Efficient, potent, and specific near-universal base and prime editing for ELANE-mutant severe congenital neutropenia

Abstract Severe congenital neutropenia (SCN) is a life-threatening disorder of neutrophil production most frequently caused by dominant mutations in the ELANE gene. ELANE pathogenic mutations lead to the production of mutant neutrophil elastase (NE) proteins that can disrupt NE folding and trafficking, which triggers the unfolded protein response (UPR) leading to cellular apoptosis and neutrophil maturation arrest at the promyelocyte-to-myelocyte stage of neutrophil differentiation. Previously, we and others have demonstrated that introducing indels in ELANE via Cas9-mediated gene editing to activate nonsense-mediated decay (NMD) can restore normal neutrophil development, serving as a potential universal therapeutic strategy. However, nuclease editing is associated with drawbacks such as p53-dependent DNA damage responses, unintended large deletions or rearrangements resulting from double-strand breaks (DSBs), and limited multiplex potential. Here, we report efficient, potent, and specific ELANE base and prime editing (BE and PE) approaches as less genotoxic alternatives. We designed a gRNA for adenine base editing (ABE) targeting a splice site in ELANE at the intron 2–exon 3 splice acceptor site and a pegRNA targeting exon 2 to introduce premature termination codons. In CD34⁺ HSPCs, ELANE editing with ABE8e yielded 94% A>G conversion at target position A8 with 43% bystander editing at A10, while PE with PEmax-La:ELANE-pegRNA without a nicking sgRNA (PE2-type) achieved 68% precise prime edits. To model SCN in vitro, we sequentially introduced a pathogenic ELANE exon 5 mutation via Cas9 editing and therapeutic edits with Cas9, ABE8e, or PEmax-La in CD34⁺ HSPCs. Both BE and PE restored neutrophil maturation arrest by inducing NMD and reducing ELANE mRNA expression. To test the therapeutic potential for SCN, mobilized CD34⁺ HSPCs from an ELANE p.Ser126Leu mutant patient were edited ex vivo with either nuclease, BE or PE targeting ELANE (as well as mock and neutral locus edited controls). We transferred the edited cells to NBSGW mice for in vivo engraftment and multilineage differentiation. After 16 weeks, high bone marrow human chimerism (>90%) was maintained across all groups. Granulocyte engraftment was significantly improved with ELANE editing (62% for nuclease, 62% for BE and 56% for PE) versus mock (12%) and safe locus controls (9%). We performed a competitive transplant with ABE8e:ELANE and neutral locus edited cells. A ratio of 20:80 edited:control HSPCs produced similar ELANE edits in neutrophils as fully undiluted edited HSPCs, while edits in B cells were reduced as expected by dilution, indicating selective advantage for edited cells to bypass neutrophil maturation defect. Single cell RNA sequencing of control and ELANE edited cells showed an ER stress gene expression signature and neutrophil differentiation block only in unedited cells. Using in silico CRISPRmeand cell-based GUIDE-seq off-target nomination, we verified a single ABE8e:ELANE off-target site with 18% ± 2% editing. Previously, RNA-DNA hybrid gRNAs were described as a strategy to reduce nuclease off-target editing. We screened 37 hybrid gRNAs and found several that discriminated off-target editing. In CD34+ HSPCs, the unmodified gRNA produced 97% on-target and 30% off-target editing, while a hybrid DNA-RNA sgRNA produced 86% on-target and 0.2% off-target editing, not significantly different from an unedited control sample. For prime editing, we did not detect off-target edits in HSPCs after PEmax-La:ELANE pegRNA editing even though the same spacer produced a frequent off-target (22% edits) in a Cas9:gRNA RNP context. To evaluate whether neutrophil functions are preserved following ELANE base or prime editing, we isolated the engrafted mature human neutrophils from NBSGW mice. ELANE edited neutrophils displayed comparable oxidase activity, phagocytic activity, neutrophil extracellular trap formation, and E. coli killing capacity when compared to unedited control neutrophils, indicating preserved neutrophil function of edited cells. These results demonstrate highly efficient near-universal BE and PE approaches to ELANE gene editing of CD34+ HSPCs that lack detectable off-target effects, correct neutrophil maturation arrest, and preserve neutrophil function and HSPC engraftment for potential therapy of ELANE-mutant SCN.