Abstract
Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.
Highlights
In the last few decades, numerous studies have demonstrated the potential of using splice-switching antisense oligonucleotides (SSOs)
Splice Switching Is Dependent on PS Inclusion in 2′ -Omethyl SSOs (2OMe) Upon Lipofection in Muscle, Neuronal, Liver, and Bone Reporter Cell Lineages
To investigate the optimal modifications of PS inclusion in 2OMe to improve the splicing efficiency of SSO, 16- and 18-mer 2OMe with PS or endPS modification at a final concentration of 50 or 100 nM were transfected into the reporter cell lines by PepFect14 following our previously published protocols (Ezzat et al, 2011; Rocha et al, 2016)
Summary
In the last few decades, numerous studies have demonstrated the potential of using splice-switching antisense oligonucleotides (SSOs). SSOs are typically 15–25 mers, short oligonucleotides, designed to target pre-messenger RNA (mRNA) and modulate the splicing patterns of target transcripts by blocking Watson– Crick RNA–RNA base-pairing or RNA–protein-binding interactions (Zamecnik and Stephenson, 1978; Dominski and Kole, 1993). This function gives SSOs a solid therapeutic potential, for exon skipping in Duchenne muscular dystrophy (DMD). SSOs using 2′-O-methyl RNA (2OMe) chemistry designed to restore dystrophin were the first oligonucleotides tested in DMD (Cirak et al, 2011).
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