Unit of Gene Therapy and Stem Cell Biology, Jules-Gonin Eye Hospital, University of Lausanne, avenue de France 15, Lausanne, Switzerland.
Gene Ther. 2009 Jul;16(7):933-8. doi: 10.1038/gt.2009.41. Epub 2009 Apr 23.
We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors. A delimited space was created at 110 mum depth with a LDV femtosecond laser in pig corneas, and a HIV1-derived lentiviral vector expressing green fluorescent protein (GFP) (LV-CMV-GFP) was injected into the pocket. Corneas were subsequently dissected and kept in culture as explants. After 5 days, histological analysis of the explants revealed that the corneal pockets had closed and that the gene transfer procedure was efficient over the whole pocket area. Almost all the keratocytes were transduced in this area. Vector diffusion at right angles to the pocket's plane encompasses four (endothelium side) to 10 (epithelium side) layers of keratocytes. After 21 days, the level of transduction was similar to the results obtained after 5 days. The femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Showing the efficacy of this procedure in vivo could represent an important step toward treatment or prevention of recurrent angiogenesis of the corneal stroma.
我们研究了一种新的方法，将治疗因子递送到猪角膜基质中的基因转移。在猪角膜中，用 LDV 飞秒激光在 110 微米深度处创建一个限定的空间，并将表达绿色荧光蛋白（GFP）的 HIV1 衍生的慢病毒载体（LV-CMV-GFP）注入口袋中。随后将角膜解剖并作为外植体保持培养。5 天后，对 explants 的组织学分析表明，角膜口袋已经闭合，整个口袋区域的基因转移过程非常有效。该区域几乎所有的角膜细胞都被转导了。载体在垂直于口袋平面的方向上扩散，涵盖了 4 到 10 层角膜细胞（内皮侧）。21 天后，转导水平与 5 天后的结果相似。飞秒激光技术可实现慢病毒载体的可靠注射和扩散，从而有效地转导限定区域的基质细胞。在体内证明该方法的有效性可能是治疗或预防角膜基质中复发性血管生成的重要步骤。
