Adenoassociated Virus Serotype 9-Mediated Gene Therapy for X-Linked Adrenoleukodystrophy

1. 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
INTRODUCTION
X-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
Correspondence: Florian Eichler, Department of Neurology, Massachusetts General Hospital, 55 Fruit Street, ACC 708, Boston, Massachusetts 02114,
USA. E-mail: feichler@partners.org
Adenoassociated Virus Serotype 9-Mediated Gene
Therapy for X-Linked Adrenoleukodystrophy
Yi Gong1
, Dakai Mu1
, Shilpa Prabhakar1
, Ann Moser2
, Patricia Musolino1
, JiaQian Ren1
,
Xandra O Breakefield1
, Casey A Maguire1
and Florian S Eichler1
1
Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts, USA; 2
Peroxisome Disease Lab, Hugo W Moser Research Institute,
Baltimore, Maryland, USA
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2. © The American Society of Gene Cell Therapy
rAAV9-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.
RESULTS
rAAV-mediated transduction of ABCD1 gene in mixed
brain cell culture from Abcd1−/− mice
In 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−/− mice
After 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
ABCD1
Catalase
DAPI
β-actin
a
cb
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3. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
AAV-ABCD1 reduces VLCFA level in human X-ALD
fibroblasts
Since 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.
3
2.0
1.5
1.0
0.5
0.0
Brain glial culture
2
C26:0LPC(pmol/10ul)
C26/C22LPCratio
1
0
3
4
5
6
2
C24/C22LPCratio
1
0
WT PBS 2.5 × 10
5
2.5 × 10
4
2.5 × 10
5
AAV9-GFP AAV9-ABCD1
Abcd1
−/−
5 × 10
5
10 × 10
5
(gc/cll)
***
***
***
*
*
*
*
*
*
WT PBS 2.5 × 10
5
2.5 × 10
4
2.5 × 10
5
AAV9-GFP AAV9-ABCD1
Abcd1−/−
5 × 10
5
10 × 10
5
(gc/cll) PBS 2.5 × 10
5
5 × 10
5
10 × 10
5
2.5 × 10
5
AAV9-GFPAAV9-ABCD1
5 × 10
5
10 × 10
5
(gc/cll)
WT PBS 2.5 × 10
5
2.5 × 10
4
2.5 × 10
5
AAV9-GFP AAV9-ABCD1
Abcd1
−/−
5 × 10
5
10 × 10
5
(gc/cll)
Brain glial culture
Brain glial culture
Brain glial culture
120
80
40
Survivalrate(%)
0
a b
c
e
d
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4. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
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 AAV2
Fluc
AAV2
ABCD1
5e5/cell
PBS AAV2
ABCD1
5e5/cell
hABCD1
GAPDH
Normal ALD-GM07531 ALD-GM04904
AAV2
ABCD1
5e5/cell
***
*** *** ***
10
pmolesC26:0LPC/phospholipidfraction
5
0
2.5 × 105
AAV2-ABCD1
ALD fibroblast
5 × 105
Fibroblast
10 × 105
(gc/cll)
PBSNormal 2.5 × 10
5
AAV2-ABCD1
ALD fibroblast
5 × 105
10 × 10
5
(gc/cll)PBSNormal
2.5
***
***
**
**
* *
2.0
1.5
C26/C22LPCratio
1.0
0.5
0.0
2.5
2.0
1.5
C24/C22LPCratio
1.0
0.5
0.0
2.5 × 105
AAV2-ABCD1
ALD fibroblast
5 × 105
10 × 10
5
(gc/cll)
Fibroblast Fibroblast
a
b
d e
c
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5. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
Single intracerebroventricular and intravascular
injection of ABCD1 using AAV9 vector yields hABCD1
protein expression in the CNS
After 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 delivery
In 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 Spinal
cord
Brain
ICV
IV
Spinal cord
Kidney Lung Heart Spleen Adrenal
gland
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
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6. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
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 tissue
Following 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.
BrainSpinalcord
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
Molecular Therapy vol. 23 no. 5 may 2015 829

7. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
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.
DISCUSSION
We 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.
BrainSpinalcord
a b c d e
f g h i j
k l m n o
p q r s t
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8. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
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/C22LPCratio
0.1
0.0
0.0
0.8
1.6
***
C26:0LPC(pmol/10ul)
0.0
0.1
0.2
0.3
0.4
Abcd1−/−
Abcd1
−/−
AAV9 IV WT
GPX-1
β-actin
GPX-1proteinexpression(ratiotoactin)
2.4
0.3
0.4
0.5
0.6
0.2
C26:0FAlevel(%oftotalFA)
0.1
0.0
WT PBS AAV9-
ABCD1(ICV)
Brain
Spinal cord
Abcd1
−/−
AAV9-
ABCD1(IV)
WT PBS AAV9-
ABCD1(ICV)
Abcd1
−/−
AAV9-
ABCD1(IV)
WT PBS AAV9-
ABCD1(ICV)
Abcd1
−/−
AAV9-
ABCD1(IV)
WT PBS
Abcd1
−/−
AAV9-ABCD1 IV
Brain
Spinal cord
Plasma
Spinal cord
a b
c d
e
Molecular Therapy vol. 23 no. 5 may 2015 831

9. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
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 METHODS
Cell 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)
832 www.moleculartherapy.org vol. 23 no. 5 may 2015

10. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
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.
Molecular Therapy vol. 23 no. 5 may 2015 833

11. © The American Society of Gene Cell Therapy
rAAV9-Mediated Gene Therapy for X-ALD
SUPPLEMENTARY MATERIAL
Figure 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.
ACKNOWLEDGMENTS
We 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.
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