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Gene Therapy (2007) 14, 872–882
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ORIGINAL ARTICLE
Efficient gene transduction of neurons by lentivirus
with enhanced neuron-specific promoters
H Hioki1, H Kameda1, H Nakamura1, T Okunomiya1, K Ohira1, K Nakamura1,2, M Kuroda1, T Furuta1
and T Kaneko1,2
1
Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan and 2Core Research for
Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Japan
In the field of basic and clinical neurosciences, it is important
to develop a method for easy delivery and persistent
expression of transgene in central neurons. We firstly
generated lentiviral vectors with five kinds of neuron-specific
promoters, such as synapsin I (SYN), calcium/calmodulindependent protein kinase II, tubulin alpha I, neuron-specific
enolase and platelet-derived growth factor beta chain
promoters and then novel hybrid promoters by fusing
cytomegalovirus enhancer (E) to those neuron-specific
promoters. Neuron-specific expression of green fluorescent
protein (GFP) with those promoters was examined in vivo
by injecting the lentiviral vectors into the rat neostriatum,
thalamus and neocortex. Among all the promoters, SYN
promoter displayed the highest specificity for neuronal
expression in all the regions examined (more than 96%).
Although GFP production by the hybrid promoters was about
2–4 times larger than the non-enhanced promoters, the
neuronal specificity was significantly decreased in most
cases. However, the neuronal specificity of E/SYN hybrid
promoter exhibited the least decrease only in the thalamus.
Furthermore, the transcriptional activity and neuronal specificity of E/SYN promoter were sustained for up to 8 weeks.
Thus, lentivirus with E/SYN promoter is the best vector for
strong persistent expression in neurons.
Gene Therapy (2007) 14, 872–882. doi:10.1038/sj.gt.3302924;
published online 15 March 2007
Keywords: lentivirus; neuron specific; cytomegalovirus; enhancer; promoter; hybrid
Introduction
Gene delivery to the central nervous system with
recombinant viral vectors have aroused interest in the
field of basic and clinical neurosciences, and many kinds
of viral vectors have been developed for investigation
of neuronal morphology, gene function, modeling of
human diseases and therapeutic applications.1,2 Among
available viral vectors, lentiviral vectors offer unique
advantages of stably integrating transgene into the
genome of dividing and non-dividing cells, and providing the basis for sustained gene expression without any
toxicity and immune response. Lentiviral vectors are,
therefore, considered to be the most appropriate ones for
experiments requiring long-term gene expression and
gene therapeutic applications.
Self-inactivating and replication-defective lentiviral
vectors, derived from human immunodeficiency virus
type 1 (HIV-1), have been developed for transduction of
mammalian cells.3–6 These vectors are pseudotyped with
vesicular stomatitis virus G-protein (VSV-G), and VSV-G
achieves a broad transduction spectrum.7 Many gene
delivery vectors have used viral promoters, particularly
Correspondence: Dr T Kaneko, Department of Morphological Brain
Science, Graduate School of Medicine, Kyoto University, Kyoto 6068501, Japan.
E-mail: [email protected]
Received 24 October 2006; revised 22 December 2006; accepted 25
December 2006; published online 15 March 2007
cytomegalovirus (CMV) or rous sarcoma virus promoter,
because they have high transcription activities in all the
infected cells whatever their cell types are. Taken
together, VSV-G-pseudotyped lentiviral vectors with
CMV promoter express transgene in not only neuronal
but also glial cells in the central nervous system3–5,8 Thus,
it is indispensable to utilize neuron-specific promoters
for stable neuron-specific expression of transgene with
VSV-G-pseudotyped lentiviral vectors.
Many kinds of neuron-specific promoters have been
developed and applied to transgenic animals and viral
vectors.9 As lentiviral vectors cannot accept large genetic
sequences (up to 10 kb between the long terminal
repeats), we selected relatively small promoters. In the
present study, we used five kinds of neuron-specific
promoters; human synapsin I (SYN),10 mouse calcium/
calmodulin-dependent protein kinase II (CaMKII),11 rat
tubulin alpha I (Ta1),12 rat neuron-specific enolase
(NSE)13 and human platelet-derived growth factor-beta
chain (PDGF) promoters.14 Although these promoters are
known to be specific for neuronal expression, the
transcriptional activities are much weaker than those of
viral promoters such as CMV promoter. In the present
study, we generated novel hybrid promoters by a
combination of CMV enhancer and the neuron-specific
promoters listed above to enhance promoter activities,
and then characterized their neuronal specificities and
transcriptional activities in the rat neostriatum, thalamic
nuclei and cerebral cortex, using enhanced green fluorescent protein (GFP) as a reporter protein. The purpose of
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the present study is to explore the best promoter for
strong, persistent and specific expression of transgene in
neurons with VSV-G-pseudotyped lentiviral vectors.
Results
GFP expression by neuron-specific and enhanced
neuron-specific promoters in the rat neostriatum,
thalamus and neocortex
We generated 11 kinds of VSV-G-pseudotyped lentiviral
vectors, which express GFP under CMV, neuron-specific
and enhanced neuron-specific promoters (Figure 1, Table
1). At 1 week after injection of the lentiviral vectors into
the rat neostriatum, ventrobasal thalamic nuclei and
somatosensory cortex, GFP was visualized by immunoperoxidase staining (diaminobenzidine-4HCl (DAB)
reaction). GFP expression driven by CMV promoter
was observed in not only neuronal but also glial cells
(arrowheads and insets in Figure 2a1–a3). The immunoreactivity was very intense and detected in cell bodies
as well as distal dendrites (Figure 2a1–a3). Under SYN
promoter, GFP expression was found only in neurons,
but the immunoreactivity was conspicuously weak,
especially in the thalamus (Figure 2b1–b3). Using E/SYN
promoter, intense immunoreactivity was observed predominantly in neurons, and distal dendrites were also
well visualized (Figure 2c1–c3). These results indicate
that the neuronal specificities of SYN and E/SYN
promoters are higher than that of CMV promoter, and
the expression levels of CMV and E/SYN promoters are
stronger than that of SYN promoter. In the other neuronspecific promoters, the immunoreactivity for GFP also
became more intense but neuronal specificity was
Figure 1 Construction of pLenti6-Pro-GFP-WPRE. We firstly
introduced 11 kinds of promoters (CMV, neuron-specific and
enhanced neuron-specific promoters), GFP and WPRE into pBSII
SK (a), and then KpnI-to-NotI fragment was inserted into the KpnI/
NotI sites of pENTRIA (b). The insert (Pro-GFP-WPRE) was
transferred pLenti6 with LR recombination reaction, and resulting
to pLenti6-Pro-GFP-WPRE (c). CMV-E, the enhancer region of
human cytomegalovirus promoter; GFP, enhanced green fluorescence protein; LTR, long-terminal repeat; f, HIV-1 packaging signal;
Pro, promoter, RPE, HIV-1, Rev response element; WPRE, woodchuck hepatitis virus post-transcriptional element. See text for
further detail.
Table 1 Neuron-specific promoters used in the present study
Promoter
CMV enhancer
+
Human synapsin I
(1889–2289: M55301, GenBank)
(F: 50 -CTGCAGAGGGCCCTGCGTAT-30 , R: 50 -CGCCGCAGCGCAGATGGTCG-30 )
SYN
(401 bp)
E/SYN
(769 bp)
Mouse calcium/calmodulin-dependent protein kinase II
(7625–7988: AJ222796)
(F: 50 -ACTTGTGGACTAAGTTTGTT-30 , R: 50 -GCTGCCCCCAGAACTAGGGG-30 )
CaMKII
(364 bp)
E/CaMKII
(732 bp)
Rat tubulin alpha I
(Gloster et al.12)
(F: 50 -CCGTATTAGAAGGGATGGCT-30 , R: 50 -TTCTTACAGCGCGACTCTTA-30 )
Ta1
(1034 bp)
E/Ta1
(1402 bp)
Rat neuron-specific enolase
(Forss-Petter et al.13)
(F: 50 -GAGCTCCTCCTCTGCTCGCC-30 , R: 50 -CTCGAGGACTGCAGACTCAG-30 )
NSE
(1807 bp)
E/NSE
(2175 bp)
Human platelet-derived growth factor-beta chain
(Sasahara et al.14)
(F: 50 -GGATCCACAGTCTCCTGAGT-30 , R: 50 -CTAGGGAGGCAGCGGGGGAG-30 )
PDGF
(1468 bp)
E/PDGF
(1836 bp)
Abbreviations: CaMKII, calcium/calmodulin-dependent protein kinase II; CMV, cytomegalovirus; NSE, neuron-specific enolase; PDGF,
platelet-derived growth factor; SYN, synapsin; TaI, tubulin alpha I.
We amplified CMV promoter and five kinds of neuron-specific promoters by polymerase chain reaction with the primer sets indicated above,
and inserted them into plasmid vectors. We further developed novel hybrid promoters by fusing CMV enhancer to those neuron-specific
promoters. See text for further details.
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Figure 2 Immunoperoxidase staining for GFP at 1 week after injection of lentiviral vectors with CMV, SYN or E/SYN promoter into the rat
neostriatum, ventrobasal thalamic nuclei and somatosensory cortex. GFP expression under CMV promoter was observed in both neuronal
and glial cells (a1–a3), whereas the expression by SYN or E/SYN was neuron-specific at all the brain regions (b1–b3, c1–c3). GFP
immunoreactivity was strong under CMV or E/SYN promoter (a1–a3, c1–c3), but weak under SYN promoter (b1–b3). Arrowheads indicate
glial cells, and they were shown in insets (a1–a3). Scale bar in inset (a3) ¼ 40 mm.
decreased to some extent after addition of CMV
enhancer. Transduction efficiencies of neuron-specific
and enhanced neuron-specific promoters appeared
somewhat lower than that of CMV promoter, this
is probably because most of glial cells did not express
GFP under those promoters even though transgene
was integrated into their genomes. In addition, we
observed no remarkable differences of GFP-expressing
cell numbers between neuron-specific and enhanced
neuron-specific promoters. In the following experiments, we quantitatively analyzed neuronal specificities and expression strength of all promoters listed
in Table 1.
Neuronal specificity
At 1 week after injection of lentiviral vectors with
all promoters listed in Table 1, we randomly selected
100–200 GFP-expressing cells around the injection sites
in the rat neostriatum, ventrobasal thalamic nuclei and
somatosensory cortex, and then examined whether or
not these cells might show immunoreactivity for NeuN
(Figure 3; in the neostriatum). We compared neuronal
specificities between promoters with or without CMV
enhancer by using the two-tailed Student’s t-test (Figure
4a). After addition of CMV enhancer, the neuronal
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specificities of CaMKII, Ta1 and NSE promoters exhibited a statistically significant decrease at all the brain
regions examined, whereas that of PDGF promoter did
not display any significant change. SYN promoter
showed the highest specificity for neuronal expression
among all the promoters (more than 96%), whereas
E/SYN promoter exhibited the least decrease in neuronal
specificity only in the thalamic nuclei (P ¼ 0.042). However, the specificity of E/SYN promoter remained
relatively high at all the brain regions compared with
the other enhanced neuron-specific promoters.
We further examined time course of neuronal specificities from 3 days to 8 weeks after injection of lentiviral
vectors with CMV, SYN or E/SYN promoter into the
neostriatum (Figure 4b). The neuronal specificities of
these promoters showed no remarkable changes and
seemed constant throughout 8 weeks in the neostriatum;
the specificities of SYN and E/SYN promoters stayed
around 98%, whereas that of CMV promoter remained
about 50%.
Time course of GFP expression levels
To determine time course of GFP expression levels, we
injected lentiviral vectors with SYN or E/SYN promoter
into the rat neostriatum, allowed the animals to survive
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Figure 3 Confocal laser-scanning microscopic images of double-immunofluorescence staining for GFP and NeuN in the neostriatum. At 1
week after injection of lentiviral vectors with CMV, SYN or E/SYN promoter, GFP and NeuN were labeled with AlexaFluor 488 and
AlexaFluor 647, respectively. The, digital images were captured at about 200-mm far from injection sites. GFP was expressed in both neuronal
and glial cells under CMV promoter (a–a’’), whereas the expression by SYN or E/SYN promoter was neuron specific (b–b’’, c–c’’).
Arrowheads point to the colocalization of GFP and NeuN.
from 3 days to 8 weeks, and observed GFP-immunofluorescence (GFP-IF) or GFP-native-fluorescence (GFPNF) under confocal laser-scanning microscope. It should
be noted that GFP-IF and GFP-NF were observed mainly
in somata, whereas the fluorescence signals in dendrites
were obviously weak (Figure 5a, Figure 6b, b0 and d, d0 ),
as compared with immunoperoxidase staining (Figure
2b1–c3). We measured average intensity per pixel
of GFP-IF or GFP-NF in soma (IIF or INF (U)) with
software ImageJ, and then estimated GFP-IF or GFP-NF
in somatic volume (IIFV or INFV (106 U mm3)), presuming the neuronal cell bodies to be spherical (Figure
5a; somatic volume V ¼ 4A3/2/3p1/2 (mm3), where A is
sectional area (mm2)). We found that most GFP-IF was
actually accumulated in somata, and that IIFV reflected
about 80% of GFP produced by infected neurons (see
Supplementary Figure 1 online). The curves of I and IV
were, when normalized, similar to each other in both
GFP-IF and GFP-NF (Figure 5b,c and d,e), probably
because the vast majority of neurons in the neostriatum
is composed of one principal neuronal cell type
(medium-sized spiny neuron; X95%) and the somatic
volume of cell body was rather homogeneous in the
present study (7.271.4 (103 mm3), mean7s.d., n ¼ 270).
The increase of IIF and IIFV had considerably slowed
along the time course and seemed saturated by 8 weeks
(Figure 5b and c). On the other hand, INF and INFV were
almost linearly increased from 1 to 8 weeks after the
injection (Figure 5d and e). GFP-NF at 3 days after
the injection was so weak that we could not detect the
fluorescence signals.
To investigate the discrepancy between GFP-IF and
GFP-NF curves (Figure 5b,d and c,e), we examined the
effect of immunofluorescence staining for GFP. At 1 and
2 weeks after injection of lentiviral vectors with SYN or
E/SYN promoter into the neostriatum, INF was measured
from 15 neurons for each promoter. After immunofluorescence staining for GFP, IIF was obtained from the
identical neurons (Figure 6b, b0 and d, d0 ). A correlation
between INF and IIF was examined with linear or logistic
regression analysis. At 1 week after the injection, INF was
linearly amplified by immunofluorescence staining
(Figure 6a), but not at 2 weeks (Figure 6c). This result
indicates that when the amount of GFP surpassed certain
range, immunofluorescence staining did not linearly
amplify the fluorescence signals anymore. Thus, we
determined to compare expression strength of all
promoters listed in Table 1 at 1 week after the injection.
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brain regions examined (two-tailed Student’s t-test,
n ¼ 15, 7.9 1016ppp5.0 103). We further compared
IIF between CMV and all the hybrid promoters by
one-way analysis of variance (ANOVA) followed by
Dunnett’s post hoc test. E/Ta1 promoter displayed higher
intensity than CMV promoter in the neostriatum,
whereas E/CaMKII and E/PDGF promoters exhibited
lower intensities than CMV promoter in the thalamus
and neocortex. No significant differences were detected
between CMV and the other enhanced promoters in
the other regions examined. IIF was generally weaker in
the thalamus than in the neostriatum and neocortex.
However, this does not mean that the promoter activity
was weak in the thalamus, because IIF did not reflect the
total amount of GFP in single neurons especially when
compared with small and large neurons, such as somatic
volumes of neostriatal and thalamic neurons (7.771.6
(103 mm3) in the neostriatum and 2175.3 (103 mm3) in
the thalamus, mean7s.d., n ¼ 165).
Thus, to estimate promoter activity in neurons of
different sizes, we calculated IIFV from average IIF and
somatic volume (V). IIFV produced by all the hybrid
promoters was 1.6–3.8 times higher than non-enhanced
promoters at all the brain regions examined (Figure 7b,
two-tailed
Student’s
t-test,
n ¼ 15,
4.0 1011
pPp1.6 104). Multiple comparison tests between
CMV and all the hybrid promoters were also performed
by one-way ANOVA followed by Dennett’s post hoc test.
E/Ta1 promoter was stronger than CMV promoter in
the neostriatum, whereas E/CaMKII promoter in the
thalamus and E/PDGF promoter in the thalamus and
neocortex were weaker than CMV promoter. No significant differences were detected between CMV and the
other enhanced promoters in the other regions examined.
IIFV in the neocortex was larger than those in the
neostriatum and thalamus, suggesting that the transcriptional activity was generally stronger in the neocortex.
Discussion
Figure 4 Neuronal specificities in the rat neostriatum, ventrobasal
thalamic nuclei and somatosensory cortex. (a) We randomly
selected 100–200 GFP-expressing cells, and examined whether or
not they might show immunoreactivity for NeuN at 1 week after
viral injection. Each column represents the mean7s.d. (n ¼ 3) and
P-values were calculated by using the two-tailed Student’s t-test.
(b) Time course of neuronal specificities of CMV, SYN or E/SYN
promoter in the neostriatum. We randomly selected 100–200 GFPexpressing cells, and examined whether or not they might
show immunoreactivity for NeuN from 3 days to 8 weeks after
injection of lentiviral vectors. Each symbol represents the
mean7s.d. (n ¼ 3).
Comparisons of GFP expression levels by neuronspecific and enhanced neuron-specific promoters
At 1 week after injection of lentiviral vectors with all
promoters listed in Table 1, IIF and IIFV were measured in
the infected neurons of the rat neostriatum, ventrobasal
thalamic nuclei and layer II/III of somatosensory cortex
(Figure 7a and b), as described before (Figure 5a). We
first compared IIF between neuron-specific and enhanced
neuron-specific promoters (Figure 7a). IIF was increased
by 1.4–3.6-fold in all the enhanced promoters at all the
Gene Therapy
We developed novel hybrid promoters by fusing CMV
enhancer to neuron-specific promoters, and quantitatively examined their characteristics in vivo with VSV-Gpseudotyped lentiviral vectors derived from HIV-1. After
addition of CMV enhancer to neuron-specific promoters,
the neuronal specificities of most promoters were
significantly decreased, although the expression levels
were increased by about two- to four-fold in all the hybrid
promoters. Among all the hybrid promoters, the neuronal specificity of E/SYN promoter remained comparatively high, and the expression levels of E/SYN promoter
was almost the same as that of CMV promoter at all
the brain regions examined. These results lead to the
conclusion that E/SYN promoter might be the most
appropriate one to express a gene of interest in neurons
efficiently and specifically.
Technical consideration: quantification of promoter
activity
Transcriptional activity of promoter has been analyzed
using several reporter enzymes; chloramphenicol acetyltransferase, luciferase, alkaline phosphatase and
b-galactosidase.15 Recently, it was demonstrated that
GFP also had all the essential properties of a quantitative
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Figure 5 Time course of GFP expression levels by CMV, SYN or E/SYN promoter in the rat neostriatum. We observed GFP-IF or GFP-NF by
confocal laser-scanning microscope from 3 days to 8 weeks after injection of lentiviral vectors. Average intensity per pixel of GFP-IF or GFPNF in soma (IIF or INF (U)) and sectional area (A (mm2)) of GFP-expressing neurons were measured by using software ImageJ (a; SYN, GFP-IF,
4 weeks). Presuming the neuronal cell bodies to be spherical, we estimated somatic volume (V (mm3)) of GFP-expressing neurons and GFP-IF
or GFP-NF in somatic volume (IIFV or INFV (106 U mm3)) (a). IIF and IIFV had become saturated by 8 weeks (b, c), whereas INF and INFV
increased linearly along the time course up to 8 weeks after viral injection (d, e). Each symbol represents the mean7s.d. (n ¼ 15).
reporter protein and concluded that GFP is reliable and
useful one for analysis of promoter activity by measuring
fluorescence intensity.16 As GFP has the advantage that
promoter activity can be easily monitored in single cells
in sections, we selected GFP as a reporter protein in the
present study.
To analyze promoter activity precisely, total amount
of GFP should be wholly calculated in single cells.
Morphology of neuronal cells, however, is so complicated that it is impractical to completely estimate total
amount of GFP in single cells in sections. In the present
study, we estimated transcriptional activities by calculating IIFV from average immunofluorescence intensity per
pixel (IIF) and somatic volume (V). Although this
estimation did not include GFP in dendrites and axons,
IIF was obviously weak in those processes (Figure 5a,
Figure 6b, b0 and d, d0 ). Thus, the underestimation of GFP
amount was considered to be not so high in the present
study. Actually, GFP-IF was not detected in axonal
structures, and most GFP-IF was localized in somata.
Furthermore, GFP-IF in somatic volume reflected about
80% of GFP produced by neurons, suggesting that the
total GFP produced by neurons might be predicted by
calculating IV (see Supplementary Figure 1 online).
Under SYN and E/SYN promoters, INF and INFV were
linearly increased from 1 to 8 weeks, whereas IIF and IIFV
became saturated by 8 weeks after viral injection into the
neostriatum (Figure 5). To investigate this discrepancy,
we compared INF and IIF from identical neurons, and
analyzed their correlation at 1 or 2 weeks after viral
injection into the neostriatum (Figure 6). When INF was
about within 60 (U) in the present study, GFP-NF was
linearly amplified by immunofluorescence staining but
not at over 60 (U). This indicates that immunofluorescence staining linearly reflects the amount of antigen
within at most 1 week in the present study.
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Figure 6 A correlation between average intensities per pixel of GFP-NF and GFP-IF in soma (INF and IIF). At 1 and 2 weeks after injection of
lentiviral vectors with SYN or E/SYN promoter into the rat neostriatum, INF was measured from 15 neurons for each promoter, as in the
legend of Figure 5a. GFP was subsequently immunolabeled with AlexaFluor 488, and IIF was obtained from the identical neurons (b,b0 and
d,d0 ). A correlation between INF and IIF at 1 or 2 weeks after viral injection was analyzed by linear or logistic regression, respectively (a, c).
Figure 7 Average intensity per pixel of GFP-IF in soma (IIF) and GFP-IF in somatic volume (IIFV) in the rat neostriatum, ventrobasal thalamic
nuclei and somatosensory cortex. One week after viral injection, IIF (a) and IIFV (b) were calculated, as in the legend of Figure 5a. After
addition of CMV enhancer, IIF and IIFV displayed a statistically significant increase in all the neuron-specific promoters at all the brain regions
examined (two-tailed Student’s t-test; *, 4.0 1011 pPp5.0 103). We further performed multiple comparison tests between CMV and all
the hybrid promoters by one-way ANOVA followed by Dennett’s post hoc test (wPo 0.05; wwPo 0.01).
The effect of CMV enhancer
After addition of CMV enhancer to neuron-specific
promoters, their characteristics were markedly changed;
neuronal specificities were decreased and transcriptional
Gene Therapy
activities were drastically enhanced. Although, it has not
yet been answered how the enhancer altered characteristics of these neuron-specific promoters, a considerable
number of studies have been made on the functional
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H Hioki et al
mechanisms of CMV enhancer over the past few decades.
Several distinct sets of cellular transcription factors bind
to and stimulate the enhancer, so that it functions as
a strong transcriptional enhancer.17 Thus, it can be
assumed that transcriptional activities were augmented
but the neuronal specificities were loosened in the hybrid
promoters owing to such a functional feature of CMV
enhancer.
The potential of CMV enhancer for hybrid approach
has been demonstrated by several reports. CMV enhancer/chicken beta-actin promoter is a very strong one
and has been widely applied to transgenic animals and
viral vectors.18 A hybrid promoter by fusing CMV
enhancer to PDGF promoter was also reported, and this
hybrid promoter significantly augmented transgene
expression in neurons in vitro and in vivo, using plasmid
vectors, recombinant adeno-associated virus vectors
and baculovirus-derived vectors.19–21 Moreover, the transcriptional activity of glial fibrillary acidic protein
promoter was strikingly increased after addition of
CMV enhancer, whereas the astrocytic specificity of the
promoter remained high.22 These reports and the present
results suggest that a hybrid approach might be effective
in improvement on transcriptional activities of cellular
promoters.
Time course of GFP expression
We observed time course of transcriptional activities and
neuronal specificities of SYN and E/SYN promoters from
3 days to 8 weeks after viral injection into the neostriatum.
Surprisingly, GFP-NF linearly increased even up to 8
weeks in both the promoters, indicating that the transcriptional activities of these promoters can be maintained at
least during 8 weeks. Furthermore, the neuronal specificities of both the promoters displayed no remarkable
changes throughout 8 weeks, suggesting that neuronal
specificity is independent of time course and amount of
GFP products for at least 8 weeks. It is noteworthy that
although the transcriptional activity of E/SYN promoter
was almost as strong as that of CMV promoter, the
transcriptional activity and neuronal specificity remained
very high for up to 8 weeks. As persistent transcriptional
activity and neuronal specificity are crucial issues for
in vivo modeling and gene therapy of human neurodegenerative diseases, a combination of lentiviral vectors and
E/SYN promoter is suitable for strong persistent gene
expression in central neurons.
Prospects of VSV-G-pseudotyped lentiviral vectors
with E/SYN promoter
Lentiviral vectors are now considered to be a most
promising tool for experiments requiring long-term gene
expression and gene therapeutic applications in the
central nervous system23–25 by the following reasons:
lentiviral vectors (1) integrate transgene into the genome
of dividing and non-dividing cells; (2) enable us to
express transgene permanently in the infected cells
without immune response; (3) can be directly delivered
to specific brain region by local injection; and (4) can be
applied to mammalian species other than rats at anytime
during the lifespan of the animals. Actually, lentiviral
vectors have provided new strategies for in vivo modeling and treatments of human neurodegenerative diseases
such as Parkinson’s disease (PD).26,27
In the modeling of PD, for example, mutated alphasynuclein was overexpressed in the rat substantia nigra
by local injection of VSV-G-pseudotyped lentiviral vector
with phosphoglycerate kinase (PGK) promoter.28 After
the viral injection, most of nigral dopaminergic neurons
were selectively abolished and the immunoreactivity for
tyrosine hydroxylase (TH) was significantly decreased,
indicating that an adult onset model of PD can be
generated by direct stereotaxic injection of viral vectors
expressing a gene linked to PD. However, as PGK
promoter is one of the ubiquitous promoters, transgene
is generally expressed in both neuronal and glial cells.
Thus, it cannot be denied that the genetic model of PD
was affected by overexpression of mutated alphasynuclein in glial cells. As alpha-synuclein is produced
only in neurons, the expression should be restricted in
neurons for establishing more sophisticated animal
models and analyzing pathogenetic mechanisms more
precisely.
It was further reported that VSV-G-pseudotyped
lentivirus can achieve functional improvement in a rat
model of PD.29 In the experiment, nigral dopaminergic
neurons were damaged by administration of 6-hydroxydopamine, and then TH, aromatic amino acid dopa
decarboxylase and GTP cyclohydrolase 1 were delivered
into the dopamine-denervated striatum, using lentivirus
with CMV promoter. After the viral injection, sustained
transgene expression, dopamine production and functional improvement were observed, suggesting that this
method has the potential for gene therapy of late-stage
PD patients. Although VSV-G-pseudotyped lentiviral
vectors have been also developed for gene therapy
experiments of the other neurodegenerative diseases,
ubiquitous promoters were employed to express transgene in almost all the cases. For avoidance of unexpected
side effects caused by ectopic gene expression, it is
desirable to use tissue-specific promoters.
In the present study, we demonstrated that E/SYN
hybrid promoter is highly neuron-specific and has strong
transcriptional activity. Thus, E/SYN promoter may
replace ubiquitous promoters for gene transductions
of neurons, and VSV-G-pseudotyped lentivirus with
E/SYN promoter may accelerate the experiments for
in vivo modeling and treatments of human neurodegenerative diseases.
879
Concluding remarks
In summary, E/SYN promoter was proved to be an
excellent one for gene expression in neuronal tissues by
the following reasons; (1) the neuronal specificity was
relatively high compared with the other hybrid promoters, and kept around 98% in the neostriatum during 8
weeks; (2) the transcriptional activity was almost as
strong as that of CMV promoter, and kept for at least 8
weeks; and (3) the length of E/SYN promoter is short
(769 bp), which enable us to apply the E/SYN promoter
to many kinds of viral vectors. We expect that this novel
E/SYN promoter will be a useful tool in the field of basic
and clinical neurosciences.
Materials and methods
The experiments were conducted in accordance with the
Committees for Animal Care and Use of the Graduate
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School of Medicine at Kyoto University, and for
Recombinant DNA Study in Kyoto University. All efforts
were made to minimize animal suffering and the number
of animals used.
Plasmids construction
The lentiviral vector was derived from HIV-1 (Invitrogen, Carlsbad, CA, USA), and constructed as follows.
We used human CMV (nucleotides 1–589 of gb: U57609,
GenBank) promoter and five kinds of neuron-specific
promoters for human SYN, mouse CaMKII, rat Ta1 (a gift
from Dr Miller), rat NSE (a gift from Dr Forss-Petter and
Dr Sutcliffe) and human PDGF (a gift from Dr Sasahara
and Dr Collins) promoters (Table 1). These six promoters,
enhanced GFP (Clontech, Palo Alto, CA, USA) and
woodchuck hepatitis virus posttranscriptional regulatory
element (WPRE; nucleotides 1093–1684 of gb: U57609;
a gift from Dr Hope30) were amplified by polymerase
chain reaction (PCR), and inserted into HincII, EcoRV and
SmaI sites of pBluescript II SK (+) (pBSII SK; Stratagene,
La Jolla, CA, USA), respectively (Figure 1a). To generate
novel hybrid promoters by a combination of CMV
enhancer and neuron-specific promoters (Table 1), the
enhancer region of CMV promoter was firstly amplified
by PCR with the following primers: 50 -TAGTTATTAA
TAGTAATCAA-30 and 50 -TTTTCACGTGCCATGGTAA
TAGCGATGACT-30 (PmaCI site underlined), and subcloned into HincII site of pBSII SK. Then, five kinds of
neuron-specific promoters (Table 1), GFP and WPRE
were inserted into the PmaCI, EcoRV and SmaI sites,
respectively (Figure 1a). These totally eleven constructs
were confirmed by sequencing and named as pBSII-SKPro-GFP-WPRE. To generate a Gateway entry vector,
a KpnI-to-NotI fragment from pBSII-SK-Pro-GFP-WPRE
was inserted into the KpnI/NotI sites of pENTR1A
(Invitrogen, Figure 1b). Then, the insert from the entry
vector (Pro-GFP-WPRE) was transferred to the destination vector pLenti6/BLOCK-iT-DEST by homologous
recombination with LR clonase (Invitrogen), resulting
in pLenti6-Pro-GFP-WPRE (Figure 1c).
Production and concentration of VSV-G-pseudotyped
lentivirus
The production of VSV-G-pseudotyped lentivirus was
performed according to the manufacturer’s instructions,
ViraPower Lentiviral Expression System (Invitrogen).
The destination plasmid pLenti6-Pro-GFP-WPRE was
cotransfected with the mixture of the packaging plasmids (pLP1, pLP2 and pLP/VSVG; Invitrogen) into the
293FT producer cell line (Invitrogen), using Lipofectamine 2000 (Invitrogen). The medium was replaced at
8 h after transfection with Dulbecco’s modified Eagle’s
medium containing 10% fetal bovine serum. After 60 h
from transfection, the viral particles in the culture
supernatant were collected, filtered through 0.45-mm
filters (Millipore, Corning, NY, USA) following low
speed centrifugation (3000 g, 15 min), and then concentrated with Centricon Plus-20 (Millipore). Viral titers
(transducing units/ml) were determined by transduction
of 293FT cells with serial dilutions of the viral solution
and colony counting after blasticidin selection, and
adjusted to 1.0 106 TU/ml. The virus solution was
stored in aliquots at 801C until use for delivery to brain
tissues. This viral vector was replication deficient and
Gene Therapy
had the least chance for production of parent viral
particles in the infected cells.
Injection of viruses, fixation and immunoperoxidase
staining
Forty-one adult male Wistar rats (250–300 g; Japan SLC,
Shizuoka, Japan) were deeply anesthetized with chloral
hydrate (35 mg/100 g body weight). The virus (1.0 ml of
1.0 106 TU/ml) was stereotaxically injected by pressure
through a glass micropipette attached to Picospritzer III
(General Valve Corporation, East Hanover, NJ, USA) into
the rat neostriatum, ventrobasal thalamic nuclei and
somatosensory cortex. The rats were allowed to survive
from 3 days to 8 weeks after the injection.
The rats were deeply anesthetized again with chloral
hydrate (70 mg/100 g body weight), and perfused
transcardially with 200 ml of 5 mM phosphate-buffered
0.9% (w/v) saline (PBS; pH 7.4). The rats were further
perfused for 30 min with 200 ml of 3% (w/v) formaldehyde, 75%-saturated picric acid and 0.1 M Na2HPO4 (pH
7.0; adjusted with NaOH). The brains were removed, cut
into several blocks and post-fixed with the same fixative
above for 8 h at 41C. After cryoprotection with 30%
(w/w) sucrose in PBS, the blocks were cut into 30-mm-thick
sections on a freezing microtome. Some sections were
mounted onto gelatinized glass slides without immunostaining, and coverslipped with 50% (v/v) glycerol
and 2.5% (w/v) triethylenediamine (antifading reagent)
in PBS.
Some sections were incubated overnight with 0.2 mg/
ml affinity-purified rabbit antibody to GFP,31 and then
for 1 h with 10 mg/ml biotinylated anti-(rabbit immunoglobulin (Ig)G) donkey antibody (Chemicon, Temecula,
CA, USA). The incubation was carried out at room
temperature in PBS containing 0.3% (v/v) Triton X-100,
0.25% (w/v) l-carrageenan and 1% (v/v) donkey serum
(PBS-XCD) and followed by a rinse with PBS containing
0.3% (v/v) Triton X-100 (PBS-X). The sections were
further incubated for 1 h with avidin-biotinylated peroxidase complex (ABC-Elite; Vector Laboratories, Burlingame, CA, USA) in PBS-X. After a rinse with PBS-X,
the sections were reacted for 20–40 min with 0.02% (w/v)
DAB and 0.001% (v/v) H2O2 in 50 mM Tris-HCl (pH 7.6),
mounted onto gelatinized glass slides, dehydrated in
ethanol series, cleared in xylene and coverslipped.
Photographs were taken by the digital camera QICAM
(QIMAGING, Burnaby, BC, Canada), modified (720%
contrast enhancement) in software Canvas 8 (ACD
Systems, Saanichton, BC, Canada) and saved as 8-bit
TIFF files.
Double immunofluorescence labeling
The brain sections, which were obtained as described
above, were incubated overnight in PBS-XCD with a
mixture of 1 mg/ml anti-GFP rabbit antibody31 and 1 mg/
ml anti-NeuN mouse antibody (Chemicon). After a rinse
with PBS-X, the sections were incubated for 2 h with
5 mg/ml AlexaFluor 488-conjugated anti-(rabbit IgG)
goat antibody and 5 mg/ml AlexaFluor 647-conjugated
anti-(mouse IgG) goat antibody (Molecular Probes,
Eugene, OR, USA). The sections were mounted onto
gelatinized glass slides and coverslipped with 50% (v/v)
glycerol and 2.5% (w/v) triethylenediamine (antifading
reagent) in PBS. Digital pseudocolor images were
Lentivirus with enhanced neuron-specific promoters
H Hioki et al
captured by confocal laser-scanning microscope LSM 5
Pascal (Carl Zeiss, Oberkochen, Germany) with optical
slice thickness (Pinhole corresponding to 1 airy unit),
using a 40 objective lens (Plan-NEOFLUAR, NA ¼ 0.75,
Carl Zeiss). AlexaFluor 488 and 647 were excited
with 488- and 633-nm laser beams and observed through
510–530- and X650-nm emission filters, respectively. The
images were modified (720% contrast enhancement) in
software Canvas 8 and saved as 8-bit TIFF files.
Measuring fluorescence intensity
GFP-IF or GFP-NF was observed under confocal laserscanning microscope LSM 5 Pascal as described above.
The digital images were captured in the rat neostriatum,
ventrobasal thalamic nuclei and layer II/III of somatosensory cortex and saved as 12-bit TIFF files (without
contrast enhancement). We measured average intensity
of florescence per pixel (I (U)), sectional area (A (mm2))
with software ImageJ (http://rsb.info.nih.gov/ij). To
keep the condition for taking digital images constant,
we adjusted laser power, gain and offset each time by
monitoring intensity of fluorescence beads (#F-14791,
Molecular Probes).
Statistics
Two-tailed Student’s t-test, one-way ANOVA followed
by Dennett’s post hoc test, and linear or logistic regression
analysis were performed by using software Excel
(Microsoft Corporation, Redmond, WA, USA), Prism
(Graphpad Software Inc., San Diego, CA, USA)
and DeltaGraph (RockWare Inc., Golden, CO, USA),
respectively.
Acknowledgements
This research was supported by Grant-in-Aid for
Scientific Research 18700341, 16200025, 17650100 and
Grant-in-Aid for Scientific Research on Priority Areas
System study on higher-order brain functions 17022020
from The Ministry of Education, Culture, Sports, Science
and Technology of Japan (MEXT).
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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)
Gene Therapy