CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia

Masoud Nasri; Malte U Ritter; Perihan Mir; Benjamin Dannenmann; Masako M Kaufmann; Patricia Arreba-Tutusaus; Yun Xu; Natalia Borbaran-Bravo; Maksim Klimiankou; Claudia Lengerke; Cornelia Zeidler; Toni Cathomen; Karl Welte; Julia Skokowa

doi:10.1016/j.ymthe.2024.03.037

CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia

Mol Ther. 2024 Mar 30:S1525-0016(24)00215-6. doi: 10.1016/j.ymthe.2024.03.037. Online ahead of print.

Affiliations

¹ Department of Oncology, Hematology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, 72076 Tübingen, Germany. Electronic address: masoud.nasri@med.uni-tuebingen.de.
² Department of Oncology, Hematology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, 72076 Tübingen, Germany. Electronic address: malte.ritter@med.uni-tuebingen.de.
³ Department of Oncology, Hematology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, 72076 Tübingen, Germany.
⁴ Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany; Spemann Graduate School of Biology and Medicine, University of Freiburg, 79106 Freiburg, Germany.
⁵ Department of Oncology, Hematology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, 72076 Tübingen, Germany; Pediatric Hematology and Oncology, Hannover Medical School, 30625 Hannover, Germany.
⁶ Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.
⁷ Department of Oncology, Hematology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, 72076 Tübingen, Germany; Department of Pediatric Hematology, Oncology and Bone Marrow Transplantation, Children`s Hospital, University Hospital Tübingen, 72076 Tübingen, Germany.
⁸ Department of Oncology, Hematology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, 72076 Tübingen, Germany; Gene and RNA Therapy Center (GRTC), University Hospital Tübingen, 72076 Tübingen, Germany.

PMID: 38556793
DOI: 10.1016/j.ymthe.2024.03.037

Abstract

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (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 ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter 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 vitro and 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 translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Keywords: CRISPR Cas9 nickase; ELANE mutations; SpCas9D10A; bone marrow failure syndromes; gene editing; gene therapy; in vivo model of severe congenital neutropenia; inhibition of ELANE expression; neutrophil elastase; severe congenital neutropenia.