A Comprehensive Analysis of Differential Cellular Effects of B-domain-deleted and Full-length FVIII Expression in HEK293 Cells

Gene therapy has emerged as a promising approach for Hemophilia A and B, enabling sustained production of clotting factors and reducing reliance on frequent FVIII infusions. While Hemophilia B therapies targeting Factor IX show durable outcomes, Hemophilia A treatments using B-domain-deleted Factor VIII (BDD-FVIII), a truncated form adapted for adeno-associated virus delivery, often show reduced persistence. This study investigates cellular and molecular mechanisms underlying this disparity by comparing BDD-FVIII with full-length FVIII (FL-FVIII). <br> Using stably transfected HEK293 cells, we evaluated expression and secretion levels. BDD-FVIII showed improved expression, secretion and lower endoplasmic reticulum (ER) retention, likely due to its smaller size and reduced interaction with chaperones such as Calnexin and LMAN1. <br> We further conducted immunofluorescence co-localization studies, targeted chemical treatments, and gene knockouts of key trafficking regulators. These experiments revealed that FL-FVIII depends on classical ER-to-Golgi transport, while BDD-FVIII interacts less with early secretory pathway components. These findings demonstrate that FL- and BDD-FVIII engage distinct intracellular trafficking routes and quality control mechanisms. <br> Deletion of the FVIII B-domain significantly altered cellular behavior beyond secretion. BDD-FVIII-expressing cells showed reduced proliferation alongside activation of stress-related pathways, as confirmed by proliferation assays and RNA-seq analysis. Metabolic profiling showed that FL-FVIII imposes a greater folding and energetic burden due to its complex structure. FL-FVIII cells exhibited high mitochondrial ATP production but low residual ATP, indicating energy exhaustion. In contrast, BDD-FVIII cells maintained higher residual ATP with lower mitochondrial ATP, reflecting better energy efficiency. <br> Transcriptomic analysis revealed distinct gene expression profiles between FL-FVIII and BDD-FVIII, affecting pathways involved in cell cycle regulation, stress response, and metabolism. Notably, we identified a range of differentially regulated molecules between the two variants, including transcriptional regulators, kinases, enzymes, and chemical drugs. Among these, one compound previously used in gene therapy for Hemophilia A emerged as particularly interesting, as it has been shown to mitigate therapy-induced stress. This raises the possibility that such compounds could contribute to stabilizing BDD-FVIII by promoting a transcriptional and functional shift toward the FL-FVIII profile. These findings highlight the potential for therapeutic modulation using small molecules or kinase inhibitors to target stress-adaptive pathways. <br> Taken together, our results show that although the FVIII B-domain is not essential for coagulation, it plays a broader role in regulating secretion, metabolic homeostasis, and long-term cell adaptation. This study provides insight into the differential cellular effects of BDD- and FL-FVIII and may support the development of more targeted and personalized gene therapy strategies for Hemophilia A.