Profiling and Targeting of Regulatory RNAs to Upregulate Gene Expression

Transcription of long noncoding RNAs (lncRNAs), including enhancer RNAs (eRNAs) and promoter-associated RNAs (paRNAs), collectively termed regulatory RNAs (regRNAs), is a hallmark of active gene expression, yet it remains unknown whether regRNAs can be targeted to selectively enhance transcription in cis. We developed regRNA Capture-seq, a high-throughput method to profile regRNAs, and applied it to primary human hepatocytes, annotating thousands of regRNAs at [~]2,000 enhancers and promoters. Using this approach, we interrogated a genetically validated enhancer of the ornithine transcarbamylase (OTC) gene, mutations of which cause OTC deficiency (OTCD), the most common urea cycle disorder. Antisense oligonucleotides (ASOs) targeting enhancer-derived regRNAs led to dose-dependent upregulation of OTC in hepatocytes. Mechanistically, ASOs altered regRNA structure, elevated regRNA levels, displaced transcriptional repressors, and increased H3K27 acetylation at the targeted enhancer. This work establishes a potential therapeutic strategy for addressing haploinsufficiency and highlights regRNAs as actionable targets for ASO-mediated upregulation of gene expression.