Figure 5.
Phenotypic correction assessment of HA mice after platelet-specific gene therapy under ADC-mediated preconditioning. At least 4 months after 2bF8LV gene therapy, the bleeding phenotype in 2bF8LV-transduced recipients was assessed by a 6-hour tail-bleeding test and a needle-induced knee joint injury. For the tail-bleeding test, the tail tip was transected using a 1.6-mm-diameter template. Animals were monitored hourly, and clot time was recorded. Blood samples were collected before and after the test for blood counts. Hemoglobin level before the test was normalized to 100%. For the knee joint injury, a G30 × 1/2 needle was used to induce injury in the right knee, leaving the left knee unharmed as an intra-animal control. The diameter of the knee joint was measured using a digital micro caliper before and 24 hours after injury. The diameter of the knee joint before injury was defined as 1. WT and FVIIInull mice served as controls. (A) Percentage of animals whose tail bleeding stopped within 6 hours after tail tip transection. The statistical differences between the groups were analyzed using Fisher’s exact test. (B) Tail-bleeding time. Data from the 2 2bF8LV-transduced recipients in the (CD45.2+CD117)-ADC group that did not have sustained platelet FVIII expression are presented as a separate group. The statistical differences between the groups were analyzed using 1-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparisons test. (C) Percentage of hemoglobin remaining after the tail-bleeding test. Data from the 2 2bF8LV-transduced recipients in the (CD45.2+CD117)-ADC group that did not have sustained platelet FVIII expression are presented as a separate group. The statistical differences between the groups were analyzed using 1-way ANOVA, followed by Tukey’s multiple-comparisons test. (D) Ratio of knee joint diameter measured at 24 hours postinjury/preinjury. The statistical differences between the groups were analyzed using 2-way ANOVA, followed by Tukey’s multiple-comparisons test. Representative images of the knee joint from WT control mice (E) and FVIIInull control mice (F) 24 hours after injury. (G) Representative images of the knee joint from 2bF8LV-transduced recipients with ADC preconditioning 24 hours after injury. These data demonstrate that effective phenotypic correction is attainable in HA mice after 2bF8 lentiviral gene delivery to HSCs under a nongenotoxic hematopoietic cell–targeted ADC preconditioning. **P < .01, ***P < .001, ****P < .0001. ns, not significant.

Phenotypic correction assessment of HA mice after platelet-specific gene therapy under ADC-mediated preconditioning. At least 4 months after 2bF8LV gene therapy, the bleeding phenotype in 2bF8LV-transduced recipients was assessed by a 6-hour tail-bleeding test and a needle-induced knee joint injury. For the tail-bleeding test, the tail tip was transected using a 1.6-mm-diameter template. Animals were monitored hourly, and clot time was recorded. Blood samples were collected before and after the test for blood counts. Hemoglobin level before the test was normalized to 100%. For the knee joint injury, a G30 × 1/2 needle was used to induce injury in the right knee, leaving the left knee unharmed as an intra-animal control. The diameter of the knee joint was measured using a digital micro caliper before and 24 hours after injury. The diameter of the knee joint before injury was defined as 1. WT and FVIIInull mice served as controls. (A) Percentage of animals whose tail bleeding stopped within 6 hours after tail tip transection. The statistical differences between the groups were analyzed using Fisher’s exact test. (B) Tail-bleeding time. Data from the 2 2bF8LV-transduced recipients in the (CD45.2+CD117)-ADC group that did not have sustained platelet FVIII expression are presented as a separate group. The statistical differences between the groups were analyzed using 1-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparisons test. (C) Percentage of hemoglobin remaining after the tail-bleeding test. Data from the 2 2bF8LV-transduced recipients in the (CD45.2+CD117)-ADC group that did not have sustained platelet FVIII expression are presented as a separate group. The statistical differences between the groups were analyzed using 1-way ANOVA, followed by Tukey’s multiple-comparisons test. (D) Ratio of knee joint diameter measured at 24 hours postinjury/preinjury. The statistical differences between the groups were analyzed using 2-way ANOVA, followed by Tukey’s multiple-comparisons test. Representative images of the knee joint from WT control mice (E) and FVIIInull control mice (F) 24 hours after injury. (G) Representative images of the knee joint from 2bF8LV-transduced recipients with ADC preconditioning 24 hours after injury. These data demonstrate that effective phenotypic correction is attainable in HA mice after 2bF8 lentiviral gene delivery to HSCs under a nongenotoxic hematopoietic cell–targeted ADC preconditioning. **P < .01, ***P < .001, ****P < .0001. ns, not significant.

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