Pharmacological readthrough and base editing fornonsense mutations in an erythroid model of hereditary spherocytosis
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
Hereditary spherocytosis (HS) is the most common inherited chronic hemolytic anemia and results from defects in proteins of the erythrocyte membrane skeleton. Nonsense mutations inare a common cause of HS, yet current treatments remain largely supportive without addressing genetic defects. In this study, we identified twononsense 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 innonsense mutation-associated HS, highlighting TRIDs and ABEs as mechanistically distinct strategies with complementary streng
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 inare a common cause of HS, yet current treatments remain largely supportive without addressing genetic defects. In this study, we identified twononsense 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 innonsense 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. abs0010 Graphical abstract http://www.w3.org/1999/xlink float portrait ga1.webp undfig1 anchor portrait graphical abs0015 Using engineered erythroid K562 models carryingp.R281X or p.Q744X, Li and colleagues show that translational readthrough-inducing drugs partially restore ankyrin-1 expression and cellular phenotypes, whereas adenine base editing directly corrects these variants and restores near-wild-type phenotypes, providing proof-of concept for two molecular intervention strategies for hereditary spherocytosis. teaser abs0020