We next examined whether differences in splicing correction activity could be explained by differences in endosomal escape. All (R-X-R) 4 -PMO conjugates have therefore been synthesized as FAM-labeled derivatives and their intracellular distribution has been analyzed by fluorescence microscopy on live cells to avoid artefactual redistribution upon cell fixation. As shown in Figure 6A for the parent (R-Ahx-R) 4 -PMO-FAM conjugate, most of the material was distributed as punctate cytoplasmic material and none was detected in the nuclei. Splicing correction is probably due to the small amount of material which has escaped from the endocytic vesicles and remains undetectable by fluorescence microscopy analysis. Not surprisingly, a similar situation has been observed for other (R-X-R) 4 -PMO-FAM conjugates from our SAR studies and no concluding data have been provided by fluorescence microscopy comparative analysis (data not shown). Likewise, previous work from several groups including our own one had documented an increase in splicing correction upon treatment with endosomolytic agents such as chloroquine or calcium ions (3) . However, splicing correction never reached levels achieved with 2-OMe ON analogs transfected as lipoplexes and accordingly redistribution of the endosome-entrapped material could not be documented (21) . We now capitalize on a saponin treatment protocol which allows to gently permeabilize the plasma membrane. It was shown to open transient holes in the plasma membrane and to allow the passage of macromolecules while not damaging membranes from intracellular organelles (22) .