Homozygous mutations in HAX1 cause severe congenital neutropenia (HAX1-CN), characterized by fewer than 500 neutrophils/µl, a high risk of severe infections, and a 20% likelihood of developing MDS/AML. Currently, the only curative option is allogeneic bone marrow transplantation, which carries significant comorbidities. We aim to develop an ex vivo autologous hematopoietic stem cell gene therapy to correct the most frequent HAX1 mutation, p.W44X, found in ~80% of HAX1-CN patients.

We designed a single guide RNA targeting p.W44X and a correction template delivered via an AAV6 vector. Patient-derived hematopoietic stem and progenitor cells (HSPCs) were cultured for two days, electroporated with CRISPR/Cas9, then transduced with the AAV6 repair vector. This approach achieved ~65.8% gene correction, restoring HAX1 protein expression (Western blot).

Gene-corrected cells showed a 2.5-fold increase in neutrophil production. Corrected neutrophils performed essential functions: bacterial phagocytosis, chemotaxis, and reactive oxygen species generation. Restoration of HAX1 also rescued apoptosis resistance, as confirmed by live-cell imaging of caspase 3/7 activity. GUIDE-Seq and CAST-Seq revealed eight potential off-target sites, to be validated by targeted sequencing.

This efficient, selection-free strategy shows promise for curing HAX1-CN. Ongoing studies in immunodeficient mice will assess long-term safety and efficacy. These findings pave the way for future clinical translation, suggesting that congenital neutropenia could become routinely curable through gene therapy.