Figure 5.
Recovery from moderate FXN-mediated liver toxicity correlates with loss of transgene expression and liver regeneration
(A) Body weight curves obtained from WT untreated (n = 4), AAV9-CAG-FXN-injected (n = 8), and AAV9-CAG-FXN(N146K)-injected (n = 8) mice dosed at 1 × 1013 vg/kg. Necropsy was performed 8 weeks post-injection. All data are mean SEM. (B) Vector genome copy (VGC) in liver samples, as measured by ddPCR in WT untreated (n = 4), treated with AAV9-CAG-FXN at 1 × 1013 vg/kg, 5 weeks (n = 10) or 8 weeks (n = 8) post-injection (wpi), or with AAV9-CAG-FXN(N146K) at 1 × 1013 vg/kg, 8 wpi (n = 8). (C) mRNA expression of endogenous mouse Fxn and human FXN transgene in liver samples, as measured by ddPCR in WT untreated (n = 4), treated with AAV9-CAG-FXN at 1 × 1013 vg/kg, 5 weeks (n = 10) or 8 weeks (n = 8) post-injection (wpi), or with AAV9-CAG-FXN(N146K) at 1 × 1013 vg/kg, 8 wpi (n = 8). (D) Detection of human FXN protein by immunohistochemistry in liver section from WT mice dosed with AAV9-CAG-FXN or AAV9-CAG-FXN(N146K). Necropsy was performed 5 or 8 weeks post-injection (wpi). Scale bars, 300 μm. (E) Western blot analysis of mitochondrial and Fe-S client proteins from mouse liver extracts. Necropsy was performed 5 or 8 weeks post-injection (wpi). Vinculin (VINC) was used as a loading control. (F) Quantification of percentage of phospho-histone H3 (pHH3)-positive cells in liver sections of WT untreated (n = 7), dosed with AAV9-CAG-FXN at 1 × 1013 vg/kg, 5 weeks (n = 9) or 8 weeks (n = 7) post-injection (wpi), or dosed with AAV9-CAG-FXN(N146K) at 1 × 1013 vg/kg, 8 wpi (n = 7). (G) Representative image of pHH3 IHC (upper panel) and FXN IHC (lower panel) on consecutive liver sections of WT mouse dosed with AAV9-CAG-FXN at 1 × 1013 vg/kg. Arrows indicate corresponding pHH3-positive nuclei. Scale bars, 200 μm.