Abstract
Translational readthrough of premature termination codons (PTCs) induced by pharmacological compounds has proven to be an effective way of restoring functional protein expression and reducing symptoms in several genetic disorders. We tested the potential of different concentrations of several aminoglycosides (AAGs) for promoting PTC-readthrough in 5 genes involved in the pathogenesis of primary ciliary dyskinesia, an inherited disorder caused by the dysfunction of motile cilia and flagella. The efficiency of readthrough stimulation of PTCs cloned in dual reporter vectors was examined in 2 experimental settings: in vitro (transcription/translation system) and ex vivo (transiently transfected epithelial cell line). PTC-readthrough was observed in 5 of the 16 mutations analyzed. UGA codons were more susceptible to AAG-stimulated readthrough than UAG; no suppression of UAA was observed. The efficiency of PTC-readthrough in vitro (from less than 1% to ∼28% of the translation from the corresponding wild-type constructs) differed with the AAG type and concentration, and depended on the combination of AAG and PTC, indicating that each PTC has to be individually tested with a range of stimulating compounds. The maximal values of PTC suppression observed in the ex vivo experiments were, depending on AAG used, 3-5 times lower than the corresponding values in vitro, despite using AAG concentrations that were 2 orders of magnitude higher. This indicates that, while the in vitro system is sufficient to examine the readthrough-susceptibility of PTCs, it is not sufficient to test the compounds potential to stimulate PTC-readthrough in the living cells. Most of the tested compounds (except for G418) at their highest concentrations did not disturb ciliogenesis in the cultures of primary respiratory epithelial cells from healthy donors.