title: RNAi-mediated suppression of constitutive pulmonary gene expression by small interfering RNA in mice.
abstract: The ability of synthetic small interfering RNA (siRNA) to silence gene expression makes it a useful tool in biomedical research. However, effective and non-toxic functional siRNA delivery to mouse lungs in vivo is still a key challenge, and regulation of constitutively expressed genes is poorly characterized. Following in vitro validation of siRNA molecules, naked, stabilized siRNA (AtuRNAi) was applied intranasally (i.n.) by droplets or intratracheally (i.t.) by MicroSprayer in female C57BL/6 mice. Distribution of Cy3-tagged siRNAs was examined. Pulmonary expression of ubiquitously (lamin B1) or cell-specific (E-cadherin, VE-cadherin), constitutive genes was analysed by TaqMan-realtime-PCR. Further, formulated lipoplex-siRNA, which has enhanced transfection efficiency, was applied i.t. or intravenously (i.v.). Single i.t. as compared to i.n. application of unformulated siRNA resulted in higher delivery efficiency and homogenous pulmonary distribution. After inhalation of target-specific siRNA, reduction of epithelial E-cadherin by 21%, but no significant reduction of endothelial VE-cadherin or ubiquitously expressed lamin B1 was observed. Pharmacokinetic analysis revealed rapid transfer of intact siRNA molecules into the vascular system and accumulation in the kidneys, calling lung specificity into question. I.t. application of lipoplex-siRNA evoked inflammation. In contrast, i.v. application of lipoplex-siRNA specifically reduced expression of VE-cadherin mRNA by about 50% in lungs without evoking lung cellular influx. In conclusion, sufficient pulmonary distribution of aerosolized siRNA was attained in mice by MicroSprayer, however development of appropriate siRNA carriers is highly desirable to improve lung-specific functional inhalative siRNA delivery. Pulmonary knockdown of constitutive endothelial targets by 50% was achieved by i.v. application of lipoplex-siRNA.
authors.affiliation: Department of Internal Medicine/Infectious and Respiratory Diseases, Charité-Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany.
authors.forename: Birgitt
authors.initials: B
authors.lastname: Gutbier
authors.forename: Stefanie M
authors.initials: SM
authors.lastname: Kube
authors.forename: Katrin
authors.initials: K
authors.lastname: Reppe
authors.forename: Ansgar
authors.initials: A
authors.lastname: Santel
authors.forename: Christian
authors.initials: C
authors.lastname: Lange
authors.forename: Jörg
authors.initials: J
authors.lastname: Kaufmann
authors.forename: Norbert
authors.initials: N
authors.lastname: Suttorp
authors.forename: Martin
authors.initials: M
authors.lastname: Witzenrath
mesh_terms: D000281:Administration, Intranasal; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D015820:Cadherins / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D007249:Inflammation / Q000235:genetics; D034921:Lamin Type B / Q000235:genetics; D008168:Lung / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014132:Trachea; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D034921:Lamin Type B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000099152:Cadherin 5