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

a,Histograms of IL2RA 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 IL2RA on the surface of the T cells (No electroporation, Grey). Two days following electroporation of an RNP containing a gRNA for the site of one of the two mutations, a one base pair 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 hets (c.800 Het 2 and 3) showed slight increases in IL2RA- cells (RNP only, Blue). This modest reduction is potentially due to the gRNA targeting the C-terminus of the protein where small indels may cause less pronounced loss of surface protein expression. Surprisingly, the RNP alone resulted in IL2RA 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 percent of cells with IL2RA correction compared to RNP only could be achieved by inclusion of an ssODN HDR template sequence with the mutation correction (RNP+ssODN, Green), and further increased at this site when using a longer dsDNA HDR template to correct the mutation (RNP + dsDNA HDRT, Yellow) (Extended Data Fig. 6i). b, Amplicon sequencing was performed in select targeted patient cells. c, Stat5 phosphorylation (pStat5) in response to high dose IL-2 stimulation (200 U/mL) in targeted CD3+ T cells following 7 days of expansion post-electroporation. Increased numbers of pStat5+ cells correlated with increased IL2RA surface expression (a). d, Following 9 days of expansion post-electroporation, intracellular FoxP3 staining revealed an increased proportion of IL2RA+ 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 pmols in 1 μL water were electroporated. e, Flow cytometric analysis of GFP expression 6 days following 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 twelve healthy donors similarly edited (Fig. 1d), both c.800 heterozygotes as well as compound het 1 and 2 were within the general range observed across healthy donors, whereas compound het 3 had lower GFP expression than any healthy donor analysed. Of note, in compound het 3 HDR mediated correction at the c.530 mutation was substantially lower than the other two compound heterozygotes (Fig. 3b). IL2RA 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 het 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 het 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.