1. Volume 23, Issue 5, Pages 824-834 (May 2015)
Adenoassociated Virus Serotype 9-Mediated Gene Therapy for X-Linked Adrenoleukodystrophy 
Yi Gong, Dakai Mu, Shilpa Prabhakar, Ann Moser, Patricia Musolino, JiaQian Ren, Xandra O Breakefield, Casey A Maguire, Florian S Eichler 
Molecular Therapy 
Volume 23, Issue 5, Pages (May 2015)
DOI: /mt
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. 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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. 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 <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. 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 <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. 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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. 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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

7. 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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

8. 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 <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions