Disease Modeling of Severe Congenital Neutropenia Using CRISPR/Cas9 Gene Correction or Knockout of ELANE in Patients Derived Induced Pluripotent Stem Cells

Severe congenital neutropenia (CN) is a monogenic bone marrow failure syndrome with the frequency of 1:200,000 and is characterized by an absolute neutrophil count below 500 cells per microliter. Patients with CN suffer from severe life-threatening bacterial infections starting early after birth due to the absent or very low numbers of neutrophils in peripheral blood. While CN is a heterogeneous disease caused by many different gene mutations, autosomal-dominant ELANE mutations are the most common cause of CN. Although the majority of CN patients respond to daily treatment with granulocyte colony-stimulating factor (G-CSF), approximately 15 % do not respond at doses up to 20 μg/kg/day and approximately 20 % of G-CSF treated patients develop myelodysplasia (MDS) or acute myeloid leukemia (AML).

In the present study, we first established an efficient gene-editing platform for induced pluripotent stem cells (iPSC) of CN patients using CRISPR/Cas9 technology. The platform uses ribonucleoprotein form of CRISPR/Cas9 making the editing approach safer as it is virus- or DNA free. Also, any further selection step or introducing extra modifications in the genome of edited cells such as silent mutation are not required. We generated and characterized iPSCs from ELANE-CN patients harboring p.A57V, p.C151Y, and p.G214R mutations, that are more severe hot-spot mutations associated with G-CSF non-response or MDS/AML. We corrected each mutation followed by EB-based hematopoietic differentiation, to evaluate and compare granulocytic differentiation of CN-patient specific iPSCs, with or without ELANE mutation, in an isogenic model. To study granulocytic differentiation, we performed live cell counts, flow cytometry analysis of myeloid-specific surface marker expression, CFU assay, cell morphology of cytospin preparations and neutrophil functional tests. Our isogenic model showed that correction of ELANE mutations led to fully normalized granulocytic differentiation.

We have recently shown that CRISPR/Cas9 mediated ELANE knockout (KO) enables neutrophilic maturation of primary HSPCs and iPSCs of CN patients. We observed that granulocytic differentiation of ELANE KO iPSCs and primary HSPCs were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO neutrophils were also normal.

To model CN in silico and to reveal the key driving pathomechanisms, we designed an isogenic patient-specific disease modeling system by comparing RNA-sequencing results of CN-ELANE corrected- or CN-ELANE KO hematopoietic stem and progenitor cells (HSPCs) to the original CN-ELANE patient cells. HSPCs were derived from iPSC lines. Our analysis showed a degree of similarity in enriched pathways upon ELANE correction or ELANE KO in a patient-specific manner. Thus, upon correction of p.C151Y mutation, TNF, IL4 and IL13 signaling pathways as well as MAPK signaling, PD-1 signaling and IL10 signaling were down-regulated. Interestingly, the same pathways were down-regulated upon ELANE KO in HSPCs of the same CN patient. Correction of p.A57V mutation led to down-regulation of IL12 expression which activates STAT family. Upon ELANE KO in the cells from the same patient, IL12, IL18, and IL1-beta expression were down-regulated. We also identified common pathways enriched in most of the isogenic samples upon ELANE correction or ELANE KO like down-regulation of MAPK or IFN α/β signaling as well as down-regulation of the Rap-1 signaling pathway leading to the Erk pathway activation. Analysis of putative transcription factor binding sites (TFBSs) that are enriched in the differentially expressed gene list upon ELANE mutation correction or ELANE KO showed that transcription factors GKLF (KLF4), MAZ, Kaiso (ZBTB33) and CHURCHILL are highly enriched in UP-regulated genes, for both, correction and KO samples.

Taken together, we established a safe and efficient CRISPR/Cas9-RNP based ELANE gene-correction/knockout platform of iPSCs of ELANE-CN patients that may be used to establish an isogenic disease modeling system or provide novel stem cell-based therapy for CN patients with a high risk of leukemia development as well as for G-CSF-non-responsive patients. This platform could be also applied for other monogenic bone marrow failure syndromes.