Fig 1: 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.
Fig 2: Time- and dose-dependent correction of MCK-Fxn heart phenotype after AAV9-CAG-FXN treatment(A) Heart to body weight ratios at the time of necropsy: 57 days post-injection (dpi) for WT untreated (n = 15) and MCK-Fxn treated at 1 × 1013 vg/kg (n = 13), 14 dpi for MCK-Fxn saline group (n = 12), 26–57 dpi for MCK-Fxn treated at 3 × 1013 vg/kg (n = 11), and 20–26 dpi for MCK-Fxn treated at 1 × 1014 vg/kg (n = 12). (B) Vector genome copy (VGC) of AAV9-CAG-FXN in heart samples, as measured by ddPCR at the time of necropsy in WT untreated (n = 15), MCK-Fxn saline (n = 12), and MCK-Fxn treated at 1 × 1013 vg/kg (n = 12), 3 × 1013 vg/kg (n = 10), or 1 × 1014 vg/kg (n = 11). (C) mRNA expression of endogenous mouse Fxn and human FXN transgene in heart samples, as measured by ddPCR in WT untreated (n = 14), MCK-Fxn saline (n = 11), and MCK-Fxn treated at 1 × 1013 vg/kg (n = 13), 3 × 1013 vg/kg (n = 11), or 1 × 1014 vg/kg (n = 11). (D) Detection of Fxn transgene mRNA expression by in situ hybridization (ISH, upper panels) and human FXN protein by immunohistochemistry (IHC, lower panels) in heart sections. Scale bars, 300 µm. (E) Western blot analysis of mitochondrial and Fe-S client proteins from three mouse heart extracts from each group. Vinculin (VINC) was used as a loading control. (F) mRNA expression of cardiac dysfunction and FXN deficiency markers Nppa, Asns, Mthfd2, and Gdf15 as measured by ddPCR in WT untreated (n = 15), MCK-Fxn saline (n = 11), and MCK-Fxn treated at 1 × 1013 vg/kg (n = 13), 3 × 1013 vg/kg (n = 11), or 1 × 1014 vg/kg (n = 12). All data are mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: Transient correction of cardiac dysfunction in MCK-Fxn mice after AAV9-CAG-FXN administration(A) Upper, Experimental time course of AAV9-CAG-FXN injection and echocardiographic measurements. Lower, Body weight curves obtained from WT untreated (n = 15), MCK-Fxn saline injected (n = 12), MCK-Fxn treated at 1 × 1013 vg/kg (n = 13), 3 × 1013 vg/kg (n = 13), or 1 × 1014 vg/kg (n = 12). (B–D) Echocardiographic measurements of ejection fraction (EF) (B), fractional shortening (FS) (C), and left ventricular volume at diastole (LVVD) (D). Echocardiographic data for each group and time point are provided in Table S1. All data are mean ± SEM.
Fig 4: Toxicity associated with FXN overexpression requires FXN binding to the Fe-S cluster core complex(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 3 × 1013 vg/kg. Necropsy was performed 3 weeks post-injection. (B) Vector genome copy (VGC) in liver samples, as measured by ddPCR in WT treated with AAV9-CAG-FXN (n = 8) or AAV9-CAG-FXN(N146K) (n = 8) at 3 × 1013 vg/kg, 3 weeks post-injection. (C) mRNA expression of endogenous mouse Fxn and human FXN transgene in liver samples, as measured by ddPCR in WT treated with AAV9-CAG-FXN (n = 8) or AAV9-CAG-FXN(N146K) (n = 8) at 3 × 1013 vg/kg, 3 weeks post-injection. (D) Western blot analysis of mitochondrial and Fe-S client proteins from three mouse liver extracts, 3 weeks post-injection. Vinculin (VINC) was used as a loading control.
Fig 5: Mice requiring early euthanasia display liver pathology and Fe-S cluster deficiency(A) Vector genome copy (VGC) of AAV9-CAG-FXN in liver samples, as measured by ddPCR in WT untreated (n = 15), MCK-Fxn saline (n = 12), and MCK-Fxn treated at 1 × 1013 vg/kg (n = 13), 3 × 1013 vg/kg (n = 10), or 1 × 1014 vg/kg (n = 12). (B) mRNA expression of endogenous mouse Fxn and human FXN transgene in liver extracts, as measured by ddPCR in WT untreated (n = 15), MCK-Fxn saline (n = 12), and MCK-Fxn treated at 1 × 1013 vg/kg (n = 13), 3 × 1013 vg/kg (n = 9), or 1 × 1014 vg/kg (n = 12). (C) Detection of Fxn transgene mRNA expression by in situ hybridization (ISH, upper panels) and human FXN protein by immunohistochemistry (IHC, lower panels) in liver sections. Scale bars, 200 µm (ISH), 300 µm (IHC). (D) Measurements of liver-specific glutamate dehydrogenase (GLDH) from sera taken at time of necropsy: 57 days post-injection (dpi) for WT untreated (n = 15) and MCK-Fxn treated at 1 × 1013 vg/kg (n = 12), 14 dpi for MCK-Fxn saline group (n = 10), 26–57 dpi for MCK-Fxn treated at 3 × 1013 vg/kg (n = 11) and 20–26 dpi for MCK-Fxn treated at 1 × 1014 vg/kg (n = 13). All data are mean ± SEM. ****p < 0.0001. (E) Western blot analysis of mitochondrial and Fe-S client proteins from three mouse liver extracts from each group. Vinculin (VINC) was used as a loading control.
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