Akerberg, B. N.; Matthews, B. J.; Liu, Y.; Mathieu, C.; Williams, J.; Manska, S.; Huang, J.; Nishi, Y.; Almutairy, A.; Coughlin, E.; Golczer, G.; Waldron, S.; Pellegrino, I.; Guo, Y.; Cohick, E.; Turna, H.; Xiong, K.; Whissell, G.; Kelker, R. S.; Gamboa, M.; Lenz, E.; Jurcisin, C. J.; Pai, R.; Caravella, J. A.; Sehgal, A.; Tardiff, D. F.; Sigova, A. A.; Bumcrot, D. A.

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.