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. 2020 Mar 24;9:e52629. doi: 10.7554/eLife.52629

Figure 7. PHP.eB-iMecp2 gene transfer in Mecp2+/- females.

(a) Illustration of the experimental setting to restore the expression of Mecp2 in heterozygous (Het) animals by means of AAV-PHP.eB. (b) Mouse body weight was monitored every two weeks and represented as mean of each group. (c) General phenotypic assessment evaluated through aggregate severity score. (d) Spontaneous locomotor activity was tested in a spontaneous field arena and shown as quantification of travelled total distance. All groups of animals were tested for motor coordination using beam balance test and quantified as crossing time (e) and number of errors (f) (WT untreated [n = 8], Het treated with GFP 1×1011 vg/mouse [n = 6], Het treated with iMecp2 virus 1×1011 vg/mouse [n = 6]). (g) High magnification immunostaining for Mecp2 and V5 in cortex, striatum and cerebellum derived from WT, Mecp2+/- (Het) untreated and Het treated with PHP.eB-iMecp2 (1×1011 vg/mouse) brains. Nuclei were stained with DAPI (merge panels). (h) Bar graphs showing the fraction of V5 positive on the total DAPI positive in cortex and striatum (n = 3). (i) Western-blot analysis to quantify Mecp2 over Actin protein levels in cortex derived from WT, Het untreated and treated with iMecp2 (1×1011) animals and (j) corresponding densitometric quantification expressed in arbitrary units (right panel) (n = 3 for WT, n = 3 Het untreated; n = 3 Het treated with PHP.eB-iMecp2 1011 vg/mouse) *p<0.001 as compare to WT and Het treated with PHP.eB-iMecp2 (ANOVA-one way, Tukey’s post hoc test). Error bars, SD. **p<0.01. ANOVA-two way (b, c, e, f) or ANOVA-one way (d) with Tukey’s post hoc test.

Figure 7.

Figure 7—figure supplement 1. Characterization of iMecp2 transgene expression in brain and liver of Mecp2+/- animals.

Figure 7—figure supplement 1.

(a) Vector biodistribution (upper panel) and transgene expression (lower panel) in mice cortex and liver of wild-type female mice (WT female, n = 3), Het mice untreated (Het, n = 3) and treated with 1×1011 (n = 3) of iMecp2. Data were normalized over Mecp2 gene levels and expression of wild-type mice (WT male, n = 3), respectively. (b) Characterization of iMecp2-transduced cells in Het cortex (1×1011vg/mouse) using colocalization of V5+ cells with neurons (NeuN+) and astrocytes (Sox9). (c) Quantification of V5+ cells on total DAPI+ cells (orange bar), V5+ NeuN+ cells on total DAPI+ cells (green bar) and V5+ Sox9+ cells on total DAPI+ cells (blue bar). Dashed lines indicate the number of NeuN+ cells or Sox9+ cells on total DAPI + cells. Nuclei were stained with DAPI. All images were captured using confocal microscope. Scale bar: 50 μm. N = 3 animals per group.