Didde Haslund

GATA2 (GATA binding protein 2) deficiency is a severe immunodeficiency caused by heterozygous variants in the gene encoding the transcription factor GATA2. Ex vivo gene editing of a patient’s own CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs) could provide curative treatment. However, current methods that rely on nuclease-dependent editing face considerable challenges, including off-target effects, genotoxicity, and reduced engraftment potential. Here, we report the development of an efficient gene editing therapy for GATA2 deficiency using prime editing with a favorable safety profile in terms of off-target effects and genotoxicity. We used prime editing to correct a GATA2 c.956_962del variant in patient-derived CD34 ⁺ HSPCs, reaching up to 70% prime editing efficiency and an increase in functional GATA2 alleles from 50 to 77%. We demonstrate that prime-edited patient HSPCs showed increased engraftment potential compared with untreated cells and detected limited on-target genotoxicity and no off-target editing at the top 20 predicted sites. Short prestimulation of CD34 ⁺ HSPCs supported efficient prime editing while preserving stemness, mitigating p53 activation, and increasing multilineage engraftment. We report PASSIGE (prime editing–assisted site-specific integrase gene editing) in CD34 ⁺ HSPCs, developing a more broadly applicable, double-strand break–independent complementary DNA insertion strategy with the potential to address a large proportion of alleles causing GATA2 deficiency. Together, our results demonstrate the preclinical development of prime editing–based therapies for GATA2 deficiency in CD34 ⁺ HSPCs.

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.