Corinne Betts 1 , Amer F Saleh 2 , Andrey A Arzumanov 2 , Suzan M Hammond 1 , Caroline Godfrey 1 , Thibault Coursindel 2 , Michael J Gait 2 and Matthew JA Wood 1 Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. 26 has also been investigated, namely R6-Penetratin which contains six additional arginines. 27 Employing R6-Penetratin as the lead peptide, a series of peptide nucleic acids/PMO internalization peptides (Pips) were derived that were found to be much more stable to serum proteolysis. 28 Two such Pip peptides, Pip2a and Pip2b, conjugated to a dystrophin exon 23-specific (peptide nucleic acids) AO, were shown to be capable of inducing strong exon skipping and dystrophin positive fibres following intramuscular injection into the tibialis anterior (TA) muscle of the mdx mouse. Further optimization of this peptide series was carried out as conjugates to PMO, and Pip5e-PMO was identified as the most efficient peptide-PMO conjugate capable of inducing high levels of exon skipping and dystrophin restoration body wide, including in the heart, following a single dose intravenous administration. 29 The Pip5e structure comprises a hydrophobic core region flanked on each side by arginine-rich domains containing aminohexanoyl (X) and β-alanine (B) spacers. By analogy with the previous arginine-rich B peptide, 22 it was thought that the high arginine content of Pip5e contributed to overall delivery efficiency into all muscle tissues, whereas the hydrophobic region might be important for heart muscle delivery.