Modeling MYH11-associated aortic disease using iPSC-derived vascular smooth muscle cells for gene therapy development

Abstract Background Pathogenic variants in MYH11, encoding smooth muscle myosin heavy chain, are associated with familial aortic dissections and patent ductus arteriosus. We identified a Dutch family carrying MYH11 c.4559+1GA splice site variant with an unusual presentation of abdominal aortic dissection. While exon 32 skipping has been documented in other MYH11 splice variants, the underlying pathophysiology remains poorly understood. We hypothesize the mutant vSMC to show a migratory and proliferative (synthetic) phenotype at baseline, contributing to adverse vascular wall remodeling and aneurysm development. Purpose We established an in vitro disease model using iPSC-derived vascular smooth muscle cells (vSMCs) to investigate phenotypic differences between wild-type and mutant cells and to develop a platform for testing precision medicine approaches. Methods and Results We generated and characterized iPSCs from patients (n=3) and healthy individuals (n=2) and confirmed the presence of the pathogenic variant using Sanger sequencing. The iPSCs were differentiated into vSMCs from neural crest precursors to model the embryonic origin of the ascending aorta. Mutant vSMCs exhibited reduced expression of contractile proteins, including phosphorylated myosin light chain, transgelin (SM22α), and calponin (CNN1), as demonstrated by immunofluorescence and qPCR. Preliminary experiments revealed significantly enhanced migratory capacity in mutant cells using transwell assays (XCelligence). Single-cell nano-indentation (Pavone) confirmed decreased cellular stiffness in mutant cells, consistent with a synthetic phenotype. Conclusion and Future Directions Our iPSC-derived vSMC model recapitulates key pathological features of MYH11-associated aortopathy with findings that point towards a more synthetic phenotype of the mutant vSMCs. With the establishment of a phenotypic difference in vitro, we are now targeting mutant MYH11 splice site variants using RNA therapies. Furthermore, functional outcomes (e.g. contractility) are being using engineered vascular microtissues to evaluate contractile responses. This model offers unique insights into the pathophysiology of MYH11-related aortic disease and provides a valuable platform for developing targeted RNA therapies.