Autosomal dominant mutations in ELANE (elastase, neutrophil expressed) cause severe congenital neutropenia (CN) and cyclic neutropenia (CyN). Inhibiting ELANE expression, either by CRISPR/Cas9-mediated ELANE knockout or promoter targeting using CRISPR-Cas9 nickases, 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 ELANE 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 in vitro, with a marked improvement in ABE 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 ELANE-associated CN/CyN that addresses ELANE mutation heterogeneity.
Institute of Medical Biometry and Statistics,
Faculty of Medicine and Medical Center –
University of Freiburg
