CRISPR/Cas9-mediated ELANE mutation correction in hematopoietic stem and progenitor cells to treat Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. Here, we use CRISPR/Cas9 ribonucleoproteins and adeno-associated virus (AAV) 6 as donor template delivery system to repair the ELANE(L172P) mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used an sgRNA specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo, upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide proof of principle for using CRISPR/Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN