Pharmacological readthrough and base editing for ANK1 nonsense mutations in an erythroid model of hereditary spherocytosis
TL;DR
Findings provide exploratory proof-of-concept for targeted molecular intervention in ANK1 nonsense mutation-associated HS, highlighting TRIDs and ABEs as mechanistically distinct strategies with complementary strengths and limitations and suggesting their relative utility may depend on mutation-specific and clinical contexts.
Abstract
Hereditary spherocytosis (HS) is the most common inherited chronic hemolytic anemia and results from defects in proteins of the erythrocyte membrane skeleton. Nonsense mutations in ANK1 are a common cause of HS, yet current treatments remain largely supportive without addressing genetic defects. In this study, we identified two ANK1 nonsense mutations (p.R281X and p.Q744X) in unrelated HS pedigrees and generated K562 erythroid differentiation models using cytosine base editor-mediated knockin to evaluate potential targeted therapies. Functional analyses confirmed that both mutations markedly reduced ankyrin-1 expression and disrupted membrane-skeletal integrity. We then assessed two targeted therapeutic strategies: translational readthrough-inducing drugs (TRIDs) and adenine base editors (ABEs). Gentamicin promoted translational readthrough at both mutant sites, whereas CC-90009 showed activity primarily at p.Q744X. Both agents partially restored full-length ankyrin-1 expression, accompanied by restored membrane stability. In parallel, ABE8e-mediated correction achieved efficient (>85%) and precise genomic repair in bulk-edited K562 populations, leading to near-complete recovery of ankyrin-1 expression and membrane-skeletal integrity with minimal off-target effects. Together, these findings provide exploratory proof-of-concept for targeted molecular intervention in ANK1 nonsense mutation-associated HS, highlighting TRIDs and ABEs as mechanistically distinct strategies with complementary strengths and limitations and suggesting their relative utility may depend on mutation-specific and clinical contexts.