Transvascular delivery of small interfering
RNA to the central nervous system
Lee4, Premlata Shankar1& N. Manjunath1
(RVG) enables the transvascular delivery of small interfering RNA (siRNA) to the brain. This 29-amino-acid peptide
specifically binds to the acetylcholine receptor expressed by neuronal cells. To enable siRNA binding, a chimaeric peptide
and transduce siRNA to neuronal cells in vitro, resulting in efficient gene silencing. After intravenous injection into mice,
treatment with RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Repeated
administration of RVG-9R-bound siRNA did not induce inflammatory cytokines or anti-peptide antibodies. Thus, RVG-9R
provides a safe and noninvasive approach for the delivery of siRNA and potentially other therapeutic molecules across the
The endothelial cells of brain capillaries form extremely tight junc-
tions, providing a superfine filter that prevents the transport of most
molecules from the vasculature into the brain parenchyma1–3. To
overcome this, the conventional approach in gene therapy experi-
the brain by stereotactic surgery (reviewed in refs 2, 3). However,
these methods result only in localized delivery around the injection
site, with no widespread effects within the brain; they are also too
invasive for human therapy. If one could overcome the blood–brain
barrier (BBB), intravenous administration would provide the ideal
noninvasive means for delivery throughout the brain because of the
rich vascularity of the brain, with capillaries encasing virtually every
Because neurotropic viruses do cross the BBB to infect brain cells,
we asked whether the strategy used by viruses to enter the central
nervous system could also be used to enable delivery of siRNA to the
brain. Wechoserabies virus totest thishypothesis because it shows a
high degree of neurotropism in vivo and the cellular entry mechan-
isms have been well characterized.
RVG pseudotyping confers neuronal cell specificity
RVG interacts specifically with the nicotinic acetylcholine receptor
(AchR) on neuronal cells to enable viral entry into neuronal cells4,5.
We therefore initially tested whether pseudotyping lentivirus with
RVG, instead of the conventionally used vesicular stomatitis virus
glycoprotein (VSV-G), could confer specificity for neuronal cells.
Green fluorescent protein (GFP)-encoding lentiviral vector Lentilox
its ability to infect neuronal or non-neuronal cells. Whereas VSV-G
pseudotyped lentivirus infected both cell types, RVG pseudotyping
resulted exclusively in the infection of Neuro2a cells, not HeLa cells
(Supplementary Fig. S1a). Because RVG has been shown to mediate
retrograde axonal transport and increase the spread of a viral vector
encoding a short hairpin RNA (shFvEJ)8targeting Japanese enceph-
alitis virus (JEV) increases its antiviral efficacy. Different concentra-
tions of shFvEJlentivirus, pseudotyped with RVG or VSV-G, were
tested for protection efficacy in an intracranial JEV challenge assay8.
Whereas at a high dose (23105transducing units) both lentiviruses
afforded protection equally, at a lower dose (23103transducing
units), all mice treated with RVG-pseudotyped lentivirus survived
but all those treated with VSV-G-pseudotyped lentivirus succumbed
to JEV infection (Supplementary Fig. S1b). Taken together, these
results suggest that RVG confers neuronal cell specificity and inaddi-
the transduction of neighbouring neuronal cells.
RVG peptide binds specifically to neuronal cells
The snake-venom toxin a-bungarotoxin (BTX) specifically binds to
the AchR9, and a short (29-residue) peptide derived from RVG com-
petitively inhibits the binding of BTX to the AchR in solution10. We
reasoned that this peptide might bind specifically to neuronal cells
RVG peptide or a control peptide of similar length derived from the
rabies viral matrix protein (RV-MAT). When tested for cell binding,
RVG peptide was found to bind to the AchR-expressing Neuro2a
cells11,12but not to the receptor-negative HeLa cells, whereas RV-
MAT peptide bound to neither cell type (Fig. 1a). RVG peptide also
did not bind several other non-neuronal cells tested (Fig. 1b). To
confirm AchR-mediated binding specificity, we tested whether BTX
could inhibit RVG peptide binding to Neuro2a cells. Indeed, BTX
over, BTX was also able to displace prebound RVG from Neuro2a
cells (not shown). Next we tested whether RVG peptide could also
cells but not splenocytes bound the RVG peptide, and neither cell
1The CBR Institute for Biomedical Research and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.2Department of Internal Medicine, Roy J. and
Lucille J. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.3Research Center, Samchully Pharm. Co. Ltd., Seoul 135-735, Korea.4Department of
Bioengineering and Hanyang Fusion Materials Program, Hanyang University, Seoul 133-791, Korea.
Vol 448|5 July 2007|doi:10.1038/nature05901
type bound RV-MAT (Fig. 1d). Because AchR is widely expressed in
examined the ability of RVG peptide injected intravenously to cross
the BBB and enter brain cells. Mice were injected with biotinylated
RVG or control RV-MAT peptides and, 4h later, brain cell suspen-
sions were examined by flow cytometry. As shown in Fig. 1e, brain
cells from mice injected with RVG, but not those injected with RV-
MAT peptide, were positive for peptide uptake, indicating that the
RVG peptide might cross the BBB to enter brain cells.
RVG-9R peptide delivers siRNA to neuronal cells
Although RVG peptide can bind to neuronal cells, it does not bind
nucleic acids and therefore cannot be used to transport siRNA.
However, short, positively charged, cell-penetrating peptides bind
negatively charged nucleic acids by charge interaction14–16. A nona(L-
arginine) peptide was reported to be highly efficient in facilitating the
cellular uptake of nucleic acids, and replacement of L-arginine with
D-arginine (to form 9dR) enhanced the uptake even further17. More-
over, a cholesterol-conjugated oligo(D-arginine) has been used to
deliver siRNA to a transplanted tumour in mice18. Thus, we tested
cells. For this we used RVG-spacer-9dR (designated RVG-9R) and
control RV-MAT-spacer-9dR (RV-MAT-9R) chimaeric peptides.
Both peptides were able to bind siRNA in a dose-dependent manner
in a gel-shift assay (Fig. 2a). RVG-9R was also able to transduce fluor-
escein isothiocyanate (FITC)-labelled siRNA into neuronal cells in a
dose-dependent manner and, in agreement with siRNA binding
studies, a 1:10 molar ratio of siRNA to peptide was found optimal
for maximal transduction (Fig. 2b). To determine the neuronal spe-
cificity of siRNA delivery, Neuro2a and HeLa cells were transduced
with FITC-siRNA complexed to RVG-9R or RV-MAT-9R, and Lipo-
fectamine transfection was used as a positive control. Lipofectamine
enabled siRNA uptake by both cells, and RV-MAT-9R was unable to
transduce either cell type (Fig. 2c). In contrast, RVG-9R transduced
Thus, RVG-9R allows neuronal cell-specific siRNA delivery.
Although RVG-9R could transduce siRNA to Neuro2a cells in
the above assay, siRNA is not functional unless it is delivered into
the cytoplasm. Thus, we also assessed the gene-silencing ability of
the siRNA delivered by RVG-9R. Neuro2a cells stably expressing
high levels of GFP were transduced with anti-GFP siRNA, bound
to RVG-9R or RV-MAT-9R or transfected with siRNA by using
Lipofectamine, and GFP expression was determined 2 days later.
RV-MAT-9R-complexed siRNA was unable to decrease GFP levels,
whereas RVG-9R/siRNA silenced GFP expression to a similar extent
to Lipofectamine transfection (Fig. 2d), suggesting that the RVG-
9R-delivered siRNA was indeed functional. The RVG-9R/siRNA
Relative increase in
RVG binding (%)
RVG binding (SAPE)
RVG + BTX
Figure 1 | A short RVG peptide binds to neuronal cells in vitro and in vivo.
a, Neuro2a and HeLa cells (inset) were incubated with biotinylated RVG or
RV-MAT peptides, stained with SAPE and examined by flow cytometry.
b, Peptide binding was also tested with the indicated cell lines in triplicate.
Error bars indicate s.d. RV-MAT did not bind any of the cell lines (not
shown). c, Neuro2a cells were stained with biotinylated RVG in the absence
(red histogram) or presence (grey histograms) of decreasing concentrations
of BTX. d, Freshly isolated mouse brain (left) and spleen (right) cells were
tested for peptide binding. e, Mice were injected intravenously with
biotinylated RVG or RV-MAT peptide; 4h later, isolated brain cells were
stained with SAPE. Error bars indicate s.d. (n56).
GFP MFI (%)
siFITC (siRNA=100 pmol)
1:101:1 1:0.11:10 1:11:0.1 1:10 RVG-Bio
Figure 2 | RVG-9R peptide binds and delivers siRNA to neuronal cells in
vitro, resulting in gene silencing. a, Mobility of free or peptide-complexed
siRNA was analysed by agarose-gel electrophoresis. b, Neuro2a cells were
examined for uptake of FITC-siRNA complexed with RVG-9R at the
indicated concentrations. c, Neuro2a and HeLa (inset) cells were examined
for uptake of FITC-siRNA complexed with RVG-9R or RV-MAT-9R
peptides at a 1:10 molar ratio. Lipofectamine transfection (Lipofect.) was
used as a positive control. d, Neuro2a cells stably expressing GFP were
transduced with GFP siRNA complexed with RVG-9R or RV-MAT-9R
peptides, and GFP silencing was tested 2 days later. A representative
histogram and cumulative data from three independentexperiments(inset)
are shown. The grey filled histogram represents Neuro 2a cells not
expressing GFP. MFI, mean fluorescence intensity. Error bars indicate s.d.
NATURE|Vol 448|5 July 2007
complex was also found to be non-toxic in a 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (more
than 90% viability at 48h after treatment of Neuro2a cells with
RVG-9R at up to 25:1 peptide:siRNA ratio; data not shown).
RVG-9R enables transvascular siRNA delivery to the brain
For potential delivery in vivo, we first examined whether RVG-9R
binding protects the siRNA against degradation from serum
nucleases. Unlike naked siRNA, RVG-9R-bound siRNA was at least
partly stable for up to 8h (Supplementary Fig. S2). Next we tested
whether RVG-9R could transport siRNA to brain cells in vivo. Mice
or RV-MAT-9R; after 10h, single-cell suspensions from the brain,
spleen and liver were examined by flow cytometry. As shown in Fig.
3a,FITCfluorescence wasdetectedinthebrain onlywhenthesiRNA
spleen or liver, suggesting that RVG-9R allows specific targeting of
brain cells. The presence of FITC-positive cells in different regions
throughout the mouse brain was also confirmed by microscopic
examination of brain sections stained with anti-FITC antibody
To test brain-specific gene silencing, GFP transgenic mice were
injected intravenously with GFP siRNA bound to RVG-9R or RV-
MAT-9R on three consecutive days; their brain, spleen and liver cells
expression was much greater in the brain than in the spleen and liver
in the transgenic mice. Despite this, a significant decrease in GFP
expression was seen after treatment with RVG-9R-bound siRNA but
notwithRV-MAT-9R-bound siRNA (Fig.4a).Moreover, GFP silen-
cing was seen only in the brain and not in the liver or spleen, con-
different system, we also targeted an endogenous gene. Wild-type
Balb/c mice were injected intravenously with an siRNA targeting
the mouse gene encoding Cu-Zn superoxide dismutase 1 (SOD1;
ref. 19) complexed to RVG-9R or RV-MAT-9R, and mRNA and
protein levels of SOD1 in the brain, spleen and liver were measured
by quantitative polymerase chain reaction (PCR) and western
blotting, respectively. Although no changes in SOD1 levels were
detected in any organ in RV-MAT-9R/siRNA-treated animals, both
messenger RNA and protein levels of SOD1 were significantly
decreased in the brain, but not in other organs, in the RVG-9R/
siRNA-treated mice (Fig. 4b).
To confirm that the observed knockdown was due to specific
delivery of siRNA within the brain, we also tested for the presence
of SOD1 siRNA by northern blot analysis. siRNA was detected in the
brain but not in the spleen or liver of treated mice (Fig. 4c). Both the
gene silencing effect and siRNA detectability in the brain cells gradu-
ally decreased over a 9-day period (Fig. 4d and data not shown), in
agreement with the duration of silencing reported after local admin-
istration of siRNA in the brain20. Repeated administration of RVG-
9R/siRNA complex neither induced inflammatory cytokines nor eli-
cited an anti-peptide antibody response (Supplementary Fig. S3),
attesting to the viability of this delivery approach. Taken together,
our results show that RVG-9R enables the intravenous delivery of
siRNA to silence gene expression within the brain.
RVG-9R/siRNA treatment for viral encephalitis
We have reported that intracranial treatment with antiviral siRNAs
a noninvasive intravenous treatment method would be optimal for
Relative siRNA uptake
(fold increase in FITC MFI)
Figure 3 | RVG-9R enables transvascular delivery of siRNA to the central
nervous system. a, Mice were injected intravenously with FITC-siRNA/
peptidecomplexes,anduptake bybrain, spleenandlivercells wasexamined
by flow cytometry. Representative histograms (top) and cumulative data
(bottom) are shown. Black, RV-MAT-9R; red, RVG-9R. Error bars indicate
s.d. (n54). Three asterisks, P50.001. b, Coronal sections of brain from
FITC-siRNA/RVG-9R-injected mice (n56) were stained with anti-FITC
antibody and examined by fluorescence microscopy. Images of FITC-
positive cells in the cortex, striatum and thalamus at lower magnifications
(left panel) and higher magnifications of the boxed regions (middle panel)
sections at the higher magnification. Scale bar, 200mm.
0.64 0.75 0.85 0.96 1.04 0.51
in mRNA levels
Decrease in GFP MFI (%)
SOD-1 enzyme activity
(U per mg protein)
Figure 4 | Brain-specific gene silencing by intravenous injectionof RVG-9R/
for GFP expression. Representative histograms (top) and cumulative data
(bottom) are shown. Error bars indicate s.d. (n55); asterisks, P50.004.
lines and columns, RV-MAT-9R; red lines and columns, RVG-9R. b, Balb/c
mice were injected intravenously with SOD1 siRNA/peptide complexes, and
their brain, spleen and livers were examined for SOD1 mRNA (top) and
RVG-9R (T). Error bars indicate s.d. (n53). The numbers below the western
blot represent the ratios of band intensities of SOD-1 normalized to that of
were injected intravenously with SOD siRNA bound to RVG-9R, and the
duration of gene silencing was determined by quantification of SOD1 mRNA
levels (top)andSOD1 proteinenzymeactivity (bottom) on theindicateddays
the horizontal lines in the lower panel represent mean values.
NATURE|Vol 448|5 July 2007
clinical use. We therefore tested whether intravenous treatment with
siRNA bound to RVG-9R could protect mice from JEV-induced
encephalitis. Unlike wild-type mice, immunodeficient mice are uni-
formly susceptible to peripheral infection with flaviviruses21,22. We
therefore infected NOD/SCID mice with JEV (5LD50) intraperito-
neally followed 4h later by intravenous treatment with antiviral FvEJ
three successive days and the mice were observed for survival for at
to RV-MAT-9R or with siLuc complexed to RVG-9R all died within
10 days, showing that neither the chimaeric peptides by themselves
disease. In contrast, treatment with siFvEJcomplexed to RVG-9R
resulted in about 80% survival (Fig. 5a). The presence of siFvEJ
siRNA in the brains was also confirmed by northern blot analysis
(Fig. 5b). To rule out the possibility that non-specific interferon
(IFN) production mediated the protection observed, we measured
serum IFN levels after administration of RVG-9R/siFvEJ. Although
IFN levels were higher when mice were treated with a known
immunostimulatory siRNA23, IFN was not induced in RVG-9R/
siFvEJ-treated animals (Fig. 5c), suggesting that the protection was
mediated by RNA interference. Thus, intravenous treatment with
RVG-9R/siRNA can be used for the treatment of viral encephalitis.
Taken together, our results suggest that RVG-9R peptide may enable
transvascular delivery of siRNA to the central nervous system. The
that reported after prolonged infusion of siRNA in the central nerv-
ous system24,25. However, many aspects of this delivery system could
be refined to enhance the delivery efficacy. For instance, because
RVG-9R-bound siRNA was only partly protected against degrada-
tion in the serum (Supplementary Fig. S2), the use of chemically
stabilized siRNA26may enhance the efficacy of delivery. Moreover,
encapsulation of even a stabilized siRNA within a liposomal nano-
particle greatly enhances serum half-life and bioavailability27,28, and
liposomal and polymeric nanopraticles coated with targeting ligands
tion of these methods to generate stabilized siRNA-encapsulated
nanoparticles, coated with RVG peptide as a targeting ligand, may
provide an ideal method to enhance delivery and decrease the
requirement for siRNA and peptide for effective gene silencing.
Moreover, RVG-coated nanoparticles may also provide a method
for targeted brain delivery of other gene therapy vectors and small-
molecule drugs. Direct conjugation of siRNA to the peptide32might
be an alternative strategy to improve delivery.
Further studies to localize the presence of siRNA and gene silen-
cing in different cell types within the brain are also needed to under-
However, because RVG peptide alone (without 9R) was also detect-
able in the brain after intravenous injection (Fig. 1e), it is likely that
receptor-mediated transcytosis by means of the a7 subunit of the
AchR (which is widely expressed in the brain, including by capillary
endothelial cells13) is involved in the process. The fact that RVG-9R,
but not RV-MAT-9R, facilitated crossing of the BBB also indicates
that specific receptor binding might be important. Although cell-
penetrating peptides might also enable covalently conjugated cargo
ratio of siRNA/RVG-9R binding may be required (particularly when
port of siRNA to neuronal cells. This may explain the neuronal cell
specificity of targeting by RVG-9R. Because RVG-9R-delivered
siRNA was functional in gene silencing in multiple systems, siRNA
this happens is unclear. Similarly, siRNA complexed with protamine
has been reported to be effective in gene silencing35. Thus, although
highlights the potential of RVG-9R to mediate transvascular delivery
of siRNAs to the central nervous system. RVG-mediated delivery
might also allow the use of RNA interference for the systematic ana-
lysis of gene function in brain cells under experimental settings. In
principle, RVG-assisted delivery might also be used for the brain-
directed transport of other therapeutic molecules such as gene
therapy vectors and small-molecule drugs.
For peptide binding studies, cells were incubated for 20min with biotinylated
peptides, washed and then stained with streptavidin–phycoerythrin (SAPE).
For all siRNA delivery studies, siRNA was incubated with peptides at a 1:10
molar ratio for 10–15min at room temperature (20uC) in serum-free DMEM
medium (for in vitro studies) or 5% glucose (for in vivo studies) before use.
For all in vivo delivery experiments, mice were injected into the tail vein with
siRNA/peptide complexes in 100–200ml of 5% glucose, and the mice received
50mg of siRNA in each injection. All statistical analyses comparing groups of
mice treated with test and control peptides were performed by one-way analysis
of variance followed by Bonferroni’s post hoc test. P,0.05 was considered
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 4 January; accepted 2 May 2007.
Published online 17 June 2007.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank J. Reiser for providing the RVG construct, and
S. S. Kim, M. Kumar, S. M. Cifuni and I. Martins for technical assistance. This work
was supported by NIH grants to N.M. and P.S. P.K. was supported by a CFAR
fellowship grant, and S.K.L. was supported by a Korea Ministry of Science and
Technology grant. B.L.D. and J.L.M. were supported by NIH grants.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to N.M.
(firstname.lastname@example.org) or P.S. (email@example.com).
NATURE|Vol 448|5 July 2007
to test protection against intracranial JEV challenge in mice have been reported
previously8. Lentiviruses pseudotyped with VSV-G or RVG were generated by
transfection of the lentiviral vector plasmids together with the helper plasmid
pHR98.9DVPR (core protein) and either the pVSV-G or pLTR-RVG envelope
constructs into 293T cells. Culture supernatants were harvested after 48h, and
viral particles were concentrated by ultracentrifugation. Lentiviruses were spin-
infectedontoNeuro2a orHeLa cellsinthepresenceofPolybrene,andafter 48h
thetransductionefficiencywas determinedbyanalysingGFP expressionby flow
Peptides and siRNAs. Peptides RVG (YTIWMPENPRPGTPCDIFTNSRGK-
RASNG), RV-MAT (MNLLRKIVKNRRDEDTQKSSPASAPLDDG), RVG-9R
(YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGRRRRRRRRR) and RV-MAT-
9R (MNLLRKIVKNRRDEDTQKSSPASAPLDGGGGRRRRRRRRR) were syn-
thesized and purified by high-performance liquid chromatography at the Tufts
carboxy terminus. In RVG-9R and RV-MAT 9R peptides, the C-terminal nine
arginine residues were D-arginine.
siRNAs used in the studies included those targeting GFP (siGFP), firefly
luciferase(siLuc),theenvelope geneofJEV(siFvEJ) describedin ref.8 andthose
targeting murine Cu-Zn superoxide dismutase (SOD-1)19, and b-galactosidase
(bGal728) bearing a motif eliciting interferon production23. For some experi-
ments, siRNAwith FITC labelat the39 endof the sensestrandwas used. siRNAs
were synthesized at Samchully Pharm. Co. Ltd or obtained from Dharmacon,
Peptide binding assay. For peptide binding studies, Neuro2a, HeLa, CHO,
mouse brain, spleen or liver were used. Cells were incubated in PBS with 2.5mM
biotinylated peptides for 20min at 4uC, washed three times with PBS and then
treated with SAPE (BD Pharmingen) before analysis by flow cytometry. For
competition experiments, cells were incubated with 2.5mM biotinylated RVG
peptide in the absence or presence of different concentrations of BTX (Sigma).
EMSA. For gel mobility-shift assays, 100pmol of siRNA was incubated with
peptides at 10:1, 1:1 and 1:10 molar ratios of siRNA to peptide for 15min,
subjected to electrophoresis on 2% agarose gels and stained with ethidium
bromide. siRNA without peptide or incubated with biotinylated RVG (without
9R) served as controls.
Cytotoxicity assay. To test the cytotoxicity of RVG-9R/siRNA complexes,
Neuro2a cells (triplicates of 23105cells per well in 12-well plates) were incu-
bated with different concentrations of peptide/siRNA complexes for 24–48h
before viability was determined with a standard MTT assay.
monitored with FITC-labelled siLuc. siRNA (100pmol) was incubated with
different concentrations of RVG-9R or RV-MAT-9R in serum-free DMEM for
15min at room temperature. The complexes were then added to Neuro2a and
HeLa cell cultures (plated at 53104cells per well in 12-well plates on the
previous day). After incubation for 4h at 37uC the medium was replaced with
2ml of fresh medium supplemented with 10% fetal bovine serum (Invitrogen)
and the cells were cultured for a further 8–10h before being examined by flow
cytometry. Transfection with Lipofectamine 2000 was performed in accordance
with the manufacturer’s instructions.
Totestgenesilencing,Neuro2a cellsstably expressingGFP after transduction
with the pLL3.7 lentiviral vector were incubated with 100pmol of siGFP com-
plexedwith peptidesat a 10:1 peptide/siRNAratio and GFP expressionanalysed
48h after transduction.
Animal experiments for testing siRNA delivery and gene silencing. Balb/c,
C57BL/6-Tg(ACTB-EGFP)1Osb/J and NOD/SCID mice were purchased from
Jackson Laboratories and used at 4–6 weeks of age. All mouse experiments had
been approved by the CBR Institute (CBRI) institutional review board, and
at the CBRI.
To test peptide uptake by brain cells, 200mg of biotinylated peptides in 0.2ml
of PBS were injected into tail veins of Balb/c mice; 4h later, single-cell suspen-
sions of brains were permeabilized, treated with SAPE and analysed by flow
cytometry. For all siRNA delivery experiments, peptide/siRNA complexes (at a
and injected intravenously at 50 mg of siRNA per mouse per injection. To test
organs were harvested after a further 10h. To test GFP silencing, C57BL/6-
Tg(ACTB-EGFP)1Osb/J mice were injected with peptide/siRNA complexes on
three consecutive days and organs were harvested 2 days later. For SOD-1 silen-
cing, Balb/c mice were given three injections of siRNA/peptides at 8-h intervals
and organs were harvested at various time points. For testing protection against
JEV encephalitis, NOD/SCID mice were challenged intraperitoneally with
5LD50of JEV (LD50, the lethal dose for half of the mice, was predetermined
by using serial dilutions of the virus) 4h before treatment with intravenous
peptide/siRNA was started. The siRNA treatment was repeated at 24-h intervals
for a total of 4 days.
Staining of brain sections. Mice were injected twice with RVG-9R-bound
siRNA-FITC; brains were harvested 10–12h later. Brains were sectioned frozen
mouse anti-FITC antibodies (20mgml21; Jackson Immuno Research) or isotype
and FITC immunoreactivity was detected with Alexa-488 goat anti-mouse sec-
ondary antibodies (dilution 1:500; Invitrogen). The antibody enhancement was
performed because only one in six mice revealed FITC-positive cells by direct
SOD1 siRNA-treated mice by using an RNeasy RNA isolation kit (Qiagen). The
RNA was reverse transcribed with Superscript III and random hexamers
(Invitrogen) in accordance with the manufacturer’s protocol. Real-time PCR
was performed on 1ml of complementary DNA, or on a comparable amount of
RNA without reverse transcriptase, with the QuantiTect SYBR Green PCR kit
(Qiagen) in accordance with the manufacturer’s instructions. Amplification
conditions were as follows: 40 cycles of denaturation at 95uC for 30s, annealing
at 55uC for 30s, and extension at 72uC for 30s with a Bio-Rad iCycler. Primers
by melt-curve analysis and agarose-gel electrophoresis. SOD-1 mRNA levels
from the test animals, normalized with glyceraldehyde-3-phosphate dehydro-
genase levels, were divided by the equivalent values from untreated mice to
calculate relative changes.
Western blot analysis. Cell suspensions from mouse tissue were homogenized
Triton X-100, 0.2mM EDTA and 0.5mM dithiothreitol and protease-inhibitor
cocktail (Complete-Mini; Roche Diagnostic). The samples (10mg of protein
each) were subjected to electrophoresis on 15% SDS–polyacrylamide mini gels
(Bio-Rad) and transferred to a poly(vinylidene difluoride) membrane. The
membranewas probed with anti-b-actin antibodies(Sigma)or anti-SOD1anti-
escence western blot system (Pierce Biotechnologies). The blots were scanned
and the ratio of band intensities of SOD-1 normalized to b-actin was calculated
with Image J software.
Determination of SOD1 enzyme activity. The level of Cu/Zn SOD-1 enzyme
activity in brain tissue was measured with the SOD-1 Assay Kit-WST (Cell
Technologies, Inc.) in accordance with the manufacturer’s instructions.
Frozen brain tissues were homogenized in ice-cold sucrose buffer (50mM suc-
rose, 200mM mannitol, 1mM EDTA in 10mM Tris-HCl buffer pH7.4) and
used in ELISAafter inactivation of Mn/Fe SOD-1 with chloroform/ethanol. The
enzyme activity is denoted as units per milligram of total protein in the brain
Northern blot to detect siRNA. Small RNA (5mg) extracted from cell suspen-
sions with an miRNeasy mini kit (Qiagen) were subjected to electrophoresis on
membrane (BrightStar-plus; Ambion) and probed with sense siRNA probes as
described previously8. Antisense strand of synthetic SOD-1 siRNA (200fmol)
(first and last lanes in Fig. 4c) was used as a positive control.
or sibGal728 complexed with RVG-9R peptide. sibGal728 complexed to
Lipofectamine-2000 served as a positive control. Serum samples obtained 7h
after siRNA treatment were tested for IFN-a levels with a mouse type-I IFN
detection ELISA kit (PBL Biomedical Laboratories), in accordance with the
Serum stability. Naked and RVG-9R-complexed siRNA (100pmol) were incu-
bated at 37uC in 50% FBS (Invitrogen), 90% human serum or 90% mouse
serum. Aliquots taken at different time points were treated with Proteinase K
and frozen in 23urea TBE-loading buffer. All samples were subjected to elec-
trophoresis on 15% TBE-urea polyacrylamide gels under non-denaturing con-
ditions and detected by staining with SYBR gold.
Immunogenicity studies. Balb/c mice were injected intravenously with 50mg
of siLuc complexed to RVG-9R peptide or, for positive control, with 25mg of
trinitrophenol-conjugated keyhole-limpet haemocyanin-biotin (TNP-KLH-
biotin) peptide (Biosearch Technologies). The injection was repeated on days 3,
10 and 22, and serum samples were collected on days 21 and 30. To detect the
mice), 1:10 and 1:50 dilutions of sera were incubated in 96-well microtitre plates
coated with biotinylated RVG peptide (1mg per well). The presence of binding
horseradishperoxidase.Amousecytokine/chemokinearraykit(RayBiotechInc.) Download full-text
was used to detect a panel of 56 secreted cytokines and chemokines in the serum
15h. The manufacturer’s recommended protocol was used to perform the assay.
Quantification and statistical analysis. Western blots and cytokine array pro-
software from the National Institutes of Health (http://rsb.info.nih.gov/ij/).