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Haematologica 2020 Volume 105(3):598-609
Bone Marrow Failure
CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients
Masoud Nasri,1 Malte Ritter,1* Perihan Mir,1* Benjamin Dannenmann,1* Narges
Aghaallaei,1 Diana Amend,1 Vahagn Makaryan,2 Yun Xu,1 Breanna Fletcher,2
Regine Bernhard,1 Ingeborg Steiert,1 Karin Hähnel,1 Jürgen Berger,3 Iris Koch,3
3341 Brigitte Sailer, Katharina Hipp, Cornelia Zeidler, Maksim Klimiankou,
Baubak Bajoghli,1 David C. Dale,2 Karl Welte1,5,§ and Julia Skokowa1,§
1Department of Oncology, Hematology, Immunology, Rheumatology and Clinical Immunology, University Hospital Tübingen, Tübingen, Germany; 2Department of Medicine, University of Washington, Seattle, WA, USA; 3Max Planck Institute for Developmental Biology, Tübingen, Germany; 4Department of Oncology, Hematology, Immunology and Bone Marrow Transplantation, Hannover Medical School, Hannover, Germany and 5University Children’s Hospital Tübingen, Tübingen, Germany
*MR, PM and BD are co-second authors. §KW and JS are co-senior authors.
ABSTRACT
AAutosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 mg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. “Maturation arrest,” the failure of the marrow myeloid progenitors to form mature neutrophils, is a consis- tent feature of ELANE associated congenital neutropenia. As mutant neu- trophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and “maturation arrest” cor- rected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harbor- ing ELANE mutations as well as HL60 cells expressing mutant ELANE. We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS pro- duction, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients’ primary hematopoietic stem and progenitor cells fol- lowed by autologous transplantation may be an alternative therapy for con- genital neutropenia.
Introduction
Autosomal dominant ELANE mutations encoding neutrophil elastase (NE) are the most common cause of severe congenital neutropenia (CN), an inherited bone mar- row failure syndrome.1-3 Patients with CN suffer from severe life-threatening bac- terial infections starting early after birth due to the absence or very low numbers of neutrophils in the peripheral blood (usually less than 500 cells per mL3).
Correspondence:
JULIA SKOKOWA
Julia.Skokowa@med.uni-tuebingen.de
Received: March 14, 2019. Accepted: June 21, 2019. Pre-published: June 27, 2019.
doi:10.3324/haematol.2019.221804
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