Engineering antisense oligonucleotides for targeted mRNA degradation through lysosomal trafficking

Area of Science:

• Molecular Biology
• Drug Discovery
• Oligonucleotide Therapeutics

Background:

• Antisense oligonucleotides (ASOs) modulate gene expression by targeting mRNA.
• Current ASO efficacy is limited by the nuclear localization of RNase H, while most mRNA resides in the cytoplasm.
• This presents a challenge for developing ASO-based therapies for diseases involving cytoplasmic targets.

Purpose of the Study:

• To establish a novel mechanism for potent and targeted mRNA knockdown using a lysosomal degradation pathway.
• To develop and validate lysosomal trafficking antisense oligonucleotide (LyTON) technology for cytoplasmic mRNA degradation.
• To overcome the limitations of conventional RNase H-dependent ASOs.

Main Methods:

• Conjugation of an autophagosome-tethering compound (ATTEC) warhead (ispinesib) to RNase H-inactive, 2'-O-methylated ASOs.
• Utilizing bifunctional small molecules for lysosomal trafficking to induce degradation of cytoplasmic mRNA.
• Testing LyTON technology in various cell lines via transfection and gymnotic uptake for knockdown of multiple targets.

Main Results:

• Demonstrated significant lysosome-dependent mRNA knockdown of multiple targets using LyTON technology.
• Showcased LyTON's efficacy in various cell lines through both transfection and gymnotic uptake.
• Observed that LyTON modification enhances the knockdown efficacy of even RNase H-active ASOs.

Conclusions:

• LyTON technology enables mRNA degradation independent of RNase H recognition.
• This approach leverages enhanced chemical stability, tighter mRNA binding, and improved cell delivery profiles of ASOs.
• LyTONs expand the druggable target space for oligonucleotide therapeutics, potentially leading to new disease treatments.