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. 2015 May;23(5):824-834.
doi: 10.1038/mt.2015.6. Epub 2015 Jan 16.

Adenoassociated virus serotype 9-mediated gene therapy for x-linked adrenoleukodystrophy

Yi Gong  1 , Dakai Mu  1 , Shilpa Prabhakar  1 , Ann Moser  2 , Patricia Musolino  1 , JiaQian Ren  1 , Xandra O Breakefield  1 , Casey A Maguire  1 , Florian S Eichler  3
Affiliations

Adenoassociated virus serotype 9-mediated gene therapy for x-linked adrenoleukodystrophy

Yi Gong et al. Mol Ther. 2015 May.

Abstract

X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1-/- mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1-/- mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.

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Figures

Figure 1
Figure 1
rAAV9-mediated ABCD1 expression in mixed brain glial cell culture from Abcd1−/− mice. (a) Immunofluorescence staining of hABCD1 (red) in brain glial cell culture (from Abcd1−/− mouse) after 2.5 × 105 and 5 × 105 gc/cell rAAV9-ABCD1 transduction in vitro. (b) Western blot showing hABCD1 protein expression in mixed brain glial cell culture (from Abcd1−/− mice) after 1.25 × 105 gc/cell rAAV9-ABCD1 transduction in vitro. (c) Confocal imaging of hABCD1 (red) and catalase (green) staining in mixed brain glial cell culture (from Abcd1−/− mice) after 5 × 105 gc/cell rAAV9-ABCD1 transduction.
Figure 2
Figure 2
rAAV-ABCD1 reduces VLCFA level in mixed brain glial cell culture from Abcd1−/− mice. (a) C26:0LPC level, (b) C26/C22LPC ratio, and (c) C24/C22LPC ratio of mixed brain glial cell culture after different doses rAAV9-ABCD1 transduction. (d) Survival rate measurement after different doses rAAV9-ABCD1 and rAAV9-GFP transduction. (e) Morphologyy of mixed brain cell cultures from (Abcd1−/− mice) after different doses of rAAV9-ABCD1 transduction. Data were expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
rAAV-ABCD1 reduces VLCFA level in human X-ALD fibroblasts. (a) Western blot showing hABCD1 protein expression in normal and X-ALD patient fibroblast after 5 × 105 gc/cell rAAV2-ABCD1 transduction in vitro. PBS or rAAV2-firefly luciferase (Fluc) treated cultures served as control. (b) Immunofluoresence staining of hABCD1 (red) in X-ALD patient fibroblast after 5 × 103, 5 × 104, and 5 × 105 gc/cell rAAV2-ABCD1 transduction. (c) C26:0LPC level, (d) C26/C22 LPC ratio, and (e) C24/C22LPC ratio of X-ALD fibroblast after different doses of rAAV2-ABCD1 transduction in vitro. Data were expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4
rAAV9-mediated hABCD1 expression in Abcd1−/− mouse brain and spinal cord via both intracerebroventricular (ICV) and intravascular (IV) injection. (a) mRNA expression of Abcd1 in spinal cord tissue from both WT and Abcd1−/− mice. (b) hABCD1 expression in mouse brain, spinal cord and liver after intracerebroventricular (ICV) and intravascular (IV) delivery of rAAV9-ABCD1 into Abcd1−/− mice. (c,e) Confocal imaging showing hABCD1 expression (red) through the whole brain after (c) 1 × 1011 gc rAAV9-ABCD1 ICV injection and (e) 1 × 1012 gc rAAV9-ABCD1 IV injection. Some major structures were marked out as corpus callosum (CC), left ventricle (LV), third ventricle (V3), striatum, cerebral cortex, hippocampus, thalamus and cerebellum. (d,f) Confocal imaging showing hABCD1 expression (red) through the spinal cord after (d) 1 × 1011 gc rAAV9-ABCD1 ICV injection and (f) 1 × 1012 gc rAAV9-ABCD1 IV injection. (g) Western blot shows hABCD1 expression in different organs after 1 × 1012 gc rAAV9-ABCD1 IV injection (n = 12 for ICV and n = 6 for IV). Due to the high expression levels in peripheral organs, the brain and spinal cord ABCD1 bands are underexposed in contrast to b.
Figure 5
Figure 5
Colocalization of hABCD1 in different CNS cell types after intracerebroventricular (ICV) delivery. Upper brain: (a–j) Confocal imaging showing GFAP (green) and hABCD1 (red) staining in astrocytes at both (a) low and (f) high magnification; IBA1 (green) and hABCD1 (red) in microglia at both (b) low and (g) high magnification; Olig2 (green) and hABCD1 (red) in oligodendrocyte lineage at both (c) low and (h) high magnification; von Willebrand (green) and ABCD1 (red) in endothelial cells at both (d) low and (i) high magnification; ABCD1 (red) in neurons at (e) low and (j) high magnification following 1 × 1011 gc ICV injection into Abcd1−/− mice. Below-spinal cord: (k–r) Confocal imaging showing GFAP (green) and hABCD1 (red) staining in spinal cord astrocytes at both (k) low and (o) high magnification; IBA1 (green) and hABCD1 (red) in spinal cord microglia at both (l) low and (p) high magnification; Olig2 (green) and hABCD1 (red) in spinal cord oligodendrocyte lineage at both (m) low and (q) high magnification; von Willebrand (green) and ABCD1 (red) in brain vessel at both (n) low and (r) high magnification; (p) hABCD1 (red) in neuron indicated with white long arrow, following 1 × 1011 gc ICV injection into Abcd1−/− mice.
Figure 6
Figure 6
Colocalization of hABCD1 in different CNS cell types after intravascular (IV) delivery. Upper brain: (a–j) Confocal imaging showing GFAP (green) and hABCD1 (red) in astrocytes at both (a) low and (f) high magnification; IBA1 (green) and hABCD1 (red) in microglia at both (b) low and (g) high magnification; Olig2 (green) and hABCD1 (red) in oligodendrocyte lineage at both (c) low and (h) high magnification; von Willebrand (green) and ABCD1 (red) in endothelial cells at both (d) low and (i) high magnification; hABCD1 (red) in neurons at (e) low and (j) high magnification, following 1 × 1012 gc IV injection into Abcd1−/− mice. Below-spinal cord: (k-t) Confocal imaging showing GFAP (green) and hABCD1 (red) staining in spinal cord astrocytes at both (k) low and (p) high magnification; IBA1 (green) and hABCD1 (red) in spinal cord microglia at both (l) low and (q) high magnification; Olig2 (green) and hABCD1 (red) in spinal cord oligodendrocyte lineage at both (m) low and (r) high magnification; von Willebrand (green) and hABCD1 (red) in spinal cord vessel at both (n) low and (s) high magnification; hABCD1 (red) in neurons at (o) low and (t) high magnification, following 1 × 1012 gc IV injection into Abcd1−/− mice.
Figure 7
Figure 7
Effect of rAAV9-ABCD1 on VLCFA level and GPX-1 expression in CNS tissue. (a) C26/C22LPC ratio, (b) C26:0 fatty acid percentage in total fatty acid of brain and spinal cord tissue as well as (c) C26:0LPC level in plasma after 1 × 1011 gc ICV and 1 × 1012 gc IV injection with rAAV9-ABCD1. (d) Representative western blot imaging of GPX-1 expression in mouse spinal cord. (e) Quantification of protein expression by densitometry after normalization to β-actin by using Image J. Reductions in GPX-1 did not achieve statistical significance. Data were expressed as means ± SEM. *P < 0.05, ***P < 0.001.
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