Finotti Alessia, Borgatti Monica, Bezzerri Valentino, Nicolis Elena, Lampronti Ilaria, Dechecchi Maria, Mancini Irene, Cabrini Giulio, Saviano Michele, Avitabile Concetta, Romanelli Alessandra, Gambari Roberto
ER-GenTech and BioPharmaNet, Department of Biochemistry and Molecular Biology, Università di Ferrara, Ferrara, Italy.
Artif DNA PNA XNA. 2012 Apr-Jun;3(2):97-296. doi: 10.4161/adna.21061. Epub 2012 Apr 1.
One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3-1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.
囊性纤维化（CF）的临床特征之一是深部炎症过程，其特点是细胞因子和趋化因子的产生与释放，其中白细胞介素8（IL-8）是最重要的因子之一。因此，开发针对CF的疗法以减少CF患者气道中过度的炎症反应的兴趣日益浓厚。由于转录因子NF-κB在IL-8表达中起关键作用，转录因子诱饵（TFD）策略可能会受到关注。为了证明针对NF-κB的TFD会干扰NF-κB信号通路，我们通过染色质免疫沉淀（ChIP）证明，用铜绿假单胞菌感染的囊性纤维化IB3-1细胞的TFD寡脱氧核糖核苷酸处理会导致NF-κB因子对Il-8基因启动子的占有率降低。为了开发更稳定的治疗分子，人们考虑了基于肽核酸（PNA）的药物。在这方面，PNA-DNA-PNA（PDP）嵌合体从几个角度来看都是非常令人感兴趣的分子：（1）它们可以与脂质体和微球复合；（2）它们对脱氧核糖核酸酶、血清和细胞质提取物具有抗性；（3）它们是有效的诱饵分子。通过使用电泳迁移率变动分析和RT-PCR分析，我们已经证明：（1）PDP/PDP NF-κB诱饵嵌合体对铜绿假单胞菌感染的IB3-1细胞中促炎mRNA积累的影响与诱饵寡核苷酸的影响相同；特别是（2）PDP/PDP嵌合体是IL-8基因表达的强抑制剂；（3）与基于ODN的诱饵不同，即使在没有脂质体转染试剂保护的情况下，也能观察到PDP/PDP嵌合体的作用。我们认为，这些信息对于开发用于囊性纤维化非病毒基因治疗的稳定分子具有重大影响。
