Cancer Gene Therapy (2009) 16, 625–637
r
2009 Nature Publishing Group All rights reserved 0929-1903/09 $32.00
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ORIGINAL ARTICLE
INGN 007, an oncolytic adenovirus vector, replicates in Syrian
hamsters but not mice: comparison of biodistribution studies
B Ying1,7, K Toth2,7, JF Spencer2, J Meyer1, AE Tollefson2, D Patra2,4, D Dhar2,
EV Shashkova1,5, M Kuppuswamy2, K Doronin2,5, MA Thomas2, LA Zumstein3,6,
WSM Wold2 and DLLichtenstein1
1
VirRx Inc., St Louis, MO, USA; 2Department of Molecular Microbiology and Immunology, Saint Louis
University School of Medicine, St Louis, MO, USA and 3Introgen Therapeutics Inc., Houston, TX, USA
Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were
conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity
and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous
administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were
also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were
compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early
after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that
of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and
Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung
and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian
hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about
INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis
and oncolytic Ad vectors.
Cancer Gene Therapy (2009) 16, 625–637; doi:10.1038/cgt.2009.6; published online 6 February 2009
Keywords: adenovirus; biodistribution; oncolytic; Syrian hamster; mice; preclinical
Introduction
An emerging modality for the treatment of cancer is the
use of oncolytic (replication competent, RC) viral vectors
whose therapeutic principle is multiple rounds of lytic
vector replication resulting in widespread tumor cell
destruction.1 Vectors from many different virus families
are being explored as oncolytic agents including those
based on wild-type (wt) human adenovirus (Ad) serotype
5 (Ad5). Most oncolytic Ad vectors, including the bestcharacterized ONYX-015,2 are genetically engineered to
Correspondence: Dr DL Lichtenstein, VirRx Inc., Center for
Emerging Technologies, Suite 217, 4041 Forest Park Ave,
St Louis, MO 63108, USA.
E-mail: [email protected]
4
Current address: Department of Orthopedic Surgery, Washington
University in St Louis, St Louis, MO, USA.
5
Current address: Division of Infectious Diseases, Department of
Internal Medicine, Mayo Clinic, Rochester, MN, USA.
6
Current address: PaxVax Inc., San Diego, CA, USA.
7
These authors contributed equally to this work.
Received 11 September 2008; revised 7 November 2008; accepted 24
December 2008; published online 6 February 2009
replicate preferentially in neoplastic cells as opposed to
normal cells. However, these genetic alterations frequently attenuate vector replication, which is in opposition to the therapeutic principle. Although Ad can be
safely administered to humans,3 promising preclinical
studies have not been translated into similar efficacy in
cancer patients. Efficacy has been modest even when
combined with radiation or chemotherapy. These clinical
data underscore the need for oncolytic vectors with
improved efficacy to achieve successful clinical translation
of this new treatment.
The oncolytic Ad vector INGN 007 (VRX-007) was
designed to maximize vector replication. INGN 007,
which is based on Ad5, does not contain a genetic
alteration to restrict replication to malignant cells.
However, INGN 007 was engineered to overexpress the
Ad-encoded protein named adenovirus death protein
(ADP; formerly named E3-11.6K).4 This viral protein,
which is required for efficient release of Ad at the
culmination of the infection cycle,5–8 enhances the cell to
cell spread of vectors in which it is overexpressed and
improves efficacy in tumor xenograft models.9–15 Other
groups have also incorporated ADP into their oncolytic
Ad vectors with beneficial results.16–23
INGN 007 biodistribution in hamsters and mice
B Ying et al
626
Enhanced efficacy, however, cannot be achieved at the
expense of vector safety. The safety characteristics of a
vector are determined in part by the distribution of the
vector within the host. Biodistribution studies with RC
Ad vectors have largely been conducted in mice.24–29
However, the utility of this model with respect to RC Ad
vectors is questionable because Ad replication in normal
mouse tissues is inefficient at best.30–34 Biodistribution
and safety studies with oncolytic vectors require a
permissive, immunocompetent animal model in which
the effect of vector replication in normal host tissues and
the immune system response to infection can be examined. Although animal models other than the mouse have
been explored, these models are only semipermissive,
expensive or difficult to work with.35–43 Our laboratory
has recently developed the golden Syrian hamster
(Mesocricetus auratus) as a permissive, immunocompetent
animal model for efficacy studies of oncolytic Ad
vectors44,45 and as a model for Ad pathogenicity and
testing of anti-Ad drugs.46 We now report the first
comprehensive biodistribution study of an oncolytic Ad
vector in the Syrian hamster model. In addition, INGN
007 biodistribution was examined in C57BL/6 mice,
a model in which the biodistribution of previous Adbased vectors has been examined. The results from both
models indicate that Ad-based vectors are widely distributed shortly after intravenous administration, that
vector DNA decays rapidly but persists for as long as a
year in some organs and that INGN 007 and wt Ad5
replicate in certain Syrian hamster organs but not mouse
organs.
Materials and methods
Cells and viruses
Human HEK-293, human A549 lung carcinoma, Syrian
hamster HaK kidney and mouse mammary adenocarcinoma JC cells were grown in Dulbecco’s modified
essential medium (DMEM) supplemented with 10% fetal
bovine serum, penicillin (100 units per ml) and streptomycin (100 mg per ml). INGN 007, an oncolytic Ad
vector, has been described previously (Figures 1a and
b).11 In addition, the reference strain of wt Ad5 was used
in this study (GenBank accession number AY339865).
The replication-defective (RD) vector AdCMVpA is an
E1-deleted vector based on dl309 in which an empty
expression cassette was placed into the deleted portion of
E3 (see Figures 1a and b). Vectors and virus propagated
on HEK-293 cells were purified by column chromatography by Introgen Therapeutics Inc. (Houston, TX). All
three stocks had a concentration of greater than 1.0 1012
virus particles (vp) per ml.
Animals
A total of 200 golden Syrian hamsters (115 males and 115
females, 5–6 weeks of age and weighing 80–100 g) were
purchased from Harlan Sprague Dawley (Indianapolis,
IN) for the hamster biodistribution study. A total of 100
C57BL/6 mice (60 males and 60 females, 5–6 weeks of age
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and weighing 18–25 g) were purchased from Harlan
Sprague Dawley for the mouse biodistribution study.
All animals were housed in the Department of Comparative Medicine at the Saint Louis University School of
Medicine and were cared for in compliance with the
animal protocol approved by the Animal Care Committee
of Saint Louis University and in accordance with the
Guideline for the Care and Use of Laboratory Animals
(NIH publication number 85-23, 1996).
Syrian hamster study
One day before vector/virus injection, animals were
randomized by weight into groups of 10 animals (5 each
males and females) (Table 1). The mean body weight for
males was 96.95 g and that for females was 88.44 g.
Treatment groups consisted of INGN 007, Ad5, AdCMVpA and vehicle (10 mM Tris-HCl (pH 8.2), 10%
glycerol). On day 1, animals were anesthetized and then
administered a single intrajugular injection of vector/virus
(1.9 1012 vp per kg) or vehicle. Animals (five per sex per
group) were killed for necropsy on days 2, 7, 29, 92 and
372 (371 for females).
Mouse study
Mice were randomized by weight into groups of 10
animals (5 males and 5 females) the day before vector/
virus injection (Table 2). The mean body weight for males
was 23.27 g and that for females was 19.85 g. Treatment
groups in the mouse study were INGN 007 and vehicle
(10 mM Tris-HCl (pH 8.2), 10% glycerol). On day 1,
animals received a single intravenous (tail vein) injection
of INGN 007 (1.5 1011 vp per kg) or vehicle. Animals
(five per sex per group) were killed for necropsy on days 2,
7, 29, 92 and 365.
Necropsy, organ collection and homogenization
Groups of animals were anesthetized with CO2 and then
killed by exsanguination through cardiac puncture.
Animals were processed in the following order: vehicle
control group, AdCMVpA group (hamster study only),
Ad5 group (hamster study only) and INGN 007 group.
Samples/organs were collected in the following order:
blood, gonads, brain, lymph nodes (mesenteric), spleen,
kidneys, adrenal glands (separated from the kidneys after
dissection), bone marrow (flushed from the femur in
phosphate-buffered saline, PBS), heart, lungs/bronchi and
liver. Blood was collected in EDTA tubes and flash frozen
in liquid nitrogen. Organs were trimmed of connective
tissue and then flash frozen. In the hamster study, only a
portion of some organs was frozen as follows: the
proximal half of the right testis, the right half of the
brain and the right lateral lobe of the liver. Only a portion
of the liver (the right lateral lobe) was collected in the
mouse study. All samples were stored at 80 1C until
processed further. Samples (excluding blood and bone
marrow) were thawed and then homogenized in PBS
using 3 mm tungsten carbide beads (Qiagen, Valencia,
CA) and a bead-beater type homogenizer (TissueLyser;
Qiagen). To reduce the possibility of cross contamination
among organs from a single animal and between different
INGN 007 biodistribution in hamsters and mice
B Ying et al
627
Figure 1 Genomic structure of INGN 007, Ad5 and AdCMVpA and the QPCR assays to detect viral DNA. (a) Schematic of vectors used in this
study. The genomes of wild-type Ad5, INGN 007 and AdCMVpA are depicted. The early transcription units 1 (E1), 2 (E2), 3 (E3) and 4 (E4) are
shown as arrows. Exons 1, 2 and 3 of the tripartite leader as well as the late transcription units L1, L2, L3, L4, ADP and L5 are shown above the
Ad5 genome. The E3 region of INGN 007 is deleted and replaced by the ADP gene. AdCMVpA, which was derived from dl309, lacks all of E1 and
a portion of the E3 transcription unit. AdCMVpA does not contain a transgene. (b) Schematic of the differences between the E3 region of each of
the genomes. The amplicon of the INGN 007 QPCR assay is shown above the INGN 007 genome. The amplicon of the Ad5/AdCMVpA QPCR
assay is shown below the Ad5 genome. Performance characteristics of the INGN 007 (c) and Ad5/AdCMVpA (d) assays were derived from three
independent experiments.
Table 1 Experimental design of the Syrian hamster biodistribution
study
Group
Vector/virus
1
2
3
4
Vehicle
AdCMVpA
Ad5
INGN 007
Dose (vp per kg)
Sexes
N
—
1.9 1012
1.9 1012
1.9 1012
2
2
2
2
50
50
50
50
Table 2 Experimental design of the mouse biodistribution study
Group
Vector/virus
Dose (vp per kg)
1
2
Vehicle
INGN 007
—
1.5 1011
Sexes
N
2
2
50
50
animals, each organ was removed and trimmed with a
different set of instruments. In addition, gloves and bench
paper covering the work surface were changed between
animals, and disposable lab coats were changed between
groups.
Purification and quantification of DNA
Genomic DNA was purified from a portion of each
homogenate with a Magtration 12GC automated DNA
isolation instrument and the Magtration-MagaZorb
DNA Kit-200 High Yield (both from Precision System
Science USA Inc., Livermore, CA). DNA was eluted in
200 ml of water and then quantified in triplicate with the
Quant-iT PicoGreen dsDNA kit (Invitrogen, Carlsbad,
CA). DNA quantification assays were set up using a
Biomek 2000 Laboratory Automation Workstation
(Beckman-Coulter, Fullerton, CA) located in a biological
safety cabinet. Aerosol barrier tips were used for all
liquid-handling steps. The assays were read with a BioTek
Synergy HT (BioTek, Winooski, VT) fluorescence plate
reader outfitted with fluorescein isothiocyanate excitation
and emission filters. Software provided with the Synergy
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INGN 007 biodistribution in hamsters and mice
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628
Table 3 Real-time PCR amplicons for detection of INGN 007, Ad5 and AdCMVpA
Amplicon
Size (bp)
Function
INGN 007
139
Forward primer
Probea
Reverse primer
50 -AACGCGCCCGACCAC-30
50 -TGCTACACCCAAACAATGATGGAATCCA-30
50 -AATTCCGTCCATTTCTAGATCTCAT-30
Ad5/AdCMVpA
74
Forward primer
Probea
Reverse primer
50 -CCGGTCATTTCCTGCTCAATA-30
50 -CATTCCCCTGAACAATTGACTCTATGTGGG-30
50 -AGGTTGTAGCGCTGGAGCATA-30
a
Sequence
Probe was modified with the fluorophore 6-FAM at the 50 end and the quencher TAMRA at the 30 end.
HT plate reader was used to generate a standard curve
and calculate the concentration of each sample.
Real-time PCR assays
Two quantitative, TaqMan-based real-time PCR (QPCR)
assays were developed; one that preferentially detects
INGN 007 and one that detects both wt Ad5 and
AdCMVpA, but is incapable of detecting INGN 007.
Table 3 shows the primers and probes used for the assays.
Primers and probes were synthesized by Integrated DNA
Technologies (Coralville, IA). All hamster and mouse
samples were assayed in triplicate, one of which was
spiked with 100 copies of the appropriate viral DNA
(INGN 007 or Ad5 genomic DNA for the INGN 007 or
Ad5/AdCMVpA assays). All real-time PCR assays were
set up in 96-well PCR plates (Applied Biosystems Inc.,
Foster City, CA) using a Biomek 2000 located in a
biological safety cabinet. Assays contained 1 universal
PCR master mix (Applied Biosystems Inc.), 250 nM of
forward and reverse primers, 250 nM of probe and up to
1 mg of DNA in a total reaction volume of 50 ml. All assays
were performed using an ABI model 7500 genetic analyzer
with the following cycling parameters: 1 cycle at 50 1C for
2 min, 1 cycle at 95 1C for 10 min and 40 cycles at 95 1C for
15 s and 60 1C for 1 min. Data were analyzed using
Sequence Detection System software (Applied Biosystems
Inc.) with the threshold set to 0.128 for all INGN 007
assays or 0.200 for all Ad5/AdCMVpA assays. Controls
included with every assay consisted of a ‘no template
control’ (no DNA added), an ‘animal genomic DNA
control’ (only hamster or mouse liver genomic DNA), a
‘nontarget DNA control’ (Ad5 for INGN 007 assays and
INGN 007 for Ad5/AdCMVpA assays) and standards
from 1 102 to 1 106 copies of the appropriate viral
genomic DNA (purified INGN 007 or Ad5 viral genomic
DNA for INGN 007 or Ad5/AdCMVpA assays) diluted
in Syrian hamster (or mouse) liver genomic DNA. The
large majority of reactions contained 1 mg of organ
genomic DNA, but the data for those reactions that
contained less genomic DNA were normalized to 1 mg. All
samples in which the mean copy number of the duplicate
reactions was o100 were considered to be negative for
viral genomic DNA and were treated as having zero
copies for the purpose of calculating the average number
of copies for that particular sample type. When all organs
from a group at a particular time point were negative for
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viral genomic DNA, then that organ from that group was
not evaluated at subsequent time points.
Infectious titer assay
The level of infectious vector/virus in select Syrian
hamster organs was measured using a tissue culture
50% infectious dose (TCID50) assay. Organ homogenates
of lymph nodes (mesenteric) and QPCR-positive livers,
lungs and gonads were subjected to three freeze-thaw
cycles, sonicated for 15 min and then clarified by
centrifugation. The clarified homogenates were serially
diluted and then select dilutions were inoculated onto
HEK-293 cells in a 96-well culture dish. Seven replicates
were tested for each dilution. HEK-293 cells were used
because they complement the E1 deletion in the
AdCMVpA vector, thus allowing it to replicate. Each
individual organ homogenate was assayed in a separate
plate. Each plate also contained multiple negative (that is,
no homogenate) and positive control wells. Positive
control wells consisted of a known amount of vector/
virus spiked into the homogenate dilution; these wells
served to evaluate inhibition of vector/virus replication by
the homogenate. After 2 weeks of incubation at 37 1C,
each well was examined for cytopathic effect (CPE). Viral
titers per organ were calculated by the Reed–Muench
method taking into account the lowest dilution of
homogenate in which at least one of the spiked wells
exhibited CPE. This dilution was used to calculate the
threshold of calculability. The threshold of calculability
determined for each organ was 2.0 104 TCID50/liver,
2.8 103 TCID50/lung, 4.0 102 TCID50/ovaries,
2.4 103 TCID50/testes and 2.7 102 TCID50 per ml of
lymph node homogenate. Homogenates that yielded
positive wells (that is, wells that showed CPE) below the
threshold of calculability were scored as ‘positive but not
quantifiable’. Homogenates for which no positive wells
were evident were scored as ‘undetected’.
Immunohistochemistry
Tissue sections from the toxicology study47 were mounted
on glass slides. Deparaffinized sections were subjected to
antigen retrieval using DIVA Decloaker solution (Biocare
Medical, Concord, CA) and then stained using an
antifiber monoclonal antibody 4D2 (Lab Vision, Freemont, CA) and horseradish-peroxidase-conjugated secondary antibody (Dako, Carpinteria, CA).
INGN 007 biodistribution in hamsters and mice
B Ying et al
Infectivity ratio determination
Human A549, Syrian hamster HaK and mouse JC cells
were infected at a multiplicity of infection (MOI) of 100
plaque-forming units (PFU) per cell. After an adsorption
period of 1 h, cells were washed three times with PBS and
then supplemented with 2 ml of complete growth medium.
Cells plus medium were harvested at 0 (immediately after
the rinses following the adsorption period), 1, 2, 3 and 4
days after infection by freezing plates at 80 1C. Lysates
were subjected to three freeze-thaw cycles. DNA purified
(Magtration 12GC system) from a portion of each lysate
was quantified (Quant-iT PicoGreen dsDNA kit) and
then subjected to the Ad5/AdCMVpA QPCR assay. A
portion of each lysate was assayed by the TCID50 assay.
Both values were normalized to account for the number
of cells originally infected.
Statistical analyses
Statistical analyses were performed using nonparametric
tests due to the variance within groups. First, an analysis
of variance between groups was conducted using the
Kruskal–Wallis test. Pair-wise comparisons between the
AdCMVpA-injected group and the INGN 007- or Ad5injected groups were carried out using the Mann–Whitney
U-test.
Results
The INGN 007 and Ad5/AdCMVpA real-time PCR
assays are specific and sensitive
Two TaqMan-based QPCR assays were developed to
detect INGN 007, Ad5 and AdCMVpA. Because the
INGN 007 genome differs from the wt Ad5 genome only
by the deletion of two regions (Figures 1a and b),
sequences in INGN 007 are juxtaposed that are not
normally adjacent in Ad5. This juxtaposition forms the
basis of specificity of the INGN 007 QPCR assay because
the reverse primer spans one of the deletion junctions
(Figure 1b). As a result, the INGN 007 QPCR assay
preferentially detects INGN 007; the assay is 41000 times
less sensitive for Ad5 than for INGN 007 (data not
shown). A second QPCR assay was developed to detect
both Ad5 and AdCMVpA genomic DNA. The specificity
of this assay resides in the fact that the amplicon is present
in the Ad5 and AdCMVpA genomes but is deleted in the
INGN 007 genome (Figure 1b).
The performance characteristics of both QPCR assays
were thoroughly investigated. Both assays yielded a single
product of the appropriate size (data not shown) and were
linear in the range of 1 101 to 1 107 copies of purified
viral genomic DNA (Figures 1c and d). The Ad5/
AdCMVpA assay performed equally well with Ad5 and
AdCMVpA viral genomic DNA as the template (data not
shown). The INGN 007 assay had similar performance
characteristics in the presence of 1 mg of Syrian hamster or
mouse genomic DNA (data not shown). Furthermore,
100 copies of viral genomic DNA were reproducibly
detectable in the presence of 1 mg background genomic
DNA purified from various Syrian hamster (INGN 007
and Ad5/AdCMVpA assays) or mouse (INGN 007 assay
only) organs (data not shown). The single exception was
genomic DNA isolated from Syrian hamster lymph
nodes, which interfered with the QPCR assay (data not
shown). These data indicate that both QPCR assays are
specific, sensitive and reproducible.
Syrian hamster organs contain more DNA from
replication-competent viruses than from a replicationdefective vector
The experimental design used for the Syrian hamster
biodistribution study is shown in Table 1. After injection
(defined as day 1), animals were monitored daily for
mortality. All hamsters in the vehicle and AdCMVpA
groups remained healthy throughout the yearlong course
of study. No female hamsters died during the study but
four INGN 007-treated and five Ad5-treated male
hamsters were found dead. With one exception, the cause
of death was undetermined and the animals died on or
before day 10 of the study; one of the Ad5-treated animals
died on day 270 because of kidney failure; this death was
deemed not related to treatment.
Groups of 10 hamsters (5 males and 5 females) from
each treatment group were killed on days 2, 7, 29, 92 and
372 (371 for females). Because the day of injection was
day 1, this corresponds to 1, 6, 28, 91 and 371 (370) days
after infection. Select organs were harvested from each
hamster at the time of necropsy. Purified genomic DNA
was subjected to the INGN 007 (vehicle and INGN 007
groups) or the Ad5/AdCMVpA (vehicle, Ad5 and
AdCMVpA groups) QPCR assay. Each sample was
assayed in triplicate, one of which served as a spike
control. This control reaction assured that samples
evaluated as having fewer than 100 copies did not contain
a substance that inhibited the PCR. On the basis of the
spike controls, none of the samples exhibited inhibition in
the QPCR assay. Importantly, only a single organ from
the vehicle control group yielded a signal in the INGN
007 or Ad5/AdCMVpA assays. In addition, a positive
PCR signal was not detected in any of the ‘no template’ or
‘animal genomic DNA’ control reactions, and only a
single nontarget DNA control reaction yielded a signal.
These results indicate that there was little or no cross
contamination during vector/virus injection, necropsy, the
in-life observation period, sample processing or QPCR
assay setup.
INGN 007, Ad5 and AdCMVpA DNA were detected
in all 11 organs examined indicating that all three
genomes are widely distributed after intravenous injection
(Tables 4, 5 and 6). The primary target organ for all three
vectors/viruses was the liver because this organ contained
the most viral DNA at 1 day after injection and at all
subsequent time points (Tables 4, 5 and 6). Other organs
generally contained a much lower level of viral DNA.
Because Syrian hamsters are permissive for Ad replication, it was noteworthy that several Syrian hamster
organs contained statistically significantly more INGN
007 and Ad5 DNA than AdCMVpA DNA early after
vector/virus injection (Figures 2a and b; Tables 4, 5 and
6). In the liver, there was at least 50-fold more INGN 007
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INGN 007 biodistribution in hamsters and mice
B Ying et al
630
Table 4 Mean copy number of INGN 007 DNA in various Syrian hamster organs
Day
2
7
29
92
372d
Blood
Bone marrow
a
13 701
5918
1411
156b
0
121 515
7342c
352b
0
NT
Brain
365
769b
0
NT
NT
Heart
Adrenal glands
b
5399
11 003b
1646
736
306
b
10 496
66 159a
2093
530
160
Kidney
Liver
c
Lungs/Bronchi
c
2024
14 796b
2517
390
161
18 339 870
1 005 784a
38 146a
15 873b
1093
b
20 176
11 488b
1138b
319
295a
Spleen
Testes
Ovaries
93 970
15 439c
3364
1871b
981
101
91
28
0
NT
20 255
6794
513
26
0
Spleen
Testes
Ovaries
85 320
8406
2200
2851a
1101
0
NT
NT
NT
NT
1675
2513
854a
0
NT
Abbreviation: NT, not tested because all samples from the previous time point had o100 copies.
Copies per 1 mg of DNA.
a
Pp0.01 INGN 007 vs AdCMVpA (Mann–Whitney U-test).
b
Po0.05 INGN 007 vs AdCMVpA (Mann–Whitney U-test).
c
Pp0.001 INGN 007 vs AdCMVpA (Mann–Whitney U-test).
d
Males were killed on day 372, females were killed on day 371.
Table 5 Mean copy number of Ad5 DNA in various Syrian hamster organs
Day
2
7
29
92
372d
Blood
Bone marrow
a
15 222
5874
1296
168
0
41 202
2342c
0
NT
NT
Brain
234
173b
0
NT
NT
Heart
1837
4638a
421a
1054
68a
Adrenal glands
b
15 000
12 863c
860
732
146
Kidney
1795
5101a
1448
576
42
Liver
Lungs/Bronchi
c
22 768 720
900 544a
16 329c
12 850
420
b
10 882
5760
428c
329
117b
Abbreviation: NT, not tested because all samples from the previous time point had o100 copies.
Copies per 1 mg of DNA.
a
Pp0.01 Ad5 vs AdCMVpA (Mann–Whitney U-test).
b
Po0.05 Ad5 vs AdCMVpA (Mann–Whitney U-test).
c
Pp0.001 Ad5 vs AdCMVpA (Mann–Whitney U-test).
d
Males were killed on day 372, females were killed on day 371.
Table 6 Mean copy number of AdCMVpA DNA in various Syrian hamster organs
Day
Blood
Bone marrow
Brain
Heart
Adrenal glands
Kidney
Liver
Lungs/Bronchi
Spleen
Testes
Ovaries
2
7
29
92
372a
1300
61
0
NT
NT
13 966
4962
1541
318
20
40
0
NT
NT
NT
1596
1065
1322
562
314
2667
1033
1451
841
287
660
442
596
289
109
337 890
120 540
72 590
5891
664
3598
2216
2599
982
1079
83 730
6182
2326
1008
1437
50
0
NT
NT
NT
809
680
138
0
NT
Abbreviation: NT, not tested because all samples from the previous time point had o100 copies.
Copies per 1 mg of DNA.
a
Males were killed on day 372, females were killed on day 371.
or Ad5 DNA than AdCMVpA DNA on day 2. This
difference declined to less than 10-fold on day 7, and by
day 29 there was actually more AdCMVpA DNA than
either INGN 007 or Ad5 DNA (Figure 2a). Although the
difference between RC and RD Ad DNA levels was
particularly evident in the liver, it was also apparent in
adrenal glands, blood, heart (only day 7 for Ad5), kidneys
(only day 7 for Ad5) and lungs (only day 2 for Ad5). By
day 29, most other organs had comparable levels of viral
DNA, although there were still some statistically significant differences (in some cases there was more
AdCMVpA DNA than INGN 007 or Ad5 DNA).
With the exceptions noted below, the peak quantity of
viral genomic DNA for most organs was observed on day
2, after which the signal generally decreased at each
Cancer Gene Therapy
successive time point such that by 1 year viral DNA was
no longer detectable in blood, bone marrow (except
AdCMVpA), brain, testes and ovaries. With the RC
vector/virus, the level of viral DNA increased from day 2
to day 7 such that the peak DNA level in heart, kidneys,
adrenal glands (INGN 007 only) and ovaries (Ad5 only)
occurred on day 7. The RD vector AdCMVpA did not
exhibit this pattern; the peak viral DNA level for all
organs was day 2.
Recovery of infectious vector/virus from Syrian hamster
organs
Select organs from the Syrian hamster biodistribution
study were assessed for the presence of infectious vector/
virus using a TCID50 assay. All liver, lung and gonad
INGN 007 biodistribution in hamsters and mice
B Ying et al
631
Figure 3 Infectious titer of INGN 007, Ad5 and AdCMVpA in Syrian
hamster liver samples. A portion of each liver sample was
homogenized and then subjected to a TCID50 assay on HEK-293
cells to determine the amount of infectious vector/virus present. The
lower limit of sensitivity of the assay with liver homogenates is shown
by the horizontal dashed line. Samples that contained detectable
infectious vector/virus but whose titer could not be calculated are
shown as being positive. Samples where vector/virus was not
detectable are shown as being negative.
Figure 2 Genome copies in Syrian hamster liver and lung samples.
The number of genome copies of AdCMVpA, Ad5 and INGN 007 in
whole Syrian hamster liver (a) and lungs (b) was calculated. The
horizontal bar represents the mean copy number. Tissue samples
that were negative for viral genomic DNA were considered as zero
for the purposes of calculating the mean copy number.
samples that were positive in the PCR assay were tested
for infectious vector/virus. Infectious Ad5 and/or INGN
007 were detected in liver, lung and testes but all ovary
samples tested were negative. In contrast, none of the
liver, lung or gonad samples from AdCMVpA-injected
animals contained infectious vector, even on day 2 when
the mean copy number in liver samples was 4300 000 per
mg of genomic DNA. As anticipated from the QPCR
results, liver harbored the greatest level of infectious
INGN 007 and Ad5; however, infectious INGN 007 or
Ad5 was not detected in liver beyond day 7 (Figure 3).
Lung samples from days 2 and 7 did contain infectious
vector/virus but at a much lower level and frequency
compared to liver samples (Table 7). The testes of only
two animals were found positive, both of which were from
the INGN 007 group on day 2.
Immunohistochemical analysis of liver samples for Ad
fiber protein supported the conclusions from the infectivity assays that infectious vector/virus was present in the
liver of animals injected with the RC vector/virus but not
the RD vector. Because there was no remaining liver
samples from the biodistribution study that could be used
for immunohistochemistry, livers from the companion
toxicity47 study were used instead. The hamsters from the
toxicity study were injected with the same dose of vector/
virus and necropsied on the same day (day 2 or 1 day after
infection). Ad fiber protein was detected in hepatocytes
from INGN 007- (Figures 4c and d) and Ad5-injected
(data not shown) animals. The signal seen in these
animals likely represents newly formed vector/virus and
not simply input vector/virus inasmuch as fiber protein
was not detected in the liver sections of AdCMVpAinjected animals (Figure 4b). Also, the staining is
predominantly localized to the nuclei, the site of Ad
assembly. No signal was observed in samples from
vehicle-injected hamsters (Figure 4a) or when liver
sections from all treatment groups were stained with an
Cancer Gene Therapy
INGN 007 biodistribution in hamsters and mice
B Ying et al
632
Table 7 TCID50 assay results from Syrian hamster lung samples
Group
Day Number
Number
Titer Number Number
PCR Pos. quantifiable (TCID50 / positive negative
organ)
AdCMVpA
2
7
29
92
372
9
9
10
8
8
0
0
0
0
0
NA
NA
NA
NA
NA
0
0
0
0
0
10
8
10
7
8
Ad5
2
7
29
92
372
10
9
8
8
3
1
0
0
0
0
509
NA
NA
NA
NA
1
0
0
0
0
7
4
8
7
3
2
10
2
1
4
7
29
92
372
10
8
8
4
0
0
0
0
1800
7310
NA
NA
NA
NA
0
0
0
0
10
7
8
4
INGN 007
Abbreviation: NA, not applicable.
isotype-matched control antibody (data not shown),
demonstrating the specificity of the assay.
Lymph nodes were also assessed in the TCID50 assay
because this organ could not be examined in the QPCR
assay (see Materials and methods). Although some lymph
nodes were positive for vector/virus, none of the samples
contained sufficient vector/virus to be quantified
(Table 8). Whereas infectious vector/virus was present in
lymph nodes from all three groups on day 2, only samples
from the INGN 007 and Ad5 groups were positive on day
7. By day 29, no infectious vector/virus was present in any
of the samples examined, so additional time points were
not tested.
INGN 007 DNA is much less abundant in mouse tissues
The experimental design used for the mouse biodistribution study is shown in Table 2. After injection with vehicle
or INGN 007, animals were monitored daily for
mortality. No treatment-related mortality was observed
in this study, although two male mice from the INGN 007
group died (one on day 28 and one on day 135).
For the mouse biodistribution study, groups of 10 mice
(5 each males and 5 females) were killed on days 2, 7, 29,
92 and 365, and then purified tissue genomic DNA was
subjected to the INGN 007 QPCR assay. All of the
negative control (no template, mouse genomic DNA only
and nontarget viral DNA) reactions and all of the organs
from the vehicle control group were negative in the QPCR
assay indicating lack of cross contamination throughout
the study. Furthermore, based on the spike controls, none
of the samples in the mouse biodistribution study
exhibited inhibition in the QPCR assay.
INGN 007 DNA was widely distributed in mice on day
2. However, unlike in hamsters, INGN 007 DNA was
detected in only 10 out of 12 organs (Table 9). In
addition, several organs contained only a low level of viral
Cancer Gene Therapy
Figure 4 Immunohistochemistry of liver samples for adenovirus
(Ad) fiber protein. Liver samples from the companion toxicology
study47 were prepared for immunohistochemical staining and then
stained using an antifiber antibody. Representative fields from the
mock-injected (a), AdCMVpA-injected (b) and INGN 007-injected
(c, d) Syrian hamsters are shown. (d) A magnified portion of the
field shown in c. The black bar represents 100 mm (a, b and c) or
10 mm (d).
Table 8 Number of TCID50-positive Syrian hamster lymph node
samples
Virus per vector
Day
2
AdCMVpA
Ad5
INGN 007
a
a
2 (10)
5 (10)
3 (10)
7
29
0 (10)
1(10)
1(10)
0 (10)
0 (10)
0 (10)
() total number of hamsters tested.
DNA. With few exceptions, the level of INGN 007 DNA
decreased in each organ at each successive time point such
that by 1 year nine out of twelve organs were negative for
viral DNA. Similar to the Syrian hamster study, and in
agreement with numerous other studies in mice, the liver
was the primary target of distribution in mice
(Table 9).24,30,48–51 The viral DNA copy number diminished rapidly in the liver such that at 1 year six of the nine
liver samples were negative for INGN 007 DNA (data not
shown). Although there was a relatively high level of
INGN 007 DNA in the blood on day 2, that level
decreased dramatically by day 7 and there was no
detectable viral DNA in blood by day 29. Despite a high
mean level of INGN 007 viral DNA present in liver on
day 2, infectious vector was not detected in these samples,
nor was infectious vector detected in any day-7 liver
samples.
Infectivity ratios in vivo and in vitro are high
There was a great excess of vector/viral genomic DNA
present in Syrian hamster liver and lungs compared to the
INGN 007 biodistribution in hamsters and mice
B Ying et al
633
Table 9 Mean copy number of INGN 007 in various mouse organs
Day
Blood
2
7
29
92
365
135 990
718
0
NT
NT
Bone marrow Brain Heart Adrenal glands Kidney
771
263
245
190
0
21
0
NT
NT
NT
1485
450
0
NT
NT
0
NT
NT
NT
NT
445
66
34
43
0
Liver
1 899 103
91 456
4604
2052
60
Lungs/Bronchi Lymph nodes Spleen Testes Ovaries
1682
331
185
164
15
162
372
0
NT
NT
6952
2888
2779
1898
266
0
NT
NT
NT
NT
29
31
0
NT
NT
Abbreviation: NT, not tested.
Copies per 1 mg of DNA.
amount of infectious vector/virus present in those organs.
The mean infectivity ratio (the ratio of vector/viral DNA
to infectious vector/virus) for INGN 007 in liver was
2.4 104 and 5.0 104 on days 2 and 7, respectively. For
Ad5, the infectivity ratio was 1.3 105 and 4.5 104 on
days 2 and 7, respectively. For the few lung samples that
contained a titratable amount of vector/virus on day 2,
the ratio was 2.7 104 for INGN 007 (mean of two
samples) and 2.9 104 for Ad5 (only one sample).
To determine if the infectivity ratio was also high in
vitro, Ad5-infected cells were harvested at various times
after infection and then the samples were assayed by
QPCR to quantify the amount of genomic DNA and by
TCID50 to measure the amount of infectious virus. Cell
lines from three different species were examined: human
A549 cells that are highly permissive for Ad5 replication,
Syrian hamster HaK cells that were previously shown to
support Ad5 replication44 and mouse JC cells that were
reported to be among the more permissive mouse cell
lines.52 In A549 and HaK cells, Ad5 genomic DNA and
infectious virus were initially present at low levels,
increased dramatically over the course of 1–2 days and
then leveled off by about 2 days after infection (Figures 5a
and b). The infectivity ratio in A549 cells leveled off at
about 1 106, whereas that in HaK cells leveled off at
nearly 1 105 (Figure 5c). It is quite interesting that the
infectivity ratio for Ad5 and INGN 007 in hamsters,
which ranged from 2.4 104 to 1.3 105 on days 2 and 7,
was similar to that of Ad5 in hamster HaK cells (1 105).
With JC cells, genomic DNA increased for 2 days before
leveling off at about 1 104 genomes per cell (Figure 5a).
However, little or no infectious Ad5 was produced in JC
cells (Figure 5b) yielding a maximum infectivity ratio of
6.5 104 (Figure 5c). We could not ascertain whether the
QPCR assay detected complete genomes, merely DNA
fragments or both types of DNAs.
Discussion
Two nonclinical biodistribution studies were conducted to
examine the dissemination and pharmacokinetics of the
oncolytic Ad vector INGN 007 following intravenous
administration, one in Syrian hamsters and the other in
C57BL/6 mice. Both studies demonstrated that viral
DNA is widely distributed shortly after administration.
However, high levels of viral DNA were not sustained as
evidenced by a rapid decline in DNA levels in most organs
between days 2 and 7. Hamster blood, brain, testes,
ovaries and bone marrow (INGN 007 and Ad5 only) were
negative by 1 year after injection. The decrease in INGN
007 DNA in mouse organs was even more dramatic; seven
organs were negative by day 29 and nine out of twelve
organs were negative after 1 year.
Ad viral DNA was principally localized to the liver in
Syrian hamsters and mice, which is in agreement with
previous reports with the mouse and other animal
models.24,30,39,44,46,48,51 Using various model systems,
distribution to and uptake into liver cells has been
ascribed to direct binding to liver cells and a variety of
interactions between Ad virions and blood cells/proteins
(for review see Baker et al.53). In the Syrian hamster
model, all three vector/viral DNAs were rapidly cleared
from the blood and were most abundant in the liver.
Additional studies are needed to determine the mechanism(s) underlying rapid clearance from the blood and
distribution to the liver in this animal model.
The Syrian hamster has been described as a permissive
animal model for RC Ads and data from the hamster
study provided further evidence for this view. INGN 007
and Ad5 DNA were more abundant than that of
AdCMVpA in several hamster organs early after injection. This was particularly evident in the liver where
INGN 007 and Ad5 DNA were 450- and B7-fold more
abundant than AdCMVpA DNA on day 2 and day 7,
respectively (Tables 4, 5 and 6; Figure 2a). In addition, Ad
fiber protein, as detected by immunohistochemistry, was
only detected in INGN 007 (and Ad5)-injected hamsters
(Figure 4). Furthermore, infectious INGN 007 and Ad5,
but not AdCMVpA, were recovered from liver samples on
days 2 and 7. Interestingly, data from the hamster
biodistribution study are concordant with the extent of
liver toxicity observed in the parallel toxicology study:
INGN 007 and Ad5 resulted in more liver damage than
AdCMVpA on day 7.47 Together, these data strongly
support the liver as a site of Ad replication in Syrian
hamsters and that replication is associated with enhanced
liver damage.
Syrian hamster organs other than liver also likely
supported Ad replication. Compared to the amount of
AdCMVpA DNA, 3- and 5-fold more Ad5 and INGN
007 genomic DNA, respectively, was present in lungs on
day 2 (Tables 4, 5 and 6; Figure 2b). In addition,
infectious INGN 007 and Ad5 were detected in lungs on
day 2, whereas no infectious AdCMVpA was detected in
Cancer Gene Therapy
INGN 007 biodistribution in hamsters and mice
B Ying et al
634
Figure 5 Ratio of Ad5 genomic DNA copies to infectious virus in
different cell lines. Human A549, Syrian hamster HaK and mouse JC
cells infected at a multiplicity of infection (MOI) of 100 PFU per cell
were harvested on the indicated days. Each lysate was subjected to
the TCID50 assay (a) and the Ad5/AdCMVpA QPCR assay (b). The
Ad5 copy number was divided by the TCID50 titer and plotted vs days
post infection (c).
Cancer Gene Therapy
any lung samples (Table 7). These data suggest that the
lung is a site of replication following intravenous injection
and agree with previously published results where Ad was
administered either intratracheally or intranasally.32,44
The heart, kidneys and adrenal glands may also be a site
of replication. These organs contained more INGN 007
and Ad5 DNA than AdCMVpA DNA on day 2
(statistically significant for all three organs for INGN
007 vs AdCMVpA and for adrenal glands for Ad5 vs
AdCMVpA) (Tables 4, 5 and 6). This difference was also
apparent on day 7 (statistically significant for all three
organs and both RC viruses vs AdCMVpA) (Tables 4, 5
and 6). Furthermore, the amount of INGN 007 and Ad5
DNA increased from day 2 to day 7 in heart, kidneys and
adrenal glands (INGN 007 only). Two pieces of data
suggest that this increase likely represents replication in
these organs. First, AdCMVpA DNA did not exhibit a
similar increase. Second, levels of INGN 007 and Ad5
DNA in other organs did not increase from day 2 to day 7.
Although INGN 007 and Ad5 were able to replicate in
certain Syrian hamster organs, the burst of replication
seen early after vector/virus injection appeared to be only
transient. No infectious vector/virus was detected beyond
day 7 despite positive QPCR results, and viral DNA in all
organs was greatly diminished by day 29. Control of
vector/virus replication by the immune system probably
accounts for these results. Serum samples were not
available from the biodistribution study to confirm this
hypothesis, but data from the companion acute toxicology study demonstrated that hamsters did mount an antiAd humoral immune response by day 29.47 Further
support for this proposition comes from the observation
that Ad5 and INGN 007 exhibit enhanced replication in
immunosuppressed hamsters.45,46 Also, in studies in
which INGN 007 was injected into subcutaneous HaK
tumors growing in immunocompetent Syrian hamsters,
anti-Ad antibodies could be detected in as little as 7 days
after injection (D Dhar and W Wold, unpublished
results).
Results obtained in the Syrian hamster biodistribution
study with respect to bone marrow may help explain the
results obtained for this organ in the toxicity study.
Histopathological lesions and defects in bone marrow
function described for Ad5- and INGN 007-injected
hamsters could be attributed to replication because these
aberrations were not evident in AdCMVpA-injected
animals.47 However, the biodistribution study provided
no evidence for Ad5 or INGN 007 replication in bone
marrow, suggesting another cause for bone marrow
toxicity, such as gene expression.
In contrast to the results seen in Syrian hamsters, the
biodistribution study in mice provided no evidence that
INGN 007 was able to replicate in this species. Despite
the high burden of vector DNA in mouse liver on day 2,
infectious INGN 007 was not recovered from this organ
on day 2 or 7 (data not shown). In addition, the peak copy
number for INGN 007 for all mouse organs was on day 2
and generally decreased at each successive time point in
each organ (Table 9). It may well be that viral DNA
replicates to some extent in mouse organs, but it appears
INGN 007 biodistribution in hamsters and mice
B Ying et al
that very little if any infectious virus is produced. Viral
DNA replication without reproduction was observed in
the mouse JC cell line (Figure 5). These data support
previously published reports indicating that normal
mouse tissues are poorly permissive for Ad replication
at best.30–34
Less infectious vector/virus was detected in Syrian
hamster liver and lung than might be expected based on
the large quantity of genomic DNA detected in these
organs. The infectivity ratio in these organs was 1 104 to
1 105 genome copies per infectious particle. Similar
infectivity ratios were found in cell lines from three
different species suggesting that both in vivo and in vitro
far more viral DNA is produced than is packaged into
infectious particles.
This study expands our understanding and highlights
the utility of this new, valuable animal model for RC Ads.
Specifically, this report confirmed that Ad is capable of
replicating in Syrian hamster liver and lungs and that
other organs such as heart, kidneys and adrenal glands
may also represent a target for Ad replication. Further
studies are required to confirm these results. On the basis
of these data and the lack of evidence for replication in
normal mouse tissues, this study provides further evidence
that the Syrian hamster is the small animal model of
choice for examining Ad-mediated pathogenicity, immune
responses to RC Ads and efficacy of oncolytic Ad vectors.
Acknowledgements
This research was supported by grants CA118022,
CA108335 and CA81829 from the National Institutes of
Health to WSMW. Funding for this work was also
supported by a research and development agreement to
VirRx Inc. from Introgen Therapeutics Inc.
Disclosure/Conflict of interest
WSMW, KT, AET, KD, MK and Introgen Therapeutics
Inc. own shares of VirRx Inc.
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