Erratum: Stent-Mediated Gene Delivery for Site-Specific Transgene Administration to the Airway Epithelium and Management of Tracheobronchial Tumors

<b><i>Background:</i></b> Gene therapy is currently under investigation as a means of managing a variety of pulmonary diseases. Unfortunately, gene transfer to bronchial epithelium has been hampered by the lack of stable and efficient transduction. Recent studies have shown that gene vectors could be tethered to the metallic surfaces of intra-arterial stents. This approach enables efficacious and site-specific adenoviral gene delivery to the vascular endothelium. <b><i>Objectives:</i></b> We hypothesized that airway mesh stents impregnated with viral gene vectors could be used for local gene delivery to benign and malignant bronchial epithelium. <b><i>Methods:</i></b> Serotype 5 adenoviral vectors (Ad5, E1-/E3-) containing the reporter genes green fluorescent protein (Ad.GFP) or β-galactoside/<i>LacZ</i> (Ad.<i>LacZ</i>), or a therapeutic gene, Ad.INF-β, were coupled to either metallic mesh disks or stents via anti-Ad knob antibodies. These platforms were assessed for their ability to transfect bronchial epithelial cells from both rats and humans, as well as murine (L1C2) and human (A549) lung cancer cell lines. Gene transfer was quantified by fluorescent microscopy, scanning fluorimetry for Ad.GFP, and light microscopy studies assessing β-galactosidase staining for Ad.<i>LacZ</i>. Metallic mesh and stent-mediated gene transfer was also performed in a murine flank tumor model and in a rat endotracheal tumor model in order to evaluate the therapeutic potential. <b><i>Results:</i></b> In these studies, murine and human non-small cell lung cancer (NSCLC) cells were successfully transfected with reporter genes in vitro. Ad.<i>LacZ</i>-complexed mesh successfully transfected reporter genes into established murine flank NSCLC tumors. In addition, Ad.<i>LacZ</i>-tethered stents could effectively transfect both tracheobronchial epithelium and submucosal glands in rats. Similar epithelial transfection was achieved in ex vivo human bronchial epithelium. Pilot in vivo experimentation provided data supporting the concept that therapeutic genes could also be delivered with this technology. In additional pilot in vivo experiments, the growth of murine flank tumors was inhibited by placement of mesh disks coupled with Ad.muINF-β, and rats bearing endotracheal tumors demonstrated a trend towards prolonged survival with insertion of Ad.ratINF-β-tethered stents. <b><i>Conclusions:</i></b> Stent-mediated gene delivery successfully enabled site-specific vector administration to target rat and human airway cells in cell culture, organ culture and in vivo. Local tracheobronchial gene delivery via stents could provide a viable clinical solution for overcoming the difficulties encountered with vector delivery within the lungs, in particular by lowering requisite vector titers and by directing desired vectors to areas of interest. This strategy may prove valuable for treating tumors involving the tracheobronchial tree, as well as other nonmalignant tracheobronchial disorders.

<b><i>背景:</i></b> 基因治疗（gene therapy）目前正被探索作为多种肺部疾病的管理手段。遗憾的是，向支气管上皮（bronchial epithelium）的基因转染始终受限于缺乏稳定且高效的转导方法。近期研究表明，基因载体可锚定至动脉内支架（intra-arterial stents）的金属表面，该策略可实现针对血管内皮（vascular endothelium）的高效且位点特异性腺病毒（adenovirus）基因递送。
<b><i>研究目标:</i></b> 我们提出假说，负载病毒基因载体的气道网状支架（airway mesh stents）可用于向良性及恶性支气管上皮进行局部基因递送。
<b><i>研究方法:</i></b> 将携带报告基因（reporter genes）绿色荧光蛋白（Ad.GFP）或β-半乳糖苷酶/<i>LacZ</i>（Ad.<i>LacZ</i>）的5型腺病毒载体（Ad5, E1-/E3-），抑或治疗性基因载体Ad.INF-β，通过抗腺病毒knob抗体（anti-Ad knob antibodies）偶联至金属网状圆盘或支架。随后评估这些载体平台对大鼠、人支气管上皮细胞，以及小鼠（L1C2）和人（A549）肺癌细胞系的转染能力。基因转染效率通过荧光显微镜（fluorescent microscopy）、检测Ad.GFP的扫描荧光法（scanning fluorimetry），以及通过光镜（light microscopy）观察Ad.<i>LacZ</i>的β-半乳糖苷酶染色情况进行量化。此外，为评估该技术的治疗潜力，我们还在小鼠皮下肿瘤模型（murine flank tumor model）及大鼠气管内肿瘤模型（rat endotracheal tumor model）中开展了金属网状支架介导的基因递送实验。
<b><i>研究结果:</i></b> 本研究中，小鼠及人非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞在体外均成功被报告基因载体转染。Ad.<i>LacZ</i>偶联的网状圆盘可成功将报告基因转染至已构建的小鼠皮下NSCLC肿瘤中。此外，Ad.<i>LacZ</i>锚定的支架可有效转染大鼠的气管支气管上皮及黏膜下腺体。在离体人支气管上皮样本中也实现了相似的上皮转染效果。初步体内实验数据证实，该技术同样可递送治疗性基因。额外的体内预实验显示，植入偶联Ad.muINF-β的网状圆盘可抑制小鼠皮下肿瘤的生长；携带气管内肿瘤的大鼠在植入Ad.ratINF-β锚定的支架后，生存期呈现延长趋势。
<b><i>研究结论:</i></b> 支架介导的基因递送可成功实现位点特异性载体给药，在细胞培养、器官培养及体内模型中靶向递送大鼠及人气道细胞。通过支架实现的局部气管支气管基因递送，可解决肺内载体递送面临的诸多难题——尤其是可降低所需载体滴度（vector titers），并将目标载体精准递送至感兴趣区域。该策略有望用于治疗气管支气管树（tracheobronchial tree）相关肿瘤，以及其他非恶性气管支气管疾病。