Intrathecal Injection of Helper-Dependent Adenoviral
Vectors Results in Long-Term Transgene Expression
in Neuroependymal Cells and Neurons
Scott Dindot,1*Pasquale Piccolo,2*Nathan Grove,3Donna Palmer,3and Nicola Brunetti-Pierri2,4
Helper-dependent adenoviral (HDAd) vectors are devoid of all viral genes and result in long-term transgene
expression in the absence of chronic toxicity. Because of their ability to infect post-mitotic cells, including cells of
the central nervous system, HDAd vectors are particularly attractive for brain-directed gene therapy. In this
study, we show that intrathecal injection of HDAd results in extensive transduction of ependymal cells and
sustained expression of the transgene up to 1 year post-administration. We also demonstrate, for the first time,
the ability of HDAd injected by this route of delivery to transduce neuronal cells. The transduced neuroepithelial
cells can be potentially used to secrete therapeutic proteins into the cerebrospinal fluid and provide them via
cross-correction to nontransduced cells. Targeting of neuronal cells and long-term transgene expression make
this approach attractive for the treatment of several neurologic diseases.
therapeutic applications. Because of their ability to infect post-
mitotic cells, including cells of the CNS (Persson et al., 2006)
and to mediate long-term transgene expression, Ad-based
vectors are very attractive for brain-directed gene therapy.
Unlike the rapid decline observed in transgene expression in
peripheral organs following intravenous administration, first
generation Ad (FGAd)-mediated transduction of adult brain
cells results in long-term transgene expression (Davidson et al.,
1993; Le Gal La Salle et al., 1993). It is thought that FGAd-
mediated long-term transgene expression occurs because the
brain is relatively protected from the effects of the immune
response. Therefore, Ad injection into the brain results in an
ineffective T-cell response against brain-transduced cells (By-
rnes et al., 1996). However, the immune system can respond to
antigenic stimuli in the brain (Perry et al., 1993), and if a pe-
ripheral immune response against Ad is elicited after natural
infection or vector readministration, loss of transgene expres-
sion and chronic inflammation are observed (Thomas et al.,
denoviral (Ad) vectors for delivering genes to the
central nervous system (CNS) hold great promise for
2000). Interestingly, these problems are not seen with helper-
dependent adenoviral (HDAd) vectors (Thomas et al., 2000;
Xiong et al., 2006). For example, in naı ¨ve animals, FGAd- and
HDAd-mediated expression of b-galactosidase in the brain is
long term, but in animals immunized prior to vector delivery,
transgene expression is abolished in FGAd-injected mice but
not in the mice injected with HDAd (Thomas et al., 2000; Xiong
et al., 2006). These studies indicate that long-term HDAd-me-
that had been immunized systemically against Ad before the
delivery of HDAd into the brain (Thomas et al., 2000; Xiong
et al., 2006). Therefore, HDAd vectors are superior to FGAd
vectors for gene therapy of brain disorders and are poten-
tially useful even in patients pre-exposed to Ad (Barcia et al.,
A major issue for brain directed Ad-mediated gene ther-
apy is the route of administration. The blood–brain barrier
limits transduction of brain cells by Ad vectors adminis-
tered intravenously. For brain-directed gene therapy, it is
necessary for the vector to either cross or circumvent the
blood–brain barrier. Injecting vector directly into the brain
parenchyma has been one of the most common approaches
1College of Veterinary Medicine and Biomedical Sciences, Veterinary Pathobiology, Texas A&M University, College Station, TX
2Telethon Institute of Genetics and Medicine, 80131 Naples, Italy.
3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030.
4Department of Pediatrics, Federico II University, 80131 Naples, Italy.
*These two authors have contributed equally.
HUMAN GENE THERAPY 22:745–751 (June 2011)
ª Mary Ann Liebert, Inc.
resulting in localized transduction of the cerebral paren-
chyma because this route of vector delivery has limited
transduction not extending beyond a few millimeters from
the needle track. While this approach could still result in
clinical benefit in disorders such as Parkinson disease, in
which a discrete set of neurons is affected, correction of brain
disorders with diffuse involvement is far more complicated.
Further problems with intracerebral injections are the inva-
siveness of the procedure, the small volume of vector that
can be delivered, and the possible need for multiple sites of
Injection of viral vectors into the cerebrospinal fluid (CSF)
through injection in the cisterna magna or through lumbar
puncture might represent an alternative approach for wide-
spread CNS correction. Vector administration into the CSF
circulation may allow viral vector-mediated transduction of
neuroepithelial cells and the delivery of transgene products
to the whole CNS through the ventricular circulation (Butti
et al., 2008a). Therefore, this approach may have potential for
several clinical applications that can benefit from the ex-
pression in the CSF of a bioactive molecule (Elliger et al.,
1999, 2002; Watson et al., 2006). Intrathecal administration of
HDAd resulted in transgene expression in neuroepithelial
cells for about 3 months without systemic or local toxicity in
nonhuman primates with pre-existing anti-adenoviral im-
munity (Butti et al., 2008b).
In the present study, we analyzed in detail the cell types that
are transduced by HDAd delivered through CSF injection, and
we also investigated the duration of HDAd-mediated trans-
Materials and Methods
HDAd-CMV-LacZ bears a cytomegalovirus (CMV)-LacZ
expression cassette and HDAd-CMV-GFP contains a CMV-
GFP expression cassette. HDAd vectors were produced in 116
cells with the helper virus AdNG163 as described elsewhere
(Palmer and Ng, 2003). Helper virus contamination levels
were determined as described elsewhere and were found to
be <0.05%. DNA analyses of HDAd genomic structure was
confirmed as described elsewhere (Palmer and Ng, 2003). All
vector preparations were tested using Multi-test Limulus
Amebocyte Lysate (Pyrogent, Biowhittaker, Walkersville,
MD) for the presence of endotoxin and were found to be
below the limit of detection (endotoxin <0.5EU/ml).
Mice and injections
Nine- to 12-week-old male C57BL/6 mice (The Jackson
Laboratory, Bar Harbor, ME) were used for all the experi-
ments. Intrathecal HDAd administrations were performed in
saline solution by cisterna magna injections (Ueda et al.,
1979) in a total volume of 10ml injected in approximately
Analyses of brains
X-gal histochemistry was performed on brain tissues as
previously described (Brunetti-Pierri et al., 2004). Total pro-
teins were extracted from the brains and the b-galactosidase
activity was determined using the b-Galactosidase Enzyme
Assay System with Reporter Lysis Buffer (Promega, Madi-
son, WI) following quantification using the Micro BCA
Protein Assay Kit (Pierce, Rockford, IL).
Brain perfusion and immunofluorescence
Mice were perfused with ice-cold phosphate-buffered sa-
line (PBS) and 4% paraformaldehyde. Dissected brains were
post-fixed in 4% paraformaldehyde solution overnight and
then cryoprotected in 30% sucrose solution. Forty-five-
micrometer sections were cut on a cryostat and stored in
PBS. Sections were washed in PBS and blocked in T-TBS
(10mM Tris-HCl, pH 7.5, 0.3% Triton-X100) plus 5% normal
goat or donkey serum for 1–2hr at 48C in a humidified
chamber with gentle agitation. Anti-GFP (NB 600-308; Novus
Biologicals, Littleton, CO) was used a 1:2000 dilution, anti-
Notch1 (05-557; Millipore, Billerica, MA) at 1:250, anti-Map2
(MAB3418; Millipore) at 1:250, anti-Doublecortin (SC-8066;
Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:250, and
anti-Calbindin (C9848; Sigma) at 1:250. Sections were wa-
shed three times in T-TBS for 10min each and then incubated
with fluorescently labeled secondary antibodies (Jackson
ImmunoResearch, West Grove, PA) for 25hr at 48C in the
dark. Anti-rabbit Alexa 488, anti-mouse Alexa 557, and anti-
goat Alexa 647 were used at 1:200 dilutions. Sections were
washed in T-TBS for 20min each and mounted on glass
slides with Vectashield (Vector Laboratories, Burlingame,
CA) mounting reagent. Confocal images were obtained us-
ing a LSM 510 META NLO multiphoton microscope (Zeiss,
Vector genome copies
Total DNA was extracted from livers and brains from
HDAd-injected mice (n¼3) using phenol–chloroform ex-
traction and quantitated by absorbance at 260nm. Quanti-
tative real-time polymerase chain reaction (PCR) was
performed using the LightCycler FastStart DNA Master
SYBER Green I (Roche, Indianapolis, IN) in a total volume of
20ml with 100ng of template DNA, 1mM of each HDAd-
AGCGCG-30and 50- CCCATAAGCTCCTTTTAACTTGTT
AAAGTC-30). Cycling conditions consisted of 958C for
10min followed by 45 cycles at 958C for 10sec, 608C for 7sec,
and 728C for 20sec. Serial dilutions of a plasmid bearing the
PCR target sequence were used as a control to determine the
amounts of HDAd and results were analyzed with Light-
Cycler software version 3.5 (Roche).
Delivery of HDAd via intrathecal injection
To investigate the efficacy of transgene expression fol-
lowing intrathecal delivery of HDAd vectors, we injected
C57BL/6 mice via cisterna magna (Ueda et al., 1979) with
1?1012viral particles (vp)/kg of HDAd-CMV-LacZ vector
(Brunetti-Pierri et al., 2004) (n¼4). Mice were sacrificed 48hr
post-injection and stained with X-gal and each mouse stained
positive for LacZ expression. Macroscopic examination of
injected brains indicated the transgene was expressed in cells
lining the ventricles, the spinal cord, and the cerebellum.
Expression was also detected in the hypothalamus and in the
olfactory bulbs (Fig. 1A). As shown by b-galactosidase ac-
tivity, administration of HDAd by intrathecal injection re-
746 DINDOT ET AL.
sulted in significant cerebral transduction as compared with
the brain transduction achieved by intravenous administra-
tion of the same vector dose (Fig. 1B).
Transgene expression in the CNS
To evaluate the duration of transgene expression, we in-
jected C57BL/6 mice with either saline or with an HDAd
vector expressing green fluorescent protein (GFP) under the
control of the ubiquitous CMV promoter (HDAd-CMV-GFP)
at the dose of 1?1012vp/kg. Mice were sacrificed at 2 days
(n¼4), 6 months (n¼5), and 12 months (n¼9) post-injection,
and the brains were harvested for immunofluorescence de-
tection of GFP (Mullen et al., 1992). At 2 days post-injection,
four mice (100%) were positive for GFP expression and high
levels were detected in the molecular layer of the cerebellum
(Fig. 2A), choroid plexus (Fig. 2B), ependymal cells lining the
spinal cord and cerebellum (Fig. 2C), and in cells within
the olfactory bulb (Fig. 2D). At 6 months, four mice (80%)
were positive for GFP expression, which was detected in the
molecular layers of the cerebellum (Fig. 2E), deep cerebellar
nuclei (Fig. 2F), prefrontal cortex (Fig. 2G), and in ependymal
cells lining the lateral ventricles (Fig. 2H). At 12 months,
seven mice (78%) were positive for GFP expression, although
it appeared that there was a reduction in the amount of GFP
expression in the molecular and granular layer of the cere-
bellum (Fig. 2I), hypothalamus (Fig. 2J), spinal cord (Fig. 2K),
and in ependymal cells lining the lateral ventricles (Fig. 2L).
Collectively, these data indicate long-term expression of the
HDAd vector in various regions and cell types within the
CNS and demonstrate a minimal reduction of expression at
12 months post-injection.
Lower doses of HDAd results in reduced cellular
transduction and expression of the transgene
To examine the effects of lower vector doses on the cellular
transduction and expression of HDAd-CMV-GFP, we in-
jected mice with 1?1010vp/kg and 1?1011vp/kg of HDAd-
CMV-GFP vector. Mice were sacrificed 2 days post-injection,
and the brains were harvested for immunofluorescence de-
tection of GFP. Analysis of lateral ventricles indicated that
the dose of 1?1010vp/kg resulted in less cellular transduc-
tion and expression of GFP expression, whereas the dose of
1?1011vp/kg resulted in more cellular transduction and
high levels of GFP expression, although not equivalent to the
1?1012vp/kg (Fig. 3A, B). Collectively, these data indicate
that the level of transduction and expression of the HDAd-
CMV-GFP virus is dose dependent.
Ciliated ependymal cells and subpopulations
of neurons and neuronal precursors
are transduced by intrathecal HDAd injections
To determine the cell types transduced by intrathecal in-
jection of HDAd vectors, we performed double fluorescent
staining of HDAd-injected brains (Izant and McIntosh, 1980).
We detected GFP expression in Notch1-expressing (Carlen
et al., 2009) ciliated cells lining the lateral ventricles (Fig. 4A).
Additionally, we detected GFP expression in neuronal cells
in the hypothalamus expressing Map2, which is a marker of
mature, differentiated neurons (Menezes and Luskin, 1994)
(Fig. 4B). In the cerebellum, we detected GFP expression in
cells located adjacent to calbindin-expressing Purkinje cells
(Fig. 4C). Interestingly, these cells expressed doublecortin,
which is a marker of neuronal lineage commitment (Walker
et al., 2007) (Fig. 4D), and the doublecortin staining was ex-
clusive to these GFP-expressing cells. Transduction of neu-
ronal cells was only observed at 6 months post-injection,
whereas transduction of ependymal cells and cerebellar cells
were observed at each time point examined.
Inflammatory responses are dose
dependent in HDAd-injected mice
To determine the degree of activation of the inflammatory
response, we analyzed serum samples collected from mice at
different time points (6, 24, 48hr) post-injection to measure
interleukin (IL)-6 levels. Similarly to the intravascular de-
livery (Zhang et al., 2001), intrathecal delivery of HDAd
results in a dose-dependent increase in serum IL-6 at 6hr
post-injection (Fig. 5); however, serum IL-6 was undetectable
ral (HDAd)-injected brains. (A) X-gal staining of HDAd-
CMV-LacZ injected brains. CMV, cytomegalovirus; Cb,
cerebellum; LV, lateral ventricle; Hy, hypothalamus; OB,
olfactory bulb. (B) b-galactosidase in brains harvested 48hr
after the intravenous or intrathecal injection of HDAd-CMV-
LacZ at the dose of 1?1012vp/kg. *p <0.05 (t-test).
Gross X-gal staining of helper-dependent adenovi-
INTRATHECAL INJECTION OF HDAd VECTORS 747
at the later time points (data not shown). To evaluate
whether intrathecal injections of HDAd result in vector sys-
temic dissemination, we determined by real-time PCR the
amount of HDAd vector genomes in livers and brains of
animals 48hr post-intrathecal injections of 1?1012vp/kg of
HDAd vector. The real-time PCR showed that the amount of
vector genomes detected in the livers was below the limit of
detection (10copies of vector genome) and the vector ge-
nomes were only detected in brains of vector-injected mice
(n¼3) (Fig. 6).
In this study, we show for the first time that HDAd vec-
tors delivered by intrathecal injection result in transduction
and long-term transgene expression from neuroependymal
and neuronal cells. Intravenous administration of the HDAd-
CMV-LacZ vector indicated that the vector is not able to
cross the blood–brain barrier in significant amounts, as
shown by the minimal increase in b-galactoidase activity in
the brain (Fig. 1), whereas other tissues (particularly the
CMV-GFP in the brain. At 2 days (2D)
post-injection, GFP expression was de-
tected in the (A) cerebellum, (B) choroid
plexus, (C) ependymal cells lining the
spinal cord and cerebellum, and (D) ol-
factory bulb. At 6 months (6M), GFP ex-
cerebellum, (F) deep cerebellar nuclei, (G)
prefrontal cortex, and (H) lateral ventri-
cles. At 12 months (12M), GFP expression
was detected in the (I) cerebellum, (J)
hypothalamus, (K) spinal cord, and (L)
lateral ventricles. GFP, green fluorescent
protein; D, day; M, month; Cb, cerebel-
lum; CP, choroid plexus; SC, spinal cord;
OB, olfactory bulb; DCN, deep cerebellar
nuclei; PCtx, prefrontal cortex; Ctx, cor-
tex; LV, lateral ventricle. TOPRO3 stains
nuclei. Scale bar represents 300mm.
Expression patterns of HdAd-
HDAd-CMV-GFP. (A) Injections of
1?1010vp/kg resulted in reduced
cellular transduction and GFP ex-
pression in cells lining lateral ventri-
cles. (B) Injections of 1?1011vp/kg
resulted in increased transduction and
expression in cells lining the lateral
Scale bar represents 300mm.
Injections of lower doses of
748 DINDOT ET AL.
liver) had substantial activity (data not shown). Conversely,
in intrathecal injected animals b-galactosidase activity was
restricted to the brain.
At the microscopic level, intrathecal injection of HDAd
vectors resulted primarily in transduction of ependymal cells
lining the ventricles, which form the blood–CSF barrier
surrounding both the brain and the spinal cord (Fig. 2). In-
terestingly, we have also shown that HDAd delivered by this
route results in transduction of neurons located deep within
the cerebral parenchyma at different time points post-vector
injection. We reasoned that HDAd delivered by intrathecal
injection infects the cells lining the ventricular system, which
include a single layer of ependymal cells facing the lumen
and the cells of the subventricular zone (SVZ) lying under-
neath the ependymal layer. It is well established that the SVZ
of the lateral ventricles is a source of adult neuronal stem
cells (NSCs). NSCs in the SVZ can differentiate into neurons
in the olfactory bulbs and in the corpus callosum, as well as
in fimbria and striatum oligodendrocytes. Although the
ability of the ependyma to give rise to NSCs is still contro-
versial (Ma et al., 2009), it is becoming clear that NSCs directly
face the lateral ventricles through small apical processes
(Mirzadeh et al., 2008), from where they likely come in contact
with viral particles injected into the CSF space. Recently, also
leptomeningeal compartment has been suggested to host a
NSC niche (Bifari et al., 2009). Therefore, it is possible that
HDAd delivered by intrathecal injection may allow trans-
duction of NSCs that are subsequently found deep in the ce-
rebral parenchyma. The detection of GFPþ/Dcxþcells in the
cerebellum supports this hypothesis. In fact, Dcx expression in
the adult brain was earlier considered to be restricted to the
neuronal precursor phase of the neuronal lineage (Brown et al.,
2003), while more recently Dcxþneurosphere-forming cells
were identified in the cerebellum of adult mouse (Walker et al.,
2007). However whether Dcx expression represent just a stage
before final commitment of stem cells to the neuronal lineage
or whether this represents an entirely separate precursor
population remains controversial.
Dcx is not expressed during gliogenesis or regenerative
axonal growth (Couillard-Despres et al., 2005). For these
expressing HDAd-CMV-GFP. Double
immunofluorescence staining of GFP
and cell type specific markers in the
transduced: (A) ciliated ependymal
cells lining the lateral ventricles ex-
pressing Notch1, (B) mature neurons
expressing Map2 in the hypothala-
mus, (C) cells adjacent to Purkinje
cells in the cerebellum, and (D)
doublecortin-expressing cells in the
molecular cell layer of the cerebellum.
GFP, green fluorescent protein; Map2,
microtubule-associated protein 2; Cb,
calbindin; Dcx, doublecortin. Scale
bar represents 25mm for (A) and (D),
and 50mm for (B) and (C).
Characterization of cell types
INTRATHECAL INJECTION OF HDAd VECTORS749
reasons, it is unlikely that GFPþ/Dcxþcells result from the
damage induced by the injection.
Intra-CSF injection (both intraventricular and lumbar
puncture) of FGAd vectors expressing LacZ in nonhuman
primates resulted in high transduction efficiency of lepto-
meningeal cells as shown by tissue staining at 72hr post-
injection (Driesse et al., 1999). Microscopic examination
revealed transduction of arachnoid cells and to a lesser ex-
tent the cells of the pia mater. Ependymal and choroid plexus
cells were also transduced (Driesse et al., 1999). Duration of
transgene expression up to 3 months was observed in rhesus
macaques injected with HDAd expressing GFP by lumbar
puncture without signs of systemic or local toxicity or evi-
dence of CNS-specific immune reaction (Butti et al., 2008b;
Terashima et al., 2009). However, in these studies transduc-
tion of neurons in the cerebral parenchyma has not been
investigated. Our study also support that, similarly to in-
travascular delivery (Schnell et al., 2001; Brunetti-Pierri et al.,
2004), intrathecal injection of HDAd results in a rapid dose-
dependent acute inflammatory response (Fig. 5). This acute
response is resolved by 48hr post-injection and is likely the
result of capsid-mediated activation of the innate immunity.
Studies in large animal models are required to establish
whether this response would be clinically acceptable.
An important finding of our study is that transgene ex-
pression is long term following intrathecal injection of HDAd
vectors, at least in mice. Moreover, it indicates for the first
time that this route of delivery results in transduction of
neuronal cells. This finding is significant because targeting of
neuronal cells is important for correction of several neuro-
logic diseases and long-term expression is required for the
treatment of genetic diseases affecting the CNS. In contrast to
the limited vector distribution achieved by intracerebral in-
jection, administration of HDAd vectors into the CSF has the
potential advantage of widespread transduction along the
cells lining the CSF space. Moreover, intrathecal injection is a
far less invasive procedure than intracerebral injection and
therefore is attractive for clinical applications.
The transduced ependymal cells could be potentially used
to secrete therapeutic proteins into the CSF. As previously
shown this route of administration resulted in the production
of significant amounts of bioactive proteins which can be
exploited for multiple therapeutic purposes (Betz et al., 1995;
Furlan et al., 1998). Transduced cells could secrete the ther-
apeutic protein via the CSF for cross-correction of non-
transduced cells. Thus, this approach may be particularly
attractive for lysosomal storage diseases in which cross-
correction is mediated by the mannose-6-phosphate receptor.
It remains to be seen whether intrathecal administration of
an HDAd vector is equally effective in larger mammals
where greater diffusion distances may limit effective distri-
bution of the vector and/or its therapeutic product.
We are grateful for financial support from the MPS Society
to NB-P. Confocal microscopy was performed in the Texas
A&M University College of Veterinary Medicine & Biome-
dical Sciences Image Analysis Laboratory, supported by
NIH-NCRR (1 S10 RR22532-01).
Author Disclosure Statement
The authors declare no conflict of interest.
Barcia, C., Jimenez-Dalmaroni, M., Kroeger, K.M., et al. (2007).
One-year expression from high-capacity adenoviral vectors in
the brains of animals with pre-existing anti-adenoviral im-
munity: clinical implications. Mol. Ther. 15, 2154–2163.
Betz, A.L., Yang, G.Y., and Davidson, B.L. (1995). Attenuation of
stroke size in rats using an adenoviral vector to induce over-
expression of interleukin-1 receptor antagonist in brain.
J. Cereb. Blood Flow Metab. 15, 547–551.
Bifari, F., Decimo, I., Chiamulera, C., et al. (2009). Novel stem/
progenitor cells with neuronal differentiation potential reside
in the leptomeningeal niche. J. Cell Mol. Med. 13(9B), 3195–
Brown, J.P., Couillard-Despres, S., Cooper-Kuhn, C.M., et al.
(2003). Transient expression of doublecortin during adult
neurogenesis. J. Comp. Neurol. 467, 1–10.
various doses expressed in vp/kg of HDAd-CMV-GFP. In-
trathecal delivery of HDAd results in a dose-dependent and
transient increase in serum IL-6. *p <0.05; **p <0.005; ***p
Serum interleukin (IL)-6 at 6hr after the injection of
animals injected at the dose of 1?1012vp/kg of HDAd-
Vector genome copies in brain and liver tissues in
750DINDOT ET AL.
Brunetti-Pierri, N., Palmer, D.J., Beaudet, A.L., et al. (2004).
Acute toxicity after high-dose systemic injection of helper-
dependent adenoviral vectors into nonhuman primates. Hum.
Gene Ther. 15, 35–46.
Butti, E., Bergami, A., Recchia, A., et al. (2008a). IL4 gene de-
livery to the CNS recruits regulatory T cells and induces
clinical recovery in mouse models of multiple sclerosis. Gene
Ther. 15, 504–515.
Butti, E., Bergami, A., Recchia, A., et al. (2008b). Absence of an
intrathecal immune reaction to a helper-dependent adenoviral
vector delivered into the cerebrospinal fluid of non-human
primates. Gene Ther. 15, 233–238.
Byrnes, A.P., Wood, M.J., and Charlton, H.M. (1996). Role of T
cells in inflammation caused by adenovirus vectors in the
brain. Gene Ther. 3, 644–651.
Carlen, M., Meletis, K., Goritz, C., et al. (2009). Forebrain epen-
dymal cells are Notch-dependent and generate neuroblasts
and astrocytes after stroke. Nat. Neurosci. 12, 259–267.
Couillard-Despres, S., Winner, B., Schaubeck, S., et al. (2005).
Doublecortin expression levels in adult brain reflect neuro-
genesis. Eur. J. Neurosci. 21, 1–14.
Davidson, B.L., Allen, E.D., Kozarsky, K.F., et al. (1993). A model
system for in vivo gene transfer into the central nervous sys-
tem using an adenoviral vector. Nat. Genet. 3, 219–223.
Driesse, M.J., Kros, J.M., Avezaat, C.J., et al. (1999). Distribution
of recombinant adenovirus in the cerebrospinal fluid of non-
human primates. Hum. Gene Ther. 10, 2347–2354.
Elliger, S.S., Elliger, C.A., Aguilar, C.P., et al. (1999). Elimination
of lysosomal storage in brains of MPS VII mice treated by
intrathecal administration of an adeno-associated virus vector.
Gene Ther. 6, 1175–1178.
Elliger, S.S., Elliger, C.A., Lang, C., and Watson, G.L. (2002).
Enhanced secretion and uptake of beta-glucuronidase im-
proves adeno-associated viral-mediated gene therapy of
mucopolysaccharidosis type VII mice. Mol. Ther. 5, 617–
Furlan, R., Poliani, P.L., Galbiati, F., et al. (1998). Central nervous
system delivery of interleukin 4 by a nonreplicative herpes
simplex type 1 viral vector ameliorates autoimmune demye-
lination. Hum. Gene Ther. 9, 2605–2617.
Izant, J.G., and McIntosh, J.R. (1980). Microtubule-associated
proteins: a monoclonal antibody to MAP2 binds to differen-
tiated neurons. Proc. Natl. Acad. Sci. U. S. A. 77, 4741–4745.
Le Gal La Salle, G., Robert, J.J., Berrard, S., et al. (1993). An
adenovirus vector for gene transfer into neurons and glia in
the brain. Science 259, 988–990.
Ma, D.K., Bonaguidi, M.A., Ming, G.L., and Song, H. (2009).
Adult neural stem cells in the mammalian central nervous
system. Cell Res. 19, 672–682.
Menezes, J.R., and Luskin, M.B. (1994). Expression of neuron-
specific tubulin defines a novel population in the proliferative
layers of the developing telencephalon. J. Neurosci. 14, 5399–
Mirzadeh, Z., Merkle, F.T., Soriano-Navarro, M., et al. (2008).
Neural stem cells confer unique pinwheel architecture to the
ventricular surface in neurogenic regions of the adult brain.
Cell Stem Cell 3, 265–278.
Mullen, R.J., Buck, C.R., and Smith, A.M. (1992). NeuN, a neu-
ronal specific nuclear protein in vertebrates. Development 116,
Palmer, D., and Ng, P. (2003). Improved system for helper-
dependent adenoviral vector production. Mol. Ther. 8, 846–852.
Perry, V.H., Andersson, P.B., and Gordon, S. (1993). Macro-
phages and inflammation in the central nervous system.
Trends Neurosci. 16, 268–273.
Persson, A., Fan, X., Widegren, B., and Englund, E. (2006). Cell
type- and region-dependent coxsackie adenovirus receptor ex-
pression in the central nervous system. J. Neurooncol. 78, 1–6.
Schnell, M.A., Zhang, Y., Tazelaar, J., et al. (2001). Activation of
innate immunity in nonhuman primates following intraportal
administration of adenoviral vectors. Mol. Ther. 3, 708–722.
Terashima, T., Oka, K., Kritz, A.B., et al. (2009). DRG-targeted
helper-dependent adenoviruses mediate selective gene deliv-
ery for therapeutic rescue of sensory neuronopathies in mice.
J. Clin. Invest. 119, 2100–2112.
Thomas, C.E., Schiedner, G., Kochanek, S., et al. (2000). Periph-
eral infection with adenovirus causes unexpected long-term
brain inflammation in animals injected intracranially with
first-generation, but not with high-capacity, adenovirus vec-
tors: toward realistic long-term neurological gene therapy for
chronic diseases. Proc. Natl. Acad. Sci. U. S. A. 97, 7482–7487.
Ueda, H., Amano, H., Shiomi, H., and Takagi, H. (1979). Com-
parison of the analgesic effects of various opioid peptides by a
newly devised intracisternal injection technique in conscious
mice. Eur. J. Pharmacol. 56, 265–268.
Walker, T.L., Yasuda, T., Adams, D.J., and Bartlett, P.F. (2007).
The doublecortin-expressing population in the developing
and adult brain contains multipotential precursors in addition
to neuronal-lineage cells. J. Neurosci. 27, 3734–3742.
Watson, G., Bastacky, J., Belichenko, P., et al. (2006). Intrathecal
administration of AAV vectors for the treatment of lysosomal
storage in the brains of MPS I mice. Gene Ther. 13, 917–925.
Xiong, W., Goverdhana, S., Sciascia, S.A., et al. (2006). Reg-
ulatable gutless adenovirus vectors sustain inducible trans-
gene expression in the brain in the presence of an immune
response against adenoviruses. J. Virol. 80, 27–37.
Zhang, Y., Chirmule, N., Gao, G.P., et al. (2001). Acute cytokine
response to systemic adenoviral vectors in mice is mediated
by dendritic cells and macrophages. Mol. Ther. 3, 697–707.
Address correspondence to:
Dr. Nicola Brunetti-Pierri
Telethon Institute of Genetics and Medicine
Via P. Castellino, 111
Received for publication December 10, 2010;
accepted after revision December 22, 2010.
Published online: December 22, 2010.
INTRATHECAL INJECTION OF HDAd VECTORS751
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