CRISPR/Cas9n-mediated ELANEpromoter editing for gene therapy of severe congenital neutropenia

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANEmutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANEmRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANEpromoter TATA-box using CRISPR/Cas9D10A nickases - termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+hematopoietic stem and progenitor cells (HSPCs) in vitroand in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocation. Taken together, ex vivogene editing of ELANE-CN HSPCs using MILESTONEin the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.