Developing a universal haematopoietic stem cell gene editing therapy for XIAP deficiency

X-linked inhibitor of apoptosis (XIAP) deficiency is a rare, life-threatening inborn error of immunity, caused by mutations in the XIAP/BIRC4 gene. Allogeneic haematopoietic stem cell (HSC) transplantation can be curative, but graft versus host disease remains a significant problem. We have developed a therapeutic autologous HSC gene editing approach, thus avoiding the risk of alloreactivity and permitting the use of less toxic conditioning regimens.

A CRISPR/Cas9/AAV6 based gene editing strategy was used to achieve precise correction at the XIAP locus in human CD34+ cells. Edited patient cells were differentiated into monocytes and stimulated with MDP or LPS to assess XIAP dependent function. An in vitro colony forming unit (CFU) assay was used to assess differentiation and engraftment potential of edited CD34+ cells.

HDR editing rates of up to 60% were achieved, without impacting cell viability. Correction of XIAP patient cells resulted in restoration of XIAP protein expression and partial restoration of TNFα secretion after stimulation with MDP in corrected patient cells. In vitro preservation of clonogenic and differentiation potential was shown. Analyses of off-target endonuclease activity, through sequencing of the predicted top 10 off-target sites and karyotype analysis, are underway.

This work confirms proof-of-principle for an HSC gene editing approach as a potential treatment for XIAP deficiency. High editing rates in CD34+ cells were achieved, with low cytotoxicity and preserved in vitro differentiation potential. High levels of targeted integration were observed in XIAP patient HSCs resulting in partial restoration of XIAP protein dependent immune function in differentiated, corrected cells.