Assessment of the nuclear pore dilating agent trans-cyclohexane-1,2-diol in differentiated airway epithelium.
BACKGROUND
The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro.


METHODS
We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug).


RESULTS
TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls.


CONCLUSIONS
Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications.
Department of Gene Therapy, Imperial College London, UK. u.griesenbach@imperial.ac.uk
Uta
U
Griesenbach
Katherine M
KM
Wilson
Raymond
R
Farley
Cuixiang
C
Meng
Felix M
FM
Munkonge
Seng H
SH
Cheng
Ronald K
RK
Scheule
Eric W F W
EW
Alton
D000818:Animals; D002478:Cells, Cultured; D003510:Cyclohexanes / Q000008:administration & dosage / Q000494:pharmacology*; D003511:Cyclohexanols / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004333:Drug Administration Routes; D004848:Epithelium / Q000187:drug effects; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008168:Lung / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D009297:Nasal Mucosa / Q000187:drug effects; D022022:Nuclear Pore / Q000187:drug effects*; D012137:Respiratory System / Q000187:drug effects*; D014162:Transfection
D003510:Cyclohexanes; D003511:Cyclohexanols; D008055:Lipids; C086724:Lipofectamine; C506365:Cyclohexane; C064619:1,2-cyclohexanediol