Abstract
Splice switching oligonucleotides (SSOs) induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™”) to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe) oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS) oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.
Highlights
It has been estimated that nearly one tenth of all diseasecausing mutations are caused by single-base pair substitutions affecting pre-mRNA splicing.[1]
Due to its susceptibility to exonuclease digestion, PS internucleotide linkages are typically incorporated to stabilize against exonucleases.17 2′OMePS RNAs have been successfully used for Duchenne muscular dystrophy (DMD) exon skipping therapy both in mdx mice and in DMD patients.[18,19]
A significant number of diseases are caused by base-pair substitutions or deletions that result in altered splicing patterns.[1]
Summary
It has been estimated that nearly one tenth of all diseasecausing mutations are caused by single-base pair substitutions affecting pre-mRNA splicing.[1]. SSOs can target regions around a mutation, such as for Duchenne muscular dystrophy (DMD), whereby frame-shift deletions in the DMD gene can be bypassed by removing additional exons to create an in-frame deletion that produces a partially functional, internally truncated dystrophin protein.[7,8] It is the unique structure and function of dystrophin, with its large series of structural repeats in the central rod domain that allows this splice switching strategy to be successful.[4,9,10,11,12]
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