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. 2013 Jan;21(1):18-30.
doi: 10.1038/mt.2012.200. Epub 2012 Sep 25.

Improved Survival and Reduced Phenotypic Severity Following AAV9/MECP2 Gene Transfer to Neonatal and Juvenile Male Mecp2 Knockout Mice

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Free PMC article

Improved Survival and Reduced Phenotypic Severity Following AAV9/MECP2 Gene Transfer to Neonatal and Juvenile Male Mecp2 Knockout Mice

Kamal K E Gadalla et al. Mol Ther. .
Free PMC article

Abstract

Typical Rett syndrome (RTT) is a pediatric disorder caused by loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. The demonstrated reversibility of RTT-like phenotypes in mice suggests that MECP2 gene replacement is a potential therapeutic option in patients. We report improvements in survival and phenotypic severity in Mecp2-null male mice after neonatal intracranial delivery of a single-stranded (ss) AAV9/chicken β-actin (CBA)-MECP2 vector. Median survival was 16.6 weeks for MECP2-treated versus 9.3 weeks for green fluorescent protein (GFP)-treated mice. ssAAV9/CBA-MECP2-treated mice also showed significant improvement in the phenotype severity score, in locomotor function, and in exploratory activity, as well as a normalization of neuronal nuclear volume in transduced cells. Wild-type (WT) mice receiving neonatal injections of the same ssAAV9/CBA-MECP2 vector did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. To test a MECP2 gene replacement approach in a manner more relevant for human translation, a self-complementary (sc) adeno-associated virus (AAV) vector designed to drive MeCP2 expression from a fragment of the Mecp2 promoter was injected intravenously (IV) into juvenile (4-5 weeks old) Mecp2-null mice. While the brain transduction efficiency in juvenile mice was low (~2-4% of neurons), modest improvements in survival were still observed. These results support the concept of MECP2 gene therapy for RTT.

Figures

Figure 1
Figure 1
Widespread expression of exogenous MeCP2 across the brain following neonatal delivery via an AAV2/9 vector. (a) MECP2_e1/Myc fusion and GFP (control) constructs were cloned into AAV2 backbones under a CBA promoter. (b) Experimental plan in which male wild-type (WT) or Mecp2−/y mice were injected intracranially with AAV9/CBA-MECP2 at P0-2, scored for the progression of RTT-like signs from 3 weeks onwards and phenotyped quantitatively at weeks 8–10. (c) Representative micrograph (whole brain, parasagittal section) showing the distribution of Myc-tagged MeCP2 expression in a 12-week-old WT mouse following bilateral injection. White dashed arrow indicates approximate injection site. Insets are higher power micrographs corresponding to the box in the large image showing proportion of Myc +ve (transduced cells) relative to the NeuN-immunolabeled cell population. (d) Quantification of transduction efficiency (as a proportion of DAPI-stained nuclei) revealed widespread, mainly neuronal, distribution across a range of CNS regions. (e) Micrograph showing transduced and non-transduced cells in hippocampal area CA3 in a WT male mouse. At a cellular level, exogenous MeCP2 (revealed by Myc immunoreactivity) was localized to neuronal nuclei with a punctate distribution characteristic of proteins that colocalize with heterochromatin. (f) Injection of AAV9/MECP2 into WT mice led to augmentation of MeCP2 levels (native + transgenic) in transduced cells and enabled quantification of relative transgenic MeCP2 abundance. Blue bars show native MeCP2 levels in non-transduced cells and red bars show levels of native + transgenic MeCP2 in transduced cells. Transduced cells exhibited mean levels of anti-MeCP2 immunofluorescence that were 105–124% higher than mean basal levels in untransduced nuclei. (g) Distribution of MeCP2 level (immunofluorescence intensity) in transduced and non-transduced layer of V pyramidal cells (primary motor cortex) from four injected brains. Analysis revealed native MeCP2 levels to be tightly regulated (narrow peak) while levels in transduced cells showed a broader and positively shifted distribution. Bars: c, top, 1 mm; c, bottom, 100 µm. Error bars show mean ± SEM. AAV, adeno-associated virus; AU, arbitrary unit; CBA, chicken β-actin; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; ITR, inverted terminal repeat; NeuN, “neuronal nuclei” antigen (Rbfox3); polyA, SV40 polyadenylation signal; RTT, Rett syndrome.
Figure 2
Figure 2
Enhanced survival and reduced RTT-like signs following neonatal delivery of ssAAV9/CBA-MECP2. (a) Survival plot showing extended lifespan of Mecp2−/y mice injected with AAV9/CBA-MEPC2 (open circles, n = 14) compared with AAV9/GFP-injected controls (open squares, n = 11). The median survival period was significantly increased from 9.3 to 16.6 weeks (P < 0.0001, Gehan-Breslow-Wilcoxon test). The plot also shows absence of lethality associated with overexpression of MeCP2 in AAV9/MECP2-treated wild-type mice (closed circles, n = 12). Closed squares show data from AAV9/GFP-treated wild-type mice (n = 12). (b) Plot showing aggregate phenotype severity score in the mice used for survival analysis in a. The rate of phenotype progression in AAV9/MECP2-treated null mice was reduced compared with the aggressive phenotype trajectory seen in AAV9/GFP control-injected mice (P < 0.05, repeated measures ANOVA between weeks 3–12). In contrast, wild-type mice injected with AAV9/MECP2 and AAV9/GFP showed no difference between groups and no change in score over the study. (c) Plot showing body weight changes over time in the same mice. There was a genotype effect (repeated-measures ANOVA, P < 0.05) but no significant treatment effect. Video camera symbols indicate time points at which movies were made (see Supplementary Videos S1–S3). #Indicates two AAV9/MECP2-treated Mecp2-null mice that survived to the end of the 30-week study (see Supplementary Figure S2). Danger symbol indicates that in the null untreated group, insufficient mice were still alive to plot the mean score after 12 weeks. AAV, adeno-associated virus; ANOVA, analysis of variance; CBA, chicken β-actin; GFP, green fluorescent protein; RTT, Rett syndrome.
Figure 3
Figure 3
Improved motor but not respiratory phenotypes following neonatal delivery of MECP2. (a) Proportion of mice able to perform at two different speeds on a motorized treadmill. There was a significant overall difference in performance between groups (10 cm/second, P = 0.0013; 25 cm/second, P < 0.0001; Fisher's exact test). Post hoc pairwise comparison of AAV9/MECP2- and AAV9/GFP-treated mice showed a difference at 25 cm/second (P = 0.024), but not at 10 cm/second. (be) Open field tests performed at 9 weeks following AAV9 injection showing measures for (b) movement duration, (c) mean velocity, and (d) total distance moved (e) together with rearing frequency. (f) Representative whole-body plethysmograph traces showing regular and erratic breathing patterns/apneas (arrows) in WT and Mecp2−/y mice, respectively. (g) Apnea frequency, (h) baseline breathing frequency, (i) breathing frequency variability. Both open field and respiratory measures were assessed by two-way ANOVA with Tukey's post hoc comparisons. *P < 0.05, **P < 0.01, ns = not significant. Number of animals per genotype/treatment group are shown within each bar. AAV, adeno-associated virus; ANOVA, analysis of variance; CV, coefficient of variation; GFP, green fluorescent protein; WT, wild-type.
Figure 4
Figure 4
Increased nuclear volume in Mecp2−/y and wild-type mice after AAV9/CBA-MECP2 injection. (a,b) Representative image from the dentate gyrus of (a) wild-type and (b) Mecp2-null mouse at 12 weeks following AAV9/MECP2 injection. Sections show DAPI-stained nuclei within the stratum granulosum and transfected cells identified by anti-Myc immunostaining (arrows). (c) Nuclear volume measurements from 3D-reconstructed DAPI-labeled nuclei in wild-type mice (n = 4). (d) Nuclear volume measurements from WT (n = 122 datapoints in total from four mice) non-transfected cells and transfected and non-transfected cells from age-matched Mecp2-null mice (n = 122 datapoints in total from from three mice). All data were normalized to the mean volume in untransfected wild-type cells. Bar plots show mean ± SEM with gray dots showing individual datapoints. Statistical analysis was carried out by two-way ANOVA and Tukey's post hoc pairwise comparison. ***P < 0.001. Bar = 10 µm. AAV, adeno-associated virus; ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant; WT, wild-type.
Figure 5
Figure 5
Enhanced survival following intravenous delivery of scAAV9/MeP-MECP2 virus to juvenile mice. (a) A MECP2_e1/Myc fusion construct was cloned into a scAAV2 ITR backbone under a truncated version of the murine Mecp2 promoter. (b) Graph showing transduction efficiency in three brain areas of Mecp2-null mice (n = 3) at endpoint following tail vein injection of scAAV9/MeP-MECP2. See Supplementary Figure S3 for representative images. Plots show mean ± SEM. (c) Survival plot showing extended survival of Mecp2−/y mice injected with scAAV9/MeP-MEPC2 (purple open squares, n = 12) compared with untreated controls (green closed squares, n = 10). The median survival period in the treated mice was significantly higher than in the untreated group (15.4 versus 10.6 weeks, respectively, P < 0.01, Gehan-Breslow-Wilcoxon test). Control WT mice showed no mortality over the experimental period (closed circles, n = 8). Arrow indicates scAAV/MeP-MECP2 injection time. (d) Micrographs showing GABA immunolabeling of transduced (MeCP2-positive, arrows) and non-transduced cells (arrowheads) in layer 2/3 of motor cortex of a Mecp2-null mouse. (e) Bar plot showing elevated levels of anti-GABA immunofluorescence in transduced cells (***P < 0.001, t-test, n = 166 cells). Bar in d = 10 µm. AAV, adeno-associated virus; BGHpolyA, bovine growth hormone polyadenylation signal; GABA, γ-aminobutyric acid; ITR, inverted terminal repeat; mut ITR, mutated inverted terminal repeat; MeP, truncated murine Mecp2 promoter; WT, wild-type.
Figure 6
Figure 6
Vector biodistribution and gene expression after intravenous injection. WT C57BL/6 mice were injected intravenously at 4–5 weeks old with vehicle, 1 × 1011 vg scAAV/MeP-GFP (+GFP), or 1 × 1011 vg scAAV/MeP-MECP2 (+MECP2), then killed at 1 or 3 weeks post-injection. (a) qPCR biodistribution of GFP or MECP2 vector genomes to major organs. (b) RT-qPCR of total MECP2 (mouse + human) relative to actin. Mean ± SEM are plotted, n = 2–4, with the exception of the +GFP mice at 3 weeks (n = 1). #Not determined. AAV, adeno-associated virus; GFP, green fluorescent protein; RT-qPCR, reverse transcription-quantitative PCR; vg, vector genome; WT, wild-type.

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