A selection free ex vivo gene therapy approach to congenital neutropenia causing HAX1 mutations

Severe congenital neutropenia (CN) patients lack mature neutrophils (< 500/µl) and have a 20 % risk to develop MDS or AML. Approximately 10 % of patients carry a homozygous loss of function mutation in the HAX1 gene. With these prospects, the clinical need for a genetic cure of CN is imminent.

We developed a selection-free approach to correct the most common HAX1 mutation p.W44X, in hematopoietic stem cells (HSCs) of CN patients ex vivo. Through electroporation, HiFiCas9 and a guide RNA are delivered as RNP into the HSCs. Then a correction template is delivered by a recombinant adeno associated virus 6 (rAAV6) vector. In patient HSCs we observed 65,8 % (± 7,12 %) correction efficiency and total editing (TE) of 84,4 % (± 4,2 %) (n=5), which led to re-expression of HAX1 protein in corrected cells. The HAX1 correction rescued the maturation arrest of granulopoiesis, as assessed by in vitro differentiation of HSCs to neutrophils. We are currently assessing in vivo engraftment capacity of gene edited cells. GUIDE and CAST-Seq show a good off-target profile of our sgRNA. Our results imply that ex vivo CRISPR-based gene-editing might be a feasible and safe approach for HAX1-CN patients.

Publication History

Article published online:
17 May 2022