Gene Editing ELANE in Human Hematopoietic Stem and Progenitor Cells Reveals Disease Mechanisms and Therapeutic Strategies for Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a life-threatening disorder of insufficient granulocytes. Lifelong granulocyte colony-stimulating factor (G-CSF) injections are the mainstay of treatment, yet there remains a high risk of myelodysplastic syndrome and acute myeloid leukemia. The most common etiology of SCN is germline ELANE mutation. These dominantly acting mutations preserve expression but alter the structure of the neutrophil elastase protein product resulting in altered protein folding and/or trafficking with excess cell death at the promyelocyte/myelocyte stage of maturation. Recent advances in gene editing technologies have enabled targeted genetic modification of hematopoietic stem cells (HSCs); nonetheless genetic repair of specific disease-associated mutations remains challenging. We hypothesized that introduction of premature termination codons (PTCs) by nuclease-mediated frameshift mutations within early exons of ELANE could constitute a universal, highly efficient, simple therapeutic approach for ELANE-associated SCN. We predicted that the PTCs would trigger nonsense mediated decay (NMD) of the mutant transcript resulting in its loss of expression and thus bypassing neutrophil precursor cell death and consequent neutropenia. The mild phenotype observed in the Papillon-Lefevre syndrome, characterized by combined serine protease deficiency, suggests that isolated neutrophil elastase deficiency would not result in clinically significant immunodeficiency.

We delivered 3xNLS-SpCas9 and ELANE targeting sgRNA as ribonucleoprotein (RNP) complexes to primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and conducted in vitro neutrophil maturation culture. Introducing indels at exon 2 of ELANE efficiently triggered NMD. Edited cells were fully competent for neutrophil maturation similar to neutral locus targeted control cells. Using three human donors, we found that ELANE exon 2 edited HSPCs produced similar human bone marrow (BM) chimerism as unedited cells in NBSGW recipient mice 16 weeks following infusion. We found similar lymphoid, erythroid, and myeloid engraftment including similar fraction of human neutrophils (13.4% of total human BM cells in unedited and 13.7% in ELANE exon 2 edited, despite 97.3% on-target indel frequency and 84.3% reduction in ELANE expression in the latter). Using CD34+ HSPCs from four ELANE mutant SCN patient donors, we demonstrated that exon 2 targeting RNPs achieve highly efficient editing exceeding 95% indel frequency, trigger ELANE transcript decay, and rescue promyelocyte stage maturation arrest.

In contrast to these ameliorating early exon frameshifts, naturally occurring SCN-associated frameshifts affect late exons of ELANE, suggesting that these mutations might escape NMD. Indeed we found that targeting ELANE exon 5 in HSPCs resulted in robust indels (93.5%), preserving ELANE expression but resulting in cell death at the promyelocyte/myelocyte stages of development, recapitulating an SCN phenotype. To our surprise, we found that only -1 frameshifts and not -2 frameshifts induced by gene editing with NHEJ repair led to the SCN-like phenotype, although we noted that all 23 reported naturally occurring SCN-associated ELANE frameshift mutations result from -1 but not -2 bp frameshifts. Using xenograft of NBSGW recipients, we found that an RNP complex leading to efficient -1 frame indels in ELANE exon 5 produced profound neutrophil maturation block, with reduction from 13.4% neutrophils in controls to 0.5% neutrophils in ELANE exon 5 targeted recipients, with otherwise indistinguishable human monocyte, lymphoid, and erythroid reconstitution as compared to controls. This dramatic phenotype contrasts with mice engineered to express SCN-associated Elane mutations that do not exhibit neutropenia, indicating species differences in granulopoiesis.

Together these results support the development of ELANE early exon targeting as a highly efficient universal therapy for ELANE mutant SCN, feasible with existing gene editing technology. Moreover, by late exon ELANE gene editing we have developed a robust new model of SCN using primary human HSPCs that recapitulates neutropenia in vivo following xenotransplant, refines the molecular genetics of mutant ELANE induced neutrophil maturation arrest, and offers opportunities to explore novel therapeutic approaches.