Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia (CN). Although the majority of CN patients respond to daily granulocyte colony stimulating factor (G-CSF), approximately 15 % do not respond to this cytokine at doses up to 50 μg/kg/day and approximately 15% of G-CSF treated patients will develop myelodysplasia (MDS) or acute myeloid leukemia (AML).
“Maturation arrest,” the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated CN. Because mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and “maturation arrest” corrected by CRISPR/Cas9 mediated gene editing.
We applied CRISPR/Cas9-sgRNA ribonucleoprotein (RNP) mediated ELANE knockout (ELANE KO) in induced pluripotent stem cells (iPSC) and primary hematopoietic stem and progenitor cells (HSPC).
We used iPSC from 2 CN patients and HSPC from 4 CN patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE. We observed that granulocytic differentiation of ELANE KO iPSC and HSPCs was comparable to healthy individuals and phagocytic function of the ELANE KO neutrophils was also normal. Knock down of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils.
These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients’ HSPCs followed by autologous transplantation may be an alternative therapy for CN.