The exon skipping potential of Pip6-PMO conjugates was evaluated in differentiated mouse H2K mdx myotubes in the absence of any transfection agent (Figure 2 ) at concentrations ranging from 0.125 to 1 µmol/l. This showed that exon skipping activity in cultured muscle cells was very similar for all these constructs, including Pip5e-PMO. These results differ from the previous Pip5 series, 29 where the flanking argininerich sequences mostly contained a fixed number of arginine residues (10) but where spacings were varied through alternative placement of aminohexanoyl and β-alanine units. This resulted in small variations in exon skipping activity that correlated well with in vivo activity. In the case of Pip6 sequences, the flanking arginine-rich sequences are identical (with the exception of Pip6e, which is identical except for one arginine immediately preceding the core which is displaced into the second position of the core). The results demonstrate that cellular exon skipping activity does not depend on the sequence or length of the hydrophobic core. Note that we have previously shown that major changes in in vitro exon skipping activities are correlated instead with the total numbers of arginine residues. 33 Alterations to the Pip5e hydrophobic sequence improve splicing activity in vivo Given the potency of Pip5e-PMO in heart tissue, the aim of altering the sequence of the hydrophobic core (whilst maintaining the 5-aa length) was to identify peptides that might be more efficient at lower doses. These modifications included inversion of the hydrophobic region (Pip6a), substitution of tyrosine by isoleucine (Pip6b), substitution of glutamine in the Pip6a sequence by displacement of the arginine immediately flanking the core in the first arginine-rich flanking region (Pip6e), and a scrambled hydrophobic core sequence (Pip6f). All Pip6 peptide-PMO conjugates were administered to mdx mice as single 12.5 mg/kg intravenous injections via the tail vein and tissues were harvested 2 weeks later and assessed for activity at both RNA and protein levels.