The potential for therapeutic application of splice-switching
oligonucleotides (SSOs) to modulate
pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo
oligonucleotide uptake efficacy.
Biological activities can be significantly enhanced through the use of synthetically conjugated cationic
cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a
CPP, but here we describe the conjugation of two phosphorodiamidate
morpholino oligonucleotide (PMO) SSOs to a single
CPP for simultaneous delivery and
pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of
Duchenne muscular dystrophy. Conjugations of PMOs to a single
CPP were carried out through an
amide bond in one case and through a
triazole linkage ('click chemistry') in the other. The most active bi-specific
CPP-PMOs demonstrated comparable exon skipping levels for both
pre-mRNA targets when compared to individual
CPP-PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single
CPP are biologically effective and potentially suitable for future therapeutic exploitation.