See accompanying article 10.1002/emmm.201302857
Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies
Article first published online: 16 SEP 2013
Copyright © 2013 EMBO Molecular Medicine
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
EMBO Molecular Medicine
Volume 5, Issue 11, pages 1698–1709, November 2013
How to Cite
Markusic, D. M., Hoffman, B. E., Perrin, G. Q., Nayak, S., Wang, X., LoDuca, P. A., High, K. A. and Herzog, R. W. (2013), Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies. EMBO Mol Med, 5: 1698–1709. doi: 10.1002/emmm.201302859
- Issue published online: 4 NOV 2013
- Article first published online: 16 SEP 2013
- Manuscript Accepted: 19 AUG 2013
- Manuscript Revised: 15 AUG 2013
- Manuscript Received: 8 APR 2013
- National Institutes of Health. Grant Numbers: P01 HD078810 (KAH and RWH), R01 AI051390 (RWH), F32 HL096281 (DMM)
- Howard Hughes Medical Institute (KAH)
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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 DT’ mouse 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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