hiPSC Modeling of Lineage-Specific Smooth Muscle Cell Defects Caused by TGFBR1 A230T Variant, and its Therapeutic Implications for Loeys-Dietz Syndrome

Background: Loeys-Dietz Syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection (TAAD). Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMC) have heterogenous embryonic origins depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored. Methods: We identified an LDS family with a dominant pathogenic variant in TGFBR1 gene ( TGFBR1 A230T ) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human-induced pluripotent stem cells (hiPSC) from subject with normal aorta to generate hiPSC carrying TGFBR1 A230T , and corrected the mutation in patient-derived hiPSC using CRISPR-Cas9 gene editing. Following their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell (NCSC) lineages, we employed conventional molecular techniques and single-cell RNA-sequencing (scRNA-seq) to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments employing Activin A and rapamycin. Results: Our results indicate the TGFBR1 A230T mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in NCSC-SMC. ScRNA-seq results implicate defective differentiation even in TGFBR1 A230T/+ CPC-SMC including disruption of SMC contraction, and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. TGFBR1 A230T selectively disrupted SMAD3 and AKT activation in CPC-SMC, and led to increased cell proliferation. Consistently, scRNA-seq revealed molecular similarities between a loss-of-function SMAD3 mutation ( SMAD3 c.652delA/+ ) and TGFBR1 A230T/+ . Lastly, combination treatment with Activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression, and function; and rescued the mechanical properties of mutant CPC-SMC tissue constructs. Conclusions: This study reveals that a pathogenic TGFBR1 variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with Activin A and rapamycin that can rescue the SMC defects caused by the variant.