Figure 5.

Controlling dose and transduction time window alleviates LV toxicity in the ex vivo expansion setting

(A) Schematic representation of the experimental design for data shown in (B)–(F). Two separate transplant sessions were performed using CD34⁺ cells from the same adult mPB donor under the same protocol; each session transplanted cell products from all groups, except the EX-U2 condition, which was added during the second transplant session. Pooled results from the two sessions are shown. (B) The percentage of CD34⁺ cells measured by flow cytometry after 7 days of expansion under different conditions. Bars represent the average from the two experimental sessions. Data not shown for EX-U2 (76%) as only a single replicate was performed. (C) Relative VCN change in the EX-U condition with respect to DP_like, as determined by ddPCR on liquid culture (n = 2 experimental sessions; n = 1 for EX-U2 [4.8], data not shown). (D) Engraftment potential of mPB CD34⁺ cells in NSG mice transplanted with either (1) 200,000 cells from the DP_like-U1 condition (n = 22) or (2) 3,200,000 ex vivo-expanded cells (equivalent to 200,000 CD34⁺ cells at day 0) expanded for 7 days and transduced at the following time points: EX-U1, 24 h, n = 21; EX-U2, 48 h, n = 7; EX-U3, 72 h, n = 22. Human cell engraftment in the BM was evaluated by analyzing the proportion of CD45⁺ cells 17 weeks post injection (Kruskal-Wallis test with Dunn’s multiple comparison). (E) Intragroup LVBC sharing percentages. Each dot represents the percentage of sharing between an individual mouse vs. all other mice receiving the same HSPC product (Kruskal-Wallis test with Dunn’s multiple comparison, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (F) Bulk RNA sequencing was performed to identify DEGs between the EX-U3 and EX-U1 groups. The cnet plot shows enriched senescence terms obtained from GSEA; blue and red dot colors represent down-/upregulated genes, respectively. The dot sizes represent the number of genes that belong to the term. (G) Experimental scheme. Adult mPB CD34⁺ cells were transduced with a clinical-grade LV and either expanded (EX) or not (DP_like) using a good manufacturing practice-compliant process. The following groups were injected into NSG mice: (1) 200,000 DP_like cells (n = 14), (2) 20,000,000 ex vivo-expanded cells at high-dose EX_high (equivalent to 530,000 CD34⁺ cells at day 0; n = 12), or (3) 4,000,000 ex vivo-expanded cells low-dose EX_low (equivalent to 100,000 CD34⁺ cells at day 0; n = 14). (H) Percentage of CD34⁺ cells and subset composition measured by flow cytometry in the respective products (day 2, DP_like; day 8, EX) before xenotransplantation. (I) Human CD45⁺ cell engraftment measured in the peripheral blood (left) and BM (right) at the indicated time points (Kruskal-Wallis test with Dunn’s multiple comparison). (J) VCN determined by ddPCR on BM cells from mice of different groups (Kruskal-Wallis test with Dunn’s multiple comparison). (K) Box plots indicating number of unique integration sites normalized on VCN (Kruskal-Wallis test with Dunn’s multiple comparison). Each dot represents an individual mouse (Kruskal-Wallis test with Dunn’s multiple comparison). (L) Box plots indicating the Shannon diversity index as a measure of clonal population diversity, (Kruskal-Wallis test with Dunn’s multiple comparison). Each dot indicates an individual mouse.