Fig. 6 | Nature Communications

Fig. 6

From: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

Fig. 6

Antigen-specificity of SVP[Rapa] treatment and role of regulatory T cells. a Protocol outline. Male C57BL/6 mice (n = 5) were first injected i.v. with 4 × 1012 vg kg−1 of AAV8-luciferase vector together with SVP[Rapa] or with SVP[empty] control. Animals were then challenged i.v. on day 21 with 4 × 1012 vg kg−1 of AAV8-hF.IX vector or AAV5-hF.IX vector or injected s.c. with hF.IX in complete Freund’s adjuvant (cFA). b Anti-AAV8 IgG in animals primed with AAV8-luc and challenged with AAV8-hF.IX. c Anti-AAV5 IgG in animals primed with AAV8-luc and challenged with AAV5-hF.IX, and d anti-hF.IX IgG antibodies determined by ELISA in animals primed with AAV8-luc and challenged with hF.IX protein in cFA. e Protocol outline. Donor mice (n = 5) were left untreated or treated i.v. with 4 × 1011 vg kg−1 of AAV8-luciferase vector together with SVP[Rapa] or SVP[empty] control. 14 days later, spleens were harvested and 3 × 107 splenocytes were adoptively transferred into recipient mice (n = 5). All animals were then treated one day later with 4 × 1011 vg kg−1 of AAV8-hF.IX vector. f Anti-AAV8 IgG antibodies measured 14 days post AAV8-hF.IX vector injection and measured by ELISA. g Protocol outline. Male C57BL/6 mice (n = 5 per group) were treated i.v. with 4 × 1012 vg kg−1 of AAV8-luciferase vector together with SVP[Rapa] or SVP[empty] control on day 0, followed by Tregs depletion (anti-CD25, clone PC61, 200 µg per mouse) on days 19 and 20. All animals were then challenged with 4 × 1012 vg kg−1 of AAV8-hF.IX vector alone on day 21. h Analysis anti-AAV8 IgG antibodies measured by ELISA. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by two-way ANOVA with Tukey post hoc test in bd, by one-way ANOVA with Tukey post hoc test in f, and by non-parametric unpaired, two-tail t-test in h (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

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