Identification of a novel muscle targeting peptide in mdx mice
ABSTRACT Exon-skipping oligonucleotides are a well-researched therapeutic strategy for Duchenne's muscular dystrophy (DMD). Despite remarkable successes in animal models with intramuscular and intravenous delivery of unmodified oligonucleotides, the ability to specifically target both normal and dystrophic muscle with a simple peptide ligand could decrease the therapeutic dose required and reduce the potential for toxicity. Thus, 3 rounds of in vivo phage display utilizing a 12-mer peptide library were performed with mdx mice and a peptide motif with potential for targeting to muscle but not liver was identified. This motif was shown to have enhanced binding affinity to C2C12 myoblasts over a scrambled control peptide and in vivo application of a fluorescein-labeled peptide containing the identified motif resulted in increased specificity for the heart and quadriceps muscle after tail-vein administration in C57BL/6 mice. This work has many potential applications for oligonucleotide or drug delivery to muscle for myopathies.
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ABSTRACT: Antisense oligonucleotide (AO) - mediated splice correction therapy for Duchenne muscular dystrophy (DMD) has shown huge promise from recent phase IIb clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide (MSP) and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide-AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to morpholino oligomers (PMOs), approximately 25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas less than 2% in corresponding tissues treated with either MSP-PMO or unmodified PMO under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-PMO can be an alternative option to current AOs.Molecular Therapy (2014); doi:10.1038/mt.2014.63.Molecular Therapy 04/2014; 22(7). DOI:10.1038/mt.2014.63 · 6.43 Impact Factor
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ABSTRACT: Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.12/2013; 24(1). DOI:10.1089/nat.2013.0448