Kasai et al: Toxicology for MGH2.1
Supplemental Materials and Methods for Kasai et al.
MGH2.1 engineering. To generate MGH2.1, the GFP sequence was removed from MGH2
genome by using the “HSVQuik” method, which was detailed in Terada et al. (2006)[1]. Briefly,
employing ET-cloning and flp recombination technique, the GFP gene was removed from a
bacterial artificial chromosome (BAC) vector fHSVQuik1to obtain the fHsvQuik2 vector. E.
coli harboring fHsvQuik2 was transformed with the same shuttle vector used to generate the
MGH2 oncolytic virus. FLP-mediated, site-specific recombination between the shuttle vector
and fHsvQuik2 results in formation of a BAC vector containing MGH2.1. Vero cells are then cotransfected with this BAC vector and a Cre recombinase-expression vector. The unwanted
prokaryotic sequences are then removed from the shuttle vector backbone by Cre-loxP
recombination. All the HSV-1 viruses were propagated on Vero (African green monkey kidney)
cells (American Type Culture Collection). Virus stocks were prepared by infecting subconfluent
monolayers of Vero cells at a multiplicity of infection (MOI) of 0.02. Infected cells were
incubated at 34°C and harvested when total cytopathic effect was observed. After three freezethaw cycles, cell debris was removed by low-speed centrifugation (2,000 x g at 4°C for 15 min)
and virus was concentrated by high-speed centrifugation (45,000 x g at 4°C for 3 hours) through
a 25% sucrose cushion. The viral pellet was resuspended in phosphate buffered saline (PBS)
(Invitrogen, Carlsbad, CA) and titers were determined by plaque assays on Vero cells. Aliquots
of viral stock were stored at -80°C until use.
Southern blot analysis. Viral DNA was isolated from infected Vero cells. After treating the
lysed cell sample with EDTA, proteinase-K, and SDS, DNA was purified by repeated phenolchloroform extraction, and precipitated with ethanol. DNA was digested with HindIII (New
England Biolabs, Ipswich, MA), separated by 0.5% agarose gel electrophoresis, and transferred
to a nylon membrane (GE Healthcare Life Sciences, Pittsburgh, PA, formerly Amersham
Biosciences). Probes included the HindIII shiCE (1.4 kb), the HindIII CYP2B1 (1.4 kb), and the
SacII+BglII CYP2B1 fragment (2.2 kb) prepared from the shuttle vector. Probe labeling and
hybridization were conducted with the enhanced chemiluminescence (ECL) system (GE
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Kasai et al: Toxicology for MGH2.1
Healthcare Life Sciences, Pittsburgh, PA, formerly Amersham Biosciences) following the
manufacturer’s protocol.
LC-MS/MS conditions
A SIL-10ADvp Shimadzu HPLC system (Shimadzu, Columbia, MD) consisting of controller,
degasser, binary pump, and auto-sampler, was used for solvent and sample delivery. The
chromatographic separation was performed on an Extended C18 column at room temperature.
The mobile phases consisted of methanol (0.2% formic acid) and 10mM ammonium formate
(0.2% formic acid) pumped at a flow rate of 0.2mL/min. An API 3000 mass spectrometer with
electrospray ionization source was used for mass analysis and detection in positive ion mode.
The detector was operated at low resolution for Q1 and unit resolution for Q3 in MRM mode
using the transitions from the molecular ions to product ions at m/z 261.1/140.0 for CP,
221.4/142.0 for PM, 393.4/349.2 for SN-38, 587.5/502.4 for CPT-11, 334.0/221.0 for 4OH-CPSCZ and 261.1/154.0 for IS.
Sample Preparation for LC/MS/MS analysis
To 100 μL aliquot of mouse plasma or 200 uL brain or liver tissue extract, 10 μL I.S. solution
and 10 μL standard solutions (for plasma), or 20 μL I.S. solution and 20 μL standard solutions
(for tissue extract) were added. The mixture was vortexed for 30 seconds and loaded to preactivated Oasis MAX cartridge. After washing with 1 mL 1% NH4OH, the analytes were eluted
by 500 μL methanol (5% formic acid). The fractions were collected and dried under a mild
stream of nitrogen. The residues were then reconstituted with 100 µL 10% methanol (1% formic
acid). The resulting solution was then analyzed by LC–MS/MS.
Measurement of carboxylesterase (CE) activity and SN-38 production rate. CE activity and
conversion of CPT-11 to SN-38 was measured following Tyminski et al. (2005)[2]. Vero cells
were seeded in P-100 culture dish at the density of 3 x 106 cells. Next day, the cells were infected
with MGH2, MGH2.1 before single plaque purification (“pooled”) and three single isolate
plaques (1-1-5, 1-2-6, and 1-2-8) at MOI=0.002 and incubated for two days at 37°C with 5%
CO2. Cells were scraped into the culture medium and separated from the medium by
centrifugation. The supernatant was transferred into new tubes and used as “medium” sample.
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Kasai et al: Toxicology for MGH2.1
Cells were washed with PBS, resuspended with 50 mM Hepes buffer (pH 7.4), and sonicated.
After centrifugation, supernatant was transferred to new tubes and used as “cell extract” and
stored. CE activity was measured spectrophotometrically by incubating samples with onitrophenyl acetate as substrate. Production rate of SN-38, CPT-11 metabolite, was measured in
cultured media and cell lysate prepared as mentioned above. Samples were incubated in the
presence of CPT-11, and concentration of SN-38 was measured by high-performance liquid
chromatography.
In vitro assay of virus cytotoxicity in the presence of cyclophosphamide (CPA). CYP2B1
function was verified in terms of cytotoxicity of MGH2.1 in the presence of CPA. U251 cells
were seeded in 6-well culture plates at the density of 2 x 105 per well in DMEM supplemented
with 2%. The following day, cells were infected with virus stocks at MOI=0.01 or 0.03,
incubated at 37°C in an atmosphere containing 5% CO2 for one hour, then fresh medium
containing cyclophosphamide (CPA; Baxter, Deerfield, IL) at indicated concentrations was
added. Viral replication was stopped 3 hours later by transferring the plates to a 39.8 °C
incubator. Cytotoxicity was measured 4 days later by enumeration through a Coulter counter
(Beckman Coulter, Indianapolis, IN).
In vivo toxicity assay. In vivo toxicity was accessed in immunocompetent mice with virus
administered intrracerebrally. bBALB/c mice were obtained from Charles River Laboratories
(Wilmington, MA) or the National Cancer Institute (Frederick, Md). Oncolytic virus was
stereotactically injected into the right frontal lobe of brain (2 mm lateral and 1 mm anterior to the
bregma at a depth of 3 mm). PBS and UV-radiated MGH2.1 were used as negative and mock
control, respectively. The survival time for each group was monitored for up to 60 days after
virus injection and body weights were measured weekly. CPA (2 mg in 100 µl PBS) was
administered intraperitoneally at 1, 3, 5, and 7 days after virus injection, and CPT-11 (2 mg in
100 µl PBS) was injected intraperitoneally 1 day after virus injection. All animal studies were
performed in accordance with guidelines issued by The Ohio State University Institutional
Animal Care and Use Committee (IACUC), utilizing an approved animal protocol. Viral
inoculation and care of animals harboring virus were conducted in approved BL2 laboratory
rooms.
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Kasai et al: Toxicology for MGH2.1
Supplemental Figure for Kasai et al.
Supplementary Figure 2. Effect of intracerebrally injected MGH2.1 +/- CPA and +/CPT11 on body weights of Balb/C mice. (a) Balb/ C mice were inoculated i.c. with various
amounts of MGH2.1 or strain F and weighed weekly up to 9 weeks and (b) Balb/C mice were
intracerebrally injected with MGH2.1 (5 x 107 pfu) and/or CPA (2 mg. intraperitoneally injected
1, 3, 5, and 7 days after virus injection) and/or CPT11 (2 mg intraperitoneally injected 1 day after
virus injection). The values on the y-axes ratio represent the percent growth in average weight
compared to that before MGH2.1 injection. Each data point represents the average and error bars
indicate the standard deviation. For assessment of MGH2.1 dose effect, statistical analysis was
conducted using repeated measures ANOVA including group, time (weeks), and an interaction
between group and time variables in the model. Multiple comparisons with the control group
were adjusted for using Dunnett-Hsu method for multiple comparisons. No statistically
significant difference was observed between mock and viable-virus injected groups (p=0.9970
for 1 x 106, p=0.9036 for 1x107, p=0.6908 for 5 x 107, and p=0.1005 for 1x108).
Supplementary Figure 1. Engineering of MGH2.1. (a) Schematic represatation of wild type
HSV strain F, its derivatives MGH1, MGH2, and MGH2.1. The stick and box drawing shows the
HSV-1 genome, namely two unique segments (UL and US), each flanked by inverted repeat
elements (ab and ca). The locations of γ134.5 genes and of the gene encoding the large subunit of
ribonucleotide reductase (UL39) are shown in the map of F strain. MGH1 contains deletions in
the both copies of γ134.5 genes and the insertion of a green fluorescent protein (GFP) cDNA into
UL39. MGH2 contains the CYP2B1 transgene, transcriptionally driven by the viral IE4/5
promoter and shiCE driven by CMV promoter, both inserted into the UL39 locus. MGH2.1 was
engineered by removing the GFP sequence from MGH2. Restriction sites of HindIII, SacII and
BglII and expected fragment sizes are provided. (b) Southern blot analysis of MGH2.1 genome.
Hybridization of HindIII-digested viral DNA to a shiCE probe and to a CYP2B1 probe and
hybridization of viral DNA digested with SacII and BglII to a CYP2B1 prove. Two independent
isolates of MGH2.1 were passaged on B7 or N23 bank cell line for 20 times and viral DNA was
extracted from the first generation and every 5th generation afterward. After digestion, viral DNA
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Kasai et al: Toxicology for MGH2.1
fragments were separated on agarose gel, and CYP2B1- and shiCE-containing fragments were
detected by the probe fragments prepared by digesting the shuttle vector plasmid with the
corresponding restriction enzyme(s). The 4.8-kb HindIII fragments were detected by shiCE
probe, while the 1.4-kb HindIII fragments and 2.2-kb SacII+BglII fragments were detected by
CYP2B1 probe.
Supplementary figure 3. In vitro functional assays of transgenes (shiCE and CYP2B1) in
MGH2.1. (a) Vero cells were infected with MGH2, MGH2.1 before plaque purification
(“pooled”), and 3 single isolate plaques (1-1-5, 1-2-6, and 1-2-8), and CE activity was measured
in culture media and cell lysates. The activity is expressed as nmol o-nitrophyl produced per
minute per mg protein for cell lysate, or nmol per minute per ml for culture medium. Each plot
represents the average of triplicate samples and standard deviations were indicated by error bars.
(b) Conversion of CPT-11 to SN-38 was measured in the same samples used for CE activity
measurements. The samples were incubated with CPT-11 and concentration of SN-38 was is
expressed as pmol SN-38 produced per hour per mg protein for cell lysate, or pmol per hour per
ml for culture medium. (c) U251 cells were infected with virus stock at MPI=0.01 or 0.03 at
various concentrations of CPA, and 5 days later surviving cell number was counted. Each plot
represents the average of triplicate samples and standard deviations were indicated by error bars.
REFERENCES FOR SUPPLEMENTARY INFORMATION
1.
2.
Terada, K, Wakimoto, H, Tyminski, E, Chiocca, EA, and Saeki, Y (2006). Development
of a rapid method to generate multiple oncolytic HSV vectors and their in vivo evaluation
using syngeneic mouse tumor models. Gene therapy 13: 705-714.
Tyminski, E, et al. (2005). Brain tumor oncolysis with replication-conditional herpes
simplex virus type 1 expressing the prodrug-activating genes, CYP2B1 and secreted
human intestinal carboxylesterase, in combination with cyclophosphamide and
irinotecan. Cancer research 65: 6850-6857.
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