Knocking Down Long Noncoding RNAs Using Antisense Oligonucleotide Gapmers.

Long noncoding RNAs (lncRNAs) are transcripts of 200 nucleotides or longer that are not translated into protein. lncRNAs are highly abundant: one study estimates they are at least four times more numerous than coding RNAs in human cells. However, the functions of more than 95% of human lncRNAs remain unknown. Synthetic antisense oligonucleotides called gapmers are powerful tools for lncRNA loss-of-function studies and represent a promising therapeutic modality. Gapmers contain a central DNA region that activates RNase H-mediated RNA degradation, flanked by chemically modified wings, such as 2'-O-methyl, 2'-O-methoxyethyl, constrained ethyl (cEt), or locked nucleic acids (LNAs) that enhance stability and affinity. This mechanism makes them particularly effective for silencing nuclear-retained lncRNAs, a key advantage over cytoplasm-acting methods like RNAi. Because RNase H activity is enriched in nuclei, gapmer-based knockdown is often more effective than siRNA or RNAi for nuclear-localized targets. Therapeutically, this promise is being realized: gapmers targeting lncRNAs have progressed into clinical trials and show robust preclinical efficacy in oncology, cardiovascular disease, pulmonary fibrosis, and neurological/neuromuscular models. This chapter discusses gapmer development, practical design tips and considerations for translating lncRNA-targeted antisense oligonucleotides into next-generation RNA-targeted therapeutics.