Approaches for Systemic Delivery of Dystrophin Antisense Peptide Nucleic Acid in the mdx Mouse Model.

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 (t½ ∼ 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 (t½ ∼ 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.

In Vivo Administration of Splice Switching PNAs Using the mdx Mouse as a Model System.

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy and is caused by gene mutations that abolish production of functional dystrophin muscle protein. A promising new treatment exploits specifically targeted RNA-acting drugs that are able to partially restore the dystrophin protein. The mdx mouse model (animal model of DMD) serves as a good in vivo model for testing these antisense drugs. The simplest in vivo test, which circumvents the systemic circulation, is intramuscular administration of the compound. After 7 days it is possible to detect exon skipping by reverse transcriptase PCR, and newly synthesized dystrophin-positive fibers by immunohistochemistry and western blotting. All muscles, including the heart, are affected by the disease and must be treated. Therefore the use of antisense therapy for treatment of DMD requires systemic administration, and the model is also useful for systemic administration.

Near-Infrared In Vivo Whole-Body Fluorescence Imaging of PNA.

Using near-infrared fluorophore Alexa Fluor 680 labeled peptide nucleic acids (PNAs) the biodistribution of such antisense agents can be analyzed in real time in live mice using in vivo imaging. Using the fluorescence intensity emitted from the mouse at different time points following administration, the systemic distribution and organ accumulation of PNA can be tracked. In addition, an estimation of the body half-life of the compound can be obtained by the change in fluorescence intensity over time. With this technique, the distribution of compounds can be monitored real time, while reducing the number of animals and amount of compounds required.