Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Splice-switching antisense oligonucleotides (SSOs) offer a promising therapeutic strategy for genetic disorders like Duchenne muscular dystrophy (DMD) by correcting pre-messenger RNA (mRNA) mutations via exon skipping.
• Phosphorothioate (PS) modification is commonly used to enhance SSO stability and pharmacokinetics, but its impact on 2'-O-methyl (2OMe) SSOs' cellular uptake and splice-switching efficiency is not fully understood.

Purpose of the Study:

• To investigate the effect of PS modification on the cellular uptake and splice-switching activity of 2OMe SSOs.
• To identify proteins involved in the nuclear trafficking of 2OMe SSOs and explore their potential for modulating delivery.

Main Methods:

• Utilized H2k-mdx myoblasts and reporter cell lines (pLuc/705) to assess cellular uptake and splice switching.
• Employed photoactivatable ribonucleoside-enhanced crosslinking and proteomics to identify SSO-binding proteins.
• Used small-interfering RNA (siRNA) to ablate specific binding proteins and evaluate effects on SSO uptake and splicing.

Main Results:

• Demonstrated that PS modification significantly facilitates the cellular uptake of 2OMe SSOs in myoblasts.
• Confirmed that PS inclusion in 2OMe SSOs (2OMePS) is crucial for nuclear accumulation and high splice-switching activity across various cell lineages (muscle, neuronal, liver, bone).
• Identified several 2OMePS-binding proteins, including Ncl, and showed that Ncl ablation enhances SSO uptake and splicing activity.

Conclusions:

• PS modification enhances the nuclear delivery and splice-switching efficacy of 2OMe SSOs in diverse cell types.
• Modulating 2OMePS-binding proteins presents a potential strategy to improve SSO delivery and therapeutic outcomes for genetic diseases.