Figure 5.

Autologous HSCT using gene-corrected HSC clones is curative in an immunodeficiency mouse model

(A) Schematic of the single clone Prkdc^scid correction model.

(B) Single-cell SCID HSC expansion outcomes. Left: frequencies of phenotypic HSC populations in screened colonies (n = 384 from 3 experiments). Right: histogram of CD201⁺CD150⁺KL cell frequency. Enlarged region shows clones with ≥10% CD201⁺CD150⁺KL cells.

(C) Genotyping of candidate clones (Sanger sequencing) (n = 96 clones, 3 experiments). Only clones with at least one HDR-corrected allele were sequenced at the off-target loci.

(D) Allelic composition of the combined cell mixture at the edited Prkdc locus (n = 3).

(E) Frequencies of PB leukocytes in CB17/SCID recipients. Left: lineage distributions in treated mice (n = 3) and in recipients receiving only 2 × 10⁵ CB17/SCID whole bone marrow cells (neg. ctrl., n = 3). Right: representative FACS plots at 16 weeks post-SCT.

(F) Allele frequencies in sorted PB cells 20 weeks post-SCT (n = 3 mice from 3 experiments).

(G) Xenograft transplantation assay. A549 cells expressing the luminescent reporter Akaluc were injected subcutaneously (s.c.) and tumor growth was tracked by in vivo imaging. Left: representative images from CB17/WT, transplanted CB17/SCID, and untreated CB17/SCID mice 3 and 14 days after inoculation. Right: quantification of luminescence over a 14-day period (CB17/WT: n = 4, CB17/SCID − SCT: n = 3, CB17/SCID + SCT: n = 3). Two-way ANOVA and Tukey’s multiple comparison test.

(H) Genotyping of candidate clones (Sanger sequencing) (n = 173 clones, 2 experiments). Clones producing a single, HDR-corrected sequencing trace were checked for LD (n = 29). Prkdc^HDR/HDR, homozygous correction; Prkdc^HDR/Δ, hemizygosity.

(I) Prkdc copy-number analysis using 0.0 kb Prkdc (around cutsite) probe, quantified against reference gene.

See also Figure S5.

Error bars represent SD. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗∗p < 0.0001.