Targeted inhibition of ELANE expression using adenine base editing to treat severe congenital neutropenia

Autosomal dominant mutations in <i>ELANE</i> (elastase, neutrophil expressed) cause severe congenital neutropenia (CN) and cyclic neutropenia (CyN). Inhibiting <i>ELANE</i> expression, either by CRISPR-Cas9-mediated <i>ELANE</i> knockout or promoter targeting using CRISPR-Cas9 nickase, has emerged as a promising gene therapy strategy to restore defective granulocytic differentiation of transplantable hematopoietic stem cells from CN patients. We developed an adenine base editor (ABE)-mediated approach targeting two nucleotides in the <i>ELANE</i> promoter to suppress neutrophil elastase expression, called PRECISE. Analysis of mRNA- and protein-based delivery of ABE revealed that although both platforms were effective in editing hematopoietic stem and progenitor cells from healthy donors with over 80% editing, only protein-based ABE delivery achieved over 68% editing in CN patient cells. Interestingly, 10%-19% editing in CN patients' hematopoietic cells using ABE mRNA restored their granulocytic differentiation <i>in vitro</i>, with a marked expansion and differentiation of ABE ribonucleoprotein (RNP)-edited cells. PRECISE-edited neutrophils retained normal function, including neutrophil extracellular trap formation, oxidative burst, and phagocytosis. Genome integrity analysis showed no genomic alterations or chromosomal aberrations, and only two off-target edits confined to non-coding intronic regions. In conclusion, PRECISE represents a translationally relevant base-editing strategy for <i>ELANE</i>-associated CN and CyN that addresses <i>ELANE</i> mutation heterogeneity.