relativamente baixas de entrada dos vectores de genes resulta na obtenção de ambas as concentrações locais elevadas de vectores de genes para a interface do implante / tecido e reduzindo a cinética de their eliminação desta localidade. Como conseqüência de residência prolongada e aumento da captação pela população de células alvo, vetor imobilização minimiza propagação do vetor gene. Assim, a inoculação acidental de tecidos não-alvo é reduzida. Superfície tethering de vetores de genes em biomateriais implantáveis ​​(também denominado como a entrega do gene mediada por substrato ou entrega gene fase sólida) foi implementado em cultura de células animais e experimentos utilizando ambos específica (antígeno-anticorpo 18-20, avidina-biotina 21,22) e não-específico (23-26 carga, van der Waals) interações. A ligação covalente de vectores para a superfície do dispositivo implantado tem sido anteriormente considerado como não funcional uma vez que demasiado fortes ligações com a superfície exclui vector internalização pelas células alvo. Recentemente foi demonstrado que esta limitação pode ser superada através da utilização de espontaneamente hidrolisável reticulante utilizado como o tetdela entre a superfície metálica modificada das proteínas da cápside de stent e o vector adenoviral de 27,28. Além disso, a taxa de libertação do vetor e naturalmente o tempo de expressão do transgene in vitro e in vivo, pode ser modulada, com o uso de agentes de ligação cruzada hidrolizáveis ​​que exibem diferentes cinéticas de hidrólise 28. O presente documento fornece um protocolo detalhado para a ligação covalente reversível de vectores adenovirais para a superfície do metal activado e apresenta uma configuração experimental útil para estudar os eventos subsequentes de transdução in vitro de músculo liso e células endoteliais em cultura e in vivo no modelo da carótida de rato de angioplastia de stent .

<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

Reestenose intra-stent apresenta uma das principais complicações de procedimentos de revascularização à base de stents utilizados para restabelecer o fluxo de sangue através de segmentos criticamente estreitados de artérias coronárias e periféricas. Stents endovasculares capazes de liberação ajustável de genes com atividade anti-reestenose pode apresentar uma estratégia alternativa para actualmente utilizado stents farmacológicos. A fim de atingir tradução clínico que os stents de genes deve apresentar uma cinética previsível de libertação do gene vector imobilizada-stent e transdução específica do local da vasculatura, evitando ao mesmo tempo uma resposta inflamatória excessiva normalmente associado com os revestimentos de polímero utilizados para a retenção física do vector. Este artigo descreve uma metodologia detalhada para tethering sem casaco de vetores de genes de adenovírus para stents com base em uma ligação reversível das partículas de adenovírus para polialilamina bifosfonato (PABT) -Modified superfície de aço inoxidável via reticulantes hidrolisáveis ​​(HC). Uma família debifuncional HC (aminas e tiol-reactivo) com uma média de t 1/2 de hidrólise do éster em cadeia que varia entre 5 e 50 dias foram utilizados para ligar o vector com o stent. O procedimento de imobilização vector é tipicamente levada a cabo a cerca de 9 horas e consiste em várias etapas: 1) a incubação das amostras de metal numa solução aquosa de PABT (4 horas); 2) desprotecção de grupos tiol instalados em PABT com tris (2-carboxietil) fosfina (20 min); 3) aumento da capacidade de tiol reactivo da superfície do metal por reacção das amostras com polietilenoimina derivatizados com grupos piridilditio) (PDT (2 h); 4) a conversão dos grupos tióis a PDT com ditiotreitol (10 min); 5) alteração de adenovírus com HC (1 hora); 6) purificação de partículas de adenovírus modificados por meio de cromatografia de exclusão de tamanho da coluna (15 min) e 7) a imobilização de partículas adenovirais tiol-reactivo na superfície de aço tiolada (1 hr). Esta técnica tem uma ampla aplicabilidade potencial além stentsfacilitando a engenharia de superfícies de dispositivos bioprostéticas para melhorar sua biocompatibilidade com a entrega de genes mediada por substrato para as células de interface do material estranho implantado.

Experimentos Vector lançamento Ancoragem de vectores adenovirais para a superfície dos implantes, incluindo dispositivos de intervenção, tais como stents, aproxima-se o vector para o local da doença, evitando parcialmente a falta de orientação física dos vectores. No entanto, para ser capaz de atingir os efeitos terapêuticos através da transdução de tecido alvo, o vector tem de ser libertado a partir da superfície (Figura 2). A utilização de a...

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.

Vascular transferência de genes de Metallic Stent superfícies usando Adenoviral Vetores Tethered através hidrolisável reticulantes

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 visualizações.  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. 

Vídeo: 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.

Transferência de genes para insuficiência cardíaca isquêmica em um modelo pré-clínica

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

Medicina

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.

Mudando a direção e orientação do campo elétrico Durante Elétrica Aplicação Pulsos melhora a transferência de Gene Plasmid 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.

Injeção de sub-retiniana de Vetores da terapia genética e células-tronco no olho do rato

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 Formação em fígado de ratos diabéticos por Helper dependente de Transferência de Gene adenoviral Vector Mediada

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.

Fabricação de calibre pequeno endopróteses Usando Electrospinning e balão expansíveis 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.

Usando a técnica de manga em um modelo do rato da transplantação da aorta - um vídeo instrutivo

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.

Estudando a Diabetes através dos olhos de um peixe: Microdissection, visualização e análise do tg(fli:EGFP) adulto vasculatura da retina de Zebrafish

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.

Na Vivo Transferência de genes para o endotélio da artéria carótida comum de coelho

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.

Triagem de Detector de metais portátil para ingestão de corpo estranho metálico em crianças

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.

Modelo murino de estenose venosa central usando fístula Aortocaval com estenose de saída

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