Kidney-targeted plasmid DNA transfer by retrograde renal vein injection

肾脏靶向基因转移有可能成为扩大我们对肾脏疾病过程的理解和革新肾脏疾病治疗的最重要工具之一。先前使用非病毒载体的基因转移方法通过肾动脉、骨盆或输尿管途径施用到肾小球、肾小管或间质成纤维细胞中，已经显示出低水平表达< 1个月。肾小管周围毛细血管（PTC）网络是肾移植排斥反应和进行性肾小管间质纤维化的主要靶点之一，其代表所有进行性肾脏疾病。我们认为含有PTC的氚是基因转移的重要候选位点。为了进入PTC，我们将溶于林格氏溶液中的lacZ表达质粒逆行注射到大鼠的肾静脉中。免疫电镜分析发现lacZ仅在PTC附近的间质成纤维细胞中表达。转基因致肾毒性 ...更多信息 FER不明显。我们使用大鼠促红细胞生成素（Epo）表达质粒载体，pCAGGS-Epo，在报告基因。我们发现当DNA溶液在5秒内注射并且体积为1.0 ml时，Epo表达最大。我们观察到血清Epo水平与高达100 μg的DNA注射量之间存在剂量-反应关系。我们仅在注射pCAGGS-Epo的肾脏中通过RT-PCR检测到转基因衍生的Epo mRNA。注射100 μg pCAGGS-Epo后，血清Epo水平在第5周达到峰值208.3 ± 71.8 mU/ml，并在第24周逐渐下降至116.2 ± 38.7 mU/ml。使用较小剂量的质粒，2或30 μg的pCAGGS-Epo，观察到类似的模式。转基因衍生的Epo分泌引起显著的红细胞生成。接下来，我们测试了大鼠肾脏是否可以仅在夹闭肾静脉下靶向。当DNA溶液在2秒内注射并且体积为0.5ml时，我们获得最大Epo表达。我们观察到血清Epo水平与高达100 μg的DNA注射量之间存在剂量-反应关系。注射100 μg pCAGGS-Epo后，血清Epo水平在第3周达到峰值423.8 ± 320.1 mU/ml，并在第24周逐渐下降至161.5 ± 125.2 mU/ml。转基因衍生的Epo分泌产生显著的红细胞生成。该新技术简单，安全，并允许高水平，长期稳定的基因表达和特异性基因转移到PTC附近的成纤维细胞，这使得它特别有吸引力的潜力，未来在人类中的应用。少

Kidney-targeted gene transfer has the potential to be one of the most important tools for broadening our understanding of renal disease processes and revolutionizing the treatment of renal diseases. Previous gene-transfer methods using nonviral vectors administered via renal arterial, pelvic, or ureteric routes into the glomerulus, tubules, or interstitial fibroblasts have shown low-level expression for < 1 month. The peritubular capillaries (PTC) network is one of the main targets of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. We believe the interstitium containing the PTC is an important candidate site for gene transfer. To access the PTC, we retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats. We found lacZ expression only in the interstitial fibroblasts near the PTC of the injected kidney by immunoelectron microscopic analysis. Nephrotoxicity due to gene trans … More fer was not apparent. We used a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo, in a reporter gene. We found maximal Epo expression when the DNA solution was injected within 5 sec, and with a volume of 1.0 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 μg. We detected the transgene-derived Epo mRNA by RT-PCR only in the kidneys injected with pCAGGS-Epo. After an injection of 100 μg of pCAGGS-Epo, the serum Epo levels peaked at 208.3 ± 71.8 mU/ml at week 5, and gradually decreased to 116.2 ± 38.7 mU/ml at week 24. A similar pattern was seen using smaller doses of plasmid, 2 or 30 μg of pCAGGS-Epo. Transgene-derived Epo secretion caused significant erythropoiesis. Next, we tested whether rat kidney could be targeted under the clamping renal vein only. We obtained maximal Epo expression when the DNA solution was injected within 2 sec, and with a volume of 0.5 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 μg. After an injection of 100 μg of pCAGGS-Epo, the serum Epo levels peaked at 423.8 ± 320.1 mU/ml at week 3, and gradually decreased to 161.5 ± 125.2 mU/ml at week 24. Transgene-derived Epo secretion gave significant erythropoiesis. The novel technique is simple, safe, and allows high-level, long-term stable gene expression and specific gene transfer to the fibroblasts near the PTC, which makes it particularly appealing for potential for future applications in humans. Less