Modelling, Optimization and Comparable Efficacy of T cells
and HSC Gene Editing for treating HIGM1 (2020)
Figure 4

Figure 4 - CD40LG gene editing in long-term repopulating human HSPC.

A Schematics of experimental protocol in HSPC.

B Percentage of HDR in HSPC subpopulations at 4 days after CD40LG editing, measured by molecular analysis (ddPCR). Cells were edited with donor template either comprising only the corrective cDNA (NR; n=4), or carrying the selector cassette IRES.NGFR (NGFR; n=5) and sorted according to surface markers: CD34-; CD34+CD133-CD90-; CD34+CD133+CD90-; CD34+CD133+CD90+. CD34+CD133+CD90+/CD90- and CD34+CD133-/CD34- conditions were used as unified groups for statistical analysis. The comparisons between the groups were performed with LME models accounting for the different subpopulations included in the analysis and with random effects defined to account for the same donor. In comparison about CD34+CD133+CD90+/CD90-, the percentages were used in square root scale to meet the assumption of normality of the residuals of the model. For CD34+CD133+CD90+/CD90-, **p= 0.0035, for CD34+CD133-/CD34- p=0.2051. Median ± IQR.

C Population composition in UT or bulk edited HSPC from (B) (n=8 for each group, except UT cells from NGFR group, n=7). Paired Wilcoxon's test, to account for the same donor, with p-values adjusted with Bonferroni's correction to account for multiple testing. Analysis was performed separately for NR and NGFR groups. Mean ± SEM.

D Human CD45+ cell engraftment in PB after transplantation of HSPC edited with the two donor templates described in (B) and with (n=4) or without (n=5) GSE56. Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the first time-point (i.e. 6 weeks; *p=0.0213 and **p=0.0069). Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching the final plateau with respect to both GSE56 groups (p=0.0034 for NGFR+GSE56 and p=0.0080 for NR+GSE56). Median ± IQR.

E Percentage of HDR within human cells over time, measured by ddPCR in PB of mice from (D). Longitudinal comparisons were performed with an LME model with time treated as a continuous variable, and followed by an appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the last time-point (i.e. 20 weeks; ****p<0.0001 in both comparisons). NR+GSE56 group shows a constant behavior over time (p=0.0588). The slope of the linear trajectory over time is not significantly different between GSE56 groups (p=0.1624), indicating gene editing stability of both groups over time. Mice transplanted with HSPC treated without GSE56 shows significantly lower slope than GSE56 groups (p<0.0001 in respect of NGFR group, p=0.0054 in respect of NR group), thus highlighting decreased gene editing efficiency over time. Median ± IQR.

F, G Percentage of HDR within human cells in (F) PB, bone marrow, spleen, thymus and (G) within BM-sorted human subpopulations (CD19+, CD33+, CD3+, CD34+), measured by ddPCR in mice from (D). Median.

H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleen of mice from (D) (n=4 NGFR+, 4 NGFR-). Mean ± SEM.