OC 11.4 - Base and Prime Editors as new correction approaches for recurrent Hemophilia A Nonsense Mutations

Background: Among F8 gene alterations, nonsense mutations are responsible for a relative proportion (>20%) of severe forms of Hemophilia A (HA). Base (BE) and Prime (PE) editors are novel editing tools designed to precisely edit the genomic DNA with a single intervention, thus potentially ensuring a lifelong effect.

Aims: To challenge BE and PE on highly frequent F8 nonsense mutations ultimately reverting them and rescuing secreted and functional FVIII levels.

Methods: Expression studies in cellular models with FVIII fused with Gaussia luciferase (FVIII-GL), as sensitive luminescence-based screening system, or a B-less FVIII constructs. Evaluation of FVIII correction by Luciferase, ELISA and aPTT assays in cell media after BE and PE treatment.

Results: We selected the most frequent F8 nonsense variants (n=9) associated with severe HA and representing 403/1053 (40%) of all patients with nonsense mutations. In the initial screening phase, we exploited the FVIII-GL platform to identify, through evaluation of luciferase expression levels, BE and PE able to restore FVIII synthesis and secretion. Noticeably, the p.R355X, p.R602X, p.R814X, p.R2166X and p.R2228X variants were efficiently rescued (from barely detectable to 5-42.5% of FVIII-GL WT). To investigate the correction in the native FVIII-coding context, BE and PE were challenged in cellular models transiently or stably expressing a B-less FVIII construct. In both models, BE and PE transfection efficiently rescued FVIII secretion (10-60%) and activity (4.3-51%). Importantly, the correction was validated by DNA sequencing, which revealed the wild-type nucleotide at the targeted mutated position.

Conclusion(s): For the first time, we successfully demonstrated the BE and PE-mediated correction of highly frequent F8 nonsense mutations associated with severe HA phenotypes, thus paving the way for the development of new tailored therapeutic options for HA.