`
`Gene Therapy (2006) 13, 1731-1736
`2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00
`www.nature.com/gt
`
`SHORT COMMUNICATION
`Imaging immediate-early and strict-late promoter
`activity during oncolytic herpes simplex virus type 1
`infection and replication in tumors
`
`IAfllUI
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`66-
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`S Yamamoto', LA Deckter, K Kasai, EA Chiocca and Y Saeki
`Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, James Cancer Hospital and Solove
`Research Institute, The Ohio State University Medical Center, Columbus, OH, USA
`
`An increasing number of oncolytic viruses have been
`developed and studied for cancer therapy. In response to
`needs for non-invasive monitoring and imaging of oncolytic
`virotherapy, several different approaches, including a posi-
`tron emission tomography-based method, a method using
`secreted marker peptides, and optical
`imaging-based
`methods, have been reported. Among these modalities, we
`utilized the luciferase-based bioluminescent assay/imaging
`systems to determine the kinetics and dynamics of a
`productive viral infection. The replication cycle of herpes
`simplex virus type 1 (HSV- 1) is punctuated by a temporal
`cascade of three classes of viral genes: immediate-early (IE),
`early (E) and late (L) genes. UL39- and y,34.5-deleted,
`
`replication-conditional HSV- 1 mutants that express firefly
`luciferase under the control of the 1E4/5 or strict-late gC
`promoters were generated. These oncolytic viruses were
`examined in cultured cells and a mouse tumor model.
`IE promoter- and strict-late promoter-mediated luciferase
`expression was confirmed to indicate viral infection and
`replication, respectively. Incorporation of a strict-late promo-
`ter-driven luciferase cassette into oncolytic HS V-1 vectors
`would be useful for assessing tumor oncolysis in preclinical
`tumor treatment studies.
`Gene Therapy (2006) 13, 1731-1736. doi:10.1038/
`sj.gt.3302831; published online 27 July 2006
`
`Keywords: herpes virus; oncolytic virus; luciferase; molecular imaging; glioma; viral replication
`
`Currently, a number of oncolytic viruses of different
`origins, such as naturally occurring wild-type Newcastle
`disease virus,' attenuated strains of reovirus2
` and
`vesicular stomatitis virus,' and genetically engineered
`mutants of herpes simplex virus type 1 (HSV-1),
`adenovirus, poxvirus and measles virus" are being
`evaluated in preclinical and clinical studies.
`With increasing needs, a number of non-invasive
`methods of imaging and/or monitoring viral infection
`have been reported, including a positron emission
`tomography (PET)-based method,' a method using inert
`soluble marker peptides' as well as optical methods
`using bioluminescent'," and fluorescent reporters."
`HSV-1 is among the most extensively studied viruses
`for oncolytic virotherapy applications. HSV-1 is a large
`enveloped virus with a 152-kb, linear double-stranded
`DNA genome and possesses the ability to infect and
`replicate in a variety of human cells as well as rodent
`cells.'2'13 The virus employs a complex temporal cascade
`of gene expression during the course of its lytic infection.
`
`Correspondence: Dr Y Saeki, Dardinger Laboratory for Neuro-
`oncology and Neurosciences, Department of Neurological Surgery,
`The Ohio State University, 385B Wiseman Hall, 400 West 12th
`Avenue, Columbus, OH 43210, USA.
`E-mail: saeki,6@osu,edu
`Current address: Department of Neurosurgery, School of Medicine,
`Tokyo Medical and Dental University, Tokyo, Japan.
`Received 16 September 2005; revised 15 June 2006; accepted 20 June
`2006; published online 27 July 2006
`
`A set of immediate-early (IF or e) genes is transcription-
`ally activated shortly after viral infection, and the
`resulting gene products regulate transcriptional activities
`of viral genes and immune evasion. A second set of
`genes called early (F or J3) genes, which includes a
`number of viral encoded enzymes that involve DNA
`replication and nucleic acid metabolism, is then ex-
`pressed. Finally, a late (L or y) set of gene products is
`transcribed and translated. These encode for the struc-
`tural proteins (e.g., capsid proteins, tegument proteins
`and membrane glycoproteins) as well as non-structural
`proteins for assembly and packaging functions. A subset
`of L genes (strict-late or 72 genes), including the UL3,
`UL38, gC (UL44) and U,11 genes, is known to express
`only after viral DNA synthesis takes place.'4'15
`Combining genes encoding light-generating enzymes
`(luciferases) such as firefly luciferase (Fluc) and Renilla
`luciferase (Riuc) with the new generation of super-
`sensitive charged coupled device (CCD) cameras has
`opened the door to sensitive in viva measurements/
`imaging of gene expression in living animals.''9 We
`hypothesized that the introduction of Fluc under the
`control of a strict-late promoter would allow non-
`invasive imaging and real-time monitoring of HSV-1
`replication in vitro and in viva.
`To compare the transcriptional dynamics of IE and
`strict-late viral promoters in the context of oncolytic
`HSV-1 vectors, UL39 and y,34.5-deleted mutants expres-
`sing Fluc under the control of the viral IE4/5 promoter
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`Imaging Transcriptional activation
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`(rQ1--Fluc) or gC promoter (rQ1-y2-Fluc) were con-
`structed (Figure Ia) using the recently developed
`HSVQuik method (see Supplementary Methods).2° Cul-
`tured GIi36AEGFR human glioma cells" were infected
`with one of the luciferase-expressing viruses at a multi-
`plicity of infection (MOT) of 3, and luciferase activity was
`assayed at different time points. As shown in Figure lb,
`luciferase activity of the cells infected with rQ1-a-Fluc
`was detected as early as 2 h with a subsequent peak
`between 6 and 9 h after infection. When infected with the
`rQ1-y2-Fluc, luciferase activity was detected much later
`(9 h) and peaked at 16 h after infection. The temporal
`kinetics displayed by luciferase activity as a function of
`the IE or strict-late promoter were confirmed to reflect
`those of the endogenous IE and strict-late gene products,
`respectively (Supplementary Figure 1). We then exam-
`ined the time course copy number of viral DNA in
`
`UL
`
`IR
`
`[!
`
`TLRI
`
`1
`
`UL39 (rR, ICP6)
`
`!CP6CA -GFP
`
`FRT
`
`IoxP
`
`rQ1-cz-Fluc
`
`—?/- r01 'y2-Fluc
`
`rQ1-a-Fluc
`-•-
`—0-- rQ1-y2-Fiuc
`
`20
`15
`10
`Hours after infection
`
`25
`
`30
`
`b
`
`2.0
`
`CD
`C
`
`0
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`Hours after infection
`
`25
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`30
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`Gene Therapy
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`infected cells by quantitative real-time polymerase chain
`reaction analysis. The copy number hardly changed
`during the first 9h, whereas it dramatically increased
`between 9 and 16h after the infection (Figure Ic). The
`temporal profile of luciferase expression mediated by the
`rQI j'2-Fluc was therefore confirmed to reflect active viral
`DNA replication in GIi36AEGFR cells. The above set of
`results was replicated with two other human cancer cell
`lines, U251 glioma and HeLa cervical carcinoma cell lines
`(data not shown). When infected at a lower MOT (0.15),
`the peak of luciferase expression mediated by IE4/5 or gC
`promoter delayed significantly, suggesting that multiple
`cycles of viral replication took place at the lower MOT
`(Supplementary Figure 2).
`Next, we sought to determine whether luciferase
`expression mediated by the recombinant HSV-1 could
`be used to measure separately the initial processes of
`viral infection and the late processes of viral replication.
`Two pharmacologic agents reported to inhibit HSV-1
`replication were used. Roscovitine (Rosco) is a cycline-
`dependent kinase inhibitor and directly or indirectly
`inhibits transcription of IE and F genes as well as viral
`DNA synthesis .11,23
` Because some of the IE gene
`products are absolutely required for the subsequent
`transcription of E and L genes and viral DNA synthesis
`is required for the transcription of strict-late genes,
`Rosco-mediated inhibition of strict-late gene expression
`is more prominent than that of IE-gene expression.
`Phosphonoacetic acid (PAA), on the other hand, is an
`inhibitor of HSV-1 DNA polymerase and blocks primar-
`ily viral DNA synthesis, rather than viral gene transcrip-
`tion.23'24 We exposed Vero cells infected with either
`to Rosco and measured
`rQ1-rr-Fluc or rQ1-y2-F1uc
`
`(a) Schematics of luciferase-expressing HSV-1 constructs.
`Figure 1
`The top line represents a schematic of the wild-type HSV-1 genome.
`The second line represents increased detail of the location of the
`'/,34.5 and LI,39 (ribonucleotide reductase (rR) or ICP6) genes. The
`rHsv-QI series are derived from the F strain and possess deletions
`in both ',',34.5 and 1.1,39 genes. The luciferase-expressing recombi-
`nants are shown in the next two lines. The luciferase expression
`cassettes were inserted in the deleted U,39 locus in the reverse
`orientation. The rQ1-a-Fluc has the IE4/5 promoter whereas rQl--
`Fluc has the strict-late gC promoter transcribing firefly luciferase
`(Fluc). Both recombinants possess an ICP6Q\-GFP fusion, which is
`non-functional as rR due to a large deletion at its carboxyl terminus,
`allowing fluorescence imaging. (b) Time course luciferase expres-
`sion in vitro. G1i36AEGFR human glioma cells plated in 24-well
`plates (1 x 101 cells/well) were infected with the luciferase-expres-
`sing vectors at an MOT of 3. At 9 different time points (0, 2, 4, 6, 9,
`12, 16, 20, and 24 h after infection), infected cells were harvested in
`triplicate. Luciferase activity was measured using Firefly Luciferase
`Assay System (Promega, Madison, WI, USA) and a MicroLumat
`LB96P (Berthold Technologies, Oak Ridge, TN, USA). Data are
`presented as mean ± s.d. (photon counts/second). (c) Time course
`copy number of vector genome in vitro. Quantitative PCR for HSV-1
`genomes was performed to assess viral DNA synthesis during the
`lytic infection. Whole cellular DNA was extracted from the infected
`Gli36L\EGFR cells (1 x 106 cells/60 mm dish at an MOI of 3) using
`QlAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) at
`different time points (2, 4, 6, 9, 12, 16, and 24 h after infection). Equal
`amount of total DNA from each sample was subjected to real-time
`PCR analysis using ABI PRISM 79001-IT (Applied Biosystems, Foster
`City CA, USA). Primers and probe set for HSV-1 gD gene (5'-
`5'-TCAGGAACCCCAGGT
`CAGCCCCGCTGGAACTACTAT-3',
`TATCCT-3' and VIC-5'-ACAGCTTCAGCGCCG-3'-TAMRA) were
`used for the analysis. The measured copy number is relative to the
`copy number measured with the input viruses (Oh). Data are
`presented as mean +s.d. of triplicate samples.
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`Imaging Transcriptional activation
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`rQ1-co-Fluc
`rQ1--Fluc
`
`=
`
`a
`
`107
`
`106
`
`0
`
`101
`
`b
`
`120
`
`100
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`80
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`>.
`.2 60
`0 Ca
`0
`
`40
`
`, 1
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`o'
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`''
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`0
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`c•
`
` 1 0
`
`(l,x
`
`"P.
`
`f
`
`q
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`
`rQ1 -a-Fluc 37°C
`
`rQl-a-Fluc4O°C
`
`rQ1 -y,-Fluc 37°C
`
`r01 -y2-Fluc 40°C
`
`=
`
`=
`
`=
`
`102 r
`
`111
`
`16 hr
`4 hr
`Hours after infection
`
`Figure 2
`In vitro determination of effects of antiviral drugs and temperature on luciferase-expressing vectors, rQ1-2-Fluc and rQ1-,-Fluc.
`(a) Vero cells were infected with the indicated viruses at an MOI of 1, in the presence or absence of Roscovitine (Rosco) (30 PM). Infected cells
`were harvested 4 or 16 h later and luciferase activity was determined (mean ±s.d. of triplicate samples). *Represents P<0.01 and - represents
`P <0.001, Student's t-test. (b) Vero cells were infected with rQ1-a-Fluc () or rQ1 -y,-Fluc (Y2) at the indicated MOI in the presence or absence of
`100 pg/mI of phosphonoacetic acid (PAA). Luciferase activity was then assayed 16 h after infection. The y-axis values are expressed as the
`percentage of luciferase activity from PAA-treated cells compared to non-PAA-treated control cells (mean±s.d. of triplicate samples).
`*Represents P <0.001 compared to rQ1-','2-Fluc MOI 3 without PAA (control) and —represents P <0.005 compared to rQ1-7,-Fluc MOI 3 with
`PAA (Student's t-test). (c) The effect of temperature on a vs promoter activity is depicted. Gli36\EGFR cells were infected with either rQ1-2-
`Fluc or rQ1-y,-Fluc at an MO! of 1, incubated for 10 min at room temperature, placed at 37 or 40°C, and harvested 4 or 16 h after infection. The
`harvested samples were subjected to the luciferase activity assay. Data are presented as mean ± s.d. of triplicate samples. *Represents
`P<0.0001, Student's t-test.
`
`luciferase activity at 4 or 16 h after infection. Figure 2a
`shows that the drug suppressed luciferase activity more
`significantly when driven by the gC promoter than by the
`IE4/5 promoter. When PAA was used, luciferase activity
`mediated by rQ1-c-Fluc was not affected, whereas that
`by rQ1-/,-Fluc was significantly decreased (Figure 2b).
`To further confirm that gC promoter-driven luciferase
`activity is dependent on viral replication, we performed
`temperature shift experiments. HSV-1 strain F is known
`to carry a temperature-sensitive mutation in the 1CP4
`gene, which results in ablation of viral DNA replication
`at a non-permissive temperature (40°C).25-26 Figure 2c
`
`shows that incubation at 40°C significantly decreased gC
`promoter activity, but did not affect the IE4/5 promoter as
`much. Quantitative genomic PCR confirmed that the
`Rosco treatment and 40°C incubation inhibited viral
`DNA synthesis (data not shown). These sets of results
`thus confirmed that luciferase expression driven by
`IE4/5 and gC promoters in the context of oncolytic
`HSV mutants measures viral infection and replication,
`respectively.
`To image the temporal activity of IF and strict-late
`promoters in vivo, subcutaneous tumors were established
`in the flanks of nude mice and then inoculated with
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`rQ1-co -Fluc
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`Imaging Transcriptional activation
`S Yamamoto et al
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`ii111101
`I
`rQ1-y2-Fluc F
`
`r
`
`72hr
`
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`
`Or
`
`20hr
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`b
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`100
`
`0(5
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`Hours after injection
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`Hours after injection
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`In vivo assay of IE or strict-late promoter activation in subcutaneous flank tumors inoculated with recombinant viruses Nude mice
`Figure 3
`were used for both normal tissue and tumor models. Mice were subcutaneously injected with 5 x 10 G1i36A-H2B-RFP cells that were
`generated by stable transduction of G1i36AEGFR cells with a lentiviral vector expressing a fusion of histone 2B with red fluorescent protein
`(RFP), allowing visualization of the nuclei by their REP expression. After 14-21 days, when tumors had achieved 5-10 mm in diameter, Fluc-
`expressing viruses were inoculated into the neoplasm. Mice were given an i.p. injection of 500 pl D-luciferin sodium (100 mg/kg body weight)
`and images were acquired using a NightOWL LB981 system (Berthold Technologies). A light image of the animal was also taken in the
`chamber using dim illumination. The spatial distribution of luciferase activity within the mice was then measured by recording photon
`counts using the cooled CCD with no illumination. Following data acquisition, postprocessing and visualization were performed using the
`Berthold WinLight 32 software. For visualization purposes, bioluminescence images were merged with the corresponding white light surface
`images by using color-overlay mode of the software, permitting correlation of areas of bioluminescent activity with anatomy. (a) The
`luciferase activity was measured at the indicated time points after inoculation of either rQ1-a-Fluc (upper row) or rQ1-?2-Fluc (lower row)
`(2.0 x 10 PFU/10 l) into subcutaneous tumors (one representative animal for each vector is shown). (b) The integrated photon counts/
`second/ tumor were graphed against duration after injection of viruses. Data are presented as mean+sd. of six mice per group. (c) rQ1-a-Fluc
`(upper row) or rQ1-y2-Fluc (lower row) (2.0 x 10 PFU/10 el) were injected in the subcutaneous space of non-tumor-bearing nude mice as a
`control. Images of one representative animal for each vector are shown. (d) The integrated photon counts/second/injection site were graphed
`against duration after injection. Data are presented as mean+s.d. of six mice per group.
`
`either rQ1-z-Fluc or rQ1-y2-Fluc. Figure 3a shows the
`imaging of IF promoter-mediated versus strict-late
`promoter-mediated luciferase expression in tumors as
`a function of time after injection. Similar to the in vitro
`findings, activation of the IE4/5 promoter could be
`imaged within 4 h after infection and increased gradu-
`ally during the course of 96 h after injection. In contrast,
`activation of the gC promoter became visible later, at 20 h
`after infection, rapidly increased between the 20- to 48-h
`time points, and was still increasing at the 96-h time
`
`point. Quantitatively, luciferase activity expressed by the
`IE4/5 promoter was significantly higher, but over the
`time course of the experiment, that expressed by the gC
`promoter reached similar intensity (Figure 3b). Histolo-
`gical examination of tumors confirmed the presence of
`replicating virus with the death of infected tumor cells
`(Supplementary Figure 3). The ability of the double-
`mutant HSV-1 to differentiate its replication in normal
`cells was also analyzed (Figure 30. When rQ1-a-Fluc was
`inoculated into subcutaneous tissue, activation of the
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`1735
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`IE4/5 promoter was detected as early as 4 h post-
`injection, indicating viral transduction to normal tissue.
`However, unlike in tumor tissue, luciferase activity
`became hardly detectable within a period of 48 h. On
`the other hands, when rQ1-y2-Fluc was injected sub-
`cutaneously, minimal luciferase activity was detected
`only at 20 h after injection. Quantitatively, gC promoter-
`mediated Fluc expression was significantly lower than
`IE4/5 promoter-mediated expression, confirming that
`normal cells can be transduced with double-mutant
`oncolytic HSV-1, but viral replication in these cells is
`very much limited (Figure 3d). The tumor-selective
`replication of the 11L39- and y734.5-deleted oncolytic
`the viral replication-dependent
`HSV-1 vectors and
`activation of the gC promoter suggest that strict-late
`promoters, such as gC, UL38 and Us1l promoters, may be
`more appropriate than lE promoters to drive therapeutic
`transgenes in the context of oncolytic HSV-1 vectors,
`which was addressed by Fu et al.27 previously.
`In this study, we constructed oncolytic HSV-1 vectors
`that express Fluc under the control of a viral IF promoter
`(IE4/5 promoter) or a strict-late promoter (gC promoter),
`and demonstrated the following: (1) bioluminescence
`readouts obtained from the infected cells and mice
`provide accurate correlates of the temporal kinetics of
`the transcriptional cascade that punctuates the viral life
`cycle in vitro and in vivo; (2) the effect of drugs and
`temperature on the viral lE- and strict-late-mediated
`transcriptional activity can be assayed by biolumines-
`cence; and (3) infection/transduction and replication
`of oncolytic HSV-1 vectors can be assayed/imaged both
`in vitro and in vivo by measuring luciferase activities
`expressed under the control of the IE4/5 promoter and
`the gC promoter, respectively.
`Development of non-invasive, real-time imaging of
`viral infection and viral replication in preclinical animal
`studies would greatly facilitate and advance the research
`on oncolytic virotherapy. Having taken advantage of two
`distinct promoters, we have proven that two distinct
`biological events, HSV-1 infection and replication, can be
`separately monitored non-invasively in vivo. This meth-
`odology will now allow us to study various factors and
`conditions that determine both infection and replication
`of oncolytic HSV-1 vectors in rodent models. For example,
`effects of various drugs that affect vascular permeability
`or host immune system can be evaluated on tissue
`distribution of viruses or viral replication, respectively.
`
`Acknowledgements
`This project was supported by Grants P01 CA69246, ROI
`N541571 and ROI CA85139 to EAC and by the Dardinger
`Center Fund for Neuro-oncology Research at the James
`Cancer Hospital, the Ohio State University Medical
`Center. We wish to acknowledge Ms Rosalyn Vu and
`Ms Suzanne Camilli for editing the manuscript and Dr
`Masayuki Nitta for providing the H2B-RFP construct.
`
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`Gene Therapy
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