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Figure S1. AAV8-ApoEe/hAAT-hF9 bio-distribution and liver specific expression of hFIX protein. Vector was given by tail vein injection at 1 × 1011 vg/mouse to haemophilia B mice (C3H/HeJ F9−/−). (A) AAV8-ApoEe/hAAT-hF9 bio-distribution performed on genomic DNA isolated from liver, kidney, lung and spleen 3 weeks following IV administration in C3H/HeJ F9−/− mice (n = 3). (B) hF9 mRNA expression determined by RT-qPCR performed on total RNA isolated from liver, kidney, lung and spleen 3 weeks following IV administration in C3H/HeJ F9−/− mice (n = 3). GAPDH copy numbers in black (left axis) and hF9 copy numbers in grey (right axis). A no reverse transcriptase control was included for liver RNA samples (n = 3). (C) Representative liver section stained for hFIX (red) and F4/80 Kupffer cells and macrophages (green). (D) Representative spleen section stained for hFIX (red) and F4/80 macrophages (green).

Figure S2. Recovery of Treg following two doses of DT. (A) Gating scheme of mouse PBMC using CD3-PerCP-CY5.5 and CD4-e450 gates to select CD4 + T cells. (B) Representative dot plots used to calculate %CD25 + FoxP3 + Treg for control untreated DTR-FoxP3 mice (left column) and DT treated DTR-FoxP3 mice (right column) with day 0 as the first DT injection.

Figure S3. Residual hF.IX ASC detected in a ‘late DTmouse with delayed Treg reconstitution. (A) Flow cytometry staining of peripheral blood 14 days following late DT treatment. (B) hF.IX B-cell ELISpot on splenocytes from late DT treated mice isolated 29 days post-DT treatment.

Figure S4. Requirement for professional antigen presenting cells (APCs) for transgene product-specific CD4+ T-cell proliferation. AAV8-EF1α-ova vector was administered by tail vein injection to BALB/c mice at 1 × 1011 vg/mouse. (A) Proliferation of transferred T cells in spleens and livers of BALB/c mice. Left panel: control animal. Right panel: animal, in which Kupffer cells and M1 macrophages were inactivated using gadolinium chloride (GdCl3). (B) Proliferation of transferred T cells in spleens and livers of conditional CD11c deficient mice (CD11-DTR BALB/c mice). Left panel: control animal. Right panel: animal, in which CD11c+ DCs cells were depleted following administration of diphtheria toxin (DT). Shown are representative examples for n = 3 per experimental group. Percent cells that have or have not proliferated are indicated. No proliferation was observed in BALB/c mice that had not received AAV-ova vector (data not shown).

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