PB0717 - In vitro Rescue of a Nonsense Mutation Responsible for Severe Coagulation Factor V Deficiency Using Ribosome Readthrough Agents

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

AT

Alice M. Todaro

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

Co-Author(s)

MC

Maria Ciccone

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

Elisabetta Castoldi, PhD

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

Background: Severe coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. Fresh-frozen plasma is the mainstay of treatment, but patients with nonsense mutations might benefit from emerging molecular therapies with ribosome readthrough agents.

Aims: We investigated a 28-year-old male with FV:C < 1% and a moderate bleeding tendency due to a homozygous F5 nonsense mutation. Ribosome readthrough agents were tested for their ability to restore functional FV expression in an in vitro model of the patient’s mutation.

Methods: Following written informed consent, the patient and his parents were investigated at the DNA, mRNA and plasma level (thrombin generation). COS-1 cells transfected with wild-type or mutant FV cDNA were treated with 0-500 µM G418 (Geneticin), ELX-02, PTC-124 (Ataluren), 2,6-diaminopurine (DAP) or Amlexanox. After 48 hours, conditioned media of untreated and treated cells were assayed for FV activity by a prothrombinase-based assay.

Results: Thrombin generation was hardly detectable in the patient’s platelet-poor plasma, but measurable in platelet-rich plasma. The patient was homozygous (and his parents heterozygous) for the F5 c.3481C>T mutation, which introduces a TGA stop codon in exon 13, causing nonsense-mediated decay of the FV mRNA and predicting premature termination of translation (p.Arg1161Ter). The mutation was linked to FV Leiden on both alleles. Expression of the p.Arg1161Ter mutation in COS-1 cells resulted in minimal FV activity in the medium (0.8±0.3% of wild-type), mimicking the patient’s undetectable FV level. Treatment with G418, ELX-02 and DAP increased FV activity in a dose-dependent manner, up to 4.7 (G418), 2.8 (ELX-02) and 13.6 times (DAP) compared to untreated cells. Differently, PTC-124 and Amlexanox (alone or in combination) did not improve FV expression.

Conclusion(s): This study provides in vitro proof-of-principle for the potential of some ribosome readthrough agents to partially restore functional FV synthesis and secretion impaired by the p.Arg1161Ter mutation.