Fig. 6: Characterization of the CX3CR1 edited hHSPCs repopulating hematopoietic organs and brain of myeloablated immunodeficient recipients.

A Frequency of mock and edited (at CX3CR1 or at the AAVS1 safe harbor locus) hCD45⁺ cells detected by flow cytometry in the peripheral blood of NSG recipient mice at 4, 8, 10, and 12 weeks post-transplant. Mean values ± SD are shown. B Frequency of mock and edited hCD45⁺ cells in the bone marrow of primary and secondary recipients evaluated at 12 weeks post-transplant. Mean and individual values ± SEM are shown. C Percentage of engrafted hCD45⁺ cells expressing CX3CR1 in BM and brain of mice transplanted with mock or edited hHSPCs. Mean values ± SEM are shown. D GFP expression (left panel) and its relative mean fluorescent intensity (MFI) (right panel) in hCD45⁺ engrafted in the BM and brain of mice transplanted with CX3CR1 edited (CX exon or CX intron) or safe harbor edited (AAVS1) hHSPCs. Mean and individual values ± SEM are shown. E Percentage of targeted alleles (HDR) assessed by ddPCR in tissues retrieved from the mice transplanted with CX3CR1 edited (CX exon or CX intron) or safe harbor edited (AAVS1) hHSPCs, compared with targeting efficiency retrieved in the infused cell product (input). The human albumin gene was used for normalization. Individual replicates and mean values ± SEM are shown. F Progeny of CX3CR1 (top) or safe harbor (bottom) edited hHSPCs engrafted in the brain of transplant recipients and differentiated into MLCs. The green signal shows the expression of the GFP transgene in edited cells driven by the endogenous CX3CR1 locus (top) or the PGK promoter (bottom). Engrafted cells identified by hNuclei (red signal) express the Iba-1 marker (pink signal) as endogenous/recipient microglia cells. G Branching analysis performed on brain engrafted MLCs edited at the CX3CR1 or safe-harbor loci. The edited cells were identified through the expression of GFP. The cumulative length of branches, complexity index (CI), and covered environment area (CEA) are shown. Mean values ± SEM are reported. H Correlation between intersection radii and sum intersection parameters obtained from Sholl analysis performed on CX3CR1 or AAVS1-edited cells engrafted in the brain of transplanted mice. The vertical and horizontal lines divide the graphs into four quadrants, to describe the cells according to different grades of morphologic complexity, i.e., UR upper right quadrant, for very complex cells characterized by a high sum of intersections and a high number of intersecting radii; LL lower left quadrant, for cells with lower complexity; UL (upper left) and LR (lower right) quadrants for cells displaying intermediate complexity between the LL and the UR quadrants. In the dotted square, the histogram represents the percentage of cells retrieved in each of the four quadrants to quantify the data. In all panels, Mock and AAVS1, n = 6; CX exon, n = 9; CX intron, n = 8. Two independent experiments. Two-way ANOVA with multiple comparison by Tukey’s test in (A, C), with significant effects of treatment (****p < 0.0001) and time points (****p < 0.0001) in (A) and of treatment (****p < 0.0001) and tissue (*p < 0.05); the results of the multiple comparisons are indicated. One-way ANOVA with multiple comparison by Kruskal–Wallis test was performed in (B, D, E, G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The absence of statistical test information means not significant (ns) except for (D, panel on the right, GFP MFI) were the following comparisons (AAVS1 BM vs CX exon Br, AAVS1 BM vs CX intron Br, CX exon BM vs CX exon Br, CX exon BM vs CX intron Br, CX intron BM vs CX exon Br, CX intro BM vs CX intron Br, AAVS1 Br vs CX exon Br, AAVS1 Br vs CX intron Br) had a ***p < 0.001. In (H), distribution of ratio analysis was performed: ****p < 0.0001 in the LL and UR quadrants. Images were acquired via Zeiss 980 Confocal acquisition, 20X and 40X, Z-stack. n > 80 cells, n = 3 mice/group. Source data are provided as a Source Data file.