FIGURES AND TABLES
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Central Nervous System-directed AAV2/5-Mediated Gene Therapy Synergizes with Bone Marrow Transplantation in the Murine Model of Globoid-cell Leukodystrophy
Darshong Lin, Anthony Donsante, Shannon Macauley, Beth Levy, Carole Vogler and Mark S Sands
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Figure 1.
Donor hematopoietic chimerism. Partial donor chimerism (25–35% GFP+ cells) was observed in the bone marrow of normal (WT), BMT-treated (BMT), or combination-treated (CmTx) Twitcher mice 40 days following myeloreductive conditioning (400 rad) and injection of 106 unfractionated GFP+ bone marrow cells. The means and 1 SD are shown.
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Figure 2.
GALC activity. GALC activity, expressed as a percent normal (% normal), was measured in the brain, liver, and kidney of Twitcher mice following individual or combined therapies. The normal GALC levels were determined by the mean activity in each tissue measured in three normal animals at 38 days of age. Untreated Twitcher mice (Twi, 38 days of age, n=3) had 2.2, 0, and 0.9% GALC activity in the brain, liver, and kidney, respectively. Twitcher animals receiving BMT alone (BMT, 34–50 days of age, n=5) had GALC levels in the brain that were not significantly different from untreated Twitcher animals. However, BMT-treated animals had 6.5 and 0.6% normal levels in the liver and kidney, respectively. Intracranial injections of AAV2/5 alone (AAV2/5, 34 days of age, n=3) resulted in 217, 4.8, and 1.0% normal GALC levels in the brain, liver, and kidney, respectively. The combination-treated twitcher mice (AAV/BMT, 34 days of age, n=3) had 380, 29, and 2.6% normal GALC levels in the brain, liver, and kidney, respectively. The bars represent the mean
1SEM.
Figure 3.
GALC localization. GALC-positive (blue) cells were observed in the cerebral cortex, hippocampus, lateral ventricle, and thalamus of untreated normal mice. Numerous intensely staining GALC-positive cells were detected in the same structures from combination-treated (CmTx) and AAV2/5-treated (AAV) Twitcher mice. However, very few GALC-positive cells were observed in similar regions of either BMT-treated (BMT) or untreated (Twi) Twitcher brains.
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Figure 4.
Astrocytosis and microglial activation. Widespread macrophage/microglial activation was observed by (a) CD11b staining in the striatum and cerebellum of untreated Twitcher (Twi) mice. Similarly, astrocytosis in the cortex and striatum of untreated Twitcher mice was detected by intense immunostaining for GFAP (b). The BMT-treated (BMT) Twitcher mice had a slight decrease in CD11b and GFAP staining in the striatum and cerebellum. The AAV2/5-treated (AAV) Twitcher mice have decreased GFAP and CD11b staining in the cortex and striatum near the injection sites. However, there is no appreciable decrease in CD11b staining in the cerebellum of AAV2/5-treated animals (a, cerebellum, AAV). The decrease in both CD11b and GFAP staining in all the brain areas examined from the combination-treated (CmTx) Twitcher mice was more dramatic than either AAV2/5 or BMT alone. There was little or no CD11b or GFAP staining in the brains of normal control animals (WT).
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Figure 5.
Myelination and globoid cells. Increased numbers of PAS-positive cells (arrows, 11 cells in this panel) are present in and around the corpus callosum in untreated (Twi) Twitcher mice compared to normal (WT) animals. Comparable numbers of PAS-positive cells were observed in the corpus callosum of Twitcher animals receiving BMT alone (data not shown). There was a dramatic decrease in the number of PAS-positive cells in both the AAV2/5-treated (AAV, two positive cells) and the combination-treated (CmTx, 0 positive cells) animals. Comparable levels of myelin (luxol fast blue staining) were observed in the corpus callosum of normal and combination-treated animals. Decreased myelin staining was observed in both the untreated Twitcher and the AAV2/5-treated animals compared to normal animals.
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Figure 6.
Life span. The mean lifespans of untreated (Twi, bold solid/dotted line, n=14), BMT-treated (BMT, bold dotted line, n=15), AAV2/5-treated (AAV2/5, bold dashed line, n=15), and combination-treated (CmTx, bold solid line, n=17) Twitcher mice are 39, 44, 49, and 104 days, respectively. Fifty percent of the combination-treated animals were still alive at 111 days of age. No untreated normal (WT, fine solid line, n=12) or normal mice that received radiation alone (data not shown, n=4) died during the study.
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Figure 7.
Body weight. The body weights of all the groups of animals were indistinguishable up to 21 days of age. Beyond 28 days of age the body weights of untreated (filled triangles) and BMT-treated (open circles) Twitcher mice were identical and decreased precipitously. Beyond 35 days of age the mean body weight of the AAV2/5-treated group (open triangles) decreased rapidly. The mean body weight of the combination-treated Twitcher mice (filled diamonds) remained relatively constant for the duration of the study, albeit less than either the untreated (filled circles) or BMT-treated (open squares) normal mice.
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Figure 8.
Behavioral analysis. The combination-treated Twitcher mice (filled diamonds, n=17) performed significantly better than untreated (filled triangles, n=14), BMT-treated (open circles, n=15), or AAV2/5-treated (open triangles, n=15) Twitcher mice in the constant speed and accelerating rotarod paradigms. Both the BMT-treated and AAV2/5-treated Twitcher groups performed significantly better than the untreated Twitcher mice in both rotarod assays. There was no significant improvement in any of the treated Twitcher mice in the wire hang test when compared to untreated Twitcher mice. All the treated Twitcher mice had significant deficits compared to untreated normal mice (filled circles, n=12) in both rotarod paradigms and in the wire hang assay.
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