Fig 3. HBB gene targeted HSPCs display long-term and multi-lineage reconstitution in NSG mice.

a) 16 weeks post-transplantation, mouse bone marrow was analyzed for human cell chimerism and GFP expression by flow cytometry. Top panel: Representative FACS plot from a mouse transplanted with RNP+AAV GFP^high HSPCs showing engrafted human cells in the red gate. Bottom panel: Representative FACS plot showing GFP-expressing human cells (red gate). CD19⁺ B cells and CD33⁺ myeloid cells are backgated and shown in blue and green, respectively. b) Human engraftment in NSG mice from all experimental groups. Three different HSPC donors were used for engraftment studies (N=number of data points within group), **** p < 0.0001, ns = p ≥ 0.05, one-way ANOVA and Tukey's multiple comparison test. Bars represent median. c) Percent GFP⁺ cells in the total human population (red), CD19⁺ B cells (blue), and CD33⁺ myeloid cells (green) (N=number of data points within group), * p < 0.05, **** p < 0.0001, one-way ANOVA and Tukey's multiple comparison test for total human cells and unpaired t test with Welch's correction for B and Myeloid cells. Bars represent median. d) 12-14 weeks post-secondary transplantation human cell chimerism and GFP expression was analyzed by flow cytometry. Left panel: Representative FACS plot from a secondary mouse transplanted with RNP+AAV (top) or RNP+AAV GFP^high (bottom) cells showing engrafted human cells in the red gate. Right panel: Percent GFP⁺ human cells in the BM of secondary recipients. e) Gel images of In-Out PCRs on sorted human cells from secondary recipients to analyze on-target integrations at the 5’ and 3’ ends. Input control PCR was performed on the human CCR5 gene. Positive control is an HSPC sample targeted at HBB with SFFV-GFP-PolyA. f) 80 million mPB-derived CD34⁺ cells were electroporated with HBB-RNPs and transduced with HBB AAV6s Bulk HSPCs or HSPCs enriched for targeting (by FACS or bead-enrichment) were transplanted into the tail vein of sublethally irradiated mice. 16 weeks post transplant human cell chimerism was analyzed by flow cytometry (N=number of data points within group). g) Percent GFP⁺ and tNGFR⁺ cells in the human population was analyzed by flow cytometry (N=number of data points within group), bars represent median.