Zelenock J A, Welling T H, Sarkar R, Gordon D G, Messina L M
Department of Surgery, University of Michigan Medical School, Ann Arbor, USA.
J Vasc Surg. 1997 Jul;26(1):119-27. doi: 10.1016/s0741-5214(97)70155-1.
Vascular cells are an important target for gene transfer because of their potential to deliver gene products both locally and systemically. Direct retroviral gene transfer to vascular cells in vivo has been limited by inefficient rates of transduction. We hypothesized that vascular cell transduction efficiency (TE), during short retroviral incubation periods, is significantly improved in vitro and in vivo using centrifugation to increase viral titer. Furthermore, we hypothesized a linear relationship between concentration of viable viral particles (measured as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods. Cultured rat pulmonary artery endothelial cells (RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac artery endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (> 100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes. High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume. Titers was consistently increased tenfold, for a twentyfold increase in concentration by volume. A 20-minute incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concentration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively. A similar effect was shown using the Moloney murine leukemia-derived MFGlacZ retroviral vector, coding for Escherichia coli beta-galactosidase. Increased titer and concentration had no effect on target cell viability, as shown by trypan blue exclusion. Although RSMCs had the most cells transduced in a given incubation period (p < 0.05), RPAECs had the highest replication rate (p < 0.05), suggesting the importance of factors other than cell cycle on retroviral TEs during short, clinically relevant incubation periods. In subsequent in vivo experiments, gene transfer was achieved in the rat carotid artery during a 20-minute incubation period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury. Rats infused with virus 2 days after balloon injury exhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats. Rats infused 4 days after balloon injury exhibited hpAP activity (0 to 25 cells/section/rat) in the media and adventitia of five out of five rats. Control rats that received the balloon injury alone or the balloon injury and unconcentrated retrovirus exhibited zero hpAP activity. We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugation to increase retroviral titer, and thereby concentration of viable viral particles.
血管细胞是基因转移的重要靶点，因为它们有潜力在局部和全身递送基因产物。体内将逆转录病毒基因直接转移到血管细胞一直受到转导效率低下的限制。我们假设，在短时间逆转录病毒孵育期内，通过离心增加病毒滴度，体外和体内的血管细胞转导效率（TE）会显著提高。此外，我们假设在短孵育期内，存活病毒颗粒浓度（以集落形成单位（CFU）/细胞衡量）与逆转录病毒TE之间存在线性关系。培养的大鼠肺动脉内皮细胞（RPAECs）、大鼠主动脉平滑肌细胞（RSMCs）和人髂动脉内皮细胞（HIAECs）在10至60分钟的逆转录病毒孵育期内，TE与高浓度病毒（>100 CFU/细胞）之间显示出强相关性。通过离心并以原始体积的一小部分重悬，可实现高滴度，从而达到高浓度。滴度持续增加10倍，体积浓度增加20倍。用编码人胎盘碱性磷酸酶的莫洛尼鼠白血病衍生逆转录病毒载体pLJhpAP，以1150 CFU/细胞的浓度孵育20分钟，RPAECs、RSMCs和HIAECs的TE分别为10.6%±0.7%、40.4%±1.6%和15.1%±2.0%。使用编码大肠杆菌β-半乳糖苷酶的莫洛尼鼠白血病衍生MFGlacZ逆转录病毒载体也显示出类似效果。如台盼蓝排斥试验所示，滴度和浓度的增加对靶细胞活力没有影响。尽管在给定孵育期内RSMCs转导的细胞最多（p<0.05），但RPAECs的复制率最高（p<0.05），这表明在短的、临床相关孵育期内，除细胞周期外的其他因素对逆转录病毒TE也很重要。在随后的体内实验中，在大鼠颈动脉球囊损伤后，通过注入浓缩的pLJhpAP逆转录病毒，在20分钟孵育期内实现了基因转移。球囊损伤后2天注入病毒的大鼠，6只中有5只在新生内膜中表现出hpAP活性（0至10个细胞/切片/大鼠）。球囊损伤后4天注入病毒的大鼠，5只中有5只在中膜和外膜中表现出hpAP活性（0至25个细胞/切片/大鼠）。单独接受球囊损伤或接受球囊损伤和未浓缩逆转录病毒的对照大鼠表现出零hpAP活性。我们得出结论，在短的、临床相关孵育期内，通过离心增加逆转录病毒滴度，从而提高存活病毒颗粒浓度，可提高体外和体内逆转录病毒介导的基因转移到血管细胞的TE。
