Extended Data Fig. 8: HDR-mediated and non-HDR-mediated correction of IL2RA c.800delA frameshift loss-of-function mutation.

a, Histograms of IL-2Rα surface expression in CD3⁺ T cells in all children from a family carrying two loss-of-function IL2RA mutations, including three compound heterozygotes that express minimal amounts of IL-2Rα on the surface of the T cells (no electroporation, grey). Two days after electroporation of an RNP containing a gRNA for the site of one of the two mutations, a 1-bp deletion in the final exon of IL2RA (c.800delA) causing a run-on past the normal stop codon, CD3⁺ T cells from a healthy donor and single heterozygotes (c.800 het 2 and 3) showed slight increases in IL-2Rα⁻ cells (RNP only, blue). This modest change is potentially due to the gRNA targeting the C terminus of the protein, in which small indels may cause less pronounced loss of surface protein expression. Notably, the RNP alone resulted in IL-2Rα surface expression in almost 50% of edited T cells in all three compound heterozygotes. In cells from two of the compound heterozygous children, increases in the percentage of cells with IL-2Rα correction compared to RNP only could be achieved by inclusion of an ssODN HDR template sequence with the mutation correction (RNP plus ssODN, green), and further increased at this site when using a longer dsDNA HDR template to correct the mutation (RNP plus dsDNA HDRT, yellow) (Extended Data Fig. 6i). b, Amplicon sequencing was performed in select targeted patient cells. c, pSTAT5 in response to high dose IL-2 stimulation (200 U ml⁻¹) in targeted CD3⁺ T cells after 7 days of expansion post-electroporation. Increased numbers of pSTAT5⁺ cells correlated with increased IL-2Rα surface expression (a). d, After 9 days of expansion post-electroporation, intracellular FOXP3 staining revealed an increased proportion of IL-2Rα⁺ FOXP3⁺ cells in CD3⁺ T cells compared to no electroporation controls. Electroporations were performed according to optimized non-viral genome targeting protocol (Methods). For ssODN electroporations, 100 pmol in 1 µl water was electroporated. e, Flow cytometric analysis of GFP expression 6 days after electroporation of a positive HDR control RAB11A–GFP dsDNA HDR template into CD3⁺ T cells from the indicated patients revealed lower GFP expression in the three compound heterozygotes compared to their two c.800 heterozygote siblings. Compared to a cohort of 12 similarly edited healthy donors (Fig. 1d), both c.800 heterozygotes as well as compound heterozygotes 1 and 2 were within the general range observed across healthy donors, whereas compound heterozygote 3 had lower GFP expression than any healthy donor analysed. Of note, in compound heterozygote 3, HDR-mediated correction at the c.530 mutation was substantially lower than the other two compound heterozygotes (Fig. 3b). IL-2Rα surface expression after electroporation of the c.800delA targeting RNP alone was similar though. Compared to HDR-mediated repair, NHEJ-mediated frameshift correction at c.800delA may be less dependent on cell proliferation, consistent with compound heterozygote 3 being the only compound heterozygous patient on active immunosuppressants at the time of blood draw and T cell isolation (Supplementary Note 3). f, Altered cell-state associated with the patient’s disease could also contribute to diminished HDR rates. TIGIT and CTLA4 expression levels in non-edited, isolated CD4⁺ T cells from each indicated patient was measured by flow cytometry. Consistent with altered cell states and or/ cell populations, cells from compound heterozygote 3 had a distinct phenotype, with increased TIGIT and CTLA4 expression compared both to healthy donors, the single heterozygous family members, as well the other two compound heterozygous siblings.