Abstract Or125: Genomic Editing of a Pathogenic Mutation in the Alpha Actin Isotype 2 Gene Rescues a Severe Vascular Disease

Introduction: The pathogenic variant c.536G>A (p. R179H) of smooth muscle alpha-actin 2 ( ACTA2 ) causes multisystemic smooth muscle dysfunction syndrome (MSMDS), a severe disorder marked by widespread smooth muscle abnormalities, resulting in life-threatening aortic disease and early mortality from aneurysms. No effective treatments exist for MSMDS. CRISPR-Cas9 genome editing has emerged as an attractive method to correct and cure genetically based diseases. We hypothesize that gene editing strategies can be used to correct the ACTA2 R179H pathogenic missense mutation in smooth muscle cells (SMCs), thereby ameliorating smooth muscle diseases such as MSMDS. Methods: We generated isogenic human induced pluripotent stem cell (iPSC) lines and humanized mice carrying this pathogenic 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 a SMC-specific 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 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 ACTA2 R179H/+ 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. Conclusion: 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.