Unbiased assessment of genome integrity and purging of large deletions at the target locus upon CD4 T-cell CRISPR-Cas9 long range editing for CD40LG deficiency

Hyper IgM1 is an X-linked combined immunodeficiency, caused by CD40LG mutations, potentially treatable with CD4+ T-cell Cas9 gene editing with a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing human cells. Large deletions were common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We also validated the sensitivity of optical genome mapping for the unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.