Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

Jonas Holst Wolff, Thomas Wisbech Skov, Didde Haslund, Sofie Rahbek Dorset, Anne Louise S. Revenfeld, Clotilde Aussel, Sofie E. Jørgensen, Mette Holm, Martin K. Thomsen, Sandra Ammann, Toni Cathomen, Trine H. Mogensen, Bjarne Kuno Møller, Rasmus O. Bak and Jacob Giehm Mikkelsen ()
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Jonas Holst Wolff: Aarhus University
Thomas Wisbech Skov: Aarhus University
Didde Haslund: Aarhus University Hospital
Sofie Rahbek Dorset: Aarhus University
Anne Louise S. Revenfeld: Aarhus University Hospital
Clotilde Aussel: Medical Center – University of Freiburg
Sofie E. Jørgensen: Aarhus University Hospital
Mette Holm: Aarhus University Hospital
Martin K. Thomsen: Aarhus University
Sandra Ammann: Medical Center – University of Freiburg
Toni Cathomen: Medical Center – University of Freiburg
Trine H. Mogensen: Aarhus University
Bjarne Kuno Møller: Aarhus University Hospital
Rasmus O. Bak: Aarhus University
Jacob Giehm Mikkelsen: Aarhus University

Nature Communications, 2025, vol. 16, issue 1, 1-16

Abstract: Abstract Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34+ hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients.

Date: 2025
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DOI: 10.1038/s41467-025-62738-2

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