Targeted inhibition of ELANE expression using adenine base editing to treat severe congenital neutropenia

Betül Findik; Benjamin Dannenmann; Franka Bernhard; Masako Monika Kaufmann; Sandra Ammann; Sergey Kandabarau; Maksim Klimiankou; Fabian Mauch; Patrick Münzer; Oliver Borst; Isabel Klefenz; Doris Steinemann; Claudia Lengerke; Cornelia Zeidler; Toni Cathomen; Karl Welte; Masoud Nasri; Julia Skokowa

doi:10.1016/j.omtm.2025.101626

Targeted inhibition of ELANE expression using adenine base editing to treat severe congenital neutropenia

Mol Ther Methods Clin Dev. 2025 Nov 5;33(4):101626. doi: 10.1016/j.omtm.2025.101626. eCollection 2025 Dec 11.

Authors

Betül Findik^{1

2}, Benjamin Dannenmann^{1

2}, Franka Bernhard^{1

2}, Masako Monika Kaufmann^{3

4

5}, Sandra Ammann^{3

4}, Sergey Kandabarau^{1

2}, Maksim Klimiankou^{1

2

6}, Fabian Mauch^{7

8}, Patrick Münzer^{7

8}, Oliver Borst^{7

8}, Isabel Klefenz⁹, Doris Steinemann⁹, Claudia Lengerke^{1

10}, Cornelia Zeidler^{1

6}, Toni Cathomen^{3

4}, Karl Welte^{6

11}, Masoud Nasri^{1

2}, Julia Skokowa^{1

2

6}

Affiliations

¹ Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Tübingen, Germany.
² Faculty of Medicine University Tübingen, Gene and RNA Therapy Center (GRTC), Tübingen, Germany.
³ Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.
⁴ Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany.
⁵ Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany.
⁶ Severe Chronic Neutropenia International Registry, SCNIR, Hannover, Germany.
⁷ DFG Heisenberg Group Cardiovascular Thromboinflammation and Translational Thrombocardiology, University of Tübingen, Tübingen, Germany.
⁸ Department of Cardiology and Angiology, University Hospital Tübingen, Tübingen, Germany.
⁹ Department of Human Genetics, Hannover Medical School, Hannover, Germany.
¹⁰ German Cancer Consortium (DKTK), partner site Tübingen, a partnership between DKFZ and University Hospital Tübingen, Tübingen, Germany.
¹¹ Department of Pediatric Hematology, Oncology and Bone Marrow Transplantation, Children's Hospital, University Hospital Tübingen, Tübingen, Germany.

Abstract

Autosomal dominant mutations in ELANE (elastase, neutrophil expressed) cause severe congenital neutropenia (CN) and cyclic neutropenia (CyN). Inhibiting ELANE expression, either by CRISPR-Cas9-mediated ELANE knockout or promoter targeting using CRISPR-Cas9 nickase, has emerged as a promising gene therapy strategy to restore defective granulocytic differentiation of transplantable hematopoietic stem cells from CN patients. We developed an adenine base editor (ABE)-mediated approach targeting two nucleotides in the ELANE promoter to suppress neutrophil elastase expression, called PRECISE. Analysis of mRNA- and protein-based delivery of ABE revealed that although both platforms were effective in editing hematopoietic stem and progenitor cells from healthy donors with over 80% editing, only protein-based ABE delivery achieved over 68% editing in CN patient cells. Interestingly, 10%-19% editing in CN patients' hematopoietic cells using ABE mRNA restored their granulocytic differentiation in vitro, with a marked expansion and differentiation of ABE ribonucleoprotein (RNP)-edited cells. PRECISE-edited neutrophils retained normal function, including neutrophil extracellular trap formation, oxidative burst, and phagocytosis. Genome integrity analysis showed no genomic alterations or chromosomal aberrations, and only two off-target edits confined to non-coding intronic regions. In conclusion, PRECISE represents a translationally relevant base-editing strategy for ELANE-associated CN and CyN that addresses ELANE mutation heterogeneity.

Keywords: ELANE; NETs; adenine base editing; bone marrow failure syndrome; gene therapy; hemopoietic stem cells; neutrophil elastase; neutrophils; pre-leukemia; severe congenital neutropenia.

Abstract

Grants and funding