Mutation-specific responses to translational read-through and nonsense-mediated decay inhibition in iPSC derived liver sinusoidal endothelial cells from Haemophilia A patients

Introduction Nonsense mutations in the F8 gene are a major cause of severe Haemophilia A and correlate with an increased risk of inhibitor formation. Strategies to rescue FVIII expression via translational readthrough (RTA) and nonsense-mediated decay inhibition (NMDI) remain poorly understood in patient-specific endothelial contexts [ 1 ] [ 2 ]. Method Human iPSCs from Haemophilia A patients carrying F8 mutations (R2228X, R1960X, I22I) and one wild-type control were differentiated into liver sinusoidal endothelial-like cells (iLSECs) [ 3 ]. Cells were treated with the read-through agent PTC124 and/or the NMD inhibitor NMDI14. Transcript levels of F8 , ATF4 , CHOP and UPF1 were quantified by RT-qPCR; FVIII protein expression and secretion were assessed by immunoprecipitation, western blot, and FVIII C2-domain-specific ELISA. Results Combined RTA+NMDI treatment markedly increased F8 mRNA in R2228X cells, accompanied by detectable FVIII protein containing the C2 domain, indicating successful translational readthrough. R1960X cells showed partial F8 mRNA stabilization but misprocessed FVIII protein. UPF1 mRNA levels increased in R2228X cells upon NMD inhibition, confirming protein pathway engagement. Wild-type and I22I controls remained unaffected. Conclusion Patient-derived iLSECs provide a physiologically relevant model to dissect mutation-specific responses to NMDI / RTA therapy. The R2228X variant demonstrates partial functional rescue at both mRNA and protein levels, highlighting the potential of personalized nonsense-suppression approaches in Haemophilia A. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany