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Gene Therapy (2011), 1 - 11
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ORIGINAL ARTICLE
In utero administration of Ad5 and AAV pseudotypes to the
fetal brain leads to efficient, widespread and long-term gene
expression
AA Rahim1,6, AM Wong2,6, S Ahmadi2, K Hoefer2, SMK Buckley3, DA Hughes3, AN Nathwani3, AH Baker4, JH McVey5, JD Cooper2
and SN Waddington1
The efficient delivery of genetic material to the developing fetal brain represents a powerful research tool and a means to
supply therapy in a number of neonatal lethal neurological disorders. In this study, we have delivered vectors based upon
adenovirus serotype 5 (Ad5) and adeno-associated virus (AAV) pseudotypes 2/5, 2/8 and 2/9 expressing green fluorescent
protein to the E16 fetal mouse brain. One month post injection, widespread caudal to rostral transduction of neural cells was
observed. In discrete areas of the brain these vectors produced differential transduction patterns. AAV2/8 and 2/9 produced the
most extensive gene delivery and had similar transduction profiles. All AAV pseudotypes preferentially transduced neurons
whereas Ad5 transduced both neurons and glial cells. None of the vectors elicited any significant microglia-mediated immune
response when compared with control uninjected mice. Whole-body imaging and immunohistological evaluation of brains
9 months post injection revealed long-term expression using these non-integrating vectors. These data will be useful in
targeting genetic material to discrete or widespread areas of the fetal brain with the purpose of devising therapies for early
neonatal lethal neurodegenerative disease and for studying brain development.
Gene Therapy advance online publication, 10 November 2011; doi:10.1038/gt.2011.157
Keywords: adenovirus; adeno-associated virus; fetal brain; pseudotypes; gene delivery
INTRODUCTION
The efficient delivery of genetic material to cells of the central
nervous system (CNS) during fetal development has useful
applications and represents a powerful research tool. Transduction
of discrete areas of the brain and subsequent expression of
cytotoxic molecules, short hairpin RNA or dominant-negative
forms of proteins could mimic neurodegenerative human disease
in animals without the need to generate transgenic or knockout
models. Furthermore, transduction of specific cell types during
gestation may allow for informative studies of fetal brain
development.
A number of CNS diseases present progressive pathology
during gestation in patients and animal models.1,2 Delivery of
therapeutic genes to the fetal brain of such models would
potentially answer a number of fundamental questions that need
addressing for potential therapies to be devised. For example,
does expression of therapeutic protein during gestation increase
lifespan and is neonatal intervention too late? In the case of
neurodegenerative disease, this is of particular importance
because damaged neurons of the CNS have a very limited
capacity to regenerate. The vast majority of viral vector-mediated
gene-delivery studies to the CNS have been in adult3 - 5 and, to a
lesser extent, neonatal6 - 9 animal models. This has been done
using a broad range of viral vectors with differential serotypes and
pseudotypes resulting in variable tropisms and transduction
efficiencies. These studies have highlighted the need for specific
cellular receptors to be available on cell membranes for
transduction by a particular viral vector to occur. However, the
availability or levels of expression of such receptors has been
shown to vary significantly during different stages of development.10 Therefore, studies of transduction/tropisms in adult or
neonates may have no relevance in the fetal brain.
The concept of viral-mediated gene delivery to the fetal brain is
not a new one and dates back to the 1980s.11 While providing
interesting data, these investigations invariably included only one
viral vector type. This makes independent comparisons between
vector tropisms and gene-delivery efficiencies in the fetal brain
difficult. We have shown previously that an integration-deficient
lentivirus can mediate efficient long-term gene delivery to the
fetal brain with no significant immune response and enhanced
viral spread compared with the adult brain.12 These findings
support the use of non-integrating vectors for CNS gene delivery
to neurons given their postmitotic status and the reduced risk of
insertional mutagenesis. However, for the application of gene
delivery to the fetal brain to be fully realised, more studies are
required that address the above issues and build upon the
advantages that this technique offers. In this study, we present
data that expand upon our previous work to include other
commonly used non-integrating viral vectors and their various
pseudotypes. This provides the opportunity for direct comparisons
and selection of appropriate vectors for a variety of downstream
applications.
1
Gene Transfer Technology Group, Institute for Women’s Health, University College London, London, UK; 2Paediatric Storage Disorder Laboratory, MRC Centre for
Neurodegeneration, Institute of Psychiatry, Kings College London, London, UK; 3Department of Haematology, University College London, London, UK; 4BHF Glasgow
Cardiovascular Centre, Univeristy of Glasgow, Glasgow, UK and 5Molecular Medicine, Thrombosis Research Institute, London, UK. Correspondence: Dr SN Waddington, Institute
for Women’s Health, University College London, 86-96 Chenies Mews, London WC1E 6HX, UK.
E-mail: [email protected]
6
These authors contributed equally to this work.
Received 18 July 2010; revised 15 July 2011; accepted 17 August 2011
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
2
RESULTS
Widespread and differential patterns of gene delivery to the fetal
brain using non-integrating viral vectors
To assess the ability of a range of non-integrating viral vectors to
transduce cells of the fetal murine brain, vector preparations of
adenovirus serotype 5 (Ad5), self-complementary adeno-associated virus pseudotyped with capsids from AAV5, AAV8 and AAV9
were produced, concentrated and titered. All vectors express
enhanced green fluorescent protein (EGFP) driven by the
cytomegalovirus promoter (Figure 1). Although titres between
Ad5 and AAV vectors were not standardised, the different
serotypes of AAV were normalised to allow for direct comparisons
between the different pseudotypes. The titre of the Ad5 virus
stock used was 2 1011 viral genomes per ml and AAV2/5, AAV2/8
and AAV2/9 was 7 1011 viral genomes per ml.
In all, 5 ml of each virus (Ad5 ¼ 1 109 viral genomes and AAV2/5,
AAV2/8 and AAV2/9 ¼ 3.5 109 viral genomes) was injected, in
utero, into the brains of fetal MF1 mice at embryonic (E) day 15
(n ¼ 3 per dam). The injected mice were labelled, subcutaneously,
with colloidal carbon to differentiate them from their uninjected
littermates. Once born, these mice were reared until postgestation (P) day 25 after which they were culled and the brains
were removed, fixed and sectioned. All mice that received
injections of virus were accounted for and easily identifiable by
the colloidal carbon marking. No procedure-related mortality was
observed at any point. EGFP expression was detected using antiEGFP antibodies and 3,30 -diaminobenzidine (DAB) staining.
Examination of the brain sections by light microscopy showed
varying levels of cell transduction present in both the cerebral
hemispheres (Figure 2). One of the brains injected with AAV2/9
showed no GFP immunoreactivity and so was classified as a failed
injection. Representative images were taken from the level of the
pre-frontal cortex, striatum, caudal hippocampus and cerebellum
of uninjected control brain (Figures 2a -- d) and brains injected with
Ad5 (Figures 2e -- h), AAV2/5 (Figures 2i -- l), AAV2/8 (Figures 2m -- p)
and AAV2/9 (Figures 2q -- t). Efficient and widespread transduction
was seen in the brains transduced with AAV2/8 and AAV2/9. Gene
expression was apparent in the brains injected with Ad5 but was
visibly less widespread. In comparison, AAV2/5-transduced cells
were sparsely distributed and this vector was clearly less efficient
at transducing neural cells.
To gauge whether fetal administration can enhance dissemination of the vector through the brain compared with neonatal
administration, we administered three P1 neonates with 5 ml of
Ad5-GFP via intracranial injection. One month post injection, the
brains were harvested and analysed by immunohistochemistry for
GFP expression, as performed on the fetal-administered brains. A
direct comparison between fetal (Figures 2e -- h) and neonatal Ad5GFP-administered brains (Figures 2y -- B) revealed less GFP staining
in the latter group within the contralateral hemisphere when
compared with the fetal-injected brains. Furthermore, there was
also less spread towards rostral and caudal areas of the brain in
these neonatally injected mice. This was best exemplified in the
cerebellum where in utero injection resulted in gene expression
around the fourth ventricle but also extensive staining in the
cerebellar lobes (Figure 2h). However, neonatal administration
resulted only in staining restricted to around the fourth ventricle
(Figure 4b). The limited spread of virus in neonatal-administered
brains compared with fetal-administered brains was consistent in
all the brains examined (Supplementary Figure S1).
Further inspection of brain sections at higher magnification
revealed both stark and subtle differences in the cellular tropisms
of the different vectors. Figure 3 shows transduction patterns in
the hippocampus, somatosensory barrel field, piriform cortex and
the cerebellar lobes. The cornu ammonis (CA1 -- 3) subfields of the
hippocampus were transduced to variable levels of efficiency,
dependent upon the vector used, by Ad5 and AAV pseudotypes
Gene Therapy (2011), 1 -- 11
Deleted 3’
TRS
a
ITR
GFP
CMV
GFP
Poly A
Poly A
ITR
CMV
b
GFP
ITR
CMV
ITR
Poly A
c
ITR
Luc
CMV
ITR
Poly A
Figure 1. Schematic representation of viral vector constructs. Genedelivery and -expression studies in the fetal brain were conducted
using AAV pseudotypes 2/5, 2/8 and 2/9 and Ad5. All AAV vectors
were in the self-complementary (sc) format and encoded GFP driven
by the cytomegalovirus (CMV) promoter (a). Two Ad5 viruses were
used; one expressing GFP driven by the CMV promoter and the
other expressing luciferase (Luc), also driven by the CMV promoter
(b and c, respectively). ITR, inverted terminal repeat; Poly A,
polyadenylation sequence; TRS, terminal repeat.
2/5, 2/8 and 2/9 (Figures 3a -- d). However, while the AAV vectors
preferentially transduced neurons, Ad5 also transduced cells of
glial morphology (indicated by white arrows). Transduction
patterns in somatosensory barrel field showed subtle differences
in transduction of cortical layers. Ad5 transduction was restricted
to cortical layer II neurons and scattered glial cells (Figure 3e),
whereas AAV pseudotypes 2/5, 2/8 and 2/9 transduced neurons
throughout cortical layers II -- VI (Figures 3f -- h). These cellular
tropisms were also observed in the motor and cingulate cortex
(data not shown). Examination of the piriform cortex revealed no
transduction by Ad5 (Figure 3i). However, all three AAV serotypes
mediated gene delivery to cells in this area (Figures 3j -- l).
Differential transduction patterns were also seen in the cerebellum
(Figures 3m -- p). Ad5 transduced cells in lobes 1 -- 5 but not the
simple lobule (sim) whereas AAV2/5 transduction was restricted to
lobes 3 -- 5, but also sim. Both AAV pseudotypes 2/8 and 2/9
transduced lobes 1 -- 5 and sim.
By selecting discrete areas of these sections and measuring GFP
immunostaining intensity by threshold analysis, a semiquantitative comparison of viral vector-mediated gene expression was
conducted. GFP staining was measured in the CA1 region of the
hippocampus (CA1), striatum (CPu), primary motor cortex (M1),
medial septal nucleus (MS), piriform cortex (Pir), ventral posteromedial and ventral posterolateral thalamic nucleus (VPM/VPL)
and the cerebellar nodule (10Cb). Similar to what we had
qualitatively seen, the levels and patterns of gene expression
varied depending upon the vector used (Figure 3u). All vectors
mediated higher levels of gene expression in the CA1 region of
the hippocampus and the MS, but comparatively lower levels in
the CPu and M1. All vectors produced high levels of expression in
the Pir except for Ad5, which produced no detectable GFP
expression. Lower amounts of GFP were detected in the VPL/VPM,
except for in AAV2/5-administered brains where expression was
higher. Conversely, AAV2/5 produced the lowest levels of GFP
measured in the 10Cb when compared with the other vectors.
Interestingly, the amounts of GFP expression produced by AAV2/8
and 2/9 were comparable in all brain regions.
& 2011 Macmillan Publishers Limited
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
3
Pre-Frontal
Cortex
Striatum
Hippocampus
Cerebellum
-ve
Ad5
In utero
AAV
2/5
AAV
2/8
AAV
2/9
Neonatal
-ve
Ad5
Figure 2. Immunohistochemical detection of GFP expression 1 month post in utero and neonatal intracranial injection of viral vectors. Fetal
mice at embryonic day 15 (E15) were administered with Ad5 (1 109 vector genomes) and AAV pseudotypes (3.5 109 vector genomes)
expressing GFP via in utero intracranial injection (n ¼ 3 per dam). The mice were born and reared until post-gestation day 25 (P25). The brains
were harvested, sectioned and immunohistochemically analysed for detection of GFP. Representative images were taken from the pre-frontal
cortex, striatum, hippocampus and the cerebellum. All brains administered with virus demonstrated varying levels of GFP immunoreactivity
except for one of the AAV2/9 brains that was designated as a failed injection. Images are taken from the brain sections of uninjected mice as
negative controls (n ¼ 3) (a -- d), mice injected with Ad5 (n ¼ 3) (e -- h), AAV2/5 (n ¼ 3) (i -- l), AAV2/8 (n ¼ 3) (m -- p) and AAV2/9 (n ¼ 2) (q -- t). P1
neonatal mice were also intracranially injected with the same volume and number of viral particles of Ad5 (n ¼ 3). Age-matched uninjected
mice were used as a negative control. The mice were culled 1 month post injection and immunohistochemically stained, as above.
Representative images were taken from the brain sections of uninjected mice (u -- x) and Ad5-injected mice (y -- B).
A broader qualitative analysis of transduction patterns in
discrete areas of the brain was conducted and scored with ()
indicating no transduction, ( þ ) a few transduced cells, ( þ þ ) a
high number of transduced cells and ( þ þ þ ) a region that was
saturated with transduced cells (Table 1).
Confirmation of viral vector neural cell-type tropism by
immunofluorescence and scanning confocal microscopy
Based on the morphology of GFP immunoreactive cells, all AAV
vectors preferentially transduced neurons, but Ad5 transduced
& 2011 Macmillan Publishers Limited
cells of both neuronal and glial morphology. To confirm these cell
tropisms, brain sections were immunofluorescently stained for
both GFP- and cell-specific phenotypic markers, and examined by
multi-channel scanning confocal microscopy. Ad5-transduced
brain sections were stained with TO-PRO-3 to label cell nuclei
(Figure 4e), NeuN to label neurons (Figure 4f) and anti-GFP
antibodies to identify transduced cells (Figure 4g), revealing the
expression of GFP in a subset of NeuN-positive neurons
(Figure 4h). The larger GFP-positive cells with glial morphology
were negative for NeuN staining (Figure 4f). These GFP-positive
cells were confirmed as astrocytes by colocalisation with for the
Gene Therapy (2011), 1 - 11
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
4
x1.6
Hippocampus
S1BF
Piriform
Cortex
Cerebellar
Lobes
Ad5
1000 m
1000 m
1000 m
1000 m
AAV2/5
AAV2/8
% Immunoreactivity
AAV2/9
22.00%
20.00%
18.00%
16.00%
14.00%
12.00%
10.00%
8.00%
6.00%
4.00%
2.00%
0.00%
Ad5
AAV2/5
AAV2/8
AAV2/9
CA1
CPu
M1
MS
Pir
VPLVPM
10Cb
Figure 3. GFP expression in discrete areas of the brain. Immunohistochemically stained brains for the detection of GFP expression were
examined by light microscopy. Representative images were taken from the following discrete regions: hippocampus, somatosensory barrel
field (S1BF), piriform cortex and cerebellar lobes. Images show the regions of the brains that were administered with Ad5 (a -- e), AAV2/5 (f -- j),
AAV2/8 (k -- o) and AAV2/9 (p -- t). All vectors transduced cells of neuronal morphology although Ad5 also transduced larger glial cells, as
indicated by white arrows. A quantitative comparison of GFP expression by threshold analysis of brain sections was conducted in the
following discrete regions: CA1 region of the hippocampus (CA1), striatum (CPu), primary motor cortex (M1), medial septal nucleus (MS),
piriform cortex (Pir), VPL/VPM and the cerebellar nodule (10Cb) (u). The data are plotted as the mean±s.e.m., n ¼ 3 for Ad5-, AAV2/5- and
AAV2/8-injected brains, and n ¼ 2 for AAV2/9.
astrocyte-specific marker S100b (Figure 4l). To confirm the
predominantly neuronal tropisms of the AAV pseudotypes, brain
sections from injected mice were also immunofluorescently
stained with TO-PRO-3, NeuN and anti-GFP antibodies. Very few
GFP-positive cells were detected in sections from AAV2/5-injected
brains (Figure 4o), which reflected the low levels of GFP expression
observed with DAB staining in somatosensory barrel field
(Figure 3c) and measured in other areas of the cortex such as
M1 (Figure 3u). However, those GFP-positive cells that were
detected were also NeuN stained (Figure 4p). A similar colocalisation of GFP and NeuN staining was also evident in brains injected
with AAV2/8 and 2/9 (Figures 4p and t). In contrast, uninjected
brains showed no detectable GFP staining (Figure 4c).
Analysis of immune system activation
As immune responses to vectors and to the high levels of GFP
expression may occur, we looked for any evidence of activated
microglia in all brains using antibodies against CD68. As a positive
control, brains were taken from Ppt1/ mice that display intense
microglial activation as part of their pathological neuronal ceroid
lipofuscinosis phenotype.13 As previously reported,13 the motor
cortex of Ppt1/ mice revealed intense CD68 immunoreactivity
(Figure 5a), but there was no obvious increase in staining for this
marker of microglial activation in the brains injected with any of
Gene Therapy (2011), 1 -- 11
the viruses (Figures 5c -- f), compared with uninjected brains
(Figure 5b). Higher-power examination of the M1 motor cortex
revealed microglia in a quiescent state in uninjected and vectoradministered brains compared with activated and swollen
microglia in the Ppt1/ brains (inset boxes). To quantify the
levels of staining, threshold analysis was performed on these
CD68-stained sections. Analysis of variance followed by Bonferoni
correction revealed that microglial activation was significantly
higher in Ppt1/ mice than in uninjected or virus-injected brains.
No significant difference in the levels of microglial activation was
seen between the brains that received viruses and uninjected
control brains (Figure 5g).
To further evaluate whether perinatal administration induced
immune tolerance, fetal and neonatal mice were intracranially
injected with Ad5-GFP and uninjected mice served as naive
controls. Three months later all mice were challenged by intraperitoneal injection of Ad5-GFP. Mice that received neither intracranial nor intraperitoneal Ad5-GFP served as negative controls. One
month later mice were culled and blood plasma was collected and
analysed for antibodies against GFP using SPR-biosensor immunoassay shown in Supplementary Figure S2. There was no
evidence of anti-GFP antibodies in serum of mice challenged at
3 months following fetal or neonatal intracranial administrations
of Ad5-GFP compared with serum from uninjected unchallenged
controls. However, even naive mice challenged by intraperitoneal
& 2011 Macmillan Publishers Limited
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
5
Figure 4. Immunofluorescent and scanning confocal microscopy studies of viral vector neural cell tropisms. Brain sections from injected mice
were immunofluorescently stained using cell-specific markers and anti-GFP antibodies. Sections from uninjected brains were used as a
negative control and stained with TO-PRO-3 to label cell nuclei (blue), NeuN to label neurons (red), anti-GFP antibodies (green) and a merged
image (a -- d, respectively). No non-specific anti-GFP staining could be detected (c). Ad5-administered brain sections were stained with
TO-PRO-3 (e) and NeuN (f ). Anti-GFP staining revealed expression in two morphologically distinct cell types; the vast majority in smaller cells
with neuronal morphology and fewer larger cells with glial morphology (g). The merging of images revealed colocalisation of GFP and NeuN
resulting in yellow signal and confirming them as neurons (h). Examples of colocalisation within cells are highlighted with white arrows. The
larger glial cells were negative for NeuN staining. To identify these cells, sections were stained with TO-PRO-3 (i) and S100b to label astrocytes
(red). When images of S100b labelled cells (j) were merged with images of GFP expressing cells (k), colocalisation of signals confirmined these
cells to be astrocytes (l). AAV2/5-injected brain sections were also stained with TO-PRO-3 (m) and revealed low levels of GFP staining and
occasional bright cells (o) that colocalised with NeuN-stained cells (n) and confirmed neuronal tropism (p). AAV2/8 and AAV2/9-administered
brain sections were stained with TO-PRO-3 (q and u, respectively) and revealed strong staining with anti-GFP antibodies (s and w, respectively)
that colocalised with NeuN-stained cells (r and v, respectively) producing yellow signal (t and x, respectively) and confirming the neuronal
tropism of these vectors.
& 2011 Macmillan Publishers Limited
Gene Therapy (2011), 1 - 11
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
6
Table 1. Qualitative analysis of vector transduction patterns in
discrete areas of the brain
Ad5
AAV2/5
AAV2/8
AAV2/9
Brain regions
Cingulate cortex
Motor cortex
Somatosensory cortex
Piriform cortex
Lateral ventricles
Caudate putamen
Pyramidal hippocampal layers
Dentate gyrus
CA1-3
Oriens layer
Hypothalamus
Posterior thalamic nuclear group
Intermediodorsal thalamic nucleus
Central lateral thalamic nucleus
Lateral amygdaloid nucleus
+
+
+
+
+
+
+
++
++
+
+
+
++
+
+
+
++
+
++
++
++
++
++
++
++
+++
+
+
+
+
+++
++
+++
+
++
++
++
++
++
+++
++
++
++
++
+
+
++
+++
+++
++
Cerebellum
Lobe 1
Lobe 2
Lobe 3
Lobes 4 and 5
Sim
Facial nucleus
++
++
++
++
+
+
+
++
++
++
++
++
+
++
++
++
++
++
+
A qualitative analysis of the transduction patterns in the brains
administered with the different vectors was conducted. The levels of
transduction in discrete areas of the brain were graded as: () no
transduction, (+) a few transduced cells, (++) a high number of transduced
cells and (+++) a region that was saturated with transduced cells.
injection of Ad5-GFP developed no detectable antibody response.
As a positive control, commercial anti-GFP antibodies produced a
large shift in relative binding and enhancement in the SPR
immunoassay.
Sustained long-term expression using non-integrating viral vectors
To assess long-term vector-driven gene expression in the brain,
fetal mice were intracranially injected with AAV2/9 and Ad5
vectors expressing GFP and firefly luciferase, respectively (Figures
1a and c). The mice injected with Ad5 were imaged at P21, 2.5
months and 12 months post injection by whole body bioluminescence imaging (in vivo imaging system (IVIS); Caliper Life Sciences,
Hopkinton, MA, USA). Although there was a marked drop in the
intensity of luciferase expression between P21 and 2.5 months,
the expression was sustained and localised within the brain
(Figure 6a). The mice administered with AAV2/9 were culled 9
months post injection. The brains were harvested, fixed and
sectioned for GFP immunohistology, as described earlier. GFPstained cells were still present throughout the brain (Figures 6b -- f),
although a qualitative microscopic survey revealed a marked
decrease in expression when compared with that seen after 1
month using this vector (Figures 3p -- t). Although this general
decrease in expression was present throughout the brain, it was
region specific with the piriform cortex retaining relatively high
levels of expression compared with the somatosensory barrel field.
DISCUSSION
The efficient delivery of genetic material to the fetal brain has
many valuable downstream applications. MicroRNAs have a crucial
role in CNS development and neurodegenerative diseases.14 - 16
Expression of candidate miRNAs, through viral vector delivery to
the brain, is a method of elucidating their roles in these events.
For example, AAV-mediated overexpression of miR-134 in
Gene Therapy (2011), 1 -- 11
postnatal brains confirmed its role in dendritic arborisation of
pyramidal neurons in cortical layer V.17 Such an approach could be
used to elucidate the role of different miRNA sequences in the
developing fetal brain. Conversely, vectors expressing ‘microRNA
sponges’ could be used to inhibit miRNA sequences.18 In a similar
manner, vectors expressing short hairpin RNA could be administered to the fetal brain to knockdown a gene’s expression and
evaluate its function in development. This would have particular
relevance if the subsequent phenotype provided a useful model
for neurodegenerative disease, circumventing the need for
producing complex transgenic models. A similar strategy could
be applied by delivering a dominant-negative form of a gene.
Gene delivery to the fetal brain is also a powerful tool for
investigating the feasibility of therapeutic approaches in early
neonatal lethal neurodegenerative diseases. Acute neuronopathic
type II Gaucher disease is such an example; irreversible disease
pathology is present in the patient’s brain during gestation2,19,20
and death usually occurs before 2 years of age.21 The delivery of
therapeutic genes to the brains of mouse models of such diseases
during gestation could provide vital information; is in utero
intervention required to prevent irreversible brain damage and
whether lifespan can be enhanced? The unique advantages that
in utero gene delivery offers for the study of neonatal lethal
diseases have been discussed in the following reviews.22,23
For all the above applications to be realised, the correct genedelivery vector needs to be utilised. This requires a thorough
understanding of viral transduction efficiency, spread, tropism
(tissue type, regional and cellular) and immune response. On the
basis of our previous work using non-integrating lentiviral vectors,
the post mitotic status of neurons and reduced risk of insertional
mutagenesis, we decided to expand the study to other nonintegrating viral vectors, adenovirus and AAV.
Viral transduction is dependent upon the expression of suitable
receptors on the cell membrane. The coxsackievirus and
adenovirus receptor (CAR) is the primary site of cell interaction
for Ad5. Studies have shown that the levels of CAR expression in
the brain decline significantly from the embryonic stage to
adulthood in mice.10,24 Coxsackievirus B5, which also uses CAR as
its primary receptor, demonstrated higher levels of transduction in
embryonic immature neurons than mature neurons and correlated with the downregulation of CAR with age.25 Examination of
CAR expression in human adult brains revealed low levels to no
expression with only the choroid plexus and pituitary gland
showing higher concentrations.26 On the basis of this evidence,
Ad5 could be viewed as a poor choice for gene delivery to the
adult brain, but well suited to the fetal brain. We observed
efficient gene delivery with long-term expression detectable a
year after administration that is consistent with similar duration of
expression seen in adult mouse brains.27 However, expression was
less widespread in the brain compared with the AAV pseudotypes
and more confined to specific areas. Interestingly, gene delivery to
the cerebellum and CA1 hippocampus using Ad5 was comparable
to that observed with the most efficient AAV2/8 and AAV2/9
vectors. No significant Ad5 microglia-mediated immune response
was detected in comparison with the AAV pseudotypes and
uninjected control mice. Although transduction was predominantly neuronal, sparse astrocyte transduction could also be seen
with this vector.
The receptors for AAV serotypes are comparatively less studied,
primarily due to their more recent introduction as gene delivery
vectors. All AAV pseudotypes used in this study resulted in
widespread gene delivery with AAV2/8 and 2/9 being the most
efficient. Gene expression was detected from rostral to caudal
regions of the brain and in both the hemispheres. Furthermore,
both pseudotypes had similar transduction profiles. This may be
attributed to the common putative laminin receptor that they
share.28 More recently, N-terminal linked galactose has been
characterised as a major receptor for AAV9.29 AAV serotypes 8 and
& 2011 Macmillan Publishers Limited
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
7
Thresholding Intensity
Ppt1-/-
6.00%
5.00%
Un-Injected
Ad5
AAV2/5
AAV2/8
AAV2/9
*
4.00%
3.00%
2.00%
1.00%
0.00%
Ppt1-/-
Un-Injected
Ad5
AAV2/5
AAV2/8
AAV2/9
Figure 5. Analysis of microglia activation by CD68 immunohistochemistry. Brain sections from injected and control uninjected mice were
probed with antibodies against the activated microglia marker CD68 and detected using DAB staining. As a positive control, sections of brains
from Ppt1/ mice that are known to have an activated microglia response were also included. Representative images were taken of the M1
motor cortex from the positive control Ppt1/ mouse (a), an uninjected negative control mouse (b) and mice injected with Ad5 (c), AAV2/5
(d), AAV2/8 (e) and AAV2/9 (f ). High magnification representative images of microglia are shown in the inset boxes. Quantitative measurement
of staining intensity of the sections was conducted by threshold image analysis (g). Data are plotted as the mean±s.d., n ¼ 3 for Ad5-, AAV2/5and AAV2/8-injected brains, n ¼ 2 for AAV2/9. The Ppt1/ mice exhibited significantly higher microgliosis compared with all injected mice,
which were indistinguishable from the uninjected control (*Po0.05, one-way analysis of variance with Bonferroni post hoc correction).
9 have been shown to efficiently transduce cells in the neonatal
mouse7 and adult non-human primate brains.30 These data, taken
together with our findings, suggest that the receptor required for
AAV2/8 and 2/9 transduction is present from gestation through to
adulthood in mice.
Gene delivery was comparatively inefficient using pseudotype
AAV2/5. This is consistent with the findings of Lubansu et al.
where AAV serotype 5 was shown to be ineffective in transducing
rat embryonic (E15) ganglionic eminence cultures. These data are
in contrast to studies showing that AAV serotype 5 was efficient at
transducing cells in adult mouse31,32 and non-human primate
brain.33,34 This discrepancy suggests that the availability of AAV5binding sites, such as 2,3-linked sialic acid,35 and potential
receptors may vary from gestation through to adulthood.
The mechanism by which AAV2/8 and AAV2/9 are able to
efficiently transduce widespread areas of the brains, when
compared with Ad5 or AAV2/5, remain unclear. Two plausible
explanations for these observations can be proposed. The first is
that the receptors required for transduction by either AAV2/8 or
AAV2/9 are more prevalent and expressed at higher concentrations on the neuronal cell membranes, although this needs to be
confirmed experimentally in the fetal brain. The second possibility
is that these viruses have the ability to transport their genomes
along axonal projections to areas distal to the site of injection.
Cearley et al.36 showed that stereotaxic injection of AAV9 into the
hippocampus of adult mice brains in one hemisphere resulted in
detection of marker gene RNA and protein in the contralateral
hippocampus. Furthermore, this was also detected in other distal
& 2011 Macmillan Publishers Limited
areas of the brain that were connected to the ipsilateral injected
hippocampus via axonal projections such as the septal nuclei and
entorhinal cortex.
The potential immune response to any viral vector administered
to the fetal brain is an important consideration. Whether the
vector is being utilised to investigate the brain development or a
disease model, microglial activation is not desirable as this could
interfere with the readout and limit efficacy. Importantly, we did
not see any significant microglia activation in the brains injected
with viruses when compared with uninjected control brains.
Our SPR immunoanalysis of GFP antibody production, following
fetal and neonatal inoculation by intracranial injection of Ad5-GFP
followed by challenging via intraperitoneal injection of the vector
3 months later, was inconclusive. No anti-GFP antibodies were
detected in those mice receiving intracranial Ad5-GFP in the fetal
or neonatal period and challenged at 3 months. However, even
naive mice challenged at 3 months showed no antibody response
either. Therefore, this suggests that to interrogate the immune
status of mice injected intracranially in the fetal or neonatal
period, it would be important to choose a more immunogenic
transgene from which an immune response in challenged naive
mice is guaranteed, as we have shown for human factor IX.37
IVIS whole body imaging at P21 and 2.5 months, and 12 months
post injection revealed sustained Ad5-mediated luciferase gene
expression. Interestingly, there was a marked drop in luciferase
expression between the P21 and 2.5 months time point. Although
the reason for this is unknown apart from the possibility that this
represents real loss of expression, progressive thickening of the
Gene Therapy (2011), 1 - 11
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
AAV2/9 - 9 months
1000 m
Ad5-Luc 12 Months
Ad5-Luc 2.5 Months
Ad5-Luc P21
8
Figure 6. Long-term gene expression in brains administered with Ad5 and AAV2/9. Fetal mice administered with Ad5 expressing luciferase
and AAV2/9 expressing GFP (n ¼ 3) via in utero intracranial injection were reared after birth for periods of 1 year and 9 months, respectively.
The mice administered with Ad5 were examined using the IVIS to detect luciferase expression at P21, 2.5 months and 12 months post
injection and representative images are shown (a). Mice that received AAV2/9 were culled and the brains were harvested and sectioned for
detection of GFP expression by immunohistochemistry. Representative images are shown of lower magnification of sections showing the
cerebral cortex, hippocampus and the thalamus (b) and higher magnification images of the hippocampus (c), somatosensory barrel field
(S1BF) (d), pirifom cortex (e) and cerebellar lobes (f ).
cranium may be one explanation. An alternative explanation is
that even if gene expression was maintained at constant levels,
the increasing volume of the brain from 21 days to 12 months
would cause an increase in photon absorption, and thus a
dampening of signal. Examination of AAV2/9-mediated GFP
expression 9 months post injection also revealed GFP-positive
cells present in all areas examined at 1 month post injection.
However, there was a visible decrease in staining intensity. Given
the preferential transduction of neurons by all viruses and the
postmitotic status of these cells, we propose that methylation of
the cytomegalovirus promoter may be responsible for this
reduced GFP staining. Although the cytomegalovirus promoter is
known to induce high levels of gene expression, methylation of
CpG motifs leading to gene silencing and loss of expression over
time is well documented.38,39 Therefore, if longer periods of
sustained expression are required, then alternative, recently
described promoters such as the mammalian b-glucuronidase
minimal promoter40 or the ubiquitous chromatin-opening element39 may be more suitable. Furthermore, although we have
looked at the levels of expression mediated by each virus in
discrete areas of the brain, the actual number of viral particles in
each of these areas is unknown. This information would be useful
in gauging any differential promoter activity in terms of both
levels and duration of gene expression.
The issue of what the corresponding developmental stage in
humans is in relation to an E15 mouse is a difficult question to
Gene Therapy (2011), 1 -- 11
answer. There is little doubt that human and mouse CNS
development are vastly different and this is further complicated
by the choice of marker used as a comparison, for example,
anatomical, immunological or physiological. If we were to use an
anatomical marker as an example, then diffusion sensor magnetic
resonance imaging has shown that the cortical and subplate in
humans thickens in the second trimester of human fetal
development.41 However, this occurs later at E14-18 in the fetal
mouse gestational period.42 Again, this raises the importance of
larger animal studies, in particular non-human primates, to
provide a more representative model. However, this does not
reduce the importance of studies in mice given the wide range of
CNS disease models available.
Our data suggest greater viral dissemination through the fetal
brain than the neonatal brain. These data show that greater vector
distribution and expression can be achieved with the same
amount of vector by administration in the fetal versus neonatal
period. Although this study suggests that AAV2/8 and 2/9 are the
most suitable vectors for widespread delivery of genetic material
to mouse fetal brain, they may not be suitable for all applications.
The limited packaging capacity of self-complementary adenoassociated viruses means that insertion of large genes over
B1.4 kb would not be feasible. In such a case, adenoviral vectors
would need to be considered, given their much larger packaging
capacity. Therefore, selection of vector will ultimately depend
upon the desired application. Over the last few years, AAV9 has
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In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
received much attention, primarily due to its ability to cross the
blood -- brain barrier following intravenous administration and
mediate efficient gene delivery to the brain.43 Although this is
important, intravenous administration also results in gene delivery
to the visceral organs and is not suitable for those studies where
gene expression is required to be restricted to the brain. Future
development of the AAV9 vector may yield a version where
intravenous administration produces gene expression only within
the CNS. However, until such developments have been achieved,
intracranial administration is the most suitable approach for gene
expression restricted to the brain. We believe that this study
provides essential information on viral vector spread, tropisms,
immune tolerance and longevity of gene expression in the fetal
brain. This is vital for novel approaches in studying brain
development and neonatal lethal neurodegenerative diseases.
MATERIALS AND METHODS
Viral vector production
Ad5-GFP and Ad5-Luc were produced as previously described by Nicklin
et al.44 The titre was calculated using a micro-bicinchoninic acid assay
and following the established formula; 1 mg of protein ¼ 4 109 viral
particles.45 The self-complimentary AAV2/5-GFP and AAV2/8-GFP were
produced using the methods previously described by Nathwani et al.46
Vector genomes were calculated using the previously described slot-blot
analysis with supercoiled plasmid DNA as standards.47 The University of
Pennsylvania Vector Core Facility supplied the self-complimentary AAV2/
9-GFP virus and the details of vector production, titration, and quality
control can be found on their website (http://www.med.upenn.edu/gtp/
vectorcore/). The titres of the viral preparations used in the study were:
Ad5 ¼ 2 1011 viral genomes per ml, and AAV2/5, AAV2/8 and AAV2/
9 ¼ 7 1011 viral genomes per ml.
Fetal and neonatal intracranial injection of viruses into mice and
staining of sections
We have previously described the in utero intracranial injection technique
that was used in this study.12 Briefly, pregnant MF1 mice at 15 days
gestation were placed under isofluorane anaesthesia, and a midline
laparotomy was conducted to expose the uterus. In all, 5 ml of vector was
administered to three fetuses per dam via transuterine injection directed
towards the anterior horn of the lateral ventricle on the left side of the
brain using a 33-gauge needle (Hamilton, Reno, NV, USA). Fetuses
administered with virus were labelled by subcutaneous injection of 3 ml
of colloidal carbon ink solution in the flank to identify them from
un-administered fetuses. The laparotomy was closed using 6 -- 0 silk sutures
and the mouse was permitted to recover in a warm cage. One day post
gestation (P1), neonatal mice were also administered with 5 ml of the
Ad5-GFP vector. The mice were anaesthetised on ice for 3 min before
receiving the injection and were also directed towards the anterior horn of
the lateral ventricle on the left side of the brain using a 33-gauge needle.
The injected mice were labelled in the paw pad using colloidal carbon ink.
The injected neonates were then returned to their littermates. End-point
analysis was conducted 1 month post injection by anaesthetising fetaland neonatal-injected mice with isoflurane and performing transcardial
perfusion fixation with heparinised PBS followed by 4% paraformaldehyde.
The brains were removed and cryoprotected in 30% sucrose in 50 mM
Tris-buffered saline (TBS) before serial cryosectioning at 40 mm-thick
sections using a Microm HM 430 Freezing Microtome (Thermo Fisher
Scientific, Loughborough, UK).
Immunohistochemical staining of brain sections for detection of
GFP and CD68
Immunohistochemical staining of brain sections was used to detect GFP
expression. Endogenous peroxidase activity was depleted by incubating
sections in 1% H202 in TBS solution for 30 min and after rinsing three times
in TBS, sections were then incubated for 30 min in a solution of 15%
normal goat serum (NGS; Vector Laboratories Inc., Burlingame, CA, USA) in
& 2011 Macmillan Publishers Limited
9
TBS-T (TBS solution containing 0.3% Triton X-100) for 30 min to block nonspecific immunoglobulin binding. Subsequently sections were incubated at
4 1C overnight in rabbit anti-GFP antibodies (1:10 000; Abcam, Cambridge,
UK) in TBS-T/10% NGS. After rinsing in TBS and incubation for 2 h in goat
anti-rabbit IgG (Vector Laboratories Inc.) diluted in TBS-T/10% NGS (1:1000),
the sections were rinsed in TBS and incubated for 2 h in a 1:1000 solution of
Vectastain avidin - biotin solution (ABC; Vector Labs, Peterborough, UK) in
TBS and prepared 30 min before use. To visualise immunoreactivity sections
were rinsed and incubated in 0.05% solution of DAB containing 0.01% H202.
The staining reaction was stopped by rinsing sections in ice cold TBS and
sections were mounted onto chrome-gelatine-coated Superfrost-plus slides
(VWR, Poole, UK) and left to dry overnight before dehydrating in a series of
industrial methylated spirits, cleared in xylene for 20 min before being
coverslipped using DPX mounting medium (VWR).
Immunohistochemical detection of activated microglia was performed
via a similar protocol to reveal CD68 immunoreactivity, using normal rabbit
serum for blocking and incubation in rat anti-mouse CD68 antibodies
(1:100; AbD Serotec, Kidlington, UK) followed by rabbit anti-rat IgG diluted
in TBS-T/10% normal rabbit serum (1:1000). These sections were rinsed,
incubated in avidin - biotin solution, washed in TBS, visualised with DAB
and mounted on slides, as described above.
All DAB-stained sections were viewed under an Axioskop 2 Mot
microscope (Carl Zeiss Ltd., Hertfordshire, UK) to examine and compare
staining patterns. Representative images were captured using an Axiocam
HR camera and Axiovision 4.2 software (Carl Zeiss Ltd).
Quantitative analysis of immunohistochemical staining
The expression of GFP and CD68 was measured by quantitative thresholding image analysis as previously described13,48 with each antigen
analysed blind to the type of vector injected. Briefly, 40 non-overlapping
RGB images were captured across four consecutive sections through the
CA1 subfield of the hippocampus, caudate putamen, motor and piriform
cortex, medial septum, VPM/VPL region of the thalamus and the cerebellar
nodule. All the images were captured using a live video camera (JVC, 3CCD,
KY-F55B) mounted onto a Zeiss Axioplan microscope using a 40
objective. During the capture phase, light intensity, microscope calibration
and video camera settings were kept constant. Images were analysed for
optimal segmentation of immunoreactive profiles, which were determined
using Image-Pro Plus (Media Cybernetics, Bathesda, MD, USA). Foreground
immunostaining was accurately defined according to averaging of the
highest and lowest immunoreactivities within the sample population for a
given immunohistochemical marker (per colour/filter channel selected)
and measured on a scale from 0 (100% transmitted light) to 255 (0%
transmitted light) for each pixel. This threshold setting was then applied as
a constant to all subsequent images analysed for the vector injected or the
antigen used. Immunoreactive profiles were discriminated in this manner
to determine the specific immunoreactive area (the mean grey value
obtained by subtracting the total mean grey value from non-immunoreacted value per defined field). Macros were recorded to transfer the data
to a spreadsheet for subsequent statistical analysis. Data were separately
plotted graphically as the mean percentage area of immunoreactivity per
field±s.e.m. for each region.
Phenotypic analysis of transduced cells
Viral vector-transduced cells were assessed for phenotype via double
labelling with neuronal and astrocytic markers. Sections were blocked in
15% NGS in TBS-T and incubated overnight in rabbit anti-GFP (1:4000;
Abcam) and either mouse anti-NeuN (1:500; Millipore, Watford, UK) or
mouse anti-S100b (1:500; Dako, Ely, UK) diluted in 10% NGS in TBS-T. The
following day, sections were rinsed in TBS and incubated for 2 h with goat
anti-rabbit Alexa 488 and goat anti-mouse Alexa 546 (1:1000; Invitrogen,
Paisley UK). After rinsing, sections were counterstained with Topro-3 (1:1000;
Invitrogen), mounted onto chrome-gelatin-coated slides and coverslipped
with Fluoromount G (SouthernBiotech, Birmingham, AL, USA). Slides were
visualised with a laser scanning confocal microscope (Leica SP5, Leica
Microsystems, Milton Keynes, UK). Confocal z-stacks were captured in the
cortex to determine the phonotypic identity of transduced cells.
Gene Therapy (2011), 1 - 11
In utero administration of Ad5 and AAV pseudotypes
AA Rahim et al
10
IVIS imaging
Mice were anaesthetised with isofluorane (Abbot Laboratories, Abbot Park, IL,
USA) and injected with D-luciferin (15 mg ml1; Gold Biotechnology, St Louis,
MO, USA). The mice were imaged with a cooled charge-coupled device
(CCCD) camera (IVIS; Caliper Life Sciences) 5 min later. Following acquisition
of a grey-scale image, a bioluminescence image was captured with a 12-cm
field of view, a binning resolution factor of 8 and a 1/f stop and open filter.
Signal intensities were calculated using Living Image software (Caliper Life
Sciences) and expressed as photons per second per cm2.
SPR-biosensor immunoassay
Samples were analysed using a Biacore T-100 instrument (GE Healthcare,
Little Chalfont, UK). GFP (10179 RU) was immobilised on flow cell 2 of a
CM5 chip by amine coupling according to the manufacturer’s instructions.
Subtracted sensorgrams were generated by subtracting the signal from
flow cell 1 whose surface was subjected to a blank amine immobilisation.
Sensorgrams were generated at a flow rate of 10 ml min1 using 10 mM
Hepes pH7.4, 150 mM NaCl, 3 mM EDTA, 0.05% tween 20 as running buffer.
All serum samples were diluted 1:10 in sample diluent 25 mM TRIS pH9,
250 mM NaCl, 3 mM EDTA, 0.05% tween 20, 2 mg ml1 dextran. Each sample
was injected for 2 min and sample binding was recorded 60 s after the end
of the injection. Immediately after completion of the sample injection, the
goat anti-mouse (IgA þ IgG þ IgM; Thermo Scientific, Loughborough, UK)
was injected at a concentration of 50 mg ml1 for 2 min and the binding
response was recorded 30 s after the injection. Finally, the sensor chip
surface was regenerated between injections with a 30 s injection of glycine
pH1.5. This was followed by a 2 min stabilisation period before another
sample was injected. A mouse monoclonal anti-GFP antibody (632375;
Clontech, Mountain View, CA, USA) was used as a positive control at a
dilution of 1:100 in sample diluent.
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CONFLICT OF INTEREST
22
The authors declare no conflict of interest.
23
ACKNOWLEDGEMENTS
We thank Gennadij Raivich, University College London (UCL) for useful discussion.
The UCL-based laboratory and AHB were supported by the Biotechnology and
Biological Sciences Research Council. AAR and SNW had also received support from
the UK Gauchers Association. The King’s College London-based laboratory was
supported by the Wellcome Trust (GR079491MA), Batten Disease Family Association
and the Batten Disease Support and Research Association.
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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)
& 2011 Macmillan Publishers Limited
Gene Therapy (2011), 1 - 11