Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization. (22nd October 2021)

• Record Type: Journal Article
• Authors: Hiraoka, Haruka; Shu, Zhaoma; Tri Le, Bao; Masuda, Keiko; Nakamoto, Kosuke; Fangjie, Lyu; Abe, Naoko; Hashiya, Fumitaka; Kimura, Yasuaki; Shimizu, Yoshihiro; Veedu, Rakesh N.; Abe, Hiroshi
• Is Part Of: Chembiochem. Volume 22:Number 24(2021)
• Page Start: 3437
• Page End: 3442
• Publication Date: 2021-10-22
• Subjects: antisense, cellular uptake, disulfide, exon skipping, nuclear transport; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202100413
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane‐permeable oligonucleotides (MPONs). Time‐course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage‐dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5‐fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping. Abstract : Enhanced cellular uptake was achieved using a disulfide‐unit‐containing membrane‐permeable oligonucleotide (MPON). Long‐term incubation enabled their nuclear internalization and subsequent exon skipping.