Mapping, programming, and correcting gene regulatory sequences for Alagille Syndrome

PROJECT SUMMARY Regulatory DNA elements, including enhancers and promoters, encode multiple transcription factor binding sites (TFBS) that quantitatively tune gene expression in a cell-type specific fashion. Understanding and engineering regulatory DNA could unlock new therapeutic approaches — for example, to restore proper expression of a disease gene. Diseases of haploinsufficiency, such as Alagille Syndrome, are one example where such an approach could be transformative. Alagille Syndrome involves haploinsufficiency of JAG1, where improper dosage in vascular endothelial cells and smooth muscle cells leads to life-threatening complications including biliary atresia as well as right-sided congenital heart defects. An ideal gene therapy solution for Alagille Syndrome would be to engineer the promoter of JAG1 to turn up the production of the unaffected allele of the gene by 2-fold. Yet, our knowledge of how to program regulatory DNA to control gene expression is incomplete, in large part because we have lacked tools to accurately identify, edit, or characterize TFBS in regulatory elements in the genome. Our team has now developed innovative tools to dramatically increase the throughput of characterizing both endogenous and synthetic TFBS and their effects on gene expression in the context of Alagille Syndrome — allowing us to design DNA edits, test their impact on gene expression with high-throughput screens, and evaluate their ability to correct pathological patterns of gene expression. In Aim 1, we will build a genome-wide nucleotide-resolution map of TFBS in endothelial cells and smooth muscle cells from healthy and Alagille hiPSCs in conditions of static, physiologic, and pathological flow. In Aim 2, we will apply new pooled prime editing technologies to systematically mutate regulatory DNA sequences in the JAG1 promoter and identify TFBS that can increase JAG1 gene expression. In Aim 3, we will use CRISPR to correct JAG1 expression in hiPSCs from Alagille patients and characterize their effects on gene expression and cellular phenotypes. Together, these studies will illuminate basic mechanisms of Alagille Syndrome, test whether restoration of JAG1 function in cells from Alagille patients is sufficient to correct disease-associated cellular phenotypes, and demonstrate a new strategy to program gene expression in the human genome by combining nucleotide- resolution computational modeling with high-throughput sequence editing of endogenous gene promoters. This approach is generalizable and can be applied to other diseases where programming gene expression is desirable, such as for other haploinsufficiencies.