CRISPR/Cas9-mediated <i>ELANE</i> knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients

Abstract

A Autosomal-dominant <i>ELANE</i> mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 μg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of <i>ELANE</i> associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that <i>ELANE</i> associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated <i>ELANE</i> knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring <i>ELANE</i> mutations as well as HL60 cells expressing mutant <i>ELANE</i> We observed that granulocytic differentiation of <i>ELANE</i> knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the <i>ELANE</i> KO (knockout) neutrophils were also normal. Knockdown of <i>ELANE</i> in the mutant <i>ELANE</i> expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that <i>ex vivo</i> CRISPR/Cas9 RNP based <i>ELANE</i> knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.