The present structure-activity relationship (SAR) studies were initiated for the following additional reasons. Fluorescence microscopy evaluation of the intracellular distribution of FAM-labeled (R-Ahx-R) 4 -PMO conjugates indicated that the majority of the material was entrapped in endocytic vesicles even at concentrations leading to efficient splicing correction. It implies that the biological activity of these conjugates is due to the small (and not detectable by fluorescence microscopy) portion of material escaping from endocytic vesicles. Finally, (R-Ahx-R) 4 -PMO conjugates have shown signs of toxicity when injected to mice at >20 mg/kg dose despite their absence of cytotoxicity in cell culture experiments (12) . This presents a dosing challenge for in vivo systemic applications.