Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo.
Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo.
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阳离子脂质介导的siRNA和反义寡核苷酸在体内转移到气道上皮细胞。
DOI：
10.1186/1465-9921-7-26
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发表时间：
2006-02-15
影响因子：
5.8
通讯作者：
Alton, EWFW
中科院分区：
文献类型：
--
作者：
Griesenbach, U;Kitson, C;Garcia, SE;Farley, R;Singh, C;Somerton, L;Painter, H;Smith, RL;Gill, DR;Hyde, SC;Chow, YH;Hu, J;Gray, M;Edbrooke, M;Ogilvie, V;MacGregor, G;Scheule, RK;Cheng, SH;Caplen, NJ;Alton, EWFW
阳离子脂质Genzyme lipid（GL）67是目前体内肺基因转移的“金标准”。在此，我们评估了GL 67是否介导siRNA和asODN摄取到体内气道上皮中。将抗lacZ和ENaC（上皮钠通道）siRNA和asODN与GL 67复合，并体内施用至小鼠气道上皮。使用实时RT-PCR以及通过蛋白质表达和功能研究评估转染效率和功效。平行进行体外实验以选择最有效的寡核苷酸。在体外，GL 67有效地复合asODNs和siRNA，并且两者在呼出气冷凝物中是稳定的。重要的是，在功能性siRNA和asODN的体外选择过程中，我们注意到asODN在转染细胞的细胞核中迅速积累，而siRNA保留在细胞质中，这种模式与其假定的作用位点一致。在体内肺转染后，siRNA仅在肺泡巨噬细胞中可见，而asODN也转染肺泡上皮细胞，但没有观察到传导气道上皮细胞的显著摄取。靶向β-半乳糖苷酶的siRNA和asODN分别使K18-lacZ小鼠气道上皮中的βgal mRNA水平降低30%和60%。然而，这不足以降低蛋白质表达。为了提高气道上皮细胞的转染效率，我们使用体内小鼠鼻模型增加siRNA和asODN的接触时间。虽然高度可变和低效，气道上皮细胞转染asODN，而不是siRNA，现在看到。由于asODN更有效地转染鼻气道上皮细胞，我们评估了asODN对ENaC（囊性纤维化的潜在治疗靶点）的作用;未检测到ENaC mRNA水平或功能的降低。这项研究表明，虽然siRNA和asODNs可以抑制基因表达的培养系统和某些器官在体内，在气道上皮细胞中看到的大DNA分子的核酸转移的障碍，也可能影响小核酸分子的体内摄取的效率。
The cationic lipid Genzyme lipid (GL) 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. Anti-lacZ and ENaC (epithelial sodium channel) siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to β-galactosidase reduced βgal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. This study suggests that although siRNAs and asODNs can be developed to inhibit gene expression in culture systems and certain organs in vivo, barriers to nucleic acid transfer in airway epithelial cells seen with large DNA molecules may also affect the efficiency of in vivo uptake of small nucleic acid molecules.
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DOI：
10.1152/ajplung.1999.276.6.l1046
发表时间：
1999-06-01
影响因子：
4.9
作者：
Jain, L;Chen, XJ;Eaton, DC
通讯作者：
Eaton, DC
影响因子：
7.7
作者：
König, J;Schreiber, R;Kunzelmann, K
通讯作者：
Kunzelmann, K
影响因子：
64.8
作者：
Reddy, MM;Light, MJ;Quinton, PM
通讯作者：
Quinton, PM
影响因子：
11.4
作者：
Staub, O;Gautschi, I;Rotin, D
通讯作者：
Rotin, D
影响因子：
168.9
作者：
Alton, EWFW;Stern, M;Geddes, DM
通讯作者：
Geddes, DM