The authors do not have competing financial interests to disclose.

<ol>
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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> 제시된 프로토콜은 스테인레스 스틸 표면 무 코팅하는 아데노 바이러스 벡터를 통해 달성 가역적 부착 기판 매개 된 유전자 전달에 대한 동작 방법을 설명한다. 혈관 재 협착의 스텐트 기반 유전자 치료의 특정 목적을 위해 개발 된 반면,이 기술은 생체 재료, 바이오 메디컬 임플란트 및 유전자 치료 등의 분야에서의 더 넓은 응용을 갖는다. 제시된 연구는 유일하게 원형...

치료 적 양상과 같은 유전자 치료의 효과는 유전자 치료 벡터의 1,2 나쁨 타겟팅 용량에 의해 방해된다. 목표 위치에 유전자 발현의 하위 치료 수준에서 적절한 타겟팅 결과 부족하고, 벡터 및 부호화 치료 제품 4 모두에 대하여 면역 반응을 마운팅 할 책임을 포함한 비 - 표적 기관 03 행 벡터의 넓은 보급에 이르게 5. 하나의 가능성은 전달의 성행위를 상쇄하고 타겟팅 혈액과 림프를 통해 자신의 무료 보급을 배제 형태로 원하는 위치에 유전자 벡터를 소개하는 홍보하는 것을 의미한다. 전형적으로, 이러한 노력들은 물리적 번째 포획하여 주사 부위에서 일시적으로 유지하는 유전자 벡터 수있는 피브린, 콜라겐이나 히알루 론산 하이드로 젤 매트릭스 6-10과 혼합 바이러스 성 또는 비 바이러스 벡터 중 구성된 국소 주사 전달 시스템에 의존고분자 네트워크의 그들. 지역화 된 유전자 치료를위한 또 다른 일반적으로 인정 패러다임 주입 보철 장치 (11, 12)의 표면에 유전자 벡터의 고정화를 이용한다. 영구 이식 의료 (혈관, 기관지, 비뇨기과 및 위장 스텐트, 심장 박동기, 인공 관절, 수술 및 부인과 메시, 등.) 환자 13 수천만에서 연간 사용됩니다. 일반적으로 효과가 있지만,이 장치는 부적절 현재의 의료 관행 14-17에 의해 제어되는 합병증하는 경향이 있습니다. 이식 보철 장치는 현지화 된 유전자 치료 치료를위한 프록시 플랫폼 역할을 할 수있는 특별한 기회를 제공하고 있습니다. 약동학 관점 파묻혀의 동력학을 임플란트 / 조직 인터페이스 유전자 벡터의 높은 지방 농도 모두를 달성하고 둔화 유전자 벡터 결과 비교적 낮은 입력 용량 의료 임플란트 표면에서 유도화이 위치에서 R 제거. 대상 세포 인구에 의해 연장 된 거주지의 결과 및 향상된 흡수으로, 벡터 고정화는 유전자 벡터의 확산을 최소화 할 수 있습니다. 따라서, 비 표적 조직의 부주의 접종이 감소된다. (또한 기판 매개 유전자 전달 또는 고상 유전자 전달 되나) 이식 생체 재료에 유전자 벡터의 표면 테 더링 사용 세포 배양 및 동물 실험에서 구현되었습니다 모두 특정 (항원 - 항체 18-20, 아비딘 - 비오틴 21, 22) 그리고 23 ~ 26 (요금, 반 데르 발스) 상호 작용 비특이적. 표면이 지나치게 강한 결합이 표적 세포로 내재화 벡터 배제 이후 주입 장치의 표면에 벡터의 공유 결합은 이전에 비 기능적으로 간주되어왔다. 최근이 제한 TET로서 사용 자발적 가수 분해성 가교제의 사용을 통해 극복 될 수 있다는 것을 입증했다아데노 바이러스 벡터의 27,28 스텐트 및 캡시드 단백질의 변성 금속 표면 사이 그녀. 또한, 벡터 방출률 및 생체 외 및 생체 내에서 유전자 발현의 시간 경과 (28)의 가수 분해 반응 속도를 나타내는 다른 가수 분해성 가교제를 이용하여 변조 될 수있다. 본 논문은 활성화 된 금속 표면에 아데노 바이러스 벡터의 가역적 공유 결합에 대한 상세한 프로토콜을 제공하고 스텐트 혈관 성형술의 쥐 경동맥 모델에서 배양 평활근 및 내피 세포 및 생체 내에서 시험 관내에서 이어지는 전달 사건을 연구하는데 유용한 실험 구성 소개 .

<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

스텐트 재 협착 널리 관상 동맥 및 말초 동맥의 매우 좁은 부분을 통해 다시 설정 혈액의 흐름에 사용되는 스텐트 기반 재 시술 절차의 주요 합병증을 선물한다. 대안 전략을 제시 할 수있다 방지 재 협착 활성 유전자의 조정 릴리스 할 수있는 혈관 내 스텐트는 현재 약물 방출 스텐트를 사용합니다. 일반적으로 벡터의 물리적 포획을 위해 사용되는 폴리머 코팅과 관련된 과도한 염증 반응을 피하면서 임상 변환을 달성하기 위하여, 유전자 스텐트는 스텐트 고정화 유전자 벡터 방출 및 혈관의 부위 - 특이 적 전달의 예측 동력학을 나타낸다한다. 이 논문은 비스포스포네이트 (PABT)이 가수 분해 가교제 (HC)를 통해 스테인리스 표면을 개질 된 폴리 알릴 아민하는 아데노 바이러스 입자의 가역적 인 결합을 기반으로 스텐트에 아데노 바이러스 유전자 벡터의 무 코팅 테 더링에 대한 자세한 방법을 설명합니다. 의 가족5 내지 오십일 레인 징의 쇄상 에스테르 가수 분해의 평균 t와 2 관능 성 (아민 - 및 티올 - 반응성) HC는 스텐트 벡터를 연결하기 위해 사용되었다. 벡터 고정화 절차는 일반적으로 9 시간 이내에 수행하고 여러 단계로 구성된다 : PABT (4 시간)의 수용액에 금속 샘플 1) 배양; 트리스 (2 - 카르복시 에틸) 포스 핀 (20 분 간격)으로 설치 PABT 티올 기, 2) 탈; 피리 딜 디티 (PDT) 그룹 (2 시간)과 폴리에틸렌 이민 유도체와 샘플을 반응시켜 금속 표면의 반응성 티올 용량 3) 확장; 디티 오 트레이 톨 (10 분)에 PDT와 티올 기의 4) 변환; HC (1 시간)와 아데노 바이러스의 5) 수정; 크기 배제 칼럼 크로마토 그래피 (15 분) 및 티올 철강 표면 (1 시간)에 티올 반응성 아데노 바이러스 입자의 7)에 의해 수정 고정화 아데노 바이러스 입자의 6)의 정제. 이 기술은 스텐트를 넘어 폭 넓은 잠재적 인 응용 성을 가지고주입 된 이물질 인터페이싱 세포 기질 - 매개 유전자 전달을 통해 자신의 생체 적합성을 향상시키기 위해 판막 장치의 표면 공학을 촉진시켜.

벡터 출시 실험 이러한 혈관 내 스텐트 같은 중재 장치를 포함하는 임플란트의 표면에 아데노 바이러스 벡터의 테 더링 부분적 벡터 &#39;물리적 타겟팅의 부족을 미연에 방지, 질병 사이트 벡터 가깝다. 그러나, 표적 조직의 형질 도입을 통해 치료 효과를 달성 할 수 있도록, 벡터는 표면 (도 2)로부터 방출되어야한다. 가수 분해성 가교제의 사용은 가...

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.

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐​​트 표면에서 혈관 유전자 전송

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 조회수.  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. 

비디오: 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.

잠복기 모델에서 허혈성 심장 마비에 대한 유전자 전송

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

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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.

전기 펄스 응용 프로그램에는 방향과 전기장의 방향을 변경하면 플라스미드 유전자 전송을 향상 체외에서</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.

마우스 눈에 유전자 치료 벡터와 줄기 세포의 Subretinal 주입

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.

도우미에 의존 Adenoviral 벡터로 인한 유전자의 전송에 의한 당뇨 쥐의 간에서 네오 - 작은 섬 형성

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

마우스 동정맥루 내피에 관내 약물 전달

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. 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.

전기 방사 및 풍선 확장 베어 메탈 스텐트를 사용하여 작은 구경 스텐트 - 이식의 제작

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.

대동맥 이식-교육용 비디오의 마우스 모델에서 소매 기법을 사용 하 여

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.

물고기는 눈을 통해 당뇨병을 공부: 기정, 시각화, 및 성인 tg(fli:EGFP) Zebrafish 망막 맥 관 구조 분석

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.

Vivo에서 토끼 일반적인 경 동맥 내 피에 유전자 이동

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.

아이 들에서 금속 이물질 섭취에 대 한 휴대용 금속 탐지기 검사

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 ...

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐​​트 표면에서 혈관 유전자 전송

가수 분해 크로스 링커를 통해 아데노 바이러스 벡터 닿는를 사용하여 금속 스텐트 표면에서 혈관 유전자 전송