Most surprising however is that Pip6e-PMO did induce some dystrophin splicing and protein restoration in heart muscle as indicated by the western and qRT-PCR results (note: not significantly different in immunohistochemical staining quantification). In the Pip-6e peptide, one arginine residue is moved into the hydrophobic core, which also results in alignment of a hydrophobic X residue adjacent to the core (X-YRFLI). One might have expected heart dystrophin production to have been completely lost in this conjugate, since a cationic amino acid (arginine) is now included in the core. By contrast, such heart activity was lost for Pip6h-PMO (X-ILFRY core) and the double arginine core conjugate Pip6g-PMO (X-YRFRLI-X core). Unexpectedly, dystrophin production was also lost in quadriceps and diaphragm for both Pip6g-and Pip6h-PMO. The unanticipated inconsistencies within the activity results for Pip6e, Pip6g, and Pip6h, and the losses of activities for Pip6c-and Pip6d-PMO, are perhaps best explained by the realization that precise spacings of the arginine residue within the Pip peptides with respect to both the outer hydrophobic amino acid spacers (X and B) and the inner hydrophobic core residues may drastically alter the pharmacological properties of each conjugate. This might occur not only through alteration in cationic/hydrophobic balance but alternatively due to secondary or tertiary structure changes of the Pip-PMOs, which could in turn affect serum protein binding or another parameter that alters the circulatory half-life, or which could affect the ability to traverse barriers required to penetrate muscle tissues. Such more subtle effects will require a more wide-ranging pharmacological and biophysical study, upon which we are currently embarking.