CRISPR/Cas9 Gene-modification Platform of ELANE Mutations in iPSCs and HSPCs of Severe Congenital Neutropenia Patients

Severe congenital neutropenia (CN) is a monogenic bone marrow failure syndrome characterized by an absolute neutrophil count below 500/ul. Autosomal-dominant ELANE mutations are the common cause of CN. ca. 15% do not respond to G-CSF therapy at doses up to 50 µg/kg/day and ca. 15% of G-CSF treated patients developed MDS or AML. We aimed to develop a platform for CRISPR/Cas9 ribonucleoprotein (RNP)- or AAV- mediated gene correction or knockout of ELANE in induced pluripotent stem cells (iPSC) and primary HSPCs of CN patients. We observed that granulocytic differentiation of ELANE KO iPSC and HSPCs was comparable to healthy individuals and phagocytic function of the ELANE KO neutrophils also was normal. Simultaneously, CRISPR/Cas9 mediated correction of more severe ELANE mutations enables neutrophilic maturation of gene edited HSPCs and iPSCs of CN patients. Moreover, using whole transcriptome analysis, we identified key signaling pathways regulated by mutated ELANE in CN HSPCs. In summary, we established CRISPR/Cas9 based gene-modification platform for CN patient's HSPCs that can be also applied for the treatment of patients with other monogenic bone marrow failure Syndromes.