Research Article

Therapeutic EphA2 Gene Targeting In vivo Using Neutral

Liposomal Small Interfering RNA Delivery
1 2 3 1
Charles N. Landen Jr., Arturo Chavez-Reyes, Corazon Bucana, Rosemarie Schmandt,
4 2 1,3
Michael T. Deavers, Gabriel Lopez-Berestein, and Anil K. Sood
Departments of 1Gynecologic Oncology, 2Experimental Therapeutics, 3Cancer Biology, and 4Pathology, The University of Texas M.D.
Anderson Cancer Center, Houston, Texas

Abstract
Inducing destruction of specific mRNA using small interfering
RNA (siRNA) is a powerful tool in analysis of protein function,
but its use as a therapeutic modality has been limited by
inefficient or impractical delivery systems. We have used
siRNA incorporated into the neutral liposome 1,2-dioleoyl-
sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo
siRNA delivery. In nude mice bearing i.p. ovarian tumors,
nonsilencing siRNA tagged with the fluorochrome Alexa 555
was encapsulated into DOPC liposomes and shown to be
taken up by the tumor as well as many major organs.
Furthermore, DOPC-encapsulated siRNA targeting the onco-
protein EphA2 was highly effective in reducing in vivo EphA2
expression 48 hours after a single dose as measured by both
Western blot and immunohistochemistry. Therapy experi-
ments in an orthotopic mouse model of ovarian cancer were
initiated 1 week after injection of either HeyA8 or SKOV3ip1
cell lines. Three weeks of treatment with EphA2-targeting
siRNA-DOPC (150 Mg/kg twice weekly) reduced tumor growth
when compared with a nonsilencing siRNA (SKOV3ip1: 0.35
versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16).
When EphA2-targeting siRNA-DOPC was combined with
paclitaxel, tumor growth was dramatically reduced compared
with treatment with paclitaxel and a nonsilencing siRNA
(SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus
0.84 g; P = 0.0027). These studies show the feasibility of siRNA
as a clinically applicable therapeutic modality. (Cancer Res
2005; 65(15): 6910-8)
Introduction
Since its description in Caenorhabditis elegans (1) and mamma-
lian cells (2), use of small interfering RNA (siRNA) as a method of
gene silencing has rapidly become a powerful tool in protein
function delineation, gene discovery, and drug development (3).
The promise of specific RNA degradation has also generated much
excitement for possible use as a therapeutic modality, but in vivo
siRNA delivery has proven difficult (4). Delivery methods that are
effective for other nucleic acids are not necessarily effective for
siRNAs (5). Therefore, most studies using siRNA in vivo involve
manipulation of gene expression in a cell line before introduction
into an animal model (6, 7) or incorporation of siRNA into a viral
vector (8, 9). Delivery of ‘‘naked’’ siRNA in vivo has been restricted
to site-specific injections or through high-pressure means that are
Requests for reprints: Anil K. Sood, Departments of Gynecologic Oncology and
Cancer Biology, The University of Texas M.D. Anderson Cancer Center, 1155 Herman
Pressler, CPB6.3244, Unit 1362, Houston, TX 77030. Phone: 713-745-5266; Fax: 713-792-
7586; E-mail: asood@mdanderson.org.
I2005 American Association for Cancer Research.
not clinically practical. The only study to show orthotopic in vivo
uptake and target down-regulation of an endogenous protein after
normal systemic dosing required chemical modulation of siRNA
that will have unknown toxicities and may affect siRNA activity or
longevity (10).
We have used an ovarian cancer xenograft mouse model to
examine the efficacy of in vivo gene silencing by siRNA. Ovarian
cancer is associated with the highest mortality among all gyne-
cologic malignancies, with an estimated 22,220 cases and 16,210
deaths in the United States in 2005 (11). The majority of ovarian
cancer patients respond to initial therapy of tumor cytoreductive
surgery and platinum-based chemotherapy, but of these, f70%
will recur and succumb to disease (12). Therefore, novel thera-
peutic strategies are urgently needed to improve the outcome of
women with ovarian cancer. Fortunately, ovarian cancer has a
favorable mouse model. I.p. injected ovarian cancer cells form
tumors resembling human cancer in growth pattern, and their
response to therapy tends to be predictive of response in human
patients (13, 14).
EphA2 is a tyrosine kinase receptor in the ephrin family that plays
a key role in neuronal development (15, 16). In adults, it is expressed
to a low degree, primarily in epithelial cells (17). Several inves-
tigators have reported EphA2 overexpression in human cancers
(18–21), and we have shown that the high rate of overexpression in
ovarian cancer is associated with poor clinical outcome (22). EphA2
can function as an oncoprotein (23), and down-regulation reduces
tumorigenicity in preclinical studies of breast and pancreatic cancer
(24–26), making it an ideal therapeutic target.
We have previously used liposomes composed of the neutral
lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to de-
liver antisense oligonucleotides in vivo (27). Here, we sought to
determine the feasibility and effectiveness of delivering EphA2-
targeting siRNA in DOPC. Therapeutic delivery of siRNA directed
against EphA2 resulted in decreased protein expression in the
tumor and remarkably decreased tumor growth when combined
with chemotherapy in an orthotopic mouse model of ovarian
cancer.
Materials and Methods
Cell lines and culture. The ovarian cancer cell lines HeyA8 and
SKOV3ip1 (28) were maintained in RPMI 1640 supplemented with 15% fetal
bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas,
CA). All in vitro experiments were conducted at 60% to 80% confluence. For
in vivo injection, cells were trypsinized and centrifuged at 1,000 rpm for
7 minutes at 4jC, washed twice, and reconstituted in serum-free HBSS
(Life Technologies, Carlsbad, CA) at a concentration of 5  106 cells/mL
(SKOV3ip1) or 1.25  106 cells/mL (HeyA8) for 200 AL i.p. injections.
Small interfering RNA constructs and in vitro delivery. siRNA was
purchased from Qiagen (Valencia, CA) in three formulations. A nonsilencing
siRNA sequence, shown by BLAST search to not share sequence homology

Cancer Res 2005; 65: (15). August 1, 2005 6910 www.aacrjournals.org


with any known human mRNA (target sequence 5V-AATTCTCCGAACGTGT-
CACGT-3V) and tagged with Alexa 555, was used to determine uptake and
distribution in various tissues when given in vivo. siRNA with the target
sequence 5V-AATGACATGCCGATCTACATG-3V, designed and shown (26) to
target mRNA of the receptor tyrosine kinase EphA2, was used to down-
regulate EphA2 in vitro and in vivo. A nonsilencing siRNA construct
(sequence as above without an Alexa 555 tag) was used as control for
EphA2-targeting experiments. For in vitro delivery, siRNA (5 Ag) was incu-
bated with 30 AL RNAiFect transfection reagent (Qiagen) for 10 minutes at
room temperature and added to cells in culture at 80% confluence in 35 mm
culture plates. The medium was changed 6 hours later, and cells collected
after 48 hours as lysate for Western blot analysis.
Liposomal preparation. siRNA for in vivo delivery was either given
naked (without transfection agent), incorporated into N-[1-(2,3-dioleoylox-
yl)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP; Roche,
Indianapolis, IN), or incorporated into DOPC. DOPC and siRNA were
mixed in the presence of excess tertiary butanol at a ratio of 1:10 (w/w)
siRNA/DOPC. Tween 20 was added to the mixture in a ratio of 1:19 Tween
20:siRNA/DOPC. The mixture was vortexed, frozen in an acetone/dry ice
bath, and lyophilized. Before in vivo administration, this preparation was
hydrated with normal 0.9% saline at a concentration of 15 Ag/mL to achieve
the desired dose in 150 to 200 AL per injection. To estimate the quantity of
siRNA not taken up by liposomes, free siRNA was separated from liposomes
using 30,000 nominal molecular weight limit filter units (Millipore Corp.,
Billerica, MA). The liposomal suspension was added to the filters and
centrifuged at 5,000  g for 40 minutes at room temperature. Fractions
were collected, the material trapped in the filter was reconstituted with
0.9% saline, and siRNA of the collected fraction and the elute were
measured by spectrophotometry.
Orthotopic in vivo model of advanced ovarian cancer and tissue
processing. Female athymic nude mice (NCr-nu) were purchased from
the National Cancer Institute-Frederick Cancer Research and Develop-
ment Center (Frederick, MD) and housed in specific pathogen-free
conditions. They were cared for in accordance with guidelines set forth by
the American Association for Accreditation of Laboratory Animal Care
and the USPHS ‘‘Policy on Human Care and Use of Laboratory Animals,’’
and all studies were approved and supervised by The University of Texas
M.D. Anderson Cancer Center Institutional Animal Care and Use
Committee. Tumors were established by i.p. injection of cells prepared
as above. This model reflects the i.p. growth pattern of advanced ovarian
cancer, as intra-abdominal spread is the main mechanism of ovarian
cancer metastasis (12–14). Studies to determine uptake of single-dose
fluorescent siRNA in tissue or silencing potential of single-dose siRNA
against EphA2 were initiated once i.p. tumors reached a size of 0.5 to 1.0
cm3 as assessed by palpation (f17 days after injection). Liposomal siRNA
(5 Ag) was given as a 200 AL i.v. bolus into the tail vein under normal
pressure, and tumor and other tissues were harvested at various time
points after injection (1 hour, 6 hours, 48 hours, 4 days, 7 days, or 10
days). Tissue specimens were snap frozen for lysate preparation, fixed in
formalin for paraffin embedding, or frozen in OCT medium for frozen
slide preparation. For long-term experiments to assess tumor growth,
therapy began 1 week after tumor cell injection. Paclitaxel (100 Ag) or
vehicle was injected i.p. once weekly; siRNAs (nonspecific or EphA2
targeting, 150 Ag/kg) in liposomes, or empty liposomes, were injected
twice weekly i.v. in 150 to 200 AL volume (depending on mouse weight)
with normal pressure. Mice (n = 10 per group) were monitored for
adverse effects, and tumors were harvested after 4 weeks of therapy or
when any of the mice began to appear moribund. Mouse weight, tumor
weight, and distribution of tumor were recorded. Vital organs were also
harvested and necropsies were done by a board-certified pathologist for
evidence of tissue toxicity.
Immunofluorescence and confocal microscopy. Tissue for immuno-
fluorescence was collected from sacrificed mice, immediately placed in
OCT medium, and rapidly frozen. Frozen sections were cut at 8 Am
sections for conventional microscopy and 30 Am sections for confocal
microscopy. Tissue was fixed with acetone and either examined
immediately or costained for f4/80 (to detect scavenging macrophages)
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In vivo siRNA-Mediated Anti-EphA2 Therapy
or CD31 (to detect endothelial cells). For immunofluorescence detection,
slides were blocked with 5% normal horse serum and 1% normal goat
serum (Invitrogen, Carlsbad, CA) in PBS, exposed to 10 Ag/mL anti-f4/80
antibody (Serotec, Oxford, United Kingdom) or 0.625 Ag/mL anti-CD31
antibody in blocking solution overnight at 4jC, washed with PBS, and
exposed to 4 Ag/mL anti-rat antibody-Alexa 488 (Molecular Probes,
Eugene, OR) in blocking solution for 1 hour at room temperature. Slides
were washed with PBS, exposed to either 1.0 Ag/mL Hoescht (Molecular
Probes, in PBS) or 10 nmol/L Sytox green (Molecular Probes, in PBS) for
10 minutes to stain nuclei, washed, and covered with propylgallate and
coverslips for microscopic evaluation. Conventional microscopy was done
with a Zeiss AxioPlan 2 microscope (Carl Zeiss, Inc., Germany),
Hamamatsu ORCA-ER Digital camera (Hamamatsu Corp., Japan), and
ImagePro software (Media Cybernetics, Silver Spring, MD). Fluorescence
in three dimensions within 30 Am sections was examined with a Zeiss
LSM 510 confocal microscope and LSM 510 Image Browser software
(Carl Zeiss).
Western blot. Cultured cell lysates were prepared by washing cells
with PBS followed by incubation in modified radioimmunoprecipitation
assay buffer (RIPA) lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl,
1% Triton, 0.5% deoxycholate plus 25 Ag/mL leupeptin, 10 Ag/mL
aprotinin, 2 mmol/L EDTA, 1 mmol/L sodium orthovanadate; Sigma
Chemical Co., St. Louis, MO) for 10 minutes at 4jC. Cells were scraped
from plates and centrifuged at 13,000 rpm for 20 minutes at 4jC and the
supernatant was stored at 80jC. To prepare lysate from snap-frozen
tissue, f30 mm3 cuts of tissue were incubated on ice in RIPA for 3 hours,
mortar and pestle disrupted and homogenized, and centrifuged, and the
supernatant was stored at 80jC. Samples from three regions of the
tumor were collected and tested individually. Protein concentrations were
determined using a BCA Protein Assay Reagent kit (Pierce Biotechnology,
Rockford, IL) and subjected to 10% SDS-PAGE separation. Samples
transferred to a nitrocellulose membrane by semidry electrophoresis (Bio-
Rad Laboratories, Hercules, CA) were incubated with 0.625 Ag/mL anti-
EphA2 antibody (Upstate, Lake Placid, NY) overnight at 4jC, detected
with 1 Ag/mL horseradish peroxidase (HRP)–conjugated anti-mouse IgG
(Amersham, Piscataway, NJ), and developed using enhanced chemilumi-
nescence detection kit (Pierce Biotechnology). Membranes were tested for
h-actin (0.1 Ag/mL anti-h-actin primary antibody; Sigma Chemical) to
confirm equal loading.
Immunohistochemistry. Formalin-fixed, paraffin-embedded sections
were deparaffinized by sequential washing with xylene, 100% ethanol, 95%
ethanol, 80% ethanol, and PBS. Antigen retrieval was done by heating in
steam cooker in 0.2 mol/L Tris-HCl (pH 9.0) for 20 minutes. After cooling
and PBS wash, endogenous peroxide was blocked with 3% H2O2 in
methanol for 5 minutes. Nonspecific proteins and exposed endogenous
mouse IgG antibodies were blocked with 0.13 Ag/mL mouse IgG Fc blocker
(The Jackson Laboratory, Bar Harbor, ME) in 0.5% blocking agent (TSA
Biotin System kit, Perkin-Elmer, Boston, MA) overnight at 4jC. Slides were
incubated in primary antibody (5 Ag/mL mouse anti-EphA2 clone EA5, a
kind gift of Dr. Michael Kinch, MedImmune, Inc., Gaithersburg, MD) for
4 hours at 4jC and washed followed by 1.5 Ag/mL biotinylated horse anti-
mouse (Vector Labs, Burlingame, CA) for 1 hour at room temperature. The
secondary antibody signal was enhanced with 0.75 Ag/mL streptavidin-HRP
(DakoCytomation, Carpinteria, CA) for 30 minutes, detected with 3,3V-
diaminobenzidine (DAB; Phoenix Biotechnologies, Huntsville, AL) substrate
for 7 minutes, and counterstained with Gill no. 3 hematoxylin (Sigma
Chemical) for 20 seconds.
Microvessel density. To determine microvessel density (MVD), collected
tissue was frozen in OCT with liquid nitrogen. Eight-micron sections were
fixed in acetone for 10 minutes, washed in PBS, and blocked with 10% fish
gelatin for 10 minutes. Slides were exposed to rat anti-mouse CD31 for
2 hours at room temperature, diluted in blocking solution, and exposed,
after three PBS washes (5 minutes each), to anti-rat IgG conjugated to HRP.
HRP was detected with DAB for 10 minutes and counterstained with Gill no.
3 hematoxylin for 20 seconds. To calculate MVD, five representative
photographs were taken of each slide (1 slide per mouse, 5 slides per group),
and the number of vessels per field ( final magnification, 100) was counted
Cancer Res 2005; 65: (15). August 1, 2005
Cancer Research

by an examiner blinded to treatment group. A vessel was defined as an open

lumen with at least one adjacent CD31-positive cell. Multiple positive cells
beside a single lumen are counted as one vessel.
Statistical considerations. For in vivo therapy experiments, 10 mice
in each group were used as directed by a power analysis to detect a 50%
reduction in tumor size (h error = 0.2). Continuous variables (tumor size
and MVD) were analyzed for statistical significance (achieved if P < 0.05)
with Student’s t test for two-group comparisons and ANOVA for multiple-
group comparisons. If values were not normally distributed, the Mann-
Whitney rank sum test was used using Stata 8 software (College Station, TX).

Results
Incorporation of small interfering RNA into liposomes. An
efficient delivery vehicle is necessary for in vivo delivery. Cationic
liposomes, while efficiently taking up nucleic acids, have had
limited success for in vivo gene down-regulation perhaps because
of their affinity for endothelial cells, their stable intracellular
nature, or their failure to release siRNA contents. We selected
DOPC because we have successfully used this molecule to deliver
antisense oligonucleotides in vivo (27). When mixed together, >90%
of liposomes spontaneously incorporate fluorescently tagged siRNA
when microscopically examined for fluorescence (Fig. 1A and B).
To estimate the quantity of siRNA not taken up by liposomes, free
siRNA was separated from liposomes with siRNA by column
filtration and residual siRNA was measured by spectrophotometry.
It is estimated that f65% of the siRNA are incorporated into
liposomes. In our experience, the liposomal-nucleic acid complexes
are stable for at least 4 weeks when stored at 20jC.
Delivery of small interfering RNA into orthotopically
implanted ovarian tumor. To examine whether siRNA could be
effectively delivered into tumor cells, we used a nonsilencing siRNA
tagged with the fluorochrome Alexa 555 in DOPC complexes. Mice
with HeyA8 orthotopic tumors (15 days after i.p. inoculation of
tumor cells; Fig. 1C) were i.v. injected with 5 Ag DOPC-conjugated
nonsilencing siRNA/Alexa 555. Tumors were harvested at 1 hour
and at 4, 7, or 10 days and examined for fluorescence. As early as
1 hour after injection, punctated emissions of the siRNA were
noted in the perinuclear region of individual cells (Fig. 1E) that
were absent in the emission pattern of tumor injected with
nonfluorescent siRNA (Fig. 1D). siRNA was seen in 80% of fields Figure 1. In vivo siRNA distribution to HeyA8 i.p. tumor tissue after a single
examined (original magnification 400) and an estimated 30% of siRNA dose. Fluorescent (A) and phase (B ) view of liposomes after siRNA
incorporation. In this frame, 91% of visible liposomes contain fluorescent
all tumor cells. This distribution can be appreciated by a low-power siRNA. C, H&E stain (original magnification, 280) of HeyA8 i.p. tumor.
view (Fig. 1H), although the signal strength is weaker at this D, autofluorescence in tumor 48 hours after i.v. administration of 5 Ag
nonfluorescent control siRNA. Tumors were harvested and frozen in OCT
magnification. medium; slides were fixed in acetone and exposed to Hoescht to stain nuclei blue
To confirm that the siRNA was present in tumor cells and not before viewing. E, fluorescent emission of Alexa 555 siRNA incorporated in
simply scavenged by macrophages, separate slides were stained for DOPC within the same tumor shown in A is seen as red punctuations within the
cytoplasm. Samples were processed identically to those in B. F, samples were
f4/80 to identify scavenging macrophages. These macrophages are processed as above and additionally exposed to anti-f4/80 antibody to detect
seen to surround nests of tumor cells that contain perinuclear scavenging macrophages and Alexa 488–tagged secondary antibody. Alexa 555
siRNA and have about the same rate of siRNA uptake as tumor cells siRNA is seen in both tumor cells and surrounding macrophages. G, 30 Am
sections were examined with confocal microscopy. Photographs taken every
(Fig. 1F), suggesting that siRNA is delivered directly into the tumor 1 Am were stacked and examined from the lateral aspect. Fluorescent siRNA is
cells. To confirm that the fluorescent signal was not a contami- noted throughout the section, and high-magnification, three-dimensional
construction shows that all siRNA are perinuclear (data not shown). Fluorescent
nating secondary antibody or an artifact of processing, 30 Am Alexa 488–tagged secondary antibody (green ) is trapped on the surface, too
sections were examined with confocal microscopy. This technique large to penetrate tissue. Nuclei are labeled with Hoescht (blue ). D-G, original
permitted signal detection within the middle of tissue rather than magnification, 400. H, lower-power (original magnification, 200) view of
siRNA distribution. The fluorescent signal is weaker and more difficult to detect
from surface emissions alone. After evaluating emissions at but allows an appreciation of distribution through f30% of the tumor after a
multiple depths, a three-dimensional cross-section was created. single dose. Fluorescence is not visible at 100 magnification.
Lateral views clearly show the presence of fluorescently tagged
siRNA within tissue parenchyma (Fig. 1G). In this view, fluorescent (Hoescht) small enough to penetrate tissue. Tumors collected at
emission from macrophage staining (green) was noted only at the 4, 7, and 10 days after a single injection were also noted to retain
surface, because the detecting antibody is too large to penetrate siRNA fluorescence. However, because this fluorescently tagged
tissue. Emission from nuclear staining (blue) is the result of a dye siRNA should be a nonsilencing construct, longevity after

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Figure 2. siRNA uptake into tumors by other delivery methods. A, tumors
collected after administration of a single high-dose (10 Ag) siRNA without
transfection reagent are seen to rarely take up siRNA, but uptake is distributed
throughout the cytoplasm. B, tumors collected from mice given Alexa 555 siRNA
complexed in DOTAP were stained with CD31-Alexa 488 (green ) to identify
endothelial cells. siRNA is seen to complex near the vasculature with poor
parenchymal tumor uptake. C, tumors collected after delivery of siRNA in DOPC
are noted to have siRNA deep within the tumor, not clustered near the
vasculature (CD31 in green ). Original magnification, 400.
Figure 3. In vivo siRNA distribution to
major organs. siRNA uptake by the liver
(A-C ), kidney (D-F), and lung (G-I ).
Histologic sections from these organs were
collected after i.v. injection of 5 Ag DOPC
siRNA. A, D, and G, H&E staining of these
organs. B, E, and H, Alexa 555 siRNA
(red) within the organ parenchymal cells. In
the kidney, siRNA is noted in both tubules
and the glomerulus (bottom aspect of E ).
C, F , and I, natural autofluorescence of
each tissue after a single i.v. injection of
nonfluorescent siRNA. All pictures are
taken at original magnification (400).
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In vivo siRNA-Mediated Anti-EphA2 Therapy
administration of a mRNA-targeting construct is likely to be of
shorter duration.
Tissue distribution of small interfering RNA after delivery
by conventional methods. To compare tumor delivery of siRNA
with other methods, we injected siRNA i.v. either without a
transfection agent (naked) or complexed in DOTAP. After ad-
ministration of naked fluorescent siRNA, fluorescence was
rarely observed (2% of 40 fields, <1% of cells; Fig. 2A)
although notably present in the desired perinuclear location in
positive cells. Administration of siRNA complexed with DOTAP
showed sporadic presence of fluorescence within tumor tissue
(7% of all fields examined). However, the observed fluorescence
was primarily adjacent to CD31-positive endothelial cells
(shown green in Fig. 2B), bringing into question whether the
liposomal contents were released or trapped in the vasculature.
Tissue obtained after siRNA delivery in DOPC and stained
with CD31 showed that delivery was not restricted to the
vasculature and is efficiently delivered deep into the tumor
parenchyma. Our DOPC liposome preparation was associated
with an estimated 10-fold improvement in delivery of siRNA
compared with DOTAP and 30-fold improvement over naked
siRNA.
Tissue distribution of small interfering RNA throughout
vital organs. To examine the distribution of delivery to other
organs, sections of the liver, kidney, spleen, heart, lung, and brain
were examined for fluorescence after a single dose of Alexa 555
siRNA in DOPC. Specimens from mice treated with nonfluorescent
siRNA were examined to determine background fluorescence.
There was significant siRNA uptake and cytoplasmic distribution in
the liver, kidney, and lung (Fig. 3A-C, respectively). There was a
small amount of uptake in the heart but significantly greater than
that of untreated heart tissue. The fluorescence emitted by
endogenous protein products in the spleen, pancreas, and brain
6913 Cancer Res 2005; 65: (15). August 1, 2005
Cancer Research
made evaluation more difficult and precludes a definitive
conclusion that liposomes are incorporated in these tissues.
Similar patterns of uptake after DOTAP-complexed and naked
siRNA administration were seen in other organs as was seen in the
tumor. DOTAP complexes formed multiple large fluorescent
signals near the vasculature without perinuclear punctuations in
the liver. There was a high level of uptake in the kidney both near
the vasculature and by individual cells. Naked siRNA administra-
tion did result in uptake by a large percentage of liver and kidney
cells, but the fluorescent signal was greatly decreased compared
with uptake by DOPC-complexed siRNA.
Down-regulation of EphA2 with liposomal small interfering
RNA. We have shown previously that EphA2 is overexpressed by
a large percentage of patients with ovarian cancer and that
overexpression is predictive of poor outcome (22). Furthermore,
this protein has low relative expression in the adult and so is an
attractive tumor selective target. Therefore, we used EphA2 as a
model to test the efficacy of siRNA therapy. In vitro, both HeyA8
and SKOV3ip1 ovarian cancer cell lines transfected with EphA2
siRNA showed a 95% decrease in EphA2 expression compared with
transfection with control siRNA as determined by Western blot
analysis (data not shown). Subsequently, we tested the ability of
DOPC liposomal siRNA to silence EphA2 in an orthotopic in vivo
model. EphA2-targeting siRNA-DOPC was given to tumor-bearing
mice and tumor collected at various time points. Measurement of
EphA2 by Western blot of tumor lysate (Fig. 4A) and by
immunohistochemistry (Fig. 4D) showed that tumor collected 48
hours following administration of single-dose anti-EphA2 siRNA
Figure 4. In vivo down-regulation of EphA2 by siRNA. A, Western blot of lysate
from orthotopic tumors collected 48 hours after a single administration of control
siRNA (lanes 1 and 2 ) or EphA2-targeting (lanes 3-5 ) siRNA, each complexed
within DOPC. To control for sampling error, lanes 1a and 1b are separate
preparations from the same tumor treated with control siRNA. Similarly, lanes 5a
and 5b are separate preparations from the same tumor treated with
EphA2-targeting siRNA. Lanes 2 to 4, additional tumor-bearing mice treated with
control or EphA2-targeting siRNA-DOPC. Adjacent sections were stained by
H&E to confirm the presence of tumor. B, immunohistochemical staining for
EphA2 of tissue treated with control siRNA/DOPC. The typical cobblestoning
appearance of this overexpressed (22) membrane-bound protein is noted.
C, immunohistochemistry 48 hours after a single treatment of EphA2-targeting
siRNA without a transfection agent (‘‘naked’’) is shown and had no detectable
affect on EphA2 expression. D, treatment of EphA2-targeting siRNA
encapsulated within DOPC effectively down-regulated EphA2 expression 48
hours after a single dose. EphA2 expression is restored 1 week after a single
treatment (not pictured). B-D, original magnification, 400.
Cancer Res 2005; 65: (15). August 1, 2005
had significantly decreased EphA2 expression compared with
treatment with a nonspecific siRNA (Fig. 4B) or naked siRNA
(Fig. 4C). Expression of EphA2 remained suppressed at 4 days,
was recovering after 7 days, and had returned to normal levels
by 10 days. Therefore, we used twice-weekly dosing of anti-EphA2
siRNA for subsequent therapy experiments.
In vivo therapy experiments with liposomal anti-EphA2
small interfering RNA. Female nude mice (n = 50 per cell line, 10
per group) were injected with HeyA8 or SKOV3ip1 cells into the
peritoneal cavity. One week after tumor cell injection, animals were
randomly allocated to five treatment groups: (a) empty liposomes,
(b) nonspecific siRNA-DOPC, (c) EphA2-targeted siRNA-DOPC, (d)
paclitaxel and nonspecific siRNA-DOPC, and (e) paclitaxel and
EphA2-targeting siRNA-DOPC. After 4 weeks of therapy, the
animals were sacrificed and necropsies were done. Tumors were
excised and weighed. Treatment with anti-EphA2 siRNA, paclitaxel
plus control siRNA, and paclitaxel plus anti-EphA2 siRNA were all
effective in reducing tumor size (overall ANOVA, P < 0.001 for both
cell lines), with combination therapy leading to 86% to 91%
reduction compared with treatment with control siRNA alone
(Fig. 5A and B). Targeting EphA2 with siRNA alone diminished
tumor growth in both lines when compared with control siRNA
alone (HeyA8: 0.98 versus 1.51 g, respectively; P = 0.155; SKOV3ip1:
0.35 versus 0.70 g, respectively; P = 0.020). EphA2-targeting siRNA in
combination with paclitaxel significantly reduced tumor growth by
67% to 82% compared with nonspecific siRNA and paclitaxel
(HeyA8: 0.21 versus 0.84 g, respectively; P < 0.003; SKOV3ip1: 0.04
versus 0.22 g, respectively; P < 0.001). This pattern of tumor growth
inhibition (moderate inhibition with EphA2 targeting alone,
marked inhibition in combination with paclitaxel) is similar to
that seen with antibody-based EphA2 down-regulation in this
mouse model (data not shown).
Data from other measured variables of these therapy experi-
ments are shown in Table 1. The incidence of tumor formation was
not significantly different among the five groups in either cell line.
However, the number of nodules formed was reduced by both
EphA2 siRNA and paclitaxel individually, and further reduced by
the combination, in both cell lines. Therapy was not continued long
enough to allow development of ascites, which is typical in the
SKOV3ip1 line 5 to 6 weeks after injection.
Interestingly, administration of nonspecific siRNA-DOPC
resulted in some reduction in tumor growth, although statistically
significant only in the HeyA8 model, when compared with empty
liposomes. These data may support prior reports that siRNA
without a specific mRNA target may have nonspecific effects that
affect tumor growth (3) and further support our hypothesis that
siRNA-DOPC is delivered to the tumor parenchyma.
Effects of EphA2-targeted therapy on vascular density. To
explore the mechanisms involved in the reduced tumor formation
with this therapy, we first confirmed that EphA2 levels remained
low with long-term therapy. Tumors harvested at the conclusion of
therapy trials were subjected to immunohistochemistry for EphA2
(Fig. 6A). Both groups treated with EphA2-targeting siRNA showed
f50% decrease in distribution of expression compared with
control siRNA or control siRNA plus paclitaxel. The intensity of
staining was not significantly different among the groups,
suggesting that gene silencing is effective if delivery is achieved.
EphA2 has been shown to play a role in migration, invasion, and
angiogenesis (21). To assess possible antiangiogenic effects of
EphA2 down-regulation, tissues obtained at the conclusion of long-
term therapy trials were subjected to immunohistochemistry for
6914 www.aacrjournals.org
 In vivo siRNA-Mediated Anti-EphA2 Therapy

Figure 5. Therapeutic efficacy of siRNA-

mediated EphA2 down-regulation. A and B,
nude mice were injected i.p. with 2.5  105
HeyA8 cells (A ) or 1.0  106 SKOV3ip1
cells (B) and randomly allocated to one of
five groups, with therapy beginning 1 week
after cell injection: (a ) empty DOPC
liposomes, (b) control siRNA in DOPC,
(c ) EphA2-targeting siRNA in DOPC, (d )
paclitaxel + control siRNA in DOPC, or (e)
paclitaxel + EphA2 siRNA in DOPC. siRNA/
liposomes were injected twice weekly at
a dose of 150 Ag/kg siRNA. Paclitaxel
(100 Ag) or vehicle (first three groups) was
injected i.p. once weekly. When control
animals began to appear moribund from
tumor volume (4-5 weeks after cell
injection), all animals in an experiment were
sacrificed, and mouse weight, tumor
weight, and tumor location were recorded.
Left, mean tumor weight F SD; right,
individual tumor values. Data for HeyA8
represent the average of two identical
experiments, which individually
gave the same statistical conclusions as
the combination.

CD31, and MVD was calculated for each group. Representative most reproducible systemic delivery of siRNA in vivo has been rapid
sections are shown in Fig. 6B, with the mean number of vessels for injection of high volume of material (i.e., 2 mL into a mouse with
each group in Fig. 6C. MVD was unaffected by control siRNA (15.7 estimated 4 mL total blood volume over 5 seconds), hydrodynam-
versus 17.9; P = 0.29) but was reduced by 52% with EphA2-targeting ically forcing siRNA into the liver (29). Such a technique would not
siRNA alone (8.6 versus 17.9; P < 0.001). Paclitaxel did not affect
MVD (18.2 versus 17.9; P = 0.80). The addition of EphA2 siRNA to
paclitaxel similarly reduced MVD by 74% compared with paclitaxel Table 1. Characteristics of tumors after siRNA/paclitaxel
and control siRNA (4.7 versus 18.2; P < 0.001). therapy
No toxicities were observed by behavioral changes, such as
eating habits and mobility in animals treated with liposomal siRNA Cell line Groups Incidence No. Ps for no.
preparations, both those that are nonsilencing and those targeting (%)* nodules of nodules
EphA2. Mouse weights were not significantly different among the
SKOV3ip1 Liposomes 100 16.3 NS
five groups of animals, suggesting that eating and drinking habits
Control siRNA 100 17.8 0.004
were not affected. Organ sections were reviewed by a board- EphA2 siRNA 100 6
certified pathologist, and after 5 weeks of therapy, no histologic Paclitaxel + 100 4.3 0.001
toxicities were detected in the liver, kidney, heart, lung, or brain. control siRNA
A slight increase in the size of the white pulp of the spleen was Paclitaxel + 90 2.2
noted in all four siRNA groups, which may be indicative of a EphA2 siRNA
general inflammatory response. HeyA8 Liposomes 100 4.8 NS
Control siRNA 95 4.6 0.056
EphA2 siRNA 85 2.8
Paclitaxel + 85 2.5 0.034
Discussion
control siRNA
In this study, we describe the therapeutic delivery of gene-specific Paclitaxel + 80 1.5
siRNA into the tumor using DOPC liposomes, with subsequent EphA2 siRNA
reduced protein expression and reduced tumor growth. To the best
of our knowledge, this is the first study to collectively show the
direct delivery, gene targeting, and growth attenuation after *There was no significant difference (P < 0.05) between any two
systemic delivery of siRNA in an orthotopic model of ovarian compared groups (by Student’s t test) or all groups (by ANOVA) for
cancer. The significance of this work is that packaging of siRNA into incidence of tumor formation.
liposomes is rapidly transferable to a clinical setting. Previously, the

www.aacrjournals.org 6915 Cancer Res 2005; 65: (15). August 1, 2005

Cancer Research
Figure 6. EphA2 expression and MVD after prolonged siRNA therapy. A,
tumors collected at the conclusion of the HeyA8 therapy trials were subjected
to immunohistochemistry for EphA2. Representative sections. The distribution of
EphA2 staining in five samples of each group was quantified, with the mean
for each group shown in the bottom right of each panel. B and C, these tumors
were stained for CD31, and the number of vessels per 10 field counted as
described in Materials and Methods. Representative sections from each group
are pictured, with mean MVD pictured graphically in C .
be feasible in a clinical setting, whereas liposomes have been used
extensively clinically for chemotherapy and other delivery systems.
Because delivery in this study was efficient to other vital organs,
most notably in the liver and kidney, this method may be used in
Cancer Res 2005; 65: (15). August 1, 2005
noncancerous conditions shown to be amenable to siRNA therapy
in preclinical models, such as viral hepatitis (9, 29) and HIV (30).
However, this mode of delivery is not tissue specific, so it will be
important that the gene chosen to down-regulate with siRNA is
not crucial to function by normal cells. Alternatively, further
modifications of the liposome may allow tumor-selective delivery
(31, 32).
The first demonstration that siRNA had activity in vivo was in
the hydrodynamic injection of naked siRNA that effectively
decreased luciferase expression in the livers of mice (29). Along
with confirmatory reports of high-pressure i.v. injection (33, 34),
others have shown that siRNA has activity in vivo using delivery
in viral vectors (8, 9), retinal electroporation (35), and direct
intracellular (36), intratumoral (37), intravitreal (38), intranasal
(39), and intrathecal (40) administration. Although these methods
are useful in a preclinical setting, their delivery methods and the
climate of viral gene therapy make clinical applicability limited.
Sorensen et al. effectively reduced tumor necrosis factor-a
expression in the liver and spleen by delivering siRNA packaged in
cationic liposomes (DOTAP), protecting mice from a lethal dose of
lipopolysaccharide (41). We have found that DOTAP accumulates
near the vasculature and is preferentially taken up by the liver and
spleen, limiting its effectiveness in systemic or antitumor therapy.
Soutschek et al. have reported that siRNA conjugated with
cholesterol improved delivery to multiple organs and that down-
regulation of ApoB was achieved in liver and jejunum (10).
However, the effects of cholesterol conjugation on siRNA activity
and duration of effect, efficiency of uptake in tumors, and toxicities
are not known. Duxbury et al. have shown that systemic delivery of
naked siRNA-targeting FAK (42), EphA2 (26), or CEACAM6 (43)
down-regulated protein expression and decreased growth of a
single s.c. injected malignant pancreatic cell line. It is possible that
naked siRNA may be effectively delivered to s.c. sites, but not to
orthotopic sites, as supported by our results. To the best of our
knowledge, others have not reported successful therapy with naked
unaltered siRNA in other cancer models.
Recent studies suggest that the specificity of siRNA may not
be as absolute as initially hoped. An analysis of gene expression
profiling suggested that RNA down-regulation might occur with
as few as 11 complementary base pairs within the 21-bp siRNA
sequence (44). A recent study of commonly used siRNA sequences
found that f75% of these sequences had nonspecific targeting
(45). Therefore, in siRNA design, a BLAST search for cross-reactive
21-bp sequences is insufficient to have confidence that the mRNA
of interest is the only target. Furthermore, siRNAs may bind mRNA
of only near-perfect complementarity and prevent translation
without degradation (46). This is the mechanism used by en-
dogenously produced microRNAs (miRNA), believed to be another
method of natural regulation of gene expression (47). Crossover of
siRNA into the miRNA pathway or down-regulation by partial
homology seem to be minimal and require participation of several
siRNA sequences, but this potential should caution conclusions
made regarding the specificity of gene down-regulation. It is
difficult at this time to speculate which particular proteins could
be ‘‘off-site’’ targets of nonspecific siRNA silencing. Studies with
microarray analysis or reporter arrays may allow such projections
to be made in the future (44, 48). Another level of questionable
specificity of siRNA introduction lies in activation of the innate
immune system. siRNA therapy has, in some circumstances, been
shown to activate IFN (49, 50). Of course, in the treatment of
cancer, IFN induction may be of additional benefit, as long as
6916 www.aacrjournals.org
 In vivo siRNA-Mediated Anti-EphA2 Therapy

toxicities are limited. This is supported by our finding that therapy
with a nonspecific siRNA construct results in some reduction in
tumor growth compared with empty liposomes.
Toxicities of liposomes are believed to be limited. Liposomal
chemotherapy is routinely used in treatment of ovarian and other
cancers (51). In a phase I trial with cationic liposomes carrying a
plasmid encoding the E1A gene, fever and pain 3 hours after
treatment were the dose-limiting toxicities (52). Although this is
the best estimation of side effects we can currently predict, delivery
of siRNA is less likely to be recognized as foreign, and host
response will almost certainly differ.
The charge of the liposome affects the tissue specificity of
liposomal uptake. Macrophages seem to preferentially take up
negatively charged liposomes (53). Different malignant cell lines
have varying uptake patterns regarding positive, neutral, or
negative charges, and in vivo uptake patterns may differ further
(53). Liposomal makeup also influences cellular toxicity, with
siRNA delivery using a liposome with a higher proportion of
neutral lipids leading to less cellular toxicity without compromis-
ing ability to down-regulate gene expression in vitro (54). Clearly, a
complete understanding of the best liposomal makeup for delivery
of therapeutic substances is still evolving. It is possible that with
siRNA delivery the use of a neutral lipid, such as DOPC, allows a
balance among efficient uptake of the siRNA into a liposome at
preparation, uptake of the liposome into a cell, and breakdown of the
intracellular liposome with release of siRNA contents into the
cytoplasm.
EphA2 is an attractive target for antitumor therapies. It is
minimally expressed in adults, being limited to some epithelial
tissues (55), and the EphA2 knockout mouse is phenotypically
normal (56). However, EphA2 is overexpressed by several cancers
(21), including ovarian, in which it is associated with poorer
survival (22). Furthermore, the receptor primarily exhibits carci-
nogenic properties through high levels of the unphosphorylated
form. Therefore, decreasing total EphA2 levels are more likely to be
effective than attempts to block its activation. We have shown that
EphA2-targeting siRNA therapy leads to a decrease in MVD. Others
have seen an antiangiogenic effect with EphA2 down-regulation
(25), and we have seen this with antibody-based approaches to
decrease EphA2 expression in vivo.5 Delineation of biological
pathways dependent on EphA2 is difficult, because EphA2
modulation has little effect on monolayer cell culture properties
(21). Further studies of in vivo–treated tissues may help to define
other mechanisms affected by EphA2 overexpression.
In vivo delivery of siRNA in experimental models has been
shown to provide feasibility for use in humans. Liposomal
delivery of drugs is established and safe, and their use for siRNA
delivery may make this therapeutic modality clinically attractive.
We have shown that using DOPC-complexed siRNA allows
delivery to tumor and other tissues, with corresponding gene
targeting and reduced tumor growth. With further study and a
cautious approach, this is a model that can be taken into a
clinical setting for cancer therapy as well as for other conditions
amenable to specific gene down-regulation.
Acknowledgments
Received 2/17/2005; revised 5/2/2005; accepted 5/18/2005.
Grant support: The University of Texas M.D. Anderson Cancer Center Bettyann
Asche-Murray Fellowship Award (C.N. Landen) and Department of Defense grant
W81XWH-04-1-0227 (A.K. Sood).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
5
Unpublished data.

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