Paper 5

original article © The American Society of Gene & Cell Therapy

X-linked adrenoleukodystrophy (X-ALD) is a devastat-ing 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 recombi-nant 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 injec-tion 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.

Received 29 September 2014; accepted 29 December 2014; advance online publication 10 February 2015. doi:10.1038/mt.2015.6

INTRODUCTIONX-linked adrenoleukodystrophy (X-ALD) is a progressive neu-rodegenerative disorder caused by mutations in the ABCD1 gene localized to Xq28.1 The ABCD1 gene consists of 10 exons span-ning 19 kb of genomic DNA and encodes a peroxisomal ATP-binding cassette (ABC) transporter responsible for transport of CoA-activated very long-chain fatty acids from the cytosol into the peroxisome for degradation.1 Biochemically, X-ALD is char-acterized by elevations of saturated straight chain very long-chain fatty acids (VLCFA: C24:0 and C26:0) and monounsaturated VLCFA (C26:1) in plasma and other tissue.2,3

The clinical manifestations of X-ALD are highly variable. 60% of the X-ALD patients develop adrenomyeloneuropathy (AMN) due to an axonal degeneration of the spinal cord, whereas 35–40% of X-ALD boys develop fatal cerebral ALD (CALD), a disorder characterized by progressive cerebral demyelination and inflammation in the white matter (WM) of the brain.4,5 Current therapeutic strategies for X-ALD encompass efforts to reduce VLCFA accumulation (Lorenzo’s oil) and compensate for the loss of gene function through hematopoietic stem cell correction. So far, hematopoetic stem cell transplantation (HSCT) is the only modality that is able to halt the progressive cerebral demyelin-ation.6 However, HSCT has several limitations. The donor search can be time consuming and thus delay treatment. In addition, the progression of CNS lesions is halted, at the earliest, 6–12 months after engraftment. Other limitations of HSCT include engraft-ment problems and graft-versus-host disease. The most common phenotype of X-ALD, AMN, currently has no treatment options available.

In vivo gene therapy offers the possibility of a one-time treat-ment for an inherited disease, with the prospect of a life-long beneficial effect.7 Recombinant adenoassociated virus (rAAV) vector-mediated gene therapy has shown great promise in sev-eral clinical trials for neurological disease with sustained trans-gene expression8–10 and functional response,11,12 as well as a safe profile. While recombinant AAV serotype 2 (rAAV2) is the most widely used in clinical trials, many other serotypes have shown an enhanced ability to transduce neurons in experimental studies.13–15 Recently, many AAV serotypes, including rAAV9, have been shown to bypass the blood–brain barrier when injected intrave-nously (IV) and efficiently target cells in the central nervous sys-tem (CNS).16–18 These cells include endothelial cells, neurons and astrocytes in the brain, and motor neurons and astrocytes in the spinal cord. The ability of AAV9 to target these CNS cells makes AAV9-mediated gene correction a plausible therapy for ALD/AMN, since long-tract CNS axons and their supporting glia are the site of pathology.

We believe an approach using rAAV-mediated delivery of ABCD1 to the CNS has desirable properties. First, rAAV vec-tors yield rapid and robust transgene expression in vivo, which may reduce the lag time to halt disease progression mentioned above. Second, IV delivery of rAAV9 vectors mediates widespread

10February2015

824

834

rAAV9-Mediated Gene Therapy for X-ALD

Molecular Therapy

10.1038/mt.2015.6

original article

00may2015

23

5

29September2014

29December2014

© The American Society of Gene & Cell Therapy

Correspondence: Florian Eichler, Department of Neurology, Massachusetts General Hospital, 55 Fruit Street, ACC 708, Boston, Massachusetts 02114, USA. E-mail: [email protected]

Adenoassociated Virus Serotype 9-Mediated Gene Therapy for X-Linked AdrenoleukodystrophyYi Gong1, Dakai Mu1, Shilpa Prabhakar1, Ann Moser2, Patricia Musolino1, JiaQian Ren1, Xandra O Breakefield1, Casey A Maguire1 and Florian S Eichler1

1Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts, USA; 2Peroxisome Disease Lab, Hugo W Moser Research Institute, Baltimore, Maryland, USA

824 www.moleculartherapy.org vol. 23 no. 5, 824–834 may 2015

http://www.nature.com/doifinder/10.1038/mt.2015.6

mailto:[email protected]

© The American Society of Gene & Cell TherapyrAAV9-Mediated Gene Therapy for X-ALD

delivery to cells (primarily neurons, endothelial cells, and astro-cytes) at the sites of ALD disease pathology in the brain and spinal cord.17–19 Given the progress in rAAV gene delivery to the CNS after IV delivery, we set out to test the hypothesis that ABCD1 gene delivery would correct cells within the brain and spinal cord and biochemically ameliorate the VLCFA accumulation in Abcd1−/− mice. We see these as the first proof of concept experi-ments relevant and translatable to humans with AMN.

RESULTSrAAV-mediated transduction of ABCD1 gene in mixed brain cell culture from Abcd1−/− miceIn order to verify the expression and correct cellular localization of ABCD1 delivered by rAAV, we first performed in vitro studies using a mixed culture of primary brain glial cells from Abcd1−/− mice. We first tested an AAV9 vector encoding GFP to transduce the brain glial cell culture. GFP was readily detectable 3 days after transduc-tion (Supplementary Figure S1). Next, we transduced these cells with AAV9-ABCD1. Our immunofluorescence staining showed a dose-dependent increase in human ABCD1 protein (hABCD1) expression after rAAV9-ABCD1 transduction (Figure 1a). Western blot confirmed the expression of hABCD1 protein in cultured mouse brain glial cells after 5 day transduction (Figure 1b). Using confocal imaging, we found that hABCD1 protein mainly localized within the cytoplasm and colocalized with catalase (Figure 1c), a peroxisomal enzyme, indicating localization of hABCD1 to peroxi-somes. In our culture system, astrocytes were the major transduced

cell type (84%), followed by oligodendrocytes (6%) and finally microglia (2%) (Supplementary Figure S2a–g).

rAAV-ABCD1 reduces VLCFA level in mixed brain cell cultures from Abcd1−/− miceAfter we confirmed the expression and correct localization of hABCD1 protein, we set out to verify the effect of hABCD1 on VLCFA accumulation in transduced cells, the biochemical hall-mark of X-ALD. To this end, we measured C22:0, C24:0, and C26:0 LPC levels in brain glial cells from WT and Abcd1−/− mice. As expected, primary cultured brain glial cells from Abcd1−/− mice had around a 2.5-fold increase in C26:0 LPC compared to WT mouse brain glial cells (P < 0.001). Transduction of rAAV9-ABCD1 reduced C26:0 LPC levels of Abcd1−/− brain glial cells in a dose-dependent manner, with 5 × 105 gc/cell leading to a two-fold reduction (P < 0.001) and 1 × 106 gc/cell leading to a three-fold reduction (P < 0.001). In our control group, rAAV9-GFP did not alter the C26:0 LPC level (Figure 2a). The C26/C22 LPC ratio (Figure 2b) and C24/C22 LPC ratio (Figure 2c) showed around four- and twofold increases respectively in Abcd1−/− brain glial cells compared to those of WT control. This was significantly reduced after rAAV9-ABCD1 treatment at doses of 5 × 105 gc/cell and 1 × 106 gc/cell (P < 0.05). These data suggest that ABCD1 gene delivered by rAAV9 can be correctly expressed and result in func-tional ABCD1 protein in cell culture. Furthermore, by assessing cell morphology and viability of the transduced cells, we found no cytotoxicity (Figure 2d,e).

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 show-ing 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.

PBS AAV9-ABCD1 2.5e5/cell

PBS

AAV9-ABCD1 5e5/cell

AAV9-ABCD1

hABCD1

10 µm

ABCD1CatalaseDAPI

β-actin

a

cb

Molecular Therapy vol. 23 no. 5 may 2015 825


AAV-ABCD1 reduces VLCFA level in human X-ALD fibroblastsSince our goal is to use rAAV for X-ALD gene therapy in humans, we set out to test delivery in human cells, specifically human fibroblasts. Not surprisingly, rAAV9-GFP transduced

these cells poorly in culture (Supplementary Figure S3) as this serotype is generally much more efficient at in vivo transduc-tion. To overcome this limitation, we used rAAV serotype 2 (rAAV2) to transduce these cells, as this serotype transduces a wide variety of cultured cells (Supplementary Figure S3).

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.

32.0

1.5

1.0

0.5

0.0

Brain glial culture

2

C26

:0LP

C(p

mol

/10

ul)

C26

/C22

LP

C r

atio

1

0

3

4

5

6

2C24

/C22

LP

C r

atio

1

0

WT PBS 2.5 × 105 2.5 × 104 2.5 × 105

AAV9-GFP AAV9-ABCD1

Abcd1−/−

5 × 105 10 × 105 (gc/cll)

***

***

***

*

*

*

*

*

*

WT PBS 2.5 × 105 2.5 × 104 2.5 × 105

AAV9-GFP AAV9-ABCD1

Abcd1−/−

5 × 105 10 × 105 (gc/cll) PBS 2.5 × 105 5 × 105 10 × 105 2.5 × 105

AAV9-GFPAAV9-ABCD1

5 × 105 10 × 105 (gc/cll)

WT PBS 2.5 × 105 2.5 × 104 2.5 × 105

AAV9-GFP AAV9-ABCD1

Abcd1−/−

5 × 105 10 × 105 (gc/cll)

Brain glial culture

Brain glial culture

Brain glial culture120

80

40

Sur

viva

l rat

e (%

)

0

a b

c

e

d

826 www.moleculartherapy.org vol. 23 no. 5 may 2015


As detected by western blot in Figure 3a, before transduc-tion, X-ALD fibroblasts had a very limited level of ABCD1 protein expression compared to normal fibroblasts, but after 5 × 105 gc/cell rAAV2-ABCD1 transduction, the expression of hABCD1 was dramatically increased in X-ALD fibroblasts. Immunofluorescence experiments demonstrated that the expression of hABCD1 increased in a dose-dependent manner after rAAV2-ABCD1 transduction, and higher levels of vector led to more cytoplasmic expression of hABCD1 (Figure 3b). To ascertain the effect of hABCD1 expression upon VLCFA

accumulation, we measured C26:0 LPC levels in fibroblasts. As shown in Figure 3c, untransduced X-ALD patient fibroblasts had around four times higher C26:0 LPC levels than normal fibroblasts. Transduction with rAAV2-ABCD1 reduced C26:0 LPC levels in X-ALD fibroblasts by ~50% (P < 0.001), indicat-ing rAAV-ABCD1 was functional in cells from X-ALD patients. Higher C26/C22 (4-fold) and C24/C22 (1.6-fold) LPC levels were also observed in untransduced X-ALD fibroblasts. These LPC levels could be significantly reduced after rAAV2-ABCD1 transduction (P < 0.01)(Figure 3d,e).

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.

PBSNormal

15

PBS AAV2Fluc

AAV2ABCD15e5/cell

PBS AAV2ABCD15e5/cell

hABCD1

GAPDH

Normal ALD-GM07531 ALD-GM04904

AAV2ABCD15e5/cell

***

*** *** ***

10pm

oles

C26

:0LP

C/p

hosp

holip

id fr

actio

n

5

0

2.5 × 105

AAV2-ABCD1

ALD fibroblast

5 × 105

Fibroblast

10 × 105 (gc/cll)

PBSNormal 2.5 × 105

AAV2-ABCD1

ALD fibroblast

5 × 105 10 × 105 (gc/cll)PBSNormal

2.5***

***

**

**

* *

2.0

1.5

C26

/C22

LP

C r

atio

1.0

0.5

0.0

2.5

2.0

1.5

C24

/C22

LP

C r

atio

1.0

0.5

0.0

2.5 × 105

AAV2-ABCD1

ALD fibroblast

5 × 105 10 × 105 (gc/cll)

Fibroblast Fibroblast

a

b

d e

c



Single intracerebroventricular and intravascular injection of ABCD1 using AAV9 vector yields hABCD1 protein expression in the CNSAfter confirming the functional activity of rAAV9-ABCD1 in vitro, we sought to characterize the in vivo function of rAAV9-ABCD1 by intracerebroventricular (ICV) and intravascular (IV) delivery in Abcd1−/− mice. First, by checking the mRNA expression in the mouse spinal cord, we confirmed the absence of the Abcd1 gene in Abcd1−/− mice (Figure 4a). Both ICV and IV injection mediated successful transduction of ABCD1 gene in Abcd1−/− mouse brain and spinal cord cells. By immunoblot analysis, ICV injections of 1 × 1011 gc/mouse rAAV9-ABCD1 yielded more than twofold higher expression of hABCD1 protein in mouse brain than IV injection at 1 × 1012 gc/mouse, and in spinal cord, ICV had slightly higher expression compared to IV (Figure 4b). Not surprisingly, IV injection led to a much higher expression of hABCD1 in the liver (Figure 4b). Interestingly, we also found high expression of hABCD1 in the adrenal gland (Figure 4g).

Using immunofluorescence, we visualized the expression of hABCD1 protein throughout the mouse brain. ICV injec-tion resulted in high, focal expression (horizontal sections) close to the injection site (left ventricle). Other specific structures such as the corpus callosum (CC), striatum, cerebral cortex,

and hippocampus also had expression (Figure 4c). IV injection yielded extensive and more evenly distributed hABCD1 expres-sion throughout the whole brain (sagittal sections) including cere-bral cortex, hippocampus, thalamus and cerebellum (Figure 4e, Supplementary Figure S4), but the expression level was not as high per cell compared to expression following ICV injection. Both ICV and IV injection led to hABCD1 protein expression in spinal cord, mainly localized to gray matter (Figure 4d,f). Strong expression of hABCD1 was also detected in other organs includ-ing liver, heart and lung following IV injection, at much higher levels than in CNS tissue (Figure 4g).

Colocalization of hABCD1 in different CNS cell types after ICV and IV deliveryIn order to determine the cell types in the CNS that were tar-geted by rAAV9-ABCD1, we sectioned brain and spinal cord and performed costaining of hABCD1 with different cellular markers including GFAP (astrocyte), IBA1 (microglia), Olig2 (Oligodendrocyte), and von Willebrand factor (endothelial cells). As shown in the confocal image in Figure 5, after ICV injection, astrocytes (Figure 5a,f), microglia (Figure 5b,g) and neurons (Figure 5e,j) were the major targeted cell types in brain; oligo-dendrocytes (Figure 5c,h) and endothelial cells (Figure 5d,i)

Figure 4 rAAV9-mediated hABCD1 expression in Abcd1−/− mouse brain and spinal cord via both intracerebroventricular (ICV) and intravas-cular (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 injec-tion. 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.

Abcd1

hABCD1

WT Brain

ICV(1E11) IV(1E12)PBS

Spinal cord

Liver

Liver Brain Spinalcord

Brain

ICV

IV

Spinal cord

Kidney Lung Heart Spleen Adrenalgland

β-actin

hABCD1

β-actin

hABCD1

GAPDH

AAV9-ABCD1 IV

β-actin

hABCD1

β-actin

β-actin

Abcd1−/−

a

c d

e f

g

b

2 mm 200 µm

1 mm

200 µm



showed less hABCD1 expression. A similar expression pattern was observed in spinal cord (Figure 5k–r). After ICV injection 12% of astrocytes and 3% of microglia but no oligodendrocytes and endothelial cells were transduced in the spinal cord. IV injec-tion led to widespread hABCD1 expression in many different cell types involving astrocytes, microglia, oligodendrocytes, endothe-lial cells, and neurons in both brain (Figure 6a–j) and spinal cord (Figure 6k–t). Approximately 23% of astrocytes, 18% of microg-lia, 7% of oligodendrocytes, and 65% of vessels were transduced in the spinal cord. Furthermore, no hABCD1 signal was detected in PBS injected mouse brain (Supplementary Figure S4) and spi-nal cord (Supplementary Figure S5) tissue, indicating specific staining of hABCD1 protein expressed by rAAV9-ABCD1. This was further confirmed by a secondary antibody only staining in rAAV9-ABCD1 treated mice.

Effect of rAAV9-ABCD1 on VLCFA level and GPX-1 expression in CNS tissueFollowing IV injection of 1 × 1012 gc/mouse of rAAV9-ABCD1, we found efficient reduction of C26:0 LPC in Abcd1−/− mouse brain and spinal cord. In both compartments, there was ~40% reduc-tion after IV administration (Figure 7a, P < 0.05). Although not significant, the total C26:0 fatty acid level was reduced in both brain and spinal cord (Figure 7b). By contrast, we found no sig-nificant reductions of C26:0 LPC in brain or spinal cord following ICV injection of 1 × 1011 gc/mouse rAAV9-ABCD1 (Figure 7a,b). Neither approach was able to reduce C26:0 LPC levels in plasma (Figure 7c).

Glutathione peroxidase 1(GPX-1) is one of the main detoxi-fying enzymes that scavenge reactive oxygen species (ROS) in the CNS. This enzyme has been reported to be increased as early as 3.5 months in Abcd1−/− mouse spinal cord, which reflects a

Figure 5 Colocalization of hABCD1 in different CNS cell types after intracerebroventricular (ICV) delivery. Upper brain: (a–j) Confocal imag-ing 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.

Bra

inS

pina

l cor

d

a b c d e

f g h i j

k l m n

o p q r

1 mm 1 mm 50 µm 50 µm 50 µm

20 µm20 µm20 µm20 µm20 µm

100 µm

20 µm 20 µm 20 µm 20 µm

200 µm 50 µm 50 µm



physiological response to increased free radicals due to Abcd1 mutation.23 We also observed increased GPX-1 in Abcd1−/− mouse spinal cord tissue compared to WT mice. After IV deliv-ery of rAAV9-ABCD1 there was a trend towards reduced GPX-1 expression (Figure 7d,e), suggesting another physiological cor-rection by rAAV9-ABCD1 gene delivery.

DISCUSSIONWe report that efficient delivery of ABCD1 gene was achieved by rAAV9 both in vitro and in vivo as measured by GFP expres-sion with the control vector as well as by immunofluorescence detection of hABCD1. Immunofluorescence showed costaining of hABCD1 and catalase, indicating localization of hABCD1 to the peroxisome. Astrocytes, microglia and neurons were the

major target cell types following ICV injection, while IV injec-tion delivered to these cell types as well as microvascular endothe-lial cells and oligodendrocytes. Compared with IV injection, the expression of hABCD1 after ICV injection was more restricted to those structures close to the injected ventricle (Figure 4c) with extremely high expression adjacent to the injected left ventricle. A similar restricted distribution was observed by Meijer et al. when they delivered AAV vector encoding GFP by intraventricu-lar injection into adult nude mice.24

For various reasons, IV delivery may be more suitable than ICV for the purposes of X-ALD. While ICV injection of rAAV is a promising strategy adopted for treating lysosomal storage dis-eases that require replacement of secreted proteins,25 ABCD1 is not secreted and cannot be passed from one cell to another via

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 magnifi-cation, 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.

Bra

inS

pina

l cor

d

a b c d e

f g h i j

k l m n o

p q r s t



cross-correction. For effective ABCD1 gene therapy, direct trans-duction of the cells involved in the pathology needs to occur. In our experiment and reported previously,17,18 IV delivery led to

extensive expression in the CNS across various cell types, espe-cially microglia, astrocytes and vascular endothelial cells; oligo-dendrocytes and neurons were also targeted. This widespread

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.

0.3***

*** ***

***

*

0.2

C26

/C22

LP

C r

atio

0.1

0.0

0.0

0.8

1.6

***

C26

:0 L

PC

(pm

ol/1

0ul)

0.0

0.1

0.2

0.3

0.4

Abcd1−/− Abcd1−/− AAV9 IV WT

GPX-1

β-actin

GP

X-1

pro

tein

exp

ress

ion(

ratio

to a

ctin

)

2.4

0.3

0.4

0.5

0.6

0.2

C26

:0 F

A le

vel(%

of t

otal

FA

)

0.1

0.0

WT PBS AAV9-ABCD1(ICV)

Brain

Spinal cord

Abcd1−/−

AAV9-ABCD1(IV)


Abcd1−/−

AAV9-ABCD1(IV)


Abcd1−/−

AAV9-ABCD1(IV)

WT PBS

Abcd1−/−

AAV9-ABCD1 IV

Brain

Spinal cord

Plasma

Spinal cord

a b

c d

e



transduction of brain and spinal cord cells is likely to be important and may reflect why we observed a larger reduction in VLCFA with IV delivery versus ICV (Figure 7a). Blood–brain barrier disruption and local inflammation may further be damaging in X-ALD,26,27 and further argue against the ICV approach.

The highest expression of ABCD1 in the wild-type central nervous system is found in microglia, astrocytes and endothelial cells, while different populations of oligodendrocytes in subcor-tical white matter and cerebellum may express variable levels of ABCD1.28 In contrast, ABCD1 is barely expressed in most neuronal populations, with the exception of certain neurons in the hypothalamus, the basal nucleus of Meynert, the peri-aqueductal gray matter and the locus ceruleus.29 Currently, the exact mechanism of axonal degeneration in X-ALD and AMN disease pathology is not known. Disturbed oligodendrocyte-axon interaction is considered to be in large part responsible for axonal degeneration, as Abcd1 knockdown in oligodendro-cytes contributes to disrupted redox equilibrium and oxidative stress.30 However, oligodendrocytes are not the only culprit, as microglial apoptosis is observed in perilesional white matter in X-ALD and represents an early stage in lesion evolution.31 Finally, demonstration of decreased brain magnetic resonance perfusion may precede disruption of the blood–brain barrier and microvascular structure, suggesting that endothelial cells are another possible target cell type.27 Fortunately, we found rAAV to transduce all of these cell types after IV delivery in mice. This is consistent with reports that rAAV9 transduces neurons, astrocytes, and microglia with high efficiency in non-human primates (NHPs), suggesting the potential for direct clinical translation.18,19 Fine-tuning transcriptional activity in different populations of these cells using cell-specific pro-moters32,33 or miRNA recognition sequences in the transgene cassette34,35 may further enhance therapeutic effects and help discern the cells most important in disease progression.

Accumulation of VLCFA (in particular C26:0) is the result of the defective peroxisome transporter function of mutant ABCD1, and is acknowledged to be the biochemical hallmark of X-ALD.36,37 In this study, rAAV9-ABCD1 showed a dose-dependent effect in reducing very long chain fatty acid (C26:0 LPC, lyso-phosphati-dylcholine) levels as well as C26/C22 LPC and C24/C22 LPC ratio in Abcd1−/− mouse brain glial cell culture and X-ALD patient fibroblasts (Figures 3 and 4). IV injection of 1 × 1012 gc/mouse also reduced C26/C22 LPC ratio in Abcd1−/− mouse brain and spinal cord, indicating the functional efficacy of rAAV9-mediated ABCD1 gene delivery. Neither IV nor ICV delivery was able to reduce plasma VLCFA. As the sources of plasma VLCFA are het-erogenous including dietary origin, de novo synthesis and dimin-ished β oxidation, this may have several explanations. Of note, the poor transfection of the spleen, an important hematopoetic organ, may also have contributed (Figure 4g).

Although there is phenotypic heterogeneity in X-ALD, it is also clear that none of the phenotypes would occur in the absence of the gene defect. Hence, regardless of whether the gene is caus-ative or merely a susceptibility gene, with second hit necessary, it justifies gene correction as a means of preventing further neuro-logic sequelae. The ability of our approach to target different cell types, particularly IV administration, suggests that it could be

effective for a variety of phenotypes, including both CALD and AMN.

Interestingly, we also found efficient delivery of ABCD1 to the adrenal gland. While the ability of rAAV to transduce the adrenal gland has been reported before,18 this has so far not been reported in a disease model of clinical relevance. The adrenal cortex is reli-ably affected in humans with ALD. While adrenal insufficiency is common in humans, it does not occur in Abcd1−/− mice. However, some microscopic level changes, such as swollen and lipid droplet filled cortical cells, can be detected in mouse adrenal gland.38 These findings suggest the possibility that adrenal func-tion in humans with X-ALD may benefit from intravenous rAAV9 delivery of ABCD1.

Another attractive feature of our findings (similar to previ-ously reported ones) is the efficient transduction of various cell types in the spinal cord following IV delivery. The fact that this leads to efficient correction of VLCFA as well may allow for treat-ment of AMN, a spinal cord axonopathy that currently lacks therapeutic options. Correction of AMN is further supported by data showing strong cortical and spinal cord transduction after rAAV7 and rAAV9 delivery into the cerebrospinal fluid of nonhu-man primates.39 Kaspar et al. have recently embarked on a human spinal muscular atrophy (SMA) trial using systemically adminis-tered rAAV9, which is strongly based on the robust transduction of spinal cord by rAAV9 in mice and NHPs.17,40

Despite our promising results in the Abcd1−/− mice, signifi-cant challenges in translating these insights to humans remain. While we report VLCFA correction via rAAV9, the optimal timing and dose to achieve clinical benefit is yet unclear. A larger study is needed to address these questions in the late onset axonopathy of the Abcd1−/− mouse. Further toxicology data in humans from AAV9 trials that target spinal cord are just emerging. However, rAAV vectors have an excellent safety profile in clinical trials,10,41 and several preclinical studies have been performed with rAAV9 in NHPs.18,40,42–45 Although differences between species and cere-bral structures may influence transduction efficiency, rAAV9 has been shown to transduce similar targets in mice and nonhuman primates.17,18 Finally, pre-existing immunity in the form of neu-tralizing antibodies to the rAAV9 capsid is a major hurdle to sys-temic administration of the vector. In children with X-ALD, this may be less of an issue, as prior exposure to natural AAV9 may not have occurred. In children and adults (with AMN), prescreen-ing patients for AAV titers may allow for effective dosing. In the future, strategies which allow the vector to evade these antibodies may circumvent this issue.

In summary, we report that IV injection of rAAV9-ABCD1 reduces very long chain fatty acids in culture and in the CNS of a mouse model of X-ALD. In addition to targeting brain and spinal cord, we also found delivery of ABCD1 to the adrenal gland. This suggests broad targeting of organs involved in X-ALD, warranting further clinical testing in this devastating genetic disorder.

MATERIALS AND METHODSCell culture. Human 293T cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supple-mented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St Louis, MO)



and 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen) in a humidi-fied atmosphere supplemented with 5% CO2 at 37 °C.

Animals. Congenic C57BL/6 Abcd1−/− and wild-type C57BL/6 mice were bought from the Jackson laboratory. Abcd1−/− mice were back crossed onto a pure C57/B6 background over six generations. They were then bred from homozygous founders, and occasionally genotyped according to the protocol provided by Jackson laboratory. Mice were fed a standard diet and were maintained under a 12-hour light–dark cycle. All mice were weighed before and after injection to monitor their health condition.

Recombinant AAV9 vectors. Human ABCD1 cDNA was kindly provided by Johannes Berger, University of Vienna, and inserted into pAAV-CBA vector using flanking HindIII and XhoI restriction sites. This rAAV vec-tor contains the strong CMV enhancer/chicken β-actin hybrid promoter, a WPRE element, and SV40 and BGH poly A sequences. rAAV production has been previously described.20 Briefly, 293T were transfected with rAAV (rAAV9 rep/cap and rAAV-ITR containing transgene (ABCD1) expression plasmid (single stranded genome)) and helper plasmid (Fd6) by the cal-cium phosphate method. Seventy-two hours after transfection cells were harvested and vector purified using a standard iodixanol density gradi-ent and ultracentrifugation protocol. Iodixanol was removed and vector concentrated in PBS by diafiltration using Amicon Ultra 100 kDa MWCO centrifugal devices (Millipore, Billerica, MA). Vector was stored at −80 °C until use. rAAV titers were determined by quantative PCR and expressed as genome copies per ml (gc/ml) as described.21

Vector administration Intravascular delivery: 6- to 8-week-old male C57BL/6 Abcd1−/− mice (n = 6) were placed in a restrainer (Braintree Scientific, Braintree, MA). Next, the tail was warmed in 40 °C water for 30 seconds, before wiping the tail with 70% isopropyl alcohol pads. Using a 100–300 µl volume of viral vec-tor in PBS containing 1–3 × 1012 genome copies (gc) rAAV9-ABCD1 was slowly injected into a lateral tail vein, before gently clamping the injec-tion site until the injection site had stopped bleeding. Initially, mice were injected with rAAV9-GFP vector as a vector control, in which no hABCD1 expression was detected; in subsequent experiments, mice received PBS injection as control. After the injection, mice were returned to their cages.

Stereotactic intracerebroventricular injection of mice: 6- to 8-week-old male C57BL/6 Abcd1−/− mice (n = 12) were anesthetized using isoflurane during the whole surgical procedure and placed in a rodent stereotactic frame. rAAV9 vectors were infused into the left lateral ventricle (coordinates from Bregma in mm: AP-0.2, ML+1.0, DV-3.0) using a Harvard 22 syringe pump (Harvard Apparatus, Holliston, MA) to drive a gas-tight Hamilton syringe attached to a 33-gauge steel needle (Hamilton). Briefly, 1 × 1011 gc rAAV9-ABCD1 (10 µl) were injected at a rate of 2 µl/minute, after which the needles was left in place for 2 minutes to prevent backflow before withdrawal. The same volume of PBS was injected by the same approach in the control mice.

Tissue and plasma preparation. Fifteen days after injection, mice were anesthetized by isoflurane. Blood was collected by cardiac puncture in mice and after that mice were sacrificed by transcardial perfusion of PBS. After removal, brain, liver, spinal cord and other organ tissue were snap-frozen and stored at −80 °C until use. Parts of tissues were fixed by 4% PFA and equilibrated in 30% sucrose prior to slicing.

Lipid analysis. To assess for the presence of free very long chain fatty acids (VLCFA) and lysophosphatidylcholine (C22:0, C24:0, and C26:0 LPC) after AAV9 gene delivery, lipid analysis was performed on cell pel-lets, plasma, brain, and spinal cord. The samples were dried, weighed, and extracted by the method of Folch. The lysophosphatidylcholines were ana-lyzed by combined liquid chromatography-tandem mass spectrometry following methods previously described.22 Absolute values of C26:0 LPC as well as ratios of C26:0/C22:0 LPC and C24:0/C22:0 LPC were reported.

Mouse brain glial cell culture and AAV9 transduction. Primary brain glial cell mixed culture was performed as followed. Briefly, 2–3 day old Abcd1−/− pups (n = 4) were sacrificed; the brain was dissected and hind-brain removed. After washing twice with PBS, brain tissue was incubated with 0.05% trypsin for 10 minutes followed by 5 minutes incubation in DNaseI. Digested brain tissues were triturated using 10 ml pipette and passed over a 100 µm filter. Filtered cells were collected by centrifuge, sus-pended in DMEM containing 10% FBS and cultured in T75 flasks at 37 °C until confluent. Brain cells were passed onto six-well plates for 24 hours and transduced with different doses of rAAV9-ABCD1 for 5 days. Cells were then collected for further analysis.

Human fibroblast cell culture and AAV2 transduction. Normal human dermal fibroblasts were bought from ATCC and X-ALD patient fibroblasts were obtained from the Coriell Institute. Cells were cultured in DMEM containing 10% FBS and maintained at 37 °C until confluent, then passed and transduced with different doses of rAAV2-ABCD1 for further experi-mental analysis.

Cell viability assay. We used cell counting kit-8 (Dojindo Molecular Technologies, Rockville, MD) to measure the cell cytotoxicity after rAAV treatment. Briefly, cells were seeded in 96-well plates and transduced with different doses of rAAV9-ABCD1 as well as rAAV9-GFP for 5 days. CCK-8 solution were added to each well and incubated for 3 hours followed by absorbance measurement at 450 nm.

Western blotting. Tissue and cell lysates were prepared by using RIPA buffer (Sigma-Aldrich) with 1% Halt Protease and Phosphatase Inhibitor Cocktail (Roche, Indianapolis, IN). Protein samples were separated on NuPAGE 4–12% Bis-tris gels (Invitrogen) and transferred on PVDF mem-branes. Membranes were blocked with 5% nonfat milk in PBS contain-ing 0.05% Tween 20 and probed with antibody against human ABCD1 (Origene, Rockville, MD) and mouse GPX-1 (Abcam, Cambridge, MA) antibody diluted in blocking buffer. Anti-β-actin (Santa Cruz, Dallas, TX) was used as a protein loading control. Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo, Rockford, IL) after incubation with HRP-conjugated second antibodies.

Immunofluorescence staining and confocal microscopy imaging Cell culture imaging: Brain glial cell mixture and fibroblasts were seeded into poly-lysine coated chamber slides and transduced with rAAV-ABCD1 for 5 days. Cells were then fixed with 4% PFA for 10 minutes and permea-bilized in blocking buffer containing 0.3% Triton X and 2% goat serum for 1 hour. Cells were then costained with mouse antihuman ABCD1 antibody (Origene) and rabbit anticatalase antibody (Abcam) at 4 °C overnight. Alexa Fluor 555 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit anti-body (Invitrogen, Eugene, OR) were used as secondary antibodies. Sections were mounted in mounting medium with DAPI (Vector, Burlingame, CA) and analyzed using confocal laser microscope (Zeiss).

Tissue section imaging: Sections of brain and spinal cord (16 µm) were cut at −25 °C using cryostat (Leica) and stored at −80 °C. The immunofluorescence staining protocol was similar as described above. Sections were stained with mouse antihuman ABCD1 antibody and then costained with rabbit anti-GFAP (Dako, Carpinteria, CA), rabbit anti-IBA1 (Wako, Richmond, VA), rabbit anti-von Willebrand (Abcam), rabbit anti-Olig2 (Abcam) respectively to localize the cell type. The slides were imaged by confocal laser microscope and transduced cells counted. Estimates of ABCD1 transduced cells of each cell type were documented in 20× and 40× (for microglia) magnification images.

Statistical analysis. Results were expressed as means ± SEM and analyzed for statistical significance by ANOVA followed by Fisher’s protected least-significant difference based on two side comparisons among experimen-tal groups using SPSS program. A P < 0.05 was considered statistically significant.



SUPPLEMENTARY MATERIALFigure S1. Brain glial cell mixture culture (from Abcd1−/− mouse) transduced with rAAV9-GFP show moderate transduction efficiency.Figure S2. Targeting cell types by rAAV9-ABCD1 in mixed brain glial cell culture.Figure S3. Fibroblast from X-ALD patient transduced with either rAAV9-GFP or AAV2-GFP.Figure S4. Expression of hABCD1 at different brain locations after PBS, 1 × 1012 gc (IV) and 1 × 1011 gc (ICV) delivery of rAAV9-ABCD1 into Abcd1−/− mice (right brain sagittal section).Figure S5. Expression of hABCD1 in spinal cord after PBS, 1 × 1012 gc (IV) and 1 × 1011 gc (ICV) delivery of rAAV9-ABCD1 into Abcd1−/− mice.

ACKNOWLEDGMENTSWe thank the Leblang Charitable Foundation and NIH (R21 NINDS, R01 NINDS) for their support. CM was supported by an NIH/NINDS R21 NS081374-01 and an American Brain Tumor Association Discovery Grant. We would like to acknowledge the Neuroscience Center Nucleic Acid Quantitation Core and Image Analysis Core (supported by NINDS P30NS045776). Lastly, this work was supported in part by a research grant from the University of Pennsylvania Orphan Disease Center. We also thank Chuying Xia, Sandra Jimenez and Ankush Chandra for their assistance during mice dissection and protein verification.

REFERENCES 1. Mosser, J, Douar, AM, Sarde, CO, Kioschis, P, Feil, R, Moser, H et al. (1993). Putative

X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 361: 726–730.

2. Moser, AB, Kreiter, N, Bezman, L, Lu, S, Raymond, GV, Naidu, S et al. (1999). Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls. Ann Neurol 45: 100–110.

3. Valianpour, F, Selhorst, JJ, van Lint, LE, van Gennip, AH, Wanders, RJ and Kemp, S (2003). Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol Genet Metab 79: 189–196.

4. Ferrer, I, Aubourg, P and Pujol, A (2010). General aspects and neuropathology of X-linked adrenoleukodystrophy. Brain Pathol 20: 817–830.

5. Singh, I and Pujol, A (2010). Pathomechanisms underlying X-adrenoleukodystrophy: a three-hit hypothesis. Brain Pathol 20: 838–844.

6. Cartier, N, Hacein-Bey-Abina, S, Bartholomae, CC, Veres, G, Schmidt, M, Kutschera, I et al. (2009). Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326: 818–823.

7. Mingozzi, F and High, KA (2011). Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 12: 341–355.

8. Kaplitt, MG, Feigin, A, Tang, C, Fitzsimons, HL, Mattis, P, Lawlor, PA et al. (2007). Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet 369: 2097–2105.

9. Marks, WJ Jr, Ostrem, JL, Verhagen, L, Starr, PA, Larson, PS, Bakay, RA et al. (2008). Safety and tolerability of intraputaminal delivery of CERE-120 (adeno-associated virus serotype 2-neurturin) to patients with idiopathic Parkinson’s disease: an open-label, phase I trial. Lancet Neurol 7: 400–408.

10. Worgall, S, Sondhi, D, Hackett, NR, Kosofsky, B, Kekatpure, MV, Neyzi, N et al. (2008). Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus expressing CLN2 cDNA. Hum Gene Ther 19: 463–474.

11. Maguire, AM, High, KA, Auricchio, A, Wright, JF, Pierce, EA, Testa, F et al. (2009). Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet 374: 1597–1605.

12. Nathwani, AC, Tuddenham, EG, Rangarajan, S, Rosales, C, McIntosh, J, Linch, DC et al. (2011). Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365: 2357–2365.

13. Davidson, BL, Stein, CS, Heth, JA, Martins, I, Kotin, RM, Derksen, TA et al. (2000). Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc Natl Acad Sci USA 97: 3428–3432.

14. Taymans, JM, Vandenberghe, LH, Haute, CV, Thiry, I, Deroose, CM, Mortelmans, L et al. (2007). Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Hum Gene Ther 18: 195–206.

15. Tarantal, AF and Lee, CC (2010). Long-term luciferase expression monitored by bioluminescence imaging after adeno-associated virus-mediated fetal gene delivery in rhesus monkeys (Macaca mulatta). Hum Gene Ther 21: 143–148.

16. Manfredsson, FP, Rising, AC and Mandel, RJ (2009). AAV9: a potential blood-brain barrier buster. Mol Ther 17: 403–405.

17. Foust, KD, Nurre, E, Montgomery, CL, Hernandez, A, Chan, CM and Kaspar, BK (2009). Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27: 59–65.

18. Gray, SJ, Matagne, V, Bachaboina, L, Yadav, S, Ojeda, SR and Samulski, RJ (2011). Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther 19: 1058–1069.

19. Foust, KD, Nurre, E, Montgomery, CL, Hernandez, A, Chan, CM and Kaspar, BK (2009). Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27: 59–65.

20. Maguire, CA, Meijer, DH, LeRoy, SG, Tierney, LA, Broekman, ML, Costa, FF et al. (2008). Preventing growth of brain tumors by creating a zone of resistance. Mol Ther 16: 1695–1702.

21. Maguire, CA, Balaj, L, Sivaraman, S, Crommentuijn, MH, Ericsson, M, Mincheva-Nilsson, L et al. (2012). Microvesicle-associated AAV vector as a novel gene delivery system. Mol Ther 20: 960–971.

22. Hubbard, WC, Moser, AB, Tortorelli, S, Liu, A, Jones, D and Moser, H (2006). Combined liquid chromatography-tandem mass spectrometry as an analytical method for high throughput screening for X-linked adrenoleukodystrophy and other peroxisomal disorders: preliminary findings. Mol Genet Metab 89: 185–187.

23. Fourcade, S, López-Erauskin, J, Galino, J, Duval, C, Naudi, A, Jove, M et al. (2008). Early oxidative damage underlying neurodegeneration in X-adrenoleukodystrophy. Hum Mol Genet 17: 1762–1773.

24. Meijer, DH, Maguire, CA, LeRoy, SG and Sena-Esteves, M (2009). Controlling brain tumor growth by intraventricular administration of an AAV vector encoding IFN-beta. Cancer Gene Ther 16: 664–671.

25. Yamazaki, Y, Hirai, Y, Miyake, K and Shimada, T (2014). Targeted gene transfer into ependymal cells through intraventricular injection of AAV1 vector and long-term enzyme replacement via the CSF. Sci Rep 4: 5506.

26. Raymond, GV, Seidman, R, Monteith, TS, Kolodny, E, Sathe, S, Mahmood, A et al. (2010). Head trauma can initiate the onset of adreno-leukodystrophy. J Neurol Sci 290: 70–74.

27. Musolino, PL, Rapalino, O, Caruso, P, Caviness, VS and Eichler, FS (2012). Hypoperfusion predicts lesion progression in cerebral X-linked adrenoleukodystrophy. Brain 135(Pt 9): 2676–2683.

28. Fouquet, F, Zhou, JM, Ralston, E, Murray, K, Troalen, F, Magal, E et al. (1997). Expression of the adrenoleukodystrophy protein in the human and mouse central nervous system. Neurobiol Dis 3: 271–285.

29. Höftberger, R, Kunze, M, Weinhofer, I, Aboul-Enein, F, Voigtländer, T, Oezen, I et al. (2007). Distribution and cellular localization of adrenoleukodystrophy protein in human tissues: implications for X-linked adrenoleukodystrophy. Neurobiol Dis 28: 165–174.

30. Baarine, M, Andréoletti, P, Athias, A, Nury, T, Zarrouk, A, Ragot, K et al. (2012). Evidence of oxidative stress in very long chain fatty acid–treated oligodendrocytes and potentialization of ROS production using RNA interference-directed knockdown of ABCD1 and ACOX1 peroxisomal proteins. Neuroscience 213: 1–18.

31. Eichler, FS, Ren, JQ, Cossoy, M, Rietsch, AM, Nagpal, S, Moser, AB et al. (2008). Is microglial apoptosis an early pathogenic change in cerebral X-linked adrenoleukodystrophy? Ann Neurol 63: 729–742.

32. Dirren, E, Towne, CL, Setola, V, Redmond, DE Jr, Schneider, BL and Aebischer, P (2014). Intracerebroventricular injection of adeno-associated virus 6 and 9 vectors for cell type-specific transgene expression in the spinal cord. Hum Gene Ther 25: 109–120.

33. von Jonquieres, G, Mersmann, N, Klugmann, CB, Harasta, AE, Lutz, B, Teahan, O et al. (2013). Glial promoter selectivity following AAV-delivery to the immature brain. PLoS ONE 8: e65646.

34. Murphy, SR, Chang, CC, Dogbevia, G, Bryleva, EY, Bowen, Z, Hasan, MT et al. (2013). Acat1 knockdown gene therapy decreases amyloid-ß in a mouse model of Alzheimer’s disease. Mol Ther 21: 1497–1506.

35. Quattrocelli, M, Crippa, S, Montecchiani, C, Camps, J, Cornaglia, AI, Boldrin, L et al. (2013). Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice. J Am Heart Assoc 2: e000284.

36. Singh, I, Moser, HW, Moser, AB and Kishimoto, Y (1981). Adrenoleukodystrophy: impaired oxidation of long chain fatty acids in cultured skin fibroblasts an adrenal cortex. Biochem Biophys Res Commun 102: 1223–1229.

37. Moser, HW, Moser, AB, Frayer, KK, Chen, W, Schulman, JD, O’Neill, BP et al. (1981). Adrenoleukodystrophy: increased plasma content of saturated very long chain fatty acids. Neurology 31: 1241–1249.

38. Forss-Petter, S, Werner, H, Berger, J, Lassmann, H, Molzer, B, Schwab, MH et al. (1997). Targeted inactivation of the X-linked adrenoleukodystrophy gene in mice. J Neurosci Res 50: 829–843.

39. Samaranch, L, Salegio, EA, San Sebastian, W, Kells, AP, Bringas, JR, Forsayeth, J et al. (2013). Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates. Hum Gene Ther 24: 526–532.

40. Foust, KD, Salazar, DL, Likhite, S, Ferraiuolo, L, Ditsworth, D, Ilieva, H et al. (2013). Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS. Mol Ther 21: 2148–2159.

41. Murrey, DA, Naughton, BJ, Duncan, FJ, Meadows, AS, Ware, TA, Campbell, KJ et al. (2014). Feasibility and safety of systemic rAAV9-hNAGLU delivery for treating mucopolysaccharidosis IIIB: toxicology, biodistribution, and immunological assessments in primates. Hum Gene Ther Clin Dev 25: 72–84.

42. Samaranch, L, Salegio, EA, San Sebastian, W, Kells, AP, Foust, KD, Bringas, JR et al. (2012). Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates. Hum Gene Ther 23: 382–389.

43. Bevan, AK, Duque, S, Foust, KD, Morales, PR, Braun, L, Schmelzer, L et al. (2011). Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders. Mol Ther 19: 1971–1980.

44. Gray, SJ, Nagabhushan Kalburgi, S, McCown, TJ and Jude Samulski, R (2013). Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene Ther 20: 450–459.

45. Mattar, CN, Waddington, SN, Biswas, A, Johana, N, Ng, XW, Fisk, AS et al. (2013). Systemic delivery of scAAV9 in fetal macaques facilitates neuronal transduction of the central and peripheral nervous systems. Gene Ther 20: 69–83.


Paper 5

Education

human abcd1 gene abcd1

aav9 abcd1

xald boys

xald patients

abcd1 mice

abcd1 mouse cns

xald patient fibroblasts

aavabcd1 transduction