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

TL;DR

Researchers developed a safer gene editing strategy using D10A Cas9n to treat chronic granulomatous disease by correcting genetic defects in hematopoietic stem cells.

Contribution

A novel D10A Cas9n-based gene editing approach with no detectable off-target activity or chromosomal translocations is introduced for treating CGD.

Findings

Using high-fidelity Cas9 minimizes off-target editing and restores engraftment potential in gene-edited HSPCs.

A D10A Cas9n strategy achieves no detectable off-target activity or chromosomal translocations.

Risk assessments show the D10A Cas9n strategy offers a potentially curative treatment for CGD.

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.

Gene editing of hematopoietic stem and progenitor cells offers promise as a curative treatment for chronic granulomatous disease (CGD). Here, the authors develop a D10A Cas9n based gene editing strategy to treat CGD with no detectable off-target activity or chromosomal translocations.

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