Figure 6.
Immune responses in HA mice after platelet-specific gene therapy with hematopoietic cell–targeted ADC preconditioning. For Treg analysis, blood samples were collected from 2bF8LV-transduced recipients, and CD4+Foxp3+ Tregs were analyzed by flow cytometry. For immune response studies, ≥24 weeks after HSCT, recipients were immunized with rhF8 by IV infusion weekly for 4 weeks. One week after the last immunization, plasmas were collected, and inhibitor titers were determined by Bethesda assay. (A) Percentage of total Tregs in 2bF8LV-transduced recipients at 11 weeks after gene therapy. The statistical differences between the groups of transduced recipients and CD45.1/FVIIInull controls were analyzed using 1-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparisons test. (B) Percentage of donor-derived Tregs in 2bF8LV-transduced recipients at 11 weeks after gene therapy. The statistical differences between the groups of transduced recipients and CD45.1/FVIIInull controls were analyzed using 1-way ANOVA, followed by Dunnett’s multiple-comparisons test. (C) The anti-FVIII inhibitor titers in 2bF8LV-transduced recipients after challenge with rhF8 at a dose of 100 U/kg per week for 4 weeks. 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 2-way ANOVA, followed by Tukey’s multiple-comparisons test. (D) The anti-FVIII inhibitor titers in 2bF8LV-transduced recipients after challenge with rhF8 at a dose of 200 U/kg per week for 4 weeks. The statistical differences between the groups were analyzed using the Mann-Whitney U test. (E) The incidence of inhibitor development in 2bF8LV-transduced recipients after challenge with rhF8 at a dose of 200 U/kg per week for 4 weeks. The statistical difference between the groups was analyzed using Fisher’s exact test. These data demonstrate that 2bF8 lentiviral gene delivery to HSCs under ADC-mediated preconditioning can suppress anti-FVIII immune responses. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Immune responses in HA mice after platelet-specific gene therapy with hematopoietic cell–targeted ADC preconditioning. For Treg analysis, blood samples were collected from 2bF8LV-transduced recipients, and CD4+Foxp3+ Tregs were analyzed by flow cytometry. For immune response studies, ≥24 weeks after HSCT, recipients were immunized with rhF8 by IV infusion weekly for 4 weeks. One week after the last immunization, plasmas were collected, and inhibitor titers were determined by Bethesda assay. (A) Percentage of total Tregs in 2bF8LV-transduced recipients at 11 weeks after gene therapy. The statistical differences between the groups of transduced recipients and CD45.1/FVIIInull controls were analyzed using 1-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparisons test. (B) Percentage of donor-derived Tregs in 2bF8LV-transduced recipients at 11 weeks after gene therapy. The statistical differences between the groups of transduced recipients and CD45.1/FVIIInull controls were analyzed using 1-way ANOVA, followed by Dunnett’s multiple-comparisons test. (C) The anti-FVIII inhibitor titers in 2bF8LV-transduced recipients after challenge with rhF8 at a dose of 100 U/kg per week for 4 weeks. 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 2-way ANOVA, followed by Tukey’s multiple-comparisons test. (D) The anti-FVIII inhibitor titers in 2bF8LV-transduced recipients after challenge with rhF8 at a dose of 200 U/kg per week for 4 weeks. The statistical differences between the groups were analyzed using the Mann-Whitney U test. (E) The incidence of inhibitor development in 2bF8LV-transduced recipients after challenge with rhF8 at a dose of 200 U/kg per week for 4 weeks. The statistical difference between the groups was analyzed using Fisher’s exact test. These data demonstrate that 2bF8 lentiviral gene delivery to HSCs under ADC-mediated preconditioning can suppress anti-FVIII immune responses. *P < .05, **P < .01, ***P < .001, ****P < .0001.

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