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<p>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 <i>CYBA</i> and <i>CYBB</i> genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated <i>CYBB</i> cDNA in CD34<sup>+</sup> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月12日

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  • 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,
  • Jacob Giehm Mikkelsen

摘要

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.