Skip to main content
ISTH 2023 Main banner
Back

    Oral Communication

    Hemophilia and Rare Bleeding Disorders

    Hemophilia Basic Science - FVIII and FIX

    OC 04.2 - B-Domain deleted Factor VIII shows clear differences in intracellular processing and cellular responses when compared to full length Factor VIII

    Saturday, June 24, 2023
    13:15 – 13:30 ET
    Room: 520
    Session Available As: On-Demand

      Presenting Author(s)

    • Rawya Al Rifai

      PhD candidat
      Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
      Bonn, Nordrhein-Westfalen, Germany

      • Co-Author(s)

    • MJ

      Muhammad Ahmer Jamil

      Dr
      Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
      bonn, Nordrhein-Westfalen, Germany

    • Salime El Hazzouri

      PhD candidate
      University Clinic Bonn, Institute of experimental Haematology and Transfusion medicine
      Bonn, Nordrhein-Westfalen, Germany

    • MI

      Mira Ibrahim, MS

      PhD candidat
      Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
      bonn, Nordrhein-Westfalen, Germany

    • NN

      Nicole Nüsgen

      Medizinisch-technische Assistentin
      Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
      bonn, Nordrhein-Westfalen, Germany

    • Johannes Oldenburg, MD (he/him/his)

      Professor, Chairman and Director
      University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
      Bonn, Nordrhein-Westfalen, Germany

    • Osman EL-Maarri, Priv Doz, PhD (he/him/his)

      Priv.-Doz. Dr.
      Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
      bonn, Nordrhein-Westfalen, Germany

    Background: Gene therapy for treatment of hemophilia A has achieved good results. However, major problem remains the lack of sustainability of FVIII blood levels and the unavailability of data on the cellular response of the targeted cells. 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 overtime, this strongly demands a thorough investigation of the suspected mechanisms at the cellular level.

    Aims: this study aims to compare the cellular trafficking and response of different FL vs. BDD-FVIII-forms and the levels of expression and secretion of FVIII intracellularly.

    Methods: HEK293 clones expressing various levels of FL- and BDD-FVIII were investigated to determine the correlations between FVIII copy number and levels of expression, secretion and storage, homeostatic cellular parameters (ATPs level, cellular proliferation, and intracellular markers co-segregation/co-localization with FVIII). Effects of cells treatment with Chloroquine, Cycloheximide, glucose-deficiency and BFA (BrefeldinA) were determined. We also performed 3´RNA-seq for expression profiling.

    Results: Our results showed 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, Cycloheximide and BFA had similar effects. Co-localizations of cellular markers with FVIII showed reduced presence of FVIII-BDD in ER/ERGIC/GABARAP and GAPARAPL1/Lysosome but increased presence in GABARAPL2 and LC3B-vesicles. Bioinformatics analysis confirmed the different cellular phenotype: differentially activated pathways and processes include cytoplasm organization, energy, stress and proliferation. 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.

    Conclusion(s): Our data indicate intrinsic differences/effects in cellular response between BDD and FL FVIII expression/secretion.

    Learning Objectives:

    • Recall learn and get updated on basic research previous knowledge of FVIII
    • Learn about current general cellular stress of overexpression a gene in gene therapy
    • Identify and focus on differences in cellular stress, intracellular trafficking and secretion between FL- and BDD- FVIII in a cell system