Wolff Jonas Holst, Skov Thomas Wisbech, Haslund Didde, Dorset Sofie Rahbek, Revenfeld Anne Louise S, Aussel Clotilde, Jørgensen Sofie E, Holm Mette, Thomsen Martin K, Ammann Sandra, Cathomen Toni, Mogensen Trine H, Møller Bjarne Kuno, Bak Rasmus O, Mikkelsen Jacob Giehm
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark.
Nat Commun. 2025 Aug 12;16(1):7475. doi: 10.1038/s41467-025-62738-2.
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.
慢性肉芽肿病（CGD）是一种由NADPH氧化酶缺陷引起的严重先天性免疫缺陷病。在此，我们开发了基于CRISPR/Cas9的基因编辑策略，用于纠正导致CGD的CYBA和CYBB基因中的变异。对于X连锁CGD，我们还通过在CD34造血干细胞和祖细胞（HSPCs）中靶向整合截短的CYBB cDNA，开发了一种近乎通用的基因编辑策略。在整个过程中，脱靶编辑和染色体易位明显，这对基因编辑的HSPCs植入免疫缺陷小鼠的能力产生了负面影响。然而，通过使用高保真Cas9将脱靶编辑降至最低，我们证明了基因编辑的HSPCs多谱系植入潜力的恢复。此外，为了进一步提高安全性，我们开发了一种没有可检测到的脱靶活性或染色体易位的D10A Cas敲除编辑方法。总体而言，通过对不同基因编辑方法的风险评估，我们提出了一种基于D10A Cas9n的安全性更高的策略，为CGD患者提供了一种潜在的治愈性治疗方法。
