Peptide Conjugation of 2′-O-methyl Phosphorothioate Antisense Oligonucleotides Enhances Cardiac Uptake and Exon Skipping in mdx Mice

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.

Phosphorothioates, Essential Components of Therapeutic Oligonucleotides

“…Improving uptake by conjugation of cell penetrating peptides to the antisense oligonucleotides has been pursued for some time [106]. Peptide conjugation for delivery of an oligonucleotide to skeletal and cardiac muscle in mice apparently appears promising for the treatment of DMD [107]. A new venue is the attachment of N-acetyl galctosamine, which increases the potency of oligonucleotides in the liver, where a hepatocyte-specific receptor exists.…”

Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated MyotubesIn Vitro

“…As this aspect is relevant for future therapeutic applications, we tested effects of a selected set of oligo chemistries on gymnosis in our DM1 myogenic cell model. Four modifications for both AON sequences, CAG7 and DMD23, were analyzed: 2′- O -methyl phosphorothioate (OMePS), 2′- O -methyl phosphate (OMe), DNA phosphorothioate (PS), and OMePS, including a 5′ conjugation with a muscle-homing peptide (P4), which has been shown to enhance activity of DMD23-OMePS and CAG7-OMePS in vivo ([29]; Mulders et al , unpublished). Note that AON DMD23-PS was included to complete the set, but is, in fact, not useful for therapeutic purposes, since this AON may induce RNAse-H-dependent breakdown of Dmd pre-mRNAs instead of exon skipping.…”

“…Three types of chemical modification were used: 2′-O-methyl phosphorothioate (-OMePS), 2′-O-methyl phosphate (-OMe), and DNA phosphorothioate (-PS). Some AONs were conjugated at their 5′ or 3′ end with a Cy3 or FAM fluorophore or with P4, a muscle targeting peptide of sequence LGAQSNF [29]. Cy3-CAG3-ENA is a 5′-CAGCAGCAG-3′ AON with an ethylene-bridged nucleic acid (ENA) phosphate backbone, 5′ conjugated to Cy3.…”

“…2′-OMe, 2′- O -methyl; AON, antisense oligonucleotide; P4 = LGAQSNF [29]; PS, phosphorothioate; PO, phosphate.…”

Antisense-Mediated Exon Skipping: Networking to Meet Opportunities and to Overcome Challenges

“…A lot of work is ongoing to overcome scientific challenges, as outlined by most papers in this issue, focusing on, for example, improving antisense efficiency (Bestas et al, 2013), chemistry ( Jarver et al, 2013), and delivery of antisense oligonucleotides to muscle and heart ( Jirka et al, 2013;Falzarano et al, 2014). However, from the above it is clear that some challenges are developmental in nature.…”