Antisense-mediated exon skipping constitutes a promising new modality for treatment of Duchenne Muscular Dystrophy (DMD), which is caused by gene mutations that typically introduce a translation stop codon in the dystrophin gene, thereby abolishing production of functional dystrophin protein. The exon removal can restore translation to produce a shortened, but still partially functional dystrophin protein. Peptide nucleic acid (PNA) as a potential antisense drug has previously been shown to restore the expression of functional dystrophin by splice modulation in the mdx mouse model of DMD. In this study, we compare systemic administration of a 20-mer splice switching antisense PNA oligomer through intravenous (i.v.) and subcutaneous (s.c.) routes in the mdx mice. Furthermore, the effect of in situ forming depot technology (BEPO®) and PNA-oligonucleotide formulation was studied. In vivo fluorescence imaging analysis showed fast renal/bladder excretion of the PNA (t1/2 ∼20 min) for i.v. administration, while s.c. administration showed a two to three times slower excretion. The release from the BEPO depot exhibited biphasic kinetics with a slow release (t1/2 ∼10 days) of 50% of the dose. In all cases, some accumulation in kidneys and liver could be detected. Formulation of PNA as a duplex hybridization complex with a complementary phosphorothioate oligonucleotide increased the solubility of the PNA. However, none of these alternative administration methods resulted in significantly improved antisense activity. Therefore, either more sophisticated formulations such as designed nanoparticles or conjugation to delivery ligands must be utilized to improve both pharmacokinetics as well as tissue targeting and availability. On the other hand, the results show that s.c. and BEPO depot administration of PNA are feasible and allow easier, higher, and less frequent dosing, as well as more controlled release, which can be exploited both for animal model studies as well as eventually in the clinic in terms of dosing optimization.