Page 85 - Haematologica March 2020

ELANE knockout restores granulopoiesis in congenital neutropenia
Hematopoietic stem and progenitor cells (HSPC) of CN patients fail to differentiate into mature neutrophils. This differentiation defect can be partially restored with daily or alternate-day subcutaneous injections of recombinant human granulocyte colony stimulating factor (rhG-CSF) in supra-physiological concentrations.4 Although rhG-CSF therapy improves the life expectancy and quality of life of CN patients, a subgroup does not respond to rhG-CSF. Additionally, about 15 % of CN patients developed myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) till now.3 There is a positive correlation between a rhG-CSF dose required to achieve acceptable neutrophil counts and a cumulative incidence to develop MDS or AML in CN patients.5 Therefore, CN patients, especially patients who either require high rhG-CSF dosages (above 50 mg/kg/day) and those who do not respond at all, need alternative therapeutic options. Hematopoietic stem cell transplantation (HSCT) would be a treatment of choice in CN patients, but it is associated with many adverse events, e.g. acute or chronic graft-ver- sus-host-disease (GvHD), life-threatening infections, graft failure or graft rejection. Indeed, the overall survival of CN patients after HSCT is approximately 80 % only.
Recently established new technologies of CRISPR/Cas9-mediated gene editing in mammalian cells6,7 offer novel therapeutic options, especially for inherited monogenic disorders, including ELANE mutations associ- ated CN. In this case, CRISPR/Cas9-mediated gene correc- tion or knockout of the mutant gene in patient`s HSPC ex vivo followed by autologous transplantation of the correct- ed HSPC might be a better treatment than high dose rhG- CSF or allogeneic stem cell transplantation.
ELANE mutations induce unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in HSPC of CN patients that leads to increased apoptosis and defec- tive granulocytic differentiation.8-11 Therefore, inactivation of ELANE using CRISPR/Cas9-mediated knockout may abrogate UPR and ER stress caused by mutated ELANE with subsequent restoration of granulocytic differentia- tion. In support of this hypothesis, we recently identified a β-lactam-based inhibitor of human neutrophil elastase (NE), MK0339, which restored defective granulocytic dif- ferentiation of induced pluripotent stem cells (iPSC) and HL60 cells expressing mutated NE.12 In addition, a recent report by Nayak et al. demonstrated the restoration of the in vitro granulopoiesis of ELANE-CN patient-derived iPSC upon treatment with Sivelestat, another NE-specific small- molecule inhibitor.12,13 Moreover, the fact that individuals showing mosaicism of inherited ELANE mutations have a higher proportion of ELANE mutated mature neutrophils hematopoietic cells in the bone marrow than in the blood14,15 supports the hypothesis that inactivation of ELANE mutations will improve neutrophil differentiation.
Another possibility to correct the disease phenotype is the direct correction of the specific gene mutation by the activation of homology-directed repair (HDR) of the mutated gene allele after cutting by CRISPR/Cas9 and co- transfection with a repair template. Most CN patients har- bor inherited autosomal dominant missense or frameshift ELANE mutations that are distributed throughout all five exons and two introns.16 Therefore, CRISPR/Cas9-mediat- ed correction of ELANE mutations would need to be patient/mutation specific. Since mutated ELANE may induce UPR and ER stress in edited cells, the introduction of new indels in the ELANE gene during the process of
CRISPR/Cas9 based editing may be not beneficial for the integrity of the hematopoietic stem cell (HSC) pool.
The first pre-clinical CRISPR/Cas9-based gene therapy study of common inherited blood disorders, sickle cell dis- ease, and β-thalassemia, was reported.17,18 In these set- tings, the β-globin gene locus was inactivated by the intro- duction of deletions in autologous HSPC by CRISPR/Cas9-mediated gene editing. This was done to mimic the hereditary persistence of fetal hemoglobin mutations in HSC.17,18
Here, we describe a CRISPR/Cas9 mediated ELANE KO by electroporation of HSPC and iPSC with ELANE specific CRISPR/Cas9-sgRNA ribonucleoprotein (RNP) complex- es. ELANE KO induces granulocytic differentiation of HSPC and iPSC of CN patients harboring ELANE muta- tions without affecting their phagocytic functions. These results suggest that it may be possible to use CRISPR/Cas9 based ELANE KO in autologous HSCT as a therapy for ELANE associated neutropenia.
Methods
Patients
Three healthy donors and five severe congenital neu- tropenia patients harboring ELANE mutations (ELANE-CN) were used in the study. Bone marrow and peripheral blood samples from patients were collected in association with an annual follow-up recommended by the Severe Chronic Neutropenia International Registry. Study approval was obtained from the Ethical Review Board of the Medical Faculty, University of Tübingen. Informed written consent was obtained from all partici- pants of this study.
Cell culture
Human CD34+ HSPC were isolated from bone marrow mononuclear cell fraction using Ficoll gradient centrifuga- tion followed by magnetic bead separation using Human CD34 Progenitor Cell Isolation Kit, (Miltenyi Biotech, #130-046-703). CD34+ cells were cultured in a density of 2 x 105 cells/mL in Stemline II Hematopoietic Stem Cell Expansion medium (Sigma Aldrich, #50192) supplement- ed with 10 % FBS, 1 % penicillin/streptomycin, 1 % L-Glutamine and a cytokine cocktail consisting of 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL TPO, 50 ng/ml SCF and 50 ng/mL FLT-3L (all cytokines were purchased from R&D Systems). Human induced pluripotent stem cells (iPSC) were cultured on Geltrex LDEV-free reduced growth factor basement membrane matrix (Thermo Fisher Scientific, #A1413201) coated plates in a density of 2 x 105 cells/mL in StemFlex medium (Thermo Fisher Scientific, #A3349401) supplemented with 1 % peni- cillin/streptomycin. HL60 cells were maintained in RPMI- 1640 supplemented with 10 % fetal bovine serum (FBS) (Gemini Bio Products, West Sacramento, CA, USA), 2 mM L-glutamine, and 1 % penicillin/streptomycin (Thermo Fisher Scientific) at 37°C and 5 % CO2.
Design of the ELANE-specific guide RNA (gRNA) Specific CRISPR-RNA (crRNA) for the knockout of the ELANE gene (cut site: chr19 [CTGCGCGGAGGC- CACTTCTG, +852,969 : -852,969], NM_001972.3 Exon 2, 161 bp; NP_001963.1 p.F54) was designed using the
CCTop website.19
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