Ex vivo gene-targeted RAG2^null HSPCs correct in vivo B cell developmental block. (A) Percent of total genome editing (INDELs and HR) in RAG2^null patient-derived HSPCs, using 4 different AAV6 production lots. AAV6 lot D (asterisk marked) was used for all subsequent engraftment studies. (B) Percent human cells (CD45⁺ HLA A-B-C⁺) engrafted in BM after transplanting (22 weeks post-Tx) 0.5 x10⁶ uncorrected RAG2^null patient-derived HSPCs (n=4 mice, grey circles), 0.5 x10⁶ coRAG2-GT HSPCs (n=15 mice, half red circles) or 1.0 x10⁶ coRAG2-GT HSPCs (n=15 mice, black circles). Healthy donor (HD) HSPCs were used as control (n=6, white circles). RAG2^null patient genotype: c.296C>A; c.1342C>A. (C) FACS-based quantification is shown in (D) of large Pre-B I, small Pre-B II, immature, and mature B cells derived from coRAG2-GT HSPCs. Each population is graphed as a percent of total B cells. Bars: mean ± s.e.m. (D) Representative FACS plots of B-cell developmental stages derived from a healthy donor (left panel), RAG2^null patient (middle panel), and coRAG2-GT RAG2^null HSPCs. FACS-based quantification of percent cells in each developmental stage is shown. (E) FACS-based quantification of CD19⁺CD20⁺IgM⁺ triple-positive B cells derived from each condition tested. (F) PCR-based sequencing of immunoglobulin M (IgM) heavy chain (Vh) families from sorted triple-positive B-cells.