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Correspondingly, each vector dose group had a statistically significant difference in transgene activity from all other vector dose groups (1-way ANOVA and Bonferroni corrected pair-wise t-tests, p < 0.05).
Additionally, at each of the vector dose groups, β-galactosidase activity levels were also measured over different time points and we found that overall there was no significant association between time points and transgene activity levels (2-way ANOVA; dose p < 0.05; time p > 0.05; interaction p > 0.05).
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At week 3 the extrahepatic concentration of vector in the higher vector dose group was highest in the heart (118-fold lower than in liver).
All mice in the intermediate vector dose group tolerated the challenge with no rise in inhibitors and no indication of anaphylaxis.
One animal in the lower vector dose group, having no prior signs of toxicity, died on study at day 67 and postmortem examination revealed only chronic changes of mild myocardial fibrosis that were not believed to be large enough to be the direct cause of death.
On examining the groups exposed to the higher vector dose (see Supplementary Table S2 and Supplementary Fig. S4), on day 3 the organ with the next highest tissue concentration relative to the liver was the spleen (27-fold lower vector copies; i.e., 4.29×10 copies in the liver and 1.58×10 copies in the spleen).
Four groups of 5-week-old KO-Sgca mice (five mice per group, except four mice for the highest vector dose) were injected either with PBS or with increasing doses of rAAV [1e11, 5e11 and 1e12 viral genome (vg)].
To further characterize the vaccine-induced antibody response, we investigated the avidity and IgG subclasses of antibodies induced after both viral-vector and protein-in-adjuvant vaccinations (day 70 after viral-vector vaccination and day 62 after protein-in-adjuvant for the 2.5 μg dose groups).
In GFP and empty vector groups there were no behavioural or post-mortem changes at 3 or 6 weeks post-administration at either vector dose.
Transgene, timing of injection and vector dose are indicated in figures legends.
Co-administration of SVP[Rapa] with each dose of AAV vector appeared to be essential to completely block anti-AAV antibody responses, as repeated vector administration with no SVP[Rapa] resulted in antibody formation even when SVP[Rapa] was co-administered with the first vector dose (Supplementary Fig. 1).
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