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    Oral Communication

    Hemophilia and Rare Bleeding Disorders

    Rare Bleeding Disorders I

    OC 07.3 - In vitro and ex vivo rescue of a nonsense mutation (F5 p.Arg1161Ter) responsible for severe coagulation factor V deficiency

    Saturday, June 24, 2023
    13:30 – 13:45 ET
    Room: 524
    Session Available As: On-Demand

      Presenting Author(s)

    • Alice M. Todaro, MSc

      PhD student
      Department of Biochemistry, CARIM, Maastricht University, Maastricht (The Netherlands)
      Maastricht, Limburg, Netherlands

      • Co-Author(s)

    • CR

      Claudia M. Radu

      Assistant Professor
      Department of Medicine, Thrombotic and Haemorrhagic Diseases Unit, University of Padua Medical School, Padua (Italy)
      Padua, Veneto, Italy

    • MC

      Maria Ciccone

      Medical Doctor
      Department of Medical Sciences, Section of Haematology, Sant’Anna Hospital, Ferrara University, Ferrara (Italy)
      Ferrara, Emilia-Romagna, Italy

    • ST

      Serena Toffanin

      PhD student
      Department of Medicine, Thrombotic and Haemorrhagic Diseases Unit, University of Padua Medical School, Padua (Italy)
      Padua, Veneto, Italy

    • MS

      Maria Luisa Serino

      Assistant Professor
      Department of Medical Sciences, Section of Haematology, Sant’Anna Hospital, Ferrara University, Ferrara (Italy)
      Ferrara, Emilia-Romagna, Italy

    • Elena Campello, MD, PhD (she/her/hers)

      Assistant Professor
      Department of Medicine, University of Padova, Italy
      Padova, Veneto, Italy

    • CB

      Cristiana Bulato

      PhD
      Department of Medicine, Thrombotic and Haemorrhagic Diseases Unit, University of Padua Medical School, Padua (Italy)
      Padova, Veneto, Italy

    • BL

      Barbara Lunghi

      Research Assistant
      Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, Ferrara University, Ferrara (Italy)
      Ferrara, Emilia-Romagna, Italy

    • DG

      Donato Gemmati

      Professor
      Department of Medical Sciences, Section of Haematology, Sant’Anna Hospital, Ferrara University, Ferrara (Italy)
      Ferrara, Emilia-Romagna, Italy

    • AC

      Antonio Cuneo

      Professor
      Department of Medical Sciences, Section of Haematology, Sant’Anna Hospital, Ferrara University
      Ferrara, Emilia-Romagna, Italy

    • TH

      Tilman M. Hackeng, PhD

      Chief Scientific Officer
      Coagulation Profile, Maastricht, The Netherlands
      Maastricht, Limburg, Netherlands

    • Paolo Simioni, MD, PhD (he/him/his)

      Full Professor
      Department of Medicine, Thrombotic and Haemorrhagic Diseases Unit, University of Padua Medical School, Padua (Italy)
      Padua, Veneto, Italy

    • FB

      Francesco Bernardi

      Professor of Biochemistry
      Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, Ferrara University, Ferrara (Italy)
      Verona, Veneto, Italy

      • Co-Author(s)

    • Elisabetta Castoldi, PhD

      Associate Professor
      Department of Biochemistry, CARIM, Maastricht University, Maastricht (The Netherlands)
      Maastricht, Limburg, Netherlands

    Background: Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder that is usually managed with fresh-frozen plasma. The F5 c.3481C>T nonsense mutation, introducing a premature stop codon in exon 13 (p.Arg1161Ter), is recurrent among FV-deficient patients. Detailed DNA, mRNA and functional analyses in a symptomatic homozygous patient suggested that this mutation might respond to ribosome readthrough agents.

    Aims: To assess the correction potential and cytotoxicity of five ribosome readthrough agents with different mechanisms of action (G418, ELX-02, PTC-124, 2,6-diaminopurine and amlexanox) in in vitro and ex vivo models of the F5 p.Arg1161Ter mutation.

    Methods: In vitro model: COS-1 cells transfected with wild-type or mutant FV cDNA were treated with readthrough agents (0-500 µM) for 48 hours. FV activity in conditioned media was determined using a prothrombinase-based assay and thrombin generation in reconstituted FV-depleted plasma. Cell viability was evaluated with an XTT-based assay. Ex vivo model: Following written informed consent, ex vivo differentiated megakaryocytes from a homozygous patient were treated with each readthrough agent for 7 days and FV expression was probed by immunofluorescence with anti-FV antibodies.

    Results: COS-1 cells transfected with the FV p.Arg1161Ter cDNA secreted hardly any functional FV (FV activity: 0.8±0.3% of wild-type). Treatment with G418, ELX-02 and 2,6-diaminopurine increased FV activity in a dose-dependent manner (up to 4.7-fold, 2.8-fold and 13.6-fold, respectively), while PTC-124 and amlexanox were ineffective. All readthrough agents except ELX-02 showed some degree of cytotoxicity. Untreated patient’s megakaryocytes were negative at FV immunostaining, but turned positive upon treatment with all five readthrough agents. Notably, the patient’s megakaryocytes were also able to internalise the FV produced by readthrough-mediated rescue of the p.Arg1161Ter mutation, a necessary requirement for maintaining the platelet FV pool in vivo.

    Conclusion(s): This study provides in vitro and ex vivo proof-of-principle for the efficacy of ribosome readthrough agents in rescuing the F5 p.Arg1161Ter mutation.

    Learning Objectives:

    • Upon completion, participant will be able to clarify that a nonsense mutation not always results in a complete null mutation (natural readthrough).
    • Upon completion, participant will be able to determine favourable characteristics of a nonsense mutation to be amenable for a correction approach.
    • Upon completion, participant will be able to visualise that megakaryocytes from a FV-deficient patient are able to internalise not only exogenous FV-WT, but also mutant FV rescued with readthrough agents.