GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE269nnn/GSE269207/primaryOK200TranscriptomicsHomo sapiensExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE269207GEOGSEfalseB-Domain deleted Factor VIII exhibits notable differences in intracellular processing and cellular responses when compared to full length Factor VIIIBackground: Gene therapy for the treatment of hemophilia A have achieved good results. However, major problem is the lack of sustainability of FVIII blood levels; also no data on the cellular response of the targeted cells is available. Hepatocytes targeting and use of B-domain-deleted F8, are two non-natural phenomena that could be linked to the decrease of FVIII-blood-levels over time, this strongly demand a thorough investigation of the suspected mechanisms at the cellular level. Aim: The aims of this study are to compare the cellular trafficking and response to different forms (FL vs BDD) and to levels of expression and secretion of FVIII in the cells. Method: we established single cells HEK293 clones expressing various levels of full-length or B-domain-deleted-FVIII. We studied the correlations between FVIII copy number and levels of expression, secretion and storage as well as homeostatic cellular parameters like ATPs level, cellular proliferation, intracellular markers co-segregation and co-localization with FVIII. Effect of cells treatment with Chloroquine, Cyclohexamid, glucose deficiency and BFA were determined. In addition, we performed 3´RNA-seq for expression profiling. Results: Our results show differences in expression/copy (higher in BDD), secretion (higher in BDD) and intracellular storage (higher in FL) between BDD and FL clones. The cellular proliferation rate was higher in FL while ATP was higher in BDD clones. Chloroquine treatment lowered secretion in FL clones but not in BDDs, while starvation, Cyclohexamid and BFA had similar effect. The co-localisations of cellular markers with FVIII show reduced presence of FVIII BDD in ER/ERGIC/GABARAP and GAPARAPL1/Lysosome while higher at GABARAPL2 and LC3B vesicles is observed. Bioinformatic analysis confirmed the cellular phenotype as differentially activated pathways and processes include organization of cytoplasm, energy, stress and proliferation related. Finally, expression association with level of secretion potentials showed clear dose effect but absence of overlaps between BDD and FL clones, indicating clear divergence of response to overexpression of BDD and FL-FVIII. Conclusions: Our data indicate intrinsic differences/effects in cellular response between BDD and FL FVIII expression/secretion.2026/06/30GSE269207GSM8308609GSM8308619GSM8308608GSM8308618GSM8308607GSM8308617GSM8308606GSM8308612GSM8308601GSM8308611GSM8308600GSM8308610GSM8308599GSM8308620GSM8308616GSM8308605GSM8308615GSM8308604GSM8308614GSM8308603GSM8308613GSM83086021679120301269207Homo sapiens