Institutes of Health (CA119335 to UCSD NanoTumor Center of Excellence for Cancer Nanotechnology, CA133634 to Y. T. Liu, NS034934 to Delivery of steric block morpholino oligomers by (R-X-R)(4) peptides: structure–activity studies Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)–PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs. Protein transduction domains as penetratin or Tat 48-60 and synthetic cell penetrating peptides (CPP) as oligoarginine have generated a large interest for their seemingly unique mechanism of membrane translocation and for their capacity to transport various biomolecules across biological membranes (1) . Both assumptions have had to be re-visited since cellular uptake does involve endocytosis (2) and transport of biomolecules does not occur as efficiently as anticipated at least at low concentrations. In a series of experiments carried out independently by several groups, CPPs mentioned above turned out inefficient in transporting uncharged splice correcting oligonucleotide (ON) analogs as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO) for a large part because CPP-conjugated material remained entrapped in endocytic vesicles (3) . Accordingly, peptides or drugs (such as chloroquine) leading to endosome destabilization did significantly increase splicing correction.