Download Recombinant AAV-mediated gene delivery to the central nervous

THE JOURNAL OF GENE MEDICINE
REVIEW
J Gene Med 2004; 6: S212–S222.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.506
ARTICLE
Recombinant AAV-mediated gene delivery to the
central nervous system
L. Tenenbaum1,3 *
A. Chtarto1,3†
E. Lehtonen1,3†
T. Velu3
J. Brotchi1,2
M. Levivier1,2
1
Laboratory of Experimental
Neurosurgery, Université Libre de
Bruxelles, Hôpital Erasme, 808, Route
de Lennik, B-1070 Brussels, Belgium
2
Department of Neurosurgery,
Université Libre de Bruxelles, Hôpital
Erasme, 808, Route de Lennik,
B-1070 Brussels, Belgium
3
Institut de Recherche
Interdisciplinaire en Biologie
Humaine et Moléculaire, Université
Libre de Bruxelles, Hôpital Erasme,
808, Route de Lennik, B-1070
Brussels, Belgium
*Correspondence to: L. Tenenbaum,
U.L.B. Campus Erasme, Bâtiment C,
I.R.I.B.H.M., 808 Route de Lennik,
B-1070 Bruxelles, Belgium. E-mail:
[email protected]
† These
authors contributed equally
to this work.
Summary
Various regions of the brain have been successfully transduced by recombinant
adeno-associated virus (rAAV) vectors with no detected toxicity. When using
the cytomegalovirus immediate early (CMV) promoter, a gradual decline in
the number of transduced cells has been described. In contrast, the use of
cellular promoters such as the neuron-specific enolase promoter or hybrid
promoters such as the chicken β-actin/CMV promoter resulted in sustained
transgene expression. The cellular tropism of rAAV-mediated gene transfer
in the central nervous system (CNS) varies depending on the serotype used.
Serotype 2 vectors preferentially transduce neurons whereas rAAV5 and
rAAV1 transduce both neurons and glial cells. Recombinant AAV4-mediated
gene transfer was inefficient in neurons and glial cells of the striatum (the
only structure tested so far) but efficient in ependymal cells.
No inflammatory response has been described following rAAV2 administration to the brain. In contrast, antibodies to AAV2 capsid and transgene
product were elicited but no reduction of transgene expression was observed
and readministration of vector without loss of efficiency was possible from
3 months after the first injection.
Based on the success of pioneer work performed with marker genes, various
strategies for therapeutic gene delivery were designed. These include enzyme
replacement in lysosomal storage diseases, Canavan disease and Parkinson’s
disease; delivery of neuroprotective factors in Parkinson’s disease, Huntington
disease, Alzheimer’s disease, amyotrophic lateral sclerosis, ischemia and
spinal cord injury; as well as modulation of neurotransmission in epilepsy
and Parkinson’s disease.
Several of these strategies have demonstrated promising results in relevant
animal models. However, their implementation in the clinics will probably
require a tight regulation and a specific targeting of therapeutic gene
expression which still demands further developments of the vectors. Copyright
 2004 John Wiley & Sons, Ltd.
Keywords AAV; enzyme replacement; neuroprotection; excitability; neurological disorders
Introduction
Various regions of the brain [1–4] and spinal cord [5] have been
successfully transduced by rAAV vectors with no detected toxicity and
low immune reaction [6,7], opening the route for gene therapy of diseases affecting the central nervous system (CNS). In this article, we will
briefly review the performances of rAAV vectors in the CNS supporting
the development of the various strategical avenues using rAAV-mediated
gene delivery which have been envisaged for curing neurological diseases.
Copyright  2004 John Wiley & Sons, Ltd.
S213
AAV Vectors in the CNS
Enzyme replacement therapies in inherited disorders
affecting the brain such as mucopolysaccharidosis [8–11]
or Canavan disease [12,13] have been initially considered
as particularly challenging since, in these diseases, all the
cells harbor an enzymatic deficiency and global brain
transduction is not achievable. However, this type of
therapy has been proven to be an accessible goal in several
cases, thanks to the efficient spread of enzyme produced
by corrected cells and further uptake by deficient cells.
The first clinical trial involving neurosurgical delivery of
an AAV-2 vector has been launched for patients with
Canavan disease [14]. Vectors can also be used to supply
locally metabolic enzymes for missing neurotransmitters
in neurodegenerative diseases [15–18].
Neuroprotective gene therapy strategies rely on the
discovery of neurotrophic and growth factors able to
counteract neuronal cell death. These could be envisaged
in all the situations in which neurons gradually die, such
as neurodegenerative diseases [19–22] or ischemia [23],
as well as in situations in which regrowth of neuron fibers
is expected such as spinal cord repair [24].
Fine-tuning of neuron firing by interfering with
neurotransmission could also be used for particular
diseases such as epilespy [25]. However, this strategy
requires a very specific targeting of particular neuronal
populations and a tight regulation of gene expression to
avoid undesirable effects [26].
More detailed reviews discussing the efficiency, kinetics
and cellular tropism of rAAV-mediated gene delivery as
well as immunological aspects have recently appeared
[27,28]. In addition, specific reviews addressing particular
diseases and/or strategies are also available [29–33].
Performances of recombinant AAV
vectors in different brain regions
Vectors based on serotype 2 of AAV
Most of the in vivo studies have been performed using
AAV2 vectors containing the strong cytomegalovirus
immediate-early (CMV) promoter (see Table 1). These
vectors have generated positive results following injection into hippocampus, substantia nigra, striatum, piriform and lateral cortex, olfactory tubercle, cerebellum, inferior colliculus, globus pallidus, basal forebrain,
subthalamic nucleus, facial motor nucleus and spinal
cord [1,2,5,34–37]. Transduction efficiencies, however,
vary markedly from one region to another. In a systematic study using AAV2, efficiency of the transgene
expression in the brain was demonstrated with the following order: hippocampus, inferior colliculus, piriform
cortex > olfactory tubercle > striatum [1]. Transduction
efficiency in the striatum has been improved by coinfusing heparin with the viral suspension [38,39]. The
rationale was that, since transduced cells were observed
only in the close vicinity of the needle tract, viral particles have presumably been trapped by receptors present
at high concentrations on neurons, preventing diffusion
at distance from the injection site. Convection-enhanced
delivery, consisting of applying a pressure gradient during infusion of the viral suspension through the brain
parenchyma, was also shown to increase viral spread and
transduction efficiency [40].
Most diseases affecting the brain progress over
long periods of time and thus potential therapeutic
proteins will need to be expressed stably for months
or even years. While long-term gene expression (up
to 1 year) from the CMV promoter has been reported
[34,41], reductions in transgene expression levels over
time in different brain regions have been described
[1,2,34]. The most striking difference has been seen
between hippocampus and inferior colliculus [1,4]. Both
areas showed strong transgene expression 1 week postinjection. However, 3 months post-injection, a drastic
reduction in the number of transduced neurons (up
to 96%) was observed in the hippocampus while in
the inferior colliculus no significant decrease occurred.
The observed decline in transgene expression has been
suggested to result from: (i) loss of vector sequences in the
absence of integration; (ii) silencing of the CMV promoter
by hypermethylation [42], or (iii) loss of transduced
cells. Promoters not subjected to methylation such as
neuron-specific promoters provide a means to obtain
sustained transduction. Indeed, when a rAAV2 vector
containing the neuron-specific enolase (NSE) promoter
was injected into the rat hippocampus or substantia
nigra, both the efficiency and the persistence of transgene
expression were shown to substantially improve as
compared with the CMV promoter [2,43,44]; see also
Table 2. Regardless of the promoter used, transduction
efficiency can be increased using posttranscriptional
regulatory elements such as the Woodchuck hepatitis virus
element posttranscriptional regulator (WPRE), which
has been shown to increase the steady-state level of
Table 1. Summary of AAV2-mediated gene transfer in the rat brain
Region Time
1 week
1 month
3 months
1 year
Cell type
References
Striatum
Hippocampus
Substantia nigra
Globus pallidus
Internal capsule
+
+++
++
+
Interneurones
[1,34]
++++
++++
+
−
Gaba-ergic neurones
[1,2]
ns
++++
++
ns
Dopaminergic neurones
[1,2]
++++
++++
ns
ns
Gaba-ergic neurones
[34]
ns
++
ns
ns
Glial cells
[34]
ns: not shown.
Copyright  2004 John Wiley & Sons, Ltd.
J Gene Med 2004; 6: S212–S222.
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L. Tenenbaum et al.
Table 2. Effects of different promoters and the WPRE posttranscriptional regulatory element on rAAV2-mediated gene transfer in
the rat brain
Region
Regulatory
elements
3 days
3 weeks
3 months
9 months
References
Hippocampus
CMV
−
+++
++
−
NSE
+
++++
++++
++++
[2,46]
Substantia nigra
NSE + WPRE
CMV
NSE
ns
++++++
++++++
++++++
[45,46]
+++
++
−
ns
++++
++++
++++
[2]
Striatum
NSE + WPRE
CMV
++++++
++++++
ns
[45]
+++
++
+
NSE
ns
++++
++++
ns
[45]
NSE + WPRE
++++++
++++++
++++++
NSE: neuron-specific enolase promoter; CMV: early cytomegalovirus promoter; WPRE: Woodchuck hepatits virus posttranscriptional regulatory element.
ns, not shown.
messenger RNA and the efficiency of translation, thus
resulting in higher levels of gene expression [45]. In a
comparative study, Paterna et al. [46] injected rAAV2
vectors containing either CMV or PDGF-β promoter in
conjunction with WPRE in the substantia nigra. The vector
containing the PDGF-β promoter and WPRE resulted
in more efficient and widespread transduction than
the PDGF-β promoter alone or the two corresponding
vectors containing the CMV promoter. Accordingly, Xu
et al. [43] have reported that WPRE boosted transgene
expression by 13-fold in the striatum and by 35fold in the hippocampus. WPRE also improved (11fold) NSE promoter-driven transgene expression in the
hippocampus [46]. Transgene expression from the hybrid
CMV-chicken β-actin (CBA) promoter, consisting of a
fusion between the chicken β-actin promoter with the
CMV promoter enhancer sequences, is even more efficient
and has been shown to be stable until at least 25 months
post-injection [44]. These results show that rAAV2mediated transgene expression in the brain can be
extremely durable. Integration in the host genome [47]
and/or persistence of episomal sequences as observed in
muscle [48] and lung [49] could be responsible for this
stability.
Several studies converged to conclude that rAAV2based vectors using the CMV promoter almost exclusively
transduce neurons when delivered in the rat CNS in
regions containing mixed neuronal-glial cell populations,
transduction of astroglial cells occurring only very
infrequently (Figure 1) [1,2,34]. In accordance with
these empirical observations, studies using virus with
fluorophore-labeled viral capsids demonstrated that
binding and uptake preferentially occur in neurons [50].
However, there seems to be no absolute obstacle to glial
cell transduction. Indeed, cultured glial cells of fetal [51]
or tumoral [52,53] origin could be efficiently infected by
wt and rAAV2. Furthermore, in vivo, in a region devoid of
neurons, the internal capsule, glial cells were transduced
by a rAAV2 vector using the CMV promoter [34]. The
study by Bartlett et al. [50] shows that the presence
of receptors and co-receptors for rAAV2 on brain cells
constitutes a major clue to explain the cellular tropism
of this vector. Viral attachment and entry has been
shown to be dependent on the presence of heparan
sulfate proteoglycans (HSPG) [54] acting in synergy
Copyright  2004 John Wiley & Sons, Ltd.
Figure 1. Tropism of rAAV2 vectors for neurons in the
fetal ventral mesencephalon. (A) Fragments of E14 rat fetal
mesencephalon were incubated in a viral suspension of rAAV
type 2 expressing the green fluorescent protein (GFP) under
the control of the CMV promoter. Cells were dissociated
and stereotaxically transplanted in the striatum of adult
rats. One month after transplantation, transgene expression
was evidenced by immunofluorescence (green). (A) The vast
majority of GFP-positive cells co-labeled with NeuN (in red)
a neuronal marker. (B) No colabeling with GFAP, a marker of
astrocytes, was observed. Partially reproduced from Lehtonen
et al. [76]
with FGF-R 1 [55] and αVβ5 [56]. The study of the
biodistribution of HSPG in the brain [57] revealed a high
immunoreactivity in the substantia nigra pars compacta
(SNc) and in the hippocampus (two regions in which
rAAV2-mediated gene transfer is particularly efficient).
The distribution of FGF-R 1 in the brain might also
provide some clues: indeed olfactory tubercule, piriform
cortex, SNc, inferior coliculus and stria terminalis, but
J Gene Med 2004; 6: S212–S222.
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AAV Vectors in the CNS
not the striatum, express high levels of FGF-R 1 mRNA
[58].
The choice of the promoter may nevertheless affect
the cellular specificity of gene transfer. As expected,
when the neuron-specific enolase (NSE) promoter is
used, transduction seems to occur primarily in neurons
[2,44]. Furthermore, specific neuronal cell populations
were preferentially transduced. For example, in the
hippocampus, the majority of transduced neurons were
GABA-ergic, GAD-positive neurons [2]. However, within
a brain region devoid of neurons, the corpus callosum,
oligodendrocytes were also transduced with a vector
using the NSE promoter, although at a low efficiency
[2]. This low level of gene transfer in cells in which
the NSE promoter is normally not active possibly
reflects interference with promoter/enhancer elements
present in the viral inverted terminal repeats (ITRs)
[59,60]. In contrast, when a vector containing the
myelin basic protein (MBP) promoter, specifically active
in oligodendrocytes, was injected in the corpus callosum,
a high level of transgene expression was observed for up to
3 months [61,62]. Injections of rAAV2 vectors containing
the astrocyte-specific glial fibrillary acidic protein (GFAP)
promoter resulted in transduction of almost exclusively
neurons in the spinal cord and in the hippocampus and
of a majority of neurons with only 5% astrocytes in
the striatum [5,27]. Similarly to the unspecific activity
of the MBP promoter inserted in an AAV vector (see
above), transcriptional elements present in the ITRs might
complicate the interpretation of studies with cell-typespecific promoters. Interestingly, in lesioned brain and
spinal cord, up to 15–30% of astrocytes were transduced
[5,27]. It remains to be determined whether these data
reflect a modified pattern of virus binding and uptake in
injured brain. Indeed, the GFAP promoter is known to be
up-regulated in reactive astrocytes in response to brain
injury [63].
Although the brain is a relatively immune privileged
site, strong immune and inflammatory responses after
intracerebral injection of some viral vectors have been
reported [64,65]. In contrast, no cellular immune
response to capsid proteins or transgene product has
been described following rAAV2-mediated gene transfer
to the brain [6,7]. Nevertheless, antibodies to both capsid
proteins or transgene product appeared at low levels at
2–4 months after injection but did not correlate with
a reduction in transgene expression [6]. Furthermore,
vector readministration was shown to be possible without
loss of efficiency if the interval between injections was
sufficient (at least 3 months) [7].
Vectors based on alternative serotypes
of AAV
AAV vectors based on serotypes 1, 4 and 5 show distinct
regional patterns of transduction and different cellular
tropisms than rAAV2.
When injected in the striatum, rAAV5 vectors transduced a greater number of cells, including both neurons
Copyright  2004 John Wiley & Sons, Ltd.
and a significant proportion of astrocytes, over a larger
volume of tissue than rAAV2. Transgene-expressing cells
were also observed at distance from the delivery site in
the septal region and neocortex [3]. In contrast, rAAV4
vectors injected into the striatum appear to transduce
almost exclusively ependymal cells [3]. Since rAAV2 and
rAAV4 particles contain nearly identical ITRs [60], differences between their preferential target cells must be
accounted for by the variations in their capsids and thus
in cellular factors required for viral particle uptake and
internalization. Indeed, in contrast to rAAV2, rAAV4- and
rAAV5-mediated transduction do not require heparan sulfate proteoglycan [66] but α-2,3-O-linked sialic acid and
α-2,3- or α-2,6-N-linked sialic acid, respectively [67,68].
The membrane receptor for AAV5 was recently identified
as PDGFR-α [69]. Recombinant AAV1 also has a higher
transduction efficiency and shows a wider distribution of
transduced cells in the striatum than rAAV2 [4]. Neurons were the predominant cell type transduced but glial
and ependymal cells were also susceptible. Receptors for
AAV1 have not yet been identified.
Strategic avenues for curing
neurological diseases by
AAV-mediated gene delivery
Enzyme replacement
Enzymatic deficiencies which affect the brain could
be targets for enzyme replacement by gene therapy.
For example, lysosomal storage diseases, such as
mucopolysaccharidosis, often provoke severe neurological
degenerations. In the cases when the lacking enzyme
supplied in the parenchyma can be taken up by deficient
cells, it is feasible to deliver the missing gene to a relatively
small number of cells while engineering the construct
in order that the transgene product is secreted in the
surrounding parenchyma [8–11]; for a review, see [30].
In Canavan disease, aspartoacylase (ASPA) gene deficiency leads to accumulation of N-acetyl-L-aspartic acid
(NAA) in the brain leading to spongiform degeneration.
AAV-mediated gene delivery of ASPA, even in a relatively
low number of cells, results in sufficient reduction of the
overall NAA amounts and correction of the deficiency
throughout the brain [12–14]; for a review, see [32].
In neurodegenerative diseases, when a specific cell
population is affected, supplying missing metabolic
enzymes could constitute a compensatory therapy. For
example, pathological features of Parkinson’s disease
(PD) principally include a loss of the dopaminergic (DA)
neurons in the mesencephalic SNc that massively projects
to the striatum. This results in a severe depletion of
striatal dopamine levels, mainly responsible for the motor
symptoms associated with the disease. The biosynthetic
pathway of dopamine involves tyrosine hydroxylase (TH)mediated transformation of dietary tyrosine into L-dopa
which, in turn, is converted into dopamine by aromatic
J Gene Med 2004; 6: S212–S222.
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L. Tenenbaum et al.
acid decarboxylase (AADC) [29]. Supplying TH [15] or
AADC [16], or both [17,18], directly in the striatum using
AAV vectors has been proposed for correction of dopamine
deficiency; for a review, see [31].
Neuroprotection
Neurotrophic factors (NF) protect neurons against
apoptosis induced by various insults. However, systemic
administration of recombinant NF has been hampered
by peripheral side effects and inability to cross the
blood–brain barrier. Since the half-life of recombinant
NF is usually short, local delivery in the CNS requires
high doses to be applied in a continuous manner,
which generally provokes adverse effects. Gene therapy
provides strategies for continuous and sustained NF
delivery at physiological doses, thus avoiding side effects
A.
of high doses and risks of cerebral infections related
to continuous administration. When the pathological
features result from the degeneration of mainly one
specific and localized neuronal population, viral-vectormediated delivery of neuroprotective factors in situ seems
feasible. PD is an excellent candidate for neuroprotective
gene therapy strategies, since the main symptoms of the
disease are provoked by the death of a single neuron
cell population: the dopaminergic neurons of SNc (for a
review, see [70]). The most potent neurotrophic factor
for dopaminergic neurons identified until now is ‘glial cell
line derived neurotrophic factor’ (GDNF), a member of the
transforming growth factor-β superfamily. In addition to
its survival promoting effect, GDNF stimulates sprouting
of dopaminergic terminals. Therefore, neuroprotective
gene therapy strategies for PD using GDNF were proposed
and evaluated in animal models (see Figure 2; for a
review, see [33]). A widely used model for PD consists
6-hydroxydopamine
CPu
FTM
Intact Lesioned
side
side
FTM
SN
SN
Rotational behavior
B.
AAV-GDNF
6-hydroxydopamine
CPu
FTM
FTM
FTM
SN
SN
SN
Rotational
behavior
C.
AAV-GDNF
6-hydroxydopamine
CPu
FTM
FTM
SN
FTM
SN
SN
Symetrical
behavior
Figure 2. Neuroprotective effect of glial cell line derived neurotrophic factor (GDNF) in the 6-hydroxydopamine-induced rat model
for Parkinson’s disease. (A) Unilateral injection of 6-hydroxydopamine, a toxin which destroys dopaminergic neurones of the
substantia nigra, induces a unilateral dopaminergic denervation of the striatum called hemiparkinsonism. Functional evaluation
of the lesion is performed by intraperitoneal injection of amphetamine, a drug which stimulates dopamine release in the intact
striatum, thus inducing a rotational behavior towards the lesioned side. (B) When injected into the substantia nigra prior to
administration of 6-hydroxydopamine, GDNF protects dopaminergic cell bodies but striatal innervation is lost. Consequently, there
is no effect on toxin-induced rotational behavior. (C) When injected in the striatum, GDNF protects both dopaminergic cell bodies
in substantia nigra and dopaminergic terminals in the striatum. Consequently, asymmetrical motor symptoms do not appear. These
experiments are described in [19]
Copyright  2004 John Wiley & Sons, Ltd.
J Gene Med 2004; 6: S212–S222.
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AAV Vectors in the CNS
of the injection of 6-hydroxydopamine (6-OHDA) into
the striatum of rats which results in partial destruction
of nigral dopaminergic neurons. Unilateral injections
of the toxin produce an asymmetric and quantifiable
motor behavior [71]. This allows an easy and reliable
control of the extent of the lesion and evaluation of the
potential benefit of therapeutic treatments. Surprisingly,
rAAV2-mediated GDNF delivery in SNc resulted in cell
protection but not in behavioral improvement [72].
In contrast, rAAV2-mediated GDNF delivery in the
striatum resulted in both an increase in the number
of dopaminergic neurons and an improvement in the
behavior [19]. Combined injections of rAAV-GDNF in both
the striatum and the SNc results in a reduced behavioral
improvement as compared with injections in the striatum
only. These data suggest that, in the absence of signals
necessary to reconstitute the nigro-striatal connections,
aberrant sprouting of dopaminergic neurons locally in the
SNc out competes directed sprouting of the remaining
fibers in the striatum; for a discussion, see [33]. In
contrast to GDNF, BDNF, another neurotrophic factor
for dopaminergic neurons, does not stimulate sprouting
but increases dopamine turnover. Consistently, an rAAVNSE-BDNF vector injected in the SNc after intrastriatal
injection of 6-hydroxydopamine prevented rotational
behavior, presumably by increasing dopamine metabolism
and release by the remaining dopaminergic fibers still
innervating the striatum [41].
AAV-mediated GDNF gene delivery has also been shown
to be effective for protection of striatal neurons in a model
for Huntington disease [20], cortical neurons in a model of
brain ischemia [23], as well as spinal cord motoneurons in
a mouse model for amyotrophic lateral sclerosis [21,73].
Recombinant AAV-mediated delivery of BDNF ameliorates
chronic pain after partial injury of the spinal cord nerves
[74]. Recombinant AAV-mediated delivery of BDNF and
of neurotrophin 3 combined with a Schwann cells implant
was shown to improve hind limb function in a model of
transected spinal cord [24].
Growth factors, such as IGF1 [21] or NGF [22],
also show interesting neuroprotective activities when
delivered by AAV2 vectors respectively via retrograde
transport in the spinal cord motoneurons [21] and in
the septal area [22], which opens perspectives for the
treatment of Alzheimer’s disease (AD).
Cell replacement therapies such as transplantation of
fetal mesencephalon in PD [75] could be combined with
gene therapy. We have shown that AAV2 vectors efficiently transduce freshly explanted fetal mesencephalon.
Furthermore, transgene expression was stable in vivo after
transplantation in the rat striatum until at least 3 months
post-transplantation [76]. This data suggests that rAAV
vectors could be used to genetically modify the fetal tissue prior to transplantation in order to promote graft
survival and functionality. The graft, which survives for
at least 10 years in patients [77], is furthermore an excellent cellular vector to deliver neuroprotective factors to
the host in order to halt the progression of the disease. Current trends are focused on neural progenitors or
Copyright  2004 John Wiley & Sons, Ltd.
stem cells that might provide an alternate source of cells
for neurotransplantation. However, after transplantation,
these cells are not stable and do not differentiate into
dopaminergic neurons. Gene transfer could be used to
enhance the survival and drive the differentiation of neural progenitors. It has recently been shown that rAAV2
vectors mediate efficient and stable gene expression in
human neural progenitors both in vitro and in vivo after
transplantation in the spinal cord [78].
Neurotransmitters and neuropeptides
Another interesting therapeutic avenue consists of
overproducing neurotransmitters to inhibit the firing of
abnormally activated neurons.
Epilepsy is an ideal target disease for strategies based on
attenuation of neuron excitability. For example, galanin
expression modulates both hippocampal excitability and
predisposition to epileptic seizures by counteracting
excess excitation in response to pathological stimuli.
This property has been exploited in a new strategy
consisting of the controlled delivery of a galanin coding
sequence fused with the fibronectin secretion signal in the
hypothalamus mediated by a tetracycline-regulated AAV
vector [25].
Modulation of neurotransmitter receptors could also
be a viable strategy. For example, reducing NMDA
receptor expression attenuates epileptic seizure sensitivity
[79]. The antisense NMDA receptor (NMDAR1) when
expressed from a CMV promoter in neurons of the inferior
colliculus reduces seizure sensitivity [26]. However,
the success of this strategy relies on the transduction
of a specific subpopulation of neurons. Indeed, when
using a tetracycline-responsive promoter which has a
different cellular tropism, increased seizure sensitivity
was observed.
The lack of dopamine in the striatum of PD patients
results in a deficit of the major inhibitory inputs in
the basal ganglia which in turn results in overactive
subthalamic nucleus and firing of substantia nigra
pars reticulata (SNr) in which neurons of the STN
project. Expression of glutamic acid decarboxylase (GAD)
mediated by an AAV2 vector in the STN results in release
of inhibitory GABA and inhibition of firing in SNr, thus
mimicking STN ablation or pharmacological silencing
already used in patients [37].
Control of transgene expression
For clinical applications, it is often desirable to limit
transgene expression to a defined time frame and/or
to precisely control its level. For example, excess
of neuroprotective factors is likely to cause severe
side effects. Indeed, motor disturbances resulting from
expression of the GDNF gene in normal animals have
been described [19]. In this study, rAAV-mediated
expression of GDNF in the right striatum of healthy
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L. Tenenbaum et al.
animals resulted in an asymmetrical behavior, presumably
due to an excess of dopamine in the injected side
relative to the intact side. Lesioning of the right
SNc of the rAAV-GDNF-treated animals by unilateral
injection of 6-OHDA restored a symmetrical behavior [19]
(Figure 3). Furthermore, in a partial dopaminergic lesion,
even though GDNF gene transfer initially resulted in
behavioral correction, long-term overexpression induced
aberrant sprouting and downregulation of TH [80].
These experiments demonstrate that the amount and
period of administration of neurotrophic factors should
be adjusted by taking into account the extent of
neurodegeneration.
Several regulatable systems are available for controlled
transgene expression in the CNS (for a review, see [81]).
These utilize a transactivator (usually chimeric) that
requires the addition of a drug to bind/detach to an
inducible promoter in order to switch ON/OFF transgene
expression. Regulatable systems with demonstrated
functionality in the brain are: the RU486 (mefipristone)
[82] and the rapamycin-inducible systems [83] placed in
an HSV vector, as well as the tetracycline-inducible [53]
and repressible systems [84,85] placed in an rAAV vector.
The tetracycline-regulated system is readily applicable
in the brain. Indeed, doxycycline, a tetracycline analog,
is used in the clinics for long-term treatment of a
brain infectious disease, brucellosis [86]. An example
of doxycycline-induced EGFP gene expression in the brain
driven by an AAV vector using the tetON system is shown
in Figure 4.
In addition, in order to avoid undesirable effects,
transgene expression should be restricted to the particular
cell type targeted. For example, expression of an NMDAR1
receptor antisense (see above) in the inferior colliculus
resulted in a decrease or an increase of seizure sensitivity
depending on whether the promoter used for antisense
transcription was the CMV or the tetracycline-responsive
promoter [26].
Conclusions
The fields of application for gene delivery in the CNS
are wide and concern disabilitating and life-threatening
diseases with enormous social impact for which there
are generally no or only symptomatic treatments. Gene
therapy offers new hopes for many of these incurable
diseases. For example, inherited enzymatic deficiencies
such as muccopolysaccharidosis and Canavan disease,
which are fatal at a young age, could be ameliorated by
A.
AAV-GDNF
6-OHDA
10 weeks
1 month
Symetrical behavior
Contralateral rotations
B.
AAV-GFP
6-OHDA
1 month
Symetrical behavior
10 weeks
Ipsilateral rotations
Figure 3. (A) rAAV-mediated expression of GDNF in the right striatum of healthy animals resulted in contralateral
amphetamine-induced rotations, presumably due to an excess of dopamine in the injected side relative to the intact side.
Lesioning of the right SNc of the rAAV-GDNF-treated animals by unilateral injection of 6-OHDA restored a symmetrical behavior.
(B) In contrast, the control rAAV-EGFP-treated group shows a symmetrical behavior 1 month post-injection. In this group, 6-OHDA
injection induced ipsilateral rotations. This experiment is described in [19]
Copyright  2004 John Wiley & Sons, Ltd.
J Gene Med 2004; 6: S212–S222.
S219
AAV Vectors in the CNS
A
B
C
D
Figure 4. Doxycycline-regulated gene expression in the rat brain.
A viral suspension containing rAAV-ptetbidi –EGFP was injected
into the rat globus pallidus. One month post-surgery, native
GFP was observed on brain sections examined by fluorescence
microscopy. Images were acquired using a constant exposure
time. Animals were fed with doxycycline-supplemented food (A,
C) or unsupplemented food (B, D). Bar: 50 µm (A, B) or 100 µm
(C, D). Reproduced from Chtarto et al. [53]
enzyme replacement. However, since these enzymes do
not cross the blood–brain barrier, systemic treatments
are limited to peripheral tissues and do not treat the
neurological manifestations of the diseases ([11] and
references therein). Gene delivery offers the possibility
to deliver missing enzymes locally and continuously
in the brain. Neurodegenerative diseases such as PD,
HD, ALS and AD are currently treated by compensatory
therapies that do not slow down the degeneration of
the affected neuron cell populations. Neurotrophic factors
have been shown to efficiently prevent neuronal cell death
but their administration is difficult to implement in the
clinics because their half-life is short and they do not
cross the blood–brain barrier. Intraparenchymal delivery
of recombinant GDNF via catheterization is currently
being evaluated in PD patients [88]. However, even if
successful, risks of cerebral infections and side effects
of high doses of recombinant GDNF [89] will limit the
large-scale applicability of this therapy. In this case also,
local neurotrophic gene delivery after a single viral vector
injection could ameliorate the outcome of the therapy.
AAV vectors are excellent candidates as tools for gene
therapy of neurological diseases. Indeed, they are able
to transduce post-mitotic cells, such as neurons [47,50],
astrocytes [51] and oligodendrocytes [61], which are
the main target cells in CNS disorders and they provide
sustained, long-term gene expression [44] which is
required to treat chronic diseases. Furthermore, AAV
vectors have a good safety profile [87] since they elicit
only low titer and transient neutralizing antibodies and
Copyright  2004 John Wiley & Sons, Ltd.
no inflammation when administered in the brain [44].
In addition, vector sequence integration is a rare event
resulting in a low risk of inadvertent oncogene activation
[48].
When rAAV viral suspensions are stereotaxically infused
in the brain, the extent of the transduced area depends
on the region targeted and on the serotype used [1,3,4].
Strategies to enhance diffusion of the viral suspension
include co-infusion with heparin which competes for
AAV2 binding to heparan sulfate proteoglycans [38,39],
convection-enhanced delivery [40], and multiple injection
sites [19]. Alternative AAV serotypes offer new hopes, in
particular AAV1 [4] and AAV5 [39], which transduce
much larger areas than AAV2.
In some applications, in which the transgene product
is a diffusible factor (e.g. neurotrophic factors, secreted
enzymes that can be taken up by neighboring cells,
etc.), the particular cell type transduced might not be
important, provided expression is efficient. In contrast, the
specific transduction of particular cell types is primordial
in certain situations, for example, when inhibition of
excitability of a particular type of neuron is the aim.
This is the case when the aim is to reduce the firing of
specific neurons, e.g., by antisense blocking of the NMDA
receptor during epileptic seizures [26]. Another example
is provided by the study of Luo et al. [37] who modified
specifically the glutamatergic neurons of the subthalamic
nucleus by an AAV vector expressing GAD in order to
release inhibitory GABA in the SNr, the structure in which
these neurons project. The cellular tropism of a particular
vector results from a combination of the various limiting
steps for transduction. Uptake and internalization of viral
particles depend on the presence of receptors and coreceptors which might be absent in certain cell types
or more abundant in others. Alternative AAV serotypes
having different requirements than AAV2 are likely to
show different cellular tropisms. For example, AAV5 using
PDGFR is likely to harbor a different cellular tropism than
AAV2. When the entry of the virus is not restrictive, tissuespecific promoters might help to target a specific cell
type. For example, the use of the MBP promoter allowed
rAAV2-mediated transduction to be targeted specifically
into oligodendrocytes in the corpus callosum [61].
However, when the models are further refined to meet
clinical requirements, it will be primordial to regulate
transgene expression. This will be important, for example,
when modulating neuron excitability (e.g. by blocking
NMDA receptor [26]) or when supplying biosynthetic
enzymes for neurotransmitters (e.g. TH and AADC to
synthesize dopamine). Neuroprotection also needs fine
regulation. Indeed, most neurotrophic factors not only
protect neurons, but also modify neuronal activity. For
example, GDNF enhances neuron terminal sprouting
([33] and references therein), and BDNF accelerates
dopamine metabolism ([41] and references therein). Thus
NF need to be expressed only during a defined period
and in controlled concentrations. The use of regulatable
vectors in the brain has already been described. However,
these are still under development and currently encounter
J Gene Med 2004; 6: S212–S222.
S220
some limitations such as reaching high concentrations of
inducer in the brain, and a basal expression level too
high to avoid biological effects in the non-induced state
[53,81].
L. Tenenbaum et al.
15.
16.
Acknowledgements
E.L. and A.C. are recipients of fellowships from the Belgian
National Research Foundation (F.N.R.S./Télévie). This work
was also supported by EC grant BIO-CT97-2207, by grants
from the Belgian National Research Foundation (FNRS-FRSM,
3.4565.98 and 3.4619.00), and by grants from ‘Société Générale
de Belgique’, ‘Banque Nationale de Belgique’ and ‘BruxellesCapitale’.
17.
18.
19.
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