Skip to main content
ISTH 2023 Main banner
Back

    Oral Communication

    Hemophilia and Rare Bleeding Disorders

    Rare Bleeding Disorders I

    OC 07.1 - Autologous CRISPR/Cas9 correction of a missense variant from a patient with severe, inherited FVII deficiency

    Saturday, June 24, 2023
    13:00 – 13:15 ET
    Room: 524
    Session Available As: On-Demand

      Presenting Author(s)

    • Giacomo Roman, MSc

      PhD student
      Department of Hematology, Oslo University Hospital, Oslo, Norway
      Oslo, Oslo, Norway

      • Co-Author(s)

    • KL

      Knut H. Lauritzen

      Researcher
      Department of Hematology, Oslo University Hospital, Oslo, Norway
      Oslo, Oslo, Norway

    • SH

      Sean Harrison

      Post doc
      Oslo University Hospital
      Oslo, Oslo, Norway

    • GS

      Gareth J Sullivan

      Group leader
      Department of Pediatric Research, Oslo University Hospital, Oslo, Norway
      Oslo, Oslo, Norway

    • NI

      Nina Iversen

      Group leader
      Department of Medical Genetics, Oslo University Hospital, Oslo, Norway
      Oslo, Oslo, Norway

    • CH

      Carola Elisabeth Henriksson

      Associate Professor
      Institute of Clinical Medicine, University of Oslo, Oslo
      Oslo, Oslo, Norway

    • HG

      Heidi Glosli

      Medical Doctor
      Division of Paediatric and Adolescent Medicine Centre for Rare Disorders Oslo University Hospital Oslo Norway.
      Oslo, Oslo, Norway

    • PS

      Per Morten Sandset

      Medical Doctor
      Oslo University Hospital
      Oslo, Oslo, Norway

    • BS

      Benedicte Stavik

      Group leader
      Department of Hematology, Oslo University Hospital, Oslo, Norway
      Oslo, Oslo, Norway

    • Maria Eugenia Chollet Dugarte, MD, PhD (she/her/hers)

      Post doctoral researcher
      Department of Hematology, Research Institute of Internal Medicine, Oslo University Hospital
      Oslo, Oslo, Norway

    Background: Coagulation Factor VII (FVII) deficiency is a rare monogenic, bleeding disorder caused by mutations in the F7 gene. We have previously reported two young siblings with dramatically reduced FVII activity and a severe, life-threatening hemorrhagic phenotype, induced by a homozygous c.718G>C missense mutation leading to a glycine-to-arginine substitution at amino acid 240 (p.G240R)

    Aims: To correct the p.G240R mutation in patients’ cells by combining Induced Pluripotent Stem Cell (iPSC) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing technology

    Methods: Patient-derived Peripheral Blood Mononuclear Cells (PBMCs) were reprogrammed into iPSCs using the non-integrative CytoTune™-iPS 2.0 kit and validated for specific markers via quantitative PCR, immunofluorescence, and tri-lineage differentiation. Different candidate guide RNAs (gRNAs) were generated by IDT™ and Benchling® designing tools and nucleofected using the Neon™ Transfection System. The on-target efficiency was evaluated by T7 Endonuclease I assay and digital droplet PCR (ddPCR), while correction occurrence via Homology-directed Repair (HDR) was assessed through restriction enzyme, ddPCR and by Sanger sequencing. Written informed consent was obtained

    Results: To confirm that iPSCs were successfully reprogrammed, the consistent expression of Sox2, Oct4 and Nanog markers was verified, as well as the absence of KOS, hc-Myc and hKlf4 reprogramming vectors. Tri-lineage assays validated the differentiation of the iPSCs to definitive endoderm, neuroepithelium and functional cardiomyocytes, corroborated by expression of FoxA2 and Sox17, Pax6 and Sox2, Troponin T2, respectively. The candidate gRNAs showed an on-target efficiency up to 48%. The selected gRNA generated a HDR rate up to 20%. Restriction enzyme studies and Sanger sequencing further confirmed a successful homozygous correction

    Conclusion(s): We proved the successful correction of a severe FVII missense variant using gene editing technology, in patients’ iPSCs. This completes an important step towards obtaining autologous iPSC-derived liver cells with functional FVII and the development of a novel cell-based therapy for the treatment of FVII deficiency

    Learning Objectives:

    • Upon completion, participants will be able to identify the potential of Induced Pluripotent Stem Cell (iPSC) technology in the field of Coagulation FVII deficiency and inherited bleeding disorders, in terms of both modeling and therapeutic purposes
    • Upon completion, participants will be able to describe the main features and challenges of optimized CRISPR/Cas9 gene editing strategies to correct disease-causing mutations, in patient-derived iPSCs
    • Upon completion, participants will be able to estimate the therapeutic potential of autologous, CRISPR-edited iPSCs. Furthermore, they will be able to describe the future applications in the field of Coagulation FVII deficiency and inherited bleeding disorders, such as iPSC-derived hepatic organoids