Confinement of transgene expression to target cells is highly desirable in gene therapy. Current strategies of transcriptional targeting to tumors usually rely on tissue-specific promoters to control gene expression. However, such promoters generally have much lower activity than the constitutive viral promoters. We have explored an alternative approach, using a strict-late viral promoter (UL38p) in the context of an oncolytic herpes simplex virus (HSV) for tumor-selective gene expression. As with many DNA viruses, the genomic transcription of HSV is a tightly regulated molecular cascade in which early and late phases of gene expression are separated by viral DNA replication. In particular, some of the late transcripts are categorized as strict-late, whose expression depends rigorously on the initiation of viral DNA replication. Our in vitro and in vivo characterization showed that in normal nondividing cells, where the oncolytic HSV has limited ability to replicate, the UL38p has minimal activity. However, in tumor or cycling cells where the virus can fully replicate, transgene expression from UL38p was almost as high as from the cytomegalovirus immediate-early promoter. These results suggest that delivery of therapeutic genes driven by UL38p through an oncolytic HSV may be an effective approach to gene therapy for malignant diseases.
Many gene therapy strategies for cancer use genetic elements or toxic molecules to control or cure the disease. The success of these approaches depend largely on the tissue-specific delivery and expression of the therapeutic molecules in high levels. One way to achieve this goal is to use tumor- or tissue-specific transcriptional regulatory elements to control gene expression. Although several tissue-specific promoters have been shown to direct gene expression selectively in tumor cells originating from the same tissue,1,2,3,4 their activity is usually much weaker than the constitutive viral promoters, such as the cytomegalovirus immediate-early promoter (CMV-P) and the long terminal repeat (LTR) of retroviruses,3,5,6 resulting in poor antitumor efficacy. Although strategies such as adding CMV enhancer sequences to the upstream of the tissue-specific promoters can substantially increase promoter activity,3 modification of this type also cause the original promoters to lose their tissue specificity. Another potential concern is that tissue-specific promoters also tend to lose their tissue specificity once they are cloned into viral vectors.7
Several conditionally replicating human viruses, including herpes simplex virus type 1 (HSV-1), have been developed as a form of anticancer therapy. These so-called oncolytic viruses have been genetically modified so that they preferentially replicate and therefore kill tumor cells by a direct cytopathic effect as they replicate within the tumor cells, while being restricted in their ability to replicate in normal cells.8,9,10
As with many DNA viruses, the transcriptional program of HSV-1 is a regulated cascade in which early and late phases of gene expression are separated by viral DNA synthesis.11 The early genes are transcribed before viral DNA replication, while the late genes are expressed at high levels only after this event. Late transcripts can be further categorized as either leaky-late, which are readily detectable before the onset of viral DNA replication, or strict-late, which can be reliably detected only after such replication has begun.12,13,14 We hypothesized that the transcriptional regulatory elements of strict-late genes might be strong and tumor-specific promoters when introduced into an oncolytic HSV. It was expected that such a strict-late viral promoter would be extremely active in the tumor tissue where the oncolytic virus can fully replicate, but silent in normal cells if these were nondividing or postmitotic, since viral replication would be limited.
We therefore constructed an oncolytic HSV that contains the secreted form of the alkaline phosphatase gene (SEAP) driven by the promoter of UL38, a well-characterized strict-late gene of HSV.15,16,17 Although this promoter has very low activity in nondividing cells, in cycling cells and in the presence of lytic HSV infection, its activity is dramatically increased to a level equivalent to that of CMV-P. Results of in vivo administration of an oncolytic HSV containing this promoter cassette also demonstrated a strong tumor-selective expression property. Hence, a strict-late viral promoter in an oncolytic HSV can function as a strong tumor-selective promoter.
Construction and characterization of vectors
To facilitate the in vitro and in vivo analysis of gene expression, we initially linked the SEAP gene with the UL38 promoter (UL38p). The same SEAP gene was also linked to CMV-P as a control. To clone the gene into the oncolytic HSV, we used an enforced ligation strategy, which is more reliable and efficient for cloning foreign genes into the HSV genome than is the traditional homologous recombination. As depicted in Figure 1, fHSV-delta-pac is a bacterial artificial chromosome (BAC)-based construct that contains a mutated HSV genome, in which the diploid gene encoding γ34.5 was partly deleted, and both copies of the HSV packaging signal (pac) were completely deleted.18 Therefore, infectious HSV cannot be generated from this construct unless an intact HSV pac is provided in cis. Any virus generated from this construct will be replication selective, because of the partial deletion of both copies of the γ34.5 gene. We initially cloned the SEAP gene into intermediate plasmids, to generate pIMJ-pac-AP and pLox-AP, in which the gene was driven by either CMV-P or by UL38p. Both gene cassettes were flanked by the recognition sites of restriction endonuclease PacI. We then cut out the DNA fragments with PacI and ligated them into the unique PacI site located in the BAC sequence of fHSV-delta-pac. The ligation mixture was directly transfected into Vero cells, and the viruses grown from the cells were collected and plaque purified, to generate Baco-AP1 (containing the CMV-P-SEAP cassette) and Baco-AP2 (containing the UL38p-SEAP cassette) (Figure 1).
These two viruses were then directly compared for their growth properties in vitro. Vero cells were infected with the viruses at three different multiplicities of infection: 0.1, 1, and 5 plaque-forming units (PFU) per cell. The viruses were harvested 24 and 48 h after infection and titrated by a plaque assay. There was no significant difference in the replication of these two viruses (Table 1).
In vitro characterization of UL38 promoter activity
To determine the background level of UL38p activity and to investigate the effect of HSV replication on transactivation of the promoter, we transfected pIMJ-pac-AP (containing the CMV-P AP cassette) and pLox-AP (containing the UL38p AP cassette) DNA into either Vero cells or a human liver cancer cell line, Hep 3B, in duplicate experiments. At 16 h after transfection, one set of the transfected cells were infected with an oncolytic HSV (Baco-1), which was constructed the same way as Baco-AP1 or Baco-AP2 except that it contained the enhanced green fluorescent protein (EGFP) gene cassette instead of SEAP. The other set of transfected cells was mock infected (with medium only). After 24 h, the medium was collected from both sets of cells and the AP released in the medium was quantified. The results showed that without HSV infection, AP in the medium of both cell types transfected with pLox-AP was barely detectable (Figure 2). However, in the presence of lytic HSV infection, the AP expression increased by more than 50-fold, reaching almost half the level of the AP released from pIMJ-pac-AP-transfected cells. HSV infection also substantially increased the level of AP expression from CMV-P, likely because HSV infection transactivates CMV-P activity.19 These data demonstrate that UL38p has extremely low basal activity, which is greatly increased in the presence of lytic HSV infection.
To determine if the activity of UL38p in an oncolytic HSV is indeed HSV replication dependent, we compared the promoter activities in normal human cells in either a cycling or a quiescent state. Since oncolytic HSV replication is cell-cycle dependent and does not occur in normal nondividing cells in vivo, the arrest of normal human cells in vitro should render the virus nonreplicative in these cells. Primary human fibroblasts were plated in 12-well plates in duplicate. One set was treated with 20 μM lovastatin, a drug that induces cell-cycle arrest but does not interfere with HSV replication.20 Both arrested and untreated (ie, cycling) cells were then infected with either Baco-AP1 or Baco-AP2 at 0.1 PFU/cell. The culture medium was collected 24 h after infection and AP in the medium was quantified. The result showed that in the cycling cells, AP expression from UL38p contained in Baco-AP2 was actually slightly higher than that expressed from CMV-P contained in Baco-AP1 (Figure 3). However, when the cell cycle was arrested, UL38p activity was reduced by more than 40-fold, a very low level. Owing to the inhibitory effect of cell arrest on the replication of oncolytic HSV, AP expression from CMV-P was also reduced, but only by less than two-fold.
In vivo characterization of UL38 promoter activity in the context of oncolytic HSV
To demonstrate directly tumor-selective gene expression of UL38p in the context of an oncolytic HSV in vivo, we subcutaneously established a human liver tumor xenograft (Hep 3B) on the right flank of athymic nude mice. Once the tumor diameter reached approximately 8 mm, the viruses (Baco-AP1 or Baco-AP2) were injected intratumorally at a dose of 5 × 106 PFU. At the same time, mice that were not inoculated with tumor were injected with the same amount of viruses either by intravenous (i.v.) or intramuscular (i.m.) injection. Blood was collected at different times after virus injection, and AP release in the samples was quantified. The AP concentration in the mice injected intratumorally with either of the viruses started to increase by day 2 after virus administration, reaching a peak level by day 3. AP release started to decline therefore, but was maintained at a relatively high level for the rest of the experiment (Figure 4). AP release from Baco-AP1 was marginally higher than that from Baco-AP2 during the entire experiment except at the last time point (day 7), when it was slightly lower than the result for Baco-AP2 (P>0.05). By contrast, there were significant differences in AP release on days 2, 3, and 4 after the viruses were injected i.v. (P<0.01). Intravenous injection of Baco-AP2 produced only a slight increase of AP expression in the blood samples taken on days 2 and 3. By day 4 after virus administration, the release of AP had returned to the background level, where it remained for the rest of the experiment. However, i.v. injection of the same amount of Baco-AP1 produced much higher AP release in the serum before day 5 after virus inoculation. AP in the blood of mice receiving either of the viruses i.m. stayed at the basal level for the entire experimental period, probably because of poor transduction of myoblasts in adult mice by HSV vectors. Together, these results demonstrate that in the context of an oncolytic HSV, UL38p can direct strong tumor-selective gene expression after its in vivo administration.
Oncolytic viruses developed from HSV have shown great promise for treating solid tumors and are currently in clinical trials for patients with brain tumors or melanoma.21,22,23 Oncolytic HSVs may also be useful vectors for delivering therapeutic genes in cancer treatment. Compared to traditional defective viral vectors, conditionally replicating vectors have several advantages. First, unlike the defective vectors, which are merely functioning as a delivery vehicle, oncolytic HSVs themselves possess a very high therapeutic index against tumors. Any antitumor effect from the delivered therapeutic gene should be additive, leading to an improved result overall. One such example is our recent demonstration that incorporation of cell-membrane fusion function into an oncolytic HSV can dramatically enhance the antitumor effect of the virus.24 Second, as oncolytic viruses have the ability to spread through tumor tissues, they can deliver the therapeutic genes to a larger area of tumor mass than defective viral vectors. Third, the ability of the virus to replicate in the tumor may prolong the period of gene expression. In support of this potential benefit, our results showed that after intratumor delivery of oncolytic HSV, the AP expression remained at a relatively high level over 1 week, in contrast to gene expression from defective HSV vectors, which usually persists for only 24–48 h after intratumor injection.25
Since most of the therapeutic genes used for antitumor therapy are also potentially toxic to normal cells, uncontrolled expression of these genes, even in the context of a tumor-restricted oncolytic virus, still poses a safety concern. This is particularly true when systemic administration is required (eg, for metastatic diseases). One way to minimize this concern is to use a tumor- or tissue-specific promoter to control gene expression. While several tissue-specific promoters have been described for tumor-specific gene expression, they generally have much lower activity than viral promoters. Here, we show that a strict-late viral promoter is a strong and tumor-specific promoter in the context of an oncolytic HSV. This property was demonstrated by both in vitro and in vivo characterization of the promoter of the strict-late gene UL38 of HSV. UL38p has very low basal activity which is maintained in nondividing cells infected with an oncolytic HSV carrying the UL38p cassette. However, in cycling cells, where the oncolytic virus can replicate, UL38p showed a similar level of activity to that of CMV-P. The cell-cycle-dependent property of UL38p was also demonstrated in vivo. Intratumor injection of both Baco-AP1 and Baco-AP2 produced high levels of AP expression, which peaked at day 3 and then remained at a relatively high level for the rest of the experiment. This pattern of gene expression correlates well with the reported growth curve of oncolytic HSV in tumor masses in vivo.26 On the other hand, only transient, low-level expression of AP was detected after i.v. injection of Baco-PA2, in contrast to the abundant AP release after i.v. administration of Baco-AP1. The transient and low-level release of AP after i.v. injection of Baco-AP2 was probably from certain dividing cells, such as epithelial cells and fibroblasts, which might have infected after systemic delivery of the virus. However, since the majority of viral particles are distributed to liver after systemic delivery,27,28 this pattern of AP expression implies that UL38p in the context of an oncolytic HSV has minimal activity in normal hepatocytes. Both viruses were also delivered intramuscularly to target the mostly postmitotic myoblasts. However, neither virus produced any appreciable AP release after this route of injection. This is probably because not enough myoblasts were transduced by either of the viruses, as it has been reported that the mature basal lamina of muscles from adult mice can prevent HSV from efficiently infecting the myofibers.29,30
Although the UL38p-AP expression cassette was inserted into the BAC sequence contained in the virus, which is distant from the promoter's natural location, the activity and its strict-late expression profile associated with the promoter seem to be well maintained. It has been reported that when a UL38p-β-galactosidase gene cassette was inserted into the glycoprotein C (gC) location in the viral genome, the promoter was significantly less active than in its normal location.17 In contrast, promoter activity was comparable to the wild-type value when the same gene cassette was inserted into the repeated region of the viral genome. Despite the influence of genomic location on UL38p activity, the kinetics of expression in either location mirrors the wild-type UL38 strict-late kinetics of expression.17 Furthermore, the low level of UL38p activity in the gC location could be partially alleviated by the incorporation of additional DNA sequences upstream of the promoter.17 In Baco-AP2, the UL38p-AP cassette (together with the packaging signal) was inserted into the middle of the BAC sequence, so that more than 3 kb of ‘stuffer’ DNA were on each side of it. This may have protected the promoter from interference by any cis elements contained in the surrounding HSV sequences.
Besides significantly stronger gene expression, the combination of a strict-late viral promoter such as UL38p with an oncolytic HSV offers additional advantages over the conventional tumor-specific promoters in a defective viral vector. Unlike tissue-specific promoters that can be applied only to tumors arising from a defined tissue, strict-late viral promoters may be useful in a variety of tumors in which the virus can conditionally replicate. This consideration is especially important for tumors in which tumor-specific promoters have not yet been defined. Moreover, conditional activation of the UL38 promoter upon the initiation of lytic HSV infection in tumor cells may synchronize virus-induced oncolysis with the therapeutic effect of the delivered gene. Such synchronized action would be particularly advantageous in combination with immunotherapy, for example, where release of tumor antigens by viral oncolysis could be timed to coincide with the release of immune-stimulating molecules from genes inserted into the virus.
Human adenovirus has also been developed for oncolytic purposes, and adenovirus-derived vectors have also been widely used as gene delivery vehicles. Since adenovirus is a DNA virus, its mode of gene expression is similar to that of HSV, in which late gene expression also begins only with the onset of viral DNA replication.31 Hence, the strategy of using a strict-late promoter as described for HSV should apply equally well to oncolytic adenoviruses. Indeed, insertion of the TNF gene into the E3B region of an oncolytic adenovirus converted the E3 transcriptional machinery into late-gene expression.32 Expression of the TNF gene from such a virus was subsequently shown to depend on the initiation of viral DNA replication. It would be informative to characterize fully this virus (ONXY-321), or a recombinant oncolytic adenovirus containing a transgene driven by the major late promoter, in the context of an oncolytic adenovirus in tumor and nontumor tissues.
Materials and methods
Vero cells (African green monkey kidney fibroblasts), human embryonic fibroblasts (HF 333.We), and human liver cancer cells (Hep 3B) were obtained from the American Type Culture Collection. They were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
The promoter and enhancer region (das) of UL38, which has been well defined,15 was amplified from HSV-1 DNA with the following pair of primers: forward 5′-IndexTermGTGGGTTGCGGACTTTCTGC-3′ and reverse 5′-IndexTermACACTCACGCAAGGCGGAAC-3′. The amplified promoter sequence was cloned into the unique NcoI site of plox-3 H, which lies next to a copy of the HSV packaging signal flanked by recognition sites for the restriction enzyme PacI (X Zhang et al, unpublished data) to generate plox-UL38p. The gene encoding the secreted form of alkaline phosphatase (SEAP) was cut out from pSEAP-control (CLONTECH Laboratories, Inc., Palo Alto, CA, USA) with HpaI and HindIII, and cloned into the unique BglII site of plox-UL38p through blunt-end ligation. Thus, in the new plasmid, designated pLox-AP, the SEAP gene is driven by the UL38 promoter. PIMJ-pac-AP was constructed by inserting the same AP gene (HpaI–HindIII fragment of pSEAP-control) downstream of the CMV-P contained in pIMJ-pac, which lies next to a copy of HSV pac flanked by PacI recognition sites (Fu and Zhang, unpublished data). Pure plasmid DNA was obtained by alkaline lysis of bacterial culture and purified by QIAGEN-tip 500 column (QIAGEN, Valencia, CA, USA).
Generation of oncolytic HSV containing AP gene cassettes
The AP gene cassette together with the pac sequence were cut out from either pLox-AP or pIMJ-pac-AP with PacI and gel purified. They were then cloned into fHSV-delta-pac for the generation of infectious oncolytic HSV through an enforced ligation strategy. As illustrated in Figure 1, fHSV-delta-pac is a bacterial artificial chromosome (BAC)-based HSV construct, in which the BAC sequence is inserted into the nucleotide 98 968 and 102 732 region of the HSV genome.18 As the diploid gene encoding γ34.5 and both copies of HSV packaging signal (pac) have been deleted from the HSV sequence contained in fHSV-delta-pac,18 infectious HSV cannot be generated from this construct unless an intact HSV pac is provided in cis, and the generated virus will be replication-conditional as a result of deletion of both copies of the γ34.5 gene. The DNA fragments containing the EGFP or AP gene cassette, together with the pac, were cut out with PacI and ligated into the unique PacI site located in the BAC sequence of fHSV-delta-pac. The ligation mixture was directly transfected into Vero cells using Lipofectamine (Gibco-BRL) and incubated for 3–5 days to generate infectious virus. The viruses, designated Baco-AP1 (containing the CMV-P-AP cassette) and Baco-AP2 (containing the UL38p-AP cassette), were subsequently plaque purified. The presence of AP gene in the viruses was confirmed by detection of AP expression. To generate large viral stocks, we infected Vero cells with each of the viruses at 0.01 plaque-forming unit (PFU) per cell. The viruses were harvested 2 days later and subjected to three cycles of freeze–thaw, followed by one cycle of sonication. Cell debris was removed by low-speed centrifugation (2000 g at 4°C for 10 min), and the virus stocks were stored at −80°C.
Transfection and infection of mammalian cells in vitro for quantification of AP release
For in vitro plasmid DNA transfection, Vero and Hep 3B cells were seeded in six-well plates 1 day earlier at 2 × 105 cells/well and incubated at 37°C in a 5% CO2 atmosphere. The plasmid DNA (2 μg) of pLox-AP or pIMJ-pac-AP was mixed with 5 μl of lipofectamine (GibcoBRL) according to the manufacturer's instruction. Before being applied to cells, the liposome-formulated DNA was added to 1 ml of DMEM without serum. The cells (about 70% confluent) were exposed to the DNA–liposome complex for 3 h at 37°C, after which the transfection mixture was replaced with 2 ml DMEM containing 10% FBS. At 16 h after transfection, the cells were either infected with 0.1 PFU/cell of Baco-1, an oncolytic HSV that was constructed in the same way as Baco-AP1 or Baco-AP2 but contains the EGFP gene cassette instead, or mock infected (with medium only). The medium was collected 24 h later for the measurement of AP release.
For in vitro characterization of Baco-AP1 and Baco-AP2, the embryonic fibroblasts were either kept in the cycling phase by growth in a medium containing 10% FBS, or were arrested with 20 μM lovastatin for 30 h in serum-free medium. Lovastatin is a chemical that induces cell-cycle arrest but does not directly interfere with HSV replication.20 However, as the replication of oncolytic HSV is cell-cycle dependent, cell-cycle arrest would lead to the inhibition of HSV DNA replication. The cells were then infected with either Baco-AP1 or Baco-AP2 at 0.1 PFU/cell, and the medium was collected 24 h after infection for the quantification of AP release.
Chemiluminescent AP assay
AP activity in culture medium or blood serum was quantified with a commercial detection kit from CLONTECH Laboratories, Inc. (Palo Alto, CA, USA). The assay was performed according to the manufacturer's instructions. Briefly, 25 μl of sample was added to 75 μl of 1 × dilution buffer. After gentle mixing, the diluted samples were incubated at 65°C for 30 min. The samples were cooled to room temperature, and 100 μl of assay buffer was added to each sample, followed by incubation for 5 min at room temperature. Finally, samples were mixed with the solution containing chemiluminescent substrate C and enhancer at a ratio of 1:20, and incubated for 30 min at room temperature. The chemiluminescent emission was detected using a luminometer (Turner Designs Instruments, Sunnyvale, CA, USA).
Six-week-old female Hsd athymic (nu/nu) mice were purchased from Harlan (Indianapolis, IN, USA). To establish human liver cancer xenografts, we cultured Hep 3B cells in standard conditions and then harvested the cells in log phase with 0.05% trypsin-EDTA. The cells were washed twice with serum-free medium before they were resuspended in PBS at a concentration of 5 × 107 cells/ml. A total of 5 × 106 cells (in a l00-μl suspension) were subcutaneously injected into the right flank of each mouse. When the tumors reached approximately 8 mm in diameter, they were injected with viruses (5 × 106 PFU in a 100 μl volume). For i.v. (tail vein) or i.m. (right hind limb) injection, mice without established tumor were injected with the same amount of viruses. Blood was withdrawn from the mice at days 1, 2, 3, 4, 5, and 7 after virus inoculation for quantification of AP release.
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We thank Yoshinaga Saeki (Massachusetts General Hospital) for the generous gift of fHSV-delta-pac and Malcolm K Brenner for support and careful reading of the manuscript.
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