Severe Congenital Neutropenia (SCN) is a rare hematological disorder characterized by profound neutrophil deficiency and recurrent infections from infancy. Over 50% of SCN cases arise from autosomal dominant mutations in the ELANE gene, which encodes neutrophil elastase (NE). Current treatments, such as granulocyte colony-stimulating factor administration and hematopoietic stem cell transplantation, often have limitations. A promising alternative is therapeutic knockout of the ELANE gene; however, double-strand break (DSB)-mediated genome editing strategies, such as Cas9, are hindered by p53 pathway activation and unintended genetic alterations, compromising genome integrity and safety. To overcome these limitations, we developed a precise base editing-mediated knockout strategy comprising three complementary approaches: (1) generation of premature termination codons (PTC), (2) splice site disruption, and (3) promoter disruption.To this end, we employed a base editing strategy to introduce PTCs by targeting conserved CAA, CAG, CGA, and TGG codons within the early exons of ELANE. A total of 23 gRNAs spanning exons 1-4 were screened in the HL-60 (human promyelocyte) cell line using three cytosine base editor (CBE) variants (BE3, TadCBE, and CBE6b), of which ten gRNAs achieved up to 95% on-target editing efficiency with minimal indel formation. This was accompanied by a corresponding reduction in ELANE mRNA and protein expression, indicative of effective ELANE knockout. Among the CBE variants tested, CBE6b exhibited the highest editing efficiency with minimal indel formation and reduced A-to-G editing, addressing key limitations associated with BE3 and TadCBE.An alternative strategy to knock out ELANE involved targeting early intronic splice donor and acceptor sites to induce aberrant splicing, resulting in PTC. Among the 28 target gRNAs tested, 18 gRNAs achieved 80-95% editing efficiency with CBE6b-NG/adenine base editor (ABE)8e. Splicing outcomes were confirmed across all targets with more than 90% reduction in the NE protein levels. To mitigate off-target risks associated with the broader PAM compatibility of ABE8e-NG, we employed efficient gRNAs compatible with ABE8e-NGG, achieving up to 80% editing with minimal bystander activity and consistent splicing disruption. To further achieve transcriptional repression of ELANE, we screened 9 gRNAs targeting the TATA box and its upstream sequence using ABE8e/TadCBE-NGG. We achieved an editing efficiency of 80-94% with ABE8e and 80-97% with CBE, with minimal indels. Within the TATA box, conversion of either of the two T's or two A's using ABE8e-NGG, as well as targeting the upstream sequence, led to effective reduction of ELANE expression.Given the limited availability of patient-derived HSPCs and to functionally validate our knockout strategy in primary cells, we employed base editing in HL-60 cell line to screen gRNAs that introduce ELANE mutations associated with misfolding and mistrafficking. Mutations such as c.-9 A>G, M1T, L47P, L59P, L172P, and disruption of intron-3AG and intron-4 AG/GT were introduced using ABE8e, while M1I, C151Y, disruption of intron-4 GT/AG, V219I, Q237Ter, and W241Ter were generated using CBE. Editing efficiencies of up to 95% with ABE and 94% with TadCBE were achieved. Based on the editing efficiency, indel rate and bystander editing, the top 5 gRNAs for ABE and the top 3 gRNAs for CBE were selected for further validation. Editing the intron-3 AG site with ABE efficiently induced exon-4 skipping at the mRNA level, disrupting a critical disulfide bond necessary for proper folding of NE, and stable NE expression was observed for c.-9 A>G, L59P, L172P, V219I, confirming successful generation of an SCN model.Based on on-target efficiency, off-target sites, indel frequency, and bystander editing, we selected the top 33 gRNAs for knockout and downregulation of ELANE, and the top 8 gRNAs for modeling SCN. Ongoing in vitro studies in healthy and patient-derived HSPCs, along with in vivo experiments, aim to functionally characterize the disease phenotype and assess the therapeutic safety of the base editing strategy for the treatment of SCN. Together, we present a safer, more efficient, and DSB-free base editing strategy for ELANE knockout that addresses key limitations of current genome editing approaches and offers a versatile platform for therapeutic gene inactivation in other monogenic disorders.

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2025
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