Abstract (from parent grant) The broad goal of this proposal it to computationally identify, validate, then target for therapeutic intervention regulatory elements within the 5' untranslated regions (UTR) of protein coding genes, known as upstream open reading frames (uORFs). In so doing, we aim to modulate the protein output from selected genes, offering a novel, translational approach with broad potential. uORFs are segments of 5′UTR mRNA sequences that can initiate and terminate translation upstream of protein-coding (CDS) start codons. Rare uORFs have been shown to produce potentially functional micro-peptides, while others affect protein expression by tuning translation rates of downstream protein-coding sequences. Our recent work showed that uORFs exhibit strong negative selection for preserving their start and stop codons, but not the encoded sequence, favoring the notion that uORFs have been retained through evolution because of their cis regulatory effect on protein gene products. Using genetic databases, we showed that variants affecting uORF start/stop codons associate with disease phenotypes (PheWAS analysis) and validated their expected effect on the protein (but not RNA) levels of the downstream genes. Potential uORFs have been identified in ~50% of all human protein-coding genes, many of which harbor more than one uORF. While blocking some uORFs decreases CDS protein output, our unpublished data show that targeting uORFs can be used to increase CDS translation. As a potential case for increasing protein levels, we focused on heritable pulmonary arterial hypertension (PAH), a fatal condition with no current cure, most commonly driven by BMPR2 haploinsufficiency. By blocking a BMPR2 uORF, we were able to increase BMPR2 protein levels by up to 220%. Based on our published and unpublished results we propose an integrative set of computational and experimental approaches to systematically detect, prioritize, validate and target uORFs for therapeutic intervention, focusing on genes associated with haploinsufficiency and ASOs as an intervention.