National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, Incheon, Korea.
Andrology. 2013 Mar;1(2):216-22. doi: 10.1111/j.2047-2927.2012.00057.x. Epub 2013 Jan 13.
A prerequisite for the successful clinical application of gene therapy in erectile dysfunction (ED) is the availability of safe and efficient gene delivery systems. The aim of this study was to examine the effectiveness of guanidinylated bioreducible polymer (GBP) polyplexes for gene delivery systems, which take advantage of the biodegradability of reducible disulfide bonds and the cell-penetrating ability of guanidine groups. For in vitro transfection experiments, we used mouse cavernous endothelial cells and A7r5 rat vascular smooth muscle cells. For in vivo experiments, we used a mouse model of hypercholesterolaemic ED in which 2-month-old male C57BL/6 mice were fed a diet containing 4% cholesterol and 1% cholic acid for 3 months. Animals or cells were treated with pCMV-Luc, poly(ethyleneimine) (PEI)25k/pCMV-Luc polyplex (weight ratio: 1) and GBP/pCMV-Luc polyplexes (weight ratio: 20, 40, 60 and 80). Gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. GBP had greater transfection efficiency as the weight ratio increased. GBP had sevenfold higher gene delivery efficiency in A7r5 cells at a weight ratio of 80 than did PEI25k. Moreover, the gene expression was more profoundly induced by GBP/pCMV-Luc than by pCMV-Luc in both the corpus cavernosum tissue of hypercholesterolaemic mice and in mouse cavernous endothelial cells, although the expression levels induced by the GBP gene delivery system were lower than those induced by the PEI25k gene delivery system. GBP revealed no considerable cytotoxicity to A7r5 cells and mouse cavernous endothelial cells (relative cell viability: 95 and 88% respectively), whereas PEI25k resulted in high cytotoxicity. Interestingly, immunofluorescent double staining revealed that luciferase expression induced by the GBP polyplex mainly overlapped with cavernous endothelial cells, but rarely with smooth muscle cells. The GBP-based non-viral gene expression system may be useful for the development of gene therapy in vasculogenic ED.
基因治疗在勃起功能障碍（ED）中的成功临床应用的前提是安全有效的基因传递系统。本研究旨在研究胍基化生物还原聚合物（GBP）多聚物作为基因传递系统的有效性，该系统利用还原型二硫键的可生物降解性和胍基的细胞穿透能力。在体外转染实验中，我们使用了小鼠海绵体内皮细胞和 A7r5 大鼠血管平滑肌细胞。在体内实验中，我们使用了一种高脂血症 ED 的小鼠模型，其中 2 个月大的雄性 C57BL/6 小鼠在 3 个月内喂食含有 4%胆固醇和 1%胆酸的饮食。动物或细胞用 pCMV-Luc、聚（亚乙基亚胺）（PEI）25k/pCMV-Luc 多聚物（重量比：1）和 GBP/pCMV-Luc 多聚物（重量比：20、40、60 和 80）处理。通过荧光素酶测定法评估基因表达，通过免疫组织化学法评估基因表达区域。随着重量比的增加，GBP 的转染效率更高。在重量比为 80 时，GBP 在 A7r5 细胞中的基因传递效率比 PEI25k 高 7 倍。此外，与 pCMV-Luc 相比，GBP/pCMV-Luc 在高脂血症小鼠的海绵体组织和小鼠海绵体内皮细胞中均能更显著地诱导基因表达，尽管 GBP 基因传递系统诱导的表达水平低于 PEI25k 基因传递系统。GBP 对 A7r5 细胞和小鼠海绵体内皮细胞无明显细胞毒性（相对细胞活力分别为 95%和 88%），而 PEI25k 则具有高细胞毒性。有趣的是，免疫荧光双重染色显示，GBP 多聚物诱导的荧光素酶表达主要与海绵体内皮细胞重叠，但很少与平滑肌细胞重叠。基于 GBP 的非病毒基因表达系统可能有助于血管性 ED 的基因治疗的发展。
