TY - JOUR
T1 - Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease
AU - Wolff, Jonas Holst
AU - Skov, Thomas Wisbech
AU - Haslund, Didde
AU - Dorset, Sofie Rahbek
AU - Revenfeld, Anne Louise S
AU - Aussel, Clotilde
AU - Jørgensen, Sofie E
AU - Holm, Mette
AU - Thomsen, Martin K
AU - Ammann, Sandra
AU - Cathomen, Toni
AU - Mogensen, Trine H
AU - Møller, Bjarne Kuno
AU - Bak, Rasmus O
AU - Mikkelsen, Jacob Giehm
N1 - © 2025. The Author(s).
PY - 2025/12
Y1 - 2025/12
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/105013021756
U2 - 10.1038/s41467-025-62738-2
DO - 10.1038/s41467-025-62738-2
M3 - Journal article
C2 - 40796771
SN - 2041-1723
VL - 16
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 7475
ER -