French B A, Mazur W, Ali N M, Geske R S, Finnigan J P, Rodgers G P, Roberts R, Raizner A E
Department of Medicine, Baylor College of Medicine, Houston, TX 77030.
Circulation. 1994 Nov;90(5):2402-13. doi: 10.1161/01.cir.90.5.2402.
Gene therapy has been proposed as a possible solution to the problem of restenosis after coronary angioplasty. The current study was undertaken to assess conventional methods of gene transfer and to develop percutaneous techniques for introducing genes directly into the coronary arteries of large mammals. Since the anticipated targets of gene therapy against restenosis include atherosclerotic and previously instrumented arteries, we also evaluated gene transfer in atherosclerotic coronary arteries and in two porcine models of restenosis: one using intracoronary stents and a second using balloon overstretch angioplasty.
The conventional method of using perforated balloon catheters to deliver Lipofectin-DNA complexes directly into the coronary arteries of intact animals was applied to 18 porcine coronary arteries including normal arteries, hypercholesterolemic arteries, and those simulating restenosis. The results of this study were consistent with previously published results indicating that only low levels of luciferase gene expression could be obtained by Lipofectin-mediated gene transfer. We therefore undertook a second, parallel study to evaluate percutaneous transluminal in vivo gene transfer using a replication-deficient adenoviral vector. A comparison of the two studies revealed that the mean level of reporter gene expression in the cohort undergoing adenoviral infection was 100-fold higher than in the cohort undergoing Lipofection. Analysis of luciferase activity over time in normal arteries revealed that recombinant gene expression was half-maximal after 1 day, peaked within 1 week, was still half-maximal at 2 weeks, and declined to low levels by 4 weeks. Histochemical analysis of coronary arteries treated with a second adenovirus expressing a nuclear-localized beta-galactosidase gene demonstrated gene transfer to a limited number of cells in the media and adventitia. Immunohistochemical analysis of Ad5-infused arteries using a monoclonal antibody directed against CD44 identified a periadventitial infiltrate composed of leukocytes.
The recombinant adenoviral vectors proved to be far more effective than Lipofectin at delivering foreign genes directly into the coronary arteries of living mammals. Furthermore, the influences of hypercholesterolemia and arterial injury appeared to have little effect on the levels of gene expression obtained using either method. The results demonstrate that low-level recombinant gene expression, the major obstacle impeding gene therapy for the prevention of restenosis, can potentially be overcome by using adenoviral vectors to mediate coronary gene transfer in vivo. The duration of gene expression provided by these vectors and their effective deployment in atherosclerotic, balloon-overstretched, and stented coronary arteries suggest that recombinant adenovirus may have potential for evaluating gene therapy in the clinically informative porcine models of coronary restenosis.
基因治疗已被提议作为解决冠状动脉血管成形术后再狭窄问题的一种可能方法。本研究旨在评估传统的基因转移方法，并开发将基因直接导入大型哺乳动物冠状动脉的经皮技术。由于针对再狭窄的基因治疗预期靶点包括动脉粥样硬化动脉和先前已进行介入操作的动脉，我们还评估了基因在动脉粥样硬化冠状动脉以及两种猪再狭窄模型中的转移情况：一种使用冠状动脉内支架，另一种使用球囊过度扩张血管成形术。
使用带孔球囊导管将脂质体 - DNA复合物直接导入完整动物冠状动脉的传统方法应用于18条猪冠状动脉，包括正常动脉、高胆固醇血症动脉以及模拟再狭窄的动脉。本研究结果与先前发表的结果一致，表明通过脂质体介导的基因转移只能获得低水平的荧光素酶基因表达。因此，我们进行了第二项平行研究，以评估使用复制缺陷型腺病毒载体进行经皮腔内体内基因转移。两项研究的比较显示，接受腺病毒感染的队列中报告基因表达的平均水平比接受脂质体转染的队列高100倍。对正常动脉中荧光素酶活性随时间的分析表明，重组基因表达在1天后达到最大值的一半，在1周内达到峰值，在2周时仍为最大值的一半，并在4周时降至低水平。用表达核定位β - 半乳糖苷酶基因的第二种腺病毒处理的冠状动脉的组织化学分析表明，基因转移到中膜和外膜的有限数量细胞中。使用针对CD44的单克隆抗体对注入Ad5的动脉进行免疫组织化学分析，发现外膜周围有由白细胞组成的浸润。
重组腺病毒载体在将外源基因直接导入活体哺乳动物冠状动脉方面被证明比脂质体有效得多。此外，高胆固醇血症和动脉损伤的影响似乎对使用任何一种方法获得的基因表达水平影响很小。结果表明，阻碍预防再狭窄基因治疗的主要障碍——低水平重组基因表达，有可能通过使用腺病毒载体在体内介导冠状动脉基因转移来克服。这些载体提供的基因表达持续时间以及它们在动脉粥样硬化、球囊过度扩张和置入支架的冠状动脉中的有效应用表明，重组腺病毒在临床上有参考价值的猪冠状动脉再狭窄模型中评估基因治疗方面可能具有潜力。
