Genomic editing of a pathogenic mutation in ACTA2 rescues multisystemic smooth muscle dysfunction syndrome

BACKGROUND: Vascular smooth muscle cells (vSMCs), the predominant cell type in the aortic wall, play a crucial role in maintaining aortic integrity, blood pressure, and cardiovascular function. vSMC contractility and function depend on smooth muscle alpha-actin 2 (ACTA2). The pathogenic variant ACTA2 c.536G>A (p. R179H) causes multisystemic smooth muscle dysfunction syndrome (MSMDS), a severe disorder marked by widespread smooth muscle abnormalities, resulting in life-threatening aortic disease and high risk early mortality from aneurysms or stroke. No effective treatments exist for MSMDS. METHODS: To develop a comprehensive therapy for MSMDS, we utilized CRISPR-Cas9 adenine base editing to correct the ACTA2 R179H mutation. We generated isogenic human induced pluripotent stem cell (iPSC) lines and humanized mice carrying this pathogenic missense mutation. iPSC-SMCs were evaluated for key functional characteristics, including proliferation, migration, and contractility. The adenine base editor (ABE) ABE8e-SpCas9-VRQR under control of either a SMC-specific promoter or a CMV promoter, and an optimized single guide RNA (sgRNA) under control of U6 promoter were delivered intravenously to humanized R179H mice using adeno-associated virus serotype 9 (AAV9) and phenotypic outcomes were evaluated. RESULTS: The R179H mutation causes a dramatic phenotypic switch in human iPSC-SMCs from a contractile to a synthetic state, a transition associated with aneurysm formation. Base editing prevented this pathogenic phenotypic switch and restored normal SMC function. In humanized mice, the ACTA2R179H/+ mutation caused widespread smooth muscle dysfunction, manifesting as decreased blood pressure, aortic dilation and dissection, bladder enlargement, gut dilation, and hydronephrosis. In vivo base editing rescued these pathological abnormalities, normalizing smooth muscle function. CONCLUSIONS: This study demonstrates the effectiveness of adenine base editing to treat MSMDS and restore aortic smooth muscle function. By correcting the ACTA2 R179H mutation, the pathogenic phenotypic shift in SMCs was prevented, key aortic smooth muscle functions were restored, and life-threatening aortic dilation and dissection were mitigated in humanized mice. These findings underscore the promise of gene-editing therapies in addressing the underlying genetic causes of smooth muscle disorders and offer a potential transformative treatment for patients facing severe vascular complications.