Abstract
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.