Figure 4.

Cumulative toxicity between proteostatic cellular stress and lentiviral transduction

(A) Colony count in CFU assays with sorted HSCs (CD34⁺CD38⁻CD90⁺CD45RA⁻CD49f⁺) or CD34⁺ (CD34⁺ 49f⁻) cells (pool of n = 4 CB donors) in presence (+) or absence (−) of 4HPR (2 μM). The mean ± SEM of four replicates (600 cells/plate) is shown (unpaired t test). (B) Colony count in CFU assays with sorted HSCs (n = 1 mPB donor) in presence (+) or absence (−) of 4HPR (2 μM) and or UM171 (35nM). The mean ± SEM of four replicates (800 cells/plate) is shown (one-way ANOVA with Bonferroni correction). (C) (Top) Experimental design. Expansion culture and/or colony assays were conducted with UM171 (UM) in presence (+) or absence (−) of 4HPR (4H). (Bottom) Colony count in CFU assays with sorted HSC (n = 1 new mPB donor) performed either on freshly sorted cells (two groups on the left) or after LV transduction and ex vivo expansion (two groups on the right), in the presence (+) or absence (−) of 4HPR (2 μM), as shown at the bottom of the graph (nd, not done). The mean ± SEM of three replicates (1,000 cells/plate) is shown. (D) Schematic representation of xenotransplantation experiments performed on the DP from an MPS patient (Pt5). (E) Human cell engraftment in the BM of NSG mice at 12 weeks after transplantation of (1) 200,000 cells from the drug product (DP) (n = 5); (2) 5,400,000 ex vivo-expanded cells EX-U (equivalent to 200,000 CD34⁺ cells at day 0, expanded for 7 days with UM171; n = 6); (3) 6,200,000 ex vivo-expanded cells EX-US (equivalent to 200,000 CD34⁺ cells at day 0, expanded for 7 days with UM171 and SR1; n = 7); (4) 1,300,000 ex vivo-expanded cells EX-US4 (equivalent to 200,000 CD34⁺ cells at day 0, expanded for 7 days with UM171, SR1, and 4HPR; n = 7); Kruskal-Wallis test with Dunn’s multiple comparison, ∗p < 0.05. (F) Human cell engraftment in the BM, 15 weeks after secondary transplantation of 1 × 10⁶ CD34⁺ cells per mouse, isolated from primary recipients described in (E). The number of mice with multilineage engraftment over the total number of transplanted mice is shown. (G) Box plots indicating the number of unique integration sites normalized on VCN retrieved from the BM of primary grafts described in (E). One-way ANOVA with Tukey’s multiple comparison test. (H) Relative abundance of integration sites in the primary recipient mice. Bottom light-gray stacks indicate the sum of integrations with <1% relative abundance, middle stacks (shades of gray) indicate integrations >1% abundance, and the top abundant integrations highlighted in blue. (I) Schematic representation of xenograft experiments performed on the progeny of sorted CD34⁺CD38⁻ cells from adult mPB testing the following 2 variables: (1) transduction (LV) or not (mock) with a purified LV vector; (2) expansion culture with or without 4HPR. (J) Human cell engraftment in the BM was evaluated by analyzing the proportion of CD45⁺ cells 16 weeks post injection. Shown is the mean ± SEM; each dot represents an individual mouse. A mouse was classified as engrafted if it contained >0.5% of hCD45⁺ cells. ns, not significant; ∗∗p < 0.01 (mock vs. LV, two-way ANOVA).