Exonic knock-out/in gene editing in hematopoietic stem and progenitor cells fully rescues RAG1 immunodeficiency [OFFTARGET]

Recombination Activating Genes (RAG) are tightly regulated during lymphoid differentiation and their mutations cause a spectrum of severe immunological disorders. Haematopoietic stem/progenitor cell (HSPC) transplantation is the treatment of choice but limited by donor availability and toxicity. To overcome these issues, we developed and validated gene editing (GE) strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR). Off-target and RNA-Seq analyses were performed on edited human and patient-derived HSPCs to assesse the genome integrity and the trascriptomic profile. Whereas integration into intron 1 achieved suboptimal levels of correction, in-frame insertion into exon 2 drove faithful recapitulation of physiologic hRAG1 expression and activity. Off-target validation was performed by deep sequencing analysis on three different mPB HD-derived HSPCs edited with gRNA#13 RNP and untreated cells were used as negative controls