Figure 1.
Toxicology and Tumorigenicity Study in a Thalassemia Murine Model
(A) Experimental plan of the toxicology and tumorigenicity study. Lin− cells from donor C57BL6/Hbbth3 mutant heterozygous (th3/+) mice were MOCK-transduced or transduced with GLOBE LV at MOI 100 and incubated overnight. The recipient mice were pre-treated with busulfan (25 mg/kg/day for 4 days). Mice were intravenously injected with a dose of 4 × 105 Lin− cells/mouse. Test group consisted of mice transplanted with GLOBE LV-transduced Lin− cells (th3/+ GLOBE LV). Control groups consisted of mice pre-treated with busulfan and transplanted with MOCK-transduced Lin− cells (th3/+ MOCK) and age-matched untreated mice (th3/+). Chimerism and cell origin were monitored using antibodies specific for CD45.1 or CD45.2 surface antigen. (B) Donor cell engraftment in male and female recipients was determined by cytofluorimetric analysis for the expression of CD45.1 in peripheral blood mononuclear cells (PBMCs) at 16 weeks and 12 months after transplantation (termination). Values are shown as mean ± SD. Student’s t test was applied (*p < 0.05, ****p < 0.0001). (C) Analysis of hematological reconstitution on peripheral blood was assessed by cytofluorimetric analysis. Percentages of myeloid and lymphoid (B and T) cells were evaluated by using antibodies against CD11b, B220, and CD3, respectively. Values are shown as mean ± SD. The GLOBE LV th3/+ group is shown in red, the MOCK th3/+ group in gray, and the th3/+ group in white. (D) VCN per genome was evaluated in GLOBE LV th3/+ group males and females. Values are shown as mean ± SD. (E) HGB concentration was evaluated in all groups of mice. Values are shown as mean ± SD. One-way ANOVA and post hoc Bonferroni’s multiple comparisons were applied (****p < 0.0001). (F) All animals were observed for signs of ill health or overt toxicity from day1 until the end of the study. Mortality was observed in the GLOBE LV th3/+ and MOCK th3/+ groups from day 153 onward. 5 deaths occurred in the GLOBE LV th3/+ group (circles). In the MOCK th3/+ group (squares), two animals died. No mortality was observed in the th3/+ group (triangles). Survival fractions were calculated using the product limit (Kaplan-Meier) method. Survival curves were compared using the log rank test (also called the Mantel-Cox test). No statistical difference was observed between the groups.