Highlights from

ASH 2019

61st Annual Meeting & Exposition of the American Society of Hematology

Orlando, Florida (USA) 7 - 10 December 2019

Gene editing in congenital neutropenia

ELANE early exon targeting is a highly efficient universal therapy for ELANE mutant severe congenital neutropenia (SCN), feasible with existing gene editing technology [1].

Patients with SCN are characterised by a high risk of myelodysplastic syndrome and acute myeloid leukaemia. Germline mutation in the ELANE gene is the most common cause of SCN, and mutations are dominant in nature, preserving gene expression while altering the structure of the neutrophil elastase protein product, which results 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 haematopoietic stem cells.

Dr Shuquan Rao (Harvard Medical School, USA) and colleagues postulated that introduction of premature termination codons by nuclease-mediated frameshift mutations within early exons of ELANE could be a simple therapeutic approach for ELANE-associated SCN. By introducing premature termination codons, the hypothesis was that nonsense mediated decay of the mutant transcript would ensue, with consequent loss of gene expression, and thereby no longer causing neutrophil precursor cell death and consequent neutropenia.

The researchers employed CRISPR-Cas9 gene editing to target ELANE in primary human CD34+ haematopoietic stem and progenitor cells in in vitro neutrophil maturation culture. The researchers first introduced early indels at exon 2 of ELANE and observed robust nonsense mediated decay, as hypothesised. Edited cells were fully competent for neutrophil maturation similar to neutral locus targeted control cells. Using 3 human donors, they showed that ELANE exon 2 edited haematopoietic stem and progenitor cells produced similar human bone marrow chimerism as unedited cells in NBSGW recipient mice 16 weeks following infusion. In a translational experiment using CD34+ haematopoietic stem and progenitor cells from 4 ELANE mutant SCN patient donors, it was demonstrated that exon 2 targeting ribonucleoproteins achieves highly efficient editing exceeding 95% indel frequency, trigger ELANE transcript decay, and rescue promyelocyte stage maturation arrest.

The authors then examined naturally occurring SCN-associated frameshifts, which affect late exons of ELANE. Targeting ELANE exon 5 in haematopoietic stem and progenitor cells resulted in robust indels (93.5%) preserving ELANE expression, but, unlike the results from targeting exon 2, it now resulted in cell death at the promyelocyte/myelocyte stages of development, recapitulating an SCN phenotype.

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, with late exon ELANE gene editing Dr Rao and colleagues have developed a robust new model of SCN using primary human haematopoietic stem and progenitor cells that recapitulates neutropenia in vivo.

Keywords: ELANE; Neutropenia; Gene Editing; CRISPR-Cas

Top image: @ iStockPhoto: ustas7777777

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