CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia

Abstract

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANE L172P mutation in SCN patientderived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, $34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.Recently, Nasri et al. 25 have shown that knock out (KO) of the ELANE gene in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 can rescue neutrophil differentiation. ELANE KO removed the deleterious effects of misfolded NE and restored mature neutrophil formation in vitro. 25 However, while an ELANE KO strategy provides a potentially promising treatment for G-CSF poor responders, it seems to favor the generation, selection, or outgrowth of edited cells that lack NE expression altogether. 25 This causes concern for human patients, since ELANE KO models

Neutrophil Elastase Defects in Congenital Neutropenia

“…An alternated approach to repair ELANE mutations was performed using two different single guide RNA (sgRNA) targeting both mutant allele and exon 4. Using this method, a group headed by Chu successfully repaired ELANE mutation in SCN patient-derived hematopoietic stem and progenitor cells together with restored neutrophil differentiation and normal elastase expression level ( 109 ).…”

Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

“…While treatment with G-CSF can improve neutrophil function and mobilization, gene editing has the potential to be curative. Tran and colleagues employed two different CRISPR/Cas9 RNPs in combination with AAV6 to repair both a specific ELANE L172P point mutation as well as targeting exon 4, in which the majority of ELANE mutations are found [ 84 ]. HDR efficiencies of 30% were achieved at exon 4 of ELANE in HSPCs from both healthy and SCN subjects, and 20% HDR-mediated correction was observed at the mutant ELANE L172P alleles in bone marrow CD34+ cells from affected patients.…”

“…Neutrophils differentiated in vitro from edited HSPCs produced similar amounts of reactive oxygen species (ROS), exhibited normal phagocytosis, efficient bacterial killing, and production of neutrophil extracellular traps (NETs). At four weeks post-transplant of humanized NOG-EXL mice (expressing human GM-CSF and IL-3), only ELANE corrected HSPCs were able to functionally differentiate into neutrophils [ 84 ] ( Table 1 ).…”

CRISPR-based strategies for targeted transgene knock-in and gene correction

“…As the majority of cells are successfully edited, no selection marker (for example, fluorescent marker or antibiotic selection) is required to screen a large number of clones to identify biallelic edited clones. Therefore, this scarless genome-editing approach is particularly useful for therapeutic applications in hard-to-transfect cells such as human pluripotent stem cells 54 , adult stem cells 28 , 55 , and primary immune cells 56 .…”