Kos Spela, Tesic Natasa, Kamensek Urska, Blagus Tanja, Cemazar Maja, Kranjc Simona, Lavrencak Jaka, Sersa Gregor
Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloska 2, 1000, Ljubljana, Slovenia.
J Membr Biol. 2015 Oct;248(5):919-28. doi: 10.1007/s00232-015-9799-4. Epub 2015 Apr 4.
In order to ensure safe, efficient and controlled gene delivery to skin, the improvement of delivery methods together with proper design of DNA is required. Non-viral delivery methods, such as gene electrotransfer, and the design of tissue-specific promoters are promising tools to ensure the safety of gene delivery to the skin. In the scope of our study, we evaluated a novel skin-specific plasmid DNA with collagen (COL) promoter, delivered to skin cells and skin tissue by gene electrotransfer. In vitro, we determined the specificity of the COL promoter in fibroblast cells. The specific expression under the control of COL promoter was obtained for the reporter gene DsRed as well as for therapeutic gene encoding cytokine IL-12. In vivo, the plasmid with COL promoter encoding the reporter gene DsRed was efficiently transfected to mouse skin. It resulted in the notable and controlled manner, however, in lower and shorter expression, compared to that obtained with ubiquitous promoter. The concentration of the IL-12 in the skin after the in vivo transfection of plasmid with COL promoter was in the same range as after the treatment in control conditions (injection of distilled water followed by the application of electric pulses). Furthermore, this gene delivery was local, restricted to the skin, without any evident systemic shedding of IL-12. Such specific targeting of skin cells, observed with tissue-specific COL promoter, would improve the effectiveness and safety of cutaneous gene therapies and DNA vaccines.
为确保向皮肤进行安全、高效且可控的基因递送，需要改进递送方法并合理设计DNA。非病毒递送方法，如基因电穿孔，以及组织特异性启动子的设计，是确保向皮肤进行基因递送安全性的有前景的工具。在我们的研究范围内，我们评估了一种带有胶原蛋白（COL）启动子的新型皮肤特异性质粒DNA，通过基因电穿孔递送至皮肤细胞和皮肤组织。在体外，我们确定了COL启动子在成纤维细胞中的特异性。在COL启动子的控制下，报告基因DsRed以及编码细胞因子IL-12的治疗性基因均获得了特异性表达。在体内，携带编码报告基因DsRed的COL启动子的质粒被有效地转染到小鼠皮肤中。然而，与使用普遍启动子相比，其表达较低且持续时间较短，但呈现出显著且可控的方式。用COL启动子的质粒进行体内转染后，皮肤中IL-12的浓度与对照条件下（注射蒸馏水后施加电脉冲）处理后的浓度处于同一范围。此外，这种基因递送是局部的，仅限于皮肤，没有任何明显的IL-12全身性释放。用组织特异性COL启动子观察到的这种对皮肤细胞的特异性靶向，将提高皮肤基因治疗和DNA疫苗的有效性和安全性。
