Allelic Variation at 9p21.3 Orchestrates Widespread RNA Splicing Shifts Governing Vascular Smooth Muscle Cell Plasticity

Genetic risk for coronary artery disease (CAD) has been linked to variants across more than 300 genomic loci. Whether and how these loci interface with RNA processing to drive disease-relevant cellular phenotypes remains unknown. Here, we applied haplotype-biased genome editing in induced pluripotent stem cells (iPSCs), followed by differentiation into vascular smooth muscle cells (VSMCs), to address how genetic variation at the strongest CAD locus - the 9p21.3 CAD risk locus - affects RNA processing and alternative splicing genome-wide. Using long-read RNA sequencing, we identified distinct allele-specific transcriptional programs driven by the two major haplotypes at 9p21.3, risk and non-risk. We unravel extensive reprogramming of mRNA splicing across the transcriptome, which leads to VSMC aberrant phenotypic modulation. The 9p21.3 risk haplotype disrupts transcript isoform expression and usage across multiple genomic loci implicated in diverse stages of atherosclerotic plaque development. We prioritized DDX5, previously implicated in CAD through GWAS. Isoform-specific modulation of DDX5 in VSMCs was sufficient to mitigate the 9p21.3 risk-associated molecular signature. Together, this work provides the first comprehensive isoform-level transcriptomic comparison of the two major haplotypes at the 9p21.3 locus and identifies a 9p21.3-DDX5 axis as a key regulator of VSMC phenotypic plasticity. These findings uncover allele-specific reprogramming of RNA splicing as a previously unrecognized mechanism underlying cardiovascular disease susceptibility and present a resource of targetable transcripts with potential relevance across vascular pathologies.