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1 Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri; 2 ML Laboratories PLC, Stephenson Building, Keele University Science Park, Keele, United Kingdom; and 3 VirRx, Inc., St. Louis, Missouri
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This general problem has stimulated interest in the use of replication-competent lytic (oncolytic) viruses (vectors) for cancer gene therapy. The life cycle of such vectors is ideal for cytoreductive genetic therapy of cancer, i.e., cell killing, regeneration of the input vector, and infection/killing of additional cells. Vector regeneration in situ is also attractive from the standpoints of safety and production because it may allow the use of lower doses of vector than with replication-defective vectors.
For this type of therapy, vector growth should be restricted to target tumor cells. Most effort has been focused on the engineering of adenovirus (Ad) type 5 (Ad5) to render its replication selective to tumor cells (reviewed in Refs. 1 and 2 ). The replacement of one or more promoters of essential Ad genes with tumor-selective promoters has been the most widely used approach (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . Attempts to obtain cancer cell specificity at the level of cell entry are also ongoing (15, 16, 17, 18, 19, 20) . Another approach exploits defects in early virus-host cell interactions that prevent apoptosis caused by Ad E1A protein expression (21, 22, 23) and drive nondividing cells into the cell cycle (4 , 24, 25, 26, 27) . Ad vectors with these defects grow poorly in normal cells but grow well in cancer cells in which these virus functions are dispensable.
Although showing promise, most of these approaches suffer from at least one disadvantage. Most commonly, the mechanism of tumor specificity used makes the vector less cytopathic (efficacy reduced). For example, deletion of the E1B 55kD gene from Ad, such as in the case of the first anticancer Ad vector, ONYX-015, reduces the efficiency of the nuclear export of viral RNAs, resulting in an overall attenuation of the virus (28) .
Various promoters have been used to restrict the replication of oncolytic Ads to tumor cells or a certain tissue type. A general problem with these vectors is that the promoters used to drive essential viral gene expression selectively in tumor cells are also active to some extent in normal tissues (29) . There is therefore a need for vectors with a greater selectivity for tumor cells and that lyse tumor cells at least as efficiently and ideally more efficiently than wild-type (wt) Ad.
A key factor affecting the efficacy of oncolytic Ad is the host immune response to vector replication. This response is highly effective, and it is thus important to maximize vector spread before it occurs. We have shown that an Ad E3-encoded protein called Adenovirus Death Protein (ADP, formerly named E3–11.6K) plays a critical role in release of Ad from infected cells (30, 31, 32) . More recently, we demonstrated that overexpression of ADP by a replicating Ad can be successfully used to increase the rate of vector spread, i.e., without aborting infection as might have been predicted (33) . Consistent with this, ADP-overexpressing oncolytic Ads exhibit markedly increased levels of antitumor efficacy, both in vitro and in vivo (4 , 24) , compared to vectors that lack ADP. These viruses have been rendered tumor selective by incorporating specific E1A mutations that abrogate binding of the E1A proteins to p300/CBP and members of the pRb family. We have demonstrated that radiation treatment enhances the antitumor effect of these tumor-selective vectors, both in vitro and in vivo (34) .
Tumor-selective replication of an ADP-overexpressing vector was also achieved by substituting the wt E4 promoter with the promoter regulating the expression of the gene for the surfactant protein B (SPB), which is active only in type II alveolar and Clara cells in the lung (4) . As the E4 proteins are required for Ad replication (35) , the replication of the resulting vector, KD1-SPB, is restricted to cells in which the SPB promoter is active (besides being restricted to cycling cells because of the deletions in the E1A gene), i.e., cells in the lung. Thus, KD1-SPB could have potential use in the treatment of lung cancer.
Efforts to understand the molecular mechanisms that underlie cancer progression have revealed a defect in the wnt signal transduction pathway (in the APC, axin, or ß-catenin genes) in most colon cancers (36) . The deregulation of wnt signaling leads to constitutive activation of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors by ß-catenin (36 , 37) . This transcription factor complex is inactive in most normal adult tissues (except hair follicles and stem cells; Ref. 38 ), but it is active in a majority of colon cancers as well as in a growing number of other types of cancer. One research group constructed a set of conditionally replicating Ads in which one or more early proteins is expressed from a synthetic promoter containing four consensus binding sites for TCF (3 , 5) . In vitro data obtained with cell lines suggest that these vectors possess a very high degree of selectivity for cancer cells with deregulated ß-catenin. A synthetic TCF-responsive promoter was also used to drive expression of the prodrug converting enzyme Escherichia coli nitroreductase in a nonreplicating Ad vector (39) .
In this report, we describe the construction and characterization of a novel oncolytic Ad vector named VRX-009 that combines both selective replication in cancer cells with deregulated wnt signaling and enhanced vector spread through overexpression of the ADP protein. VRX-009 is considerably more cytopathic than wt Ad5. The growth of VRX-009 depends on expression of Ad E4 genes from an artificial ß-catenin-dependent promoter. The combination of enhanced cell lysis activity and selectivity of this vector for ß-catenin deregulated cancer cells make VRX-009 a candidate for the therapy of a broad range of tumors with a defect in the wnt signaling pathway.
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MATERIALS AND METHODS |
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Virus Vectors.
The construction of VRX-007 is described elsewhere (33)
. In brief, this vector is derived from Ad5; it has a deletion of Ad5 bp 28,598–30,469, removing all E3 genes but the gene for the 12.5K protein. Into this deletion was inserted a copy of the adp gene such that ADP is expressed in large amounts from the major late promoter at late stages of infection (and perhaps from the E3 promoter at early stages). This allows for overexpression of ADP at the late stage of infection.
VRX-009 was constructed as follows. pdlE4prom, a plasmid containing Ad5 sequences from Ad5 bp 21,562 to the right end of the genome with the E4 promoter deleted (from Ad5 bp 35,623 to 35,775) and a Bst1107I site inserted, was generated by site-directed mutagenesis. This deletion removes the entire E4 promoter, including the TATA box. A synthetic oligonucleotide consisting of five consensus TCF binding sites and an Ad5 E1B TATA-box was inserted into the Bst1107I site, resulting in pdlE4prom-TCF. The sequence of the promoter is as follows (the TCF binding sites and the TATA-box are underlined):
5'-CCTTTGATCAATACCTTTGATCTCACCTTTGATCAAGTCCTTTGATCATACCTTTGATCTCTAAATGCACCTTTATCAGAGGGTATATAAT-3'.
A promoter with a similar sequence was characterized previously (39)
. The plasmid pdlE4prom-TCF was cotransfected with EcoRI-SpeI-digested dl327 (41)
DNA into VK10-9 cells. The cells were overlayed with agar containing DMEM, and the developing plaques were screened for the expected genotype. An isolate with the expected genome structure (Fig. 1
; verified by restriction endonuclease digestion of purified viral DNA) was plaque-purified three times. This virus isolate was named VRX-009. Large stocks of the vector were grown on W162 cells and purified on a CsCl gradient. W162 is a Vero monkey kidney cell line that stably expresses the Ad E4 proteins (42)
. VRX-009 has similar growth characteristics to wt Ad in W162 and VK10-9 cells. The vector preparations were dialyzed into physiological buffer and titered by plaque assay on VK10-9 cells. The resulting titers were in the 5 x 1010-1011 plaque-forming units (PFU)/ml range.
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Indirect Immunofluorescence.
A549 or SW480 cells were plated on glass coverslips (SW480 cells were plated on coverslips treated with poly-L-lysine). Cells were infected with 10 PFU/cell VRX-007 or VRX-009. At 2 days p.i., cells were fixed in 3.7% paraformaldehyde in PBS (room temperature) for 10 min, and then they were permeabilized with methanol (–20°C) for 6 min. After rehydration in PBS, cells were co-immunostained with a pool of mouse monoclonal antibodies specific to E1A (43)
, a rabbit polyclonal antibody to E4ORF3, a rabbit polyclonal antipeptide antibody to DNA-binding protein (DBP; a gift of Maurice Green), or a mouse monoclonal antibody to fiber (a gift of Jeff Engler). Secondary antibodies used were affinity-purified goat antirabbit IgG-FITC and goat antimouse IgG-RITC (Cappel/ICN). Images were taken on a Nikon Optiphot-2 epifluorescence microscope equipped with a Nikon DXM1200 digital camera; Nikon ACT-1 software was used for the digital images.
Cell Viability Assay.
Cells in 3.5-cm dishes were mock-infected or infected with 10 PFU/cell VRX-007 or VRX-009, respectively. At 6 days p.i., the cells were stained with trypan blue as described before (31)
. For each sample, about 400 cells were examined, and the percentage of blue cells was calculated.
Single-Step Growth Curve.
Cells in 3.5-cm dishes (eight dishes with each virus) were mock-infected or infected with 10 PFU/cell Ad5, VRX-007, or VRX-009, respectively. All dishes were washed three times with medium, and one dish from each group was harvested immediately after infection. The rest of the cells were harvested at daily intervals by freeze-thawing the contents of the dish (cells and supernatant). The lysate was clarified by low-speed centrifugation. The resulting vector stocks were titered by plaque assays in triplicate on VK10-9 cells, and the virus burst size per infected cell was calculated.
Vector Spread Assay.
A549 or SW480 cells cultured in 48-well plates were infected with a serial dilution (10–10–2 PFU/cell) of various viruses. The plates were monitored via phase-contrast microscopy, and at 7 days p.i., they were stained with crystal violet (1% crystal violet, 10% formaldehyde, and 20% ethanol) for 15 min. The excess dye was washed away with tap water, and the plates were dried at room temperature.
Tumor Xenograft Experiments.
Female nude mice (5–6 weeks old; Harlan Sprague Dawley, Indianapolis, IN) received injections s.c. into both hind flanks with 5 x 106 A549 or SW480 cells, respectively. When the tumors reached 50–150 µl, they received injections of buffer or of 3 x 108 PFU of VRX-007 or VRX-009, respectively, on 3 consecutive days, receiving a total dose of 9 x 108 PFU/tumor. Tumor sizes were measured twice weekly using a computer-linked digital caliper (Sylvac, Crissier, Switzerland) and evaluated with an in-house developed software (Mouser). The significance of the data was examined by one-way ANOVA, and pairwise comparisons were performed with Student’s t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A vector named VRX-007 was constructed previously (33)
. This vector is similar to VRX-009 except that it has the wt E4 promoter (Fig. 1)
. With both vectors, the insertion of the adp gene into the deletion in the E3 region provides for overexpression of ADP (24)
. This, in turn, results in enhanced cell lysis and vector egress (32)
. In every other respect, these vectors are equivalent to wt Ad5.
VRX-009 Selectively Expresses the E4ORF3 Protein in Cancer Cells with Deregulated wnt Signaling.
Because the TCF promoter is active only in cells with deregulated wnt signaling, we expected that the E4 proteins would be expressed by VRX-009 only in tumor cells with this molecular defect. To test this expectation, we examined VRX-009-directed synthesis of E4ORF3, a representative E4 protein (35)
, in various cell lines. VRX-007 was used as a positive control. The cells were infected with 10 PFU/cell VRX-007 or VRX-009, and cell extracts were collected on days 1 and 2 p.i. and analyzed by Western blot (Fig. 2)
. In contrast to E4ORF3, VRX-007 and VRX-009 expressed equivalent amounts of the Ad E1A protein (Fig. 2
, Lanes b–e), indicating that the efficiency of infection with both vectors is similar. As predicted, whereas VRX-007 expressed equivalent levels of E4ORF3 in all cell lines (Fig. 2
, Lanes g and h), E4ORF3 from VRX-009 was detectable only in the SW480 and LS513 colon cancer cell lines (Fig. 2
, Lanes i and j) in which the TCF promoter is active (39)
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, Lanes l–o).
To address whether the residual expression of ADP observed in VRX-009-infected A549 cells is a result of a low-level expression in all cells or due to a high-level expression in a subpopulation of cells, indirect immunofluorescence was performed. SW480 or A549 cells were infected at 10 PFU/cell with VRX-007 or VRX-009. At 48 h p.i., cells were fixed and immunostained for the E1A, E2 DBP, or fiber (a late virion structural protein) proteins. E1A proteins and DBP were equally apparent in most nuclei of both cell types at 2 days p.i. (Fig. 3, A and B)
, indicating that both vectors infect these cell lines well. The DBP pattern for most VRX-009-infected A549 cells was characteristic of early infection (before DNA replication), i.e., the nuclei were homogenously stained (Fig. 3A
, DBP). Cells were stained for E4ORF3 to determine the extent of expression from the E4 region. The wt E4 promoter in VRX-007 was active in both A549 and SW480 cells, as judged by the number of E4ORF3 positive cells (Fig. 3, A and B
, E4ORF3). However, although E4ORF3 was abundant in the nuclei of SW480 cells infected with VRX-009 (Fig. 3B)
, it was undetectable in A549 cells (Fig. 3A)
, as expected. E4 proteins facilitate productive infection and transition to late infection (during late infection the viral genome is replicated, capsid proteins, for instance fiber, are synthesized, and virions are assembled). Late protein synthesis is represented here by fiber immunofluorescence. Essentially all VRX-009-infected SW480 cells were positive for fiber (Fig. 3B
, Fiber), whereas only a minor subpopulation of VRX-009-infected A549 cells infected with VRX-009 showed fiber staining (Fig. 3A
,Fiber). We conclude that VRX-009 expresses E4 proteins (as judged from the expression of E4ORF3) only in colon cancer cells with deregulated wnt signaling.
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, whereas VRX-009 caused CPE on SW480 but not on A549 or DU145 cells (Fig. 4, C, F, and I)
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. VRX-007 was also very lytic in A549 cells. As judged from earlier studies (24)
, most of these cells were probably dead by 4 and 5 days p.i. However, more than one-half of VRX-009-infected A549 cells were still intact at 6 days p.i. (Fig. 4J)
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To further characterize the effect of the E4-TCF promoter substitution on vector replication, single-step growth assays were performed on SW480 and A549 cells. Cells were infected with 10 PFU/cell VRX-009, VRX-007, or Ad5, and then virions were extracted at consecutive days p.i. and titered on VK10-9 cells. As was shown above, the TCF promoter is active in the colon but not in the lung cell line. Ad5 and VRX-007, which have the wt E4 promoter, replicated equally well on both cell lines (Fig. 5, A and B)
. VRX-009 replicated well on SW480 cells (Fig. 5A)
, but it was severely restricted in the A549 lung cancer cells as indicated by a reduction in vector yield of about 2 orders of magnitude (Fig. 5B)
. Replication of VRX-009 in DU145 prostate cancer cells was similarly restricted (data not shown).
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VRX-009 Can Spread from Cell to Cell Effectively in SW480 but Not in A549 Cells.
To maximize the chance of achieving antitumor efficacy, a replication-competent cancer gene therapy vector should spread quickly from cell-to-cell. The overexpression of ADP by VRX-009 should lead to rapid cell lysis and vector release, resulting in an accelerated infect-release-reinfect cycle. However, in cells in which its replication is reduced, the spread of VRX-009 should be slower. To investigate this hypothesis, we performed a "vector spread assay" (24)
on SW480 and A549 cells infected with VRX-007 or VRX-009. The Ad5-derived virus named dl309, which has the E3 RID
, RIDß, and 14.7K genes deleted but expresses Ad5-like levels of ADP (33)
, was used as a wt control.
Cells in 48-well plates were infected with serial dilutions of the various viruses (10, 1, 0.1, and 0.01 PFU/cell). At 7 days p.i., the monolayers were stained with crystal violet. In this type of experiment, monolayers infected at high multiplicity of infection (i.e., 10 PFU/cell) are rapidly destroyed by the original dose of infecting virus. However, in the wells infected with low multiplicity (i.e., 0.1 or 0.01 PFU/cell), the virus must go through one or more infect-release-reinfect cycles to cause detectable CPE. In SW480 cells, both VRX-009 and VRX-007 spread faster than the wt dl309 (Fig. 5C)
. Conversely, in A549 cells, VRX-009 spread much more slowly than VRX-007 and somewhat more slowly than dl309 (Fig. 5D)
. The effect of ADP overexpression is worth noting here; VRX-007, which differs from dl309 principally in that it overexpresses ADP, spreads approximately 10 times faster than dl309 (monolayers were destroyed by one-tenth as much virus).
Our conclusion from these data is that VRX-009 can spread from cell to cell effectively in a permissive colon cancer cell line but not in a lung cancer cell line.
Expression of E4ORF3 and ADP Is Restricted in NHBE Cells and HUVEC.
Ad5 normally infects bronchial epithelial cells in the human body. To test the ability of VRX-009 to replicate in these cells, we infected them with VRX-007 or VRX-009 at 10 PFU/cell. Cell extracts were analyzed for E1A, E4ORF3, and ADP by Western blot. Both vectors expressed similar amounts of E1A (Fig. 6A
, Lanes b and c), indicating equal levels of infection. However, VRX-009 expressed barely detectable amounts of E4ORF3 and ADP compared with VRX-007 (Fig. 6A
, Lanes e, f, h, and i, respectively), indicating that expression from the TCF promoter in VRX-009 (and subsequent expression of ADP from the major late promoter) was severely reduced in NHBE cells.
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, Lanes h and j), whereas VRX-007 expressed E4ORF3 abundantly at both time points (Fig. 6B
, Lanes g and i). Significant expression of ADP was detected only at 2 days p.i. in the VRX-007 infected sample (Fig. 6B
, Lane n). At 1 d p.i. VRX-007 expressed more E1A than VRX-009, whereas at 2 days p.i. the opposite was true (Fig. 6B
, Lanes a–e); this can possibly indicate that VRX-009 reaches the late phase of replication belatedly compared with the parental virus (E1A expression decreases as the late phase of replication ensues). Alternatively, the elevated levels of E1A at late times might be the direct result of the absence of E4 proteins. NHBE cells infected with 10 PFU/cell VRX-007 or VRX-009 were also examined by indirect immunofluorescence for E1A, DBP, E4ORF3, and fiber proteins. E1A and DBP expression were used to confirm similar levels of infection. Of VRX-007-infected cells, 68% expressed E4ORF3 and 42% expressed fiber (data not shown). In contrast, only 9% of cells infected with VRX-009 expressed E4ORF3 and only 11% expressed fiber (data not shown).
These Western blot and immunofluorescence data imply that the replication of VRX-009 is much less efficient in NHBE cells than in colon cancer cells.
The ability of VRX-009 to cause CPE and replicate in HUVEC cells was also examined. HUVEC cells at about 30% confluency were mock-infected or infected with 3 PFU/cell VRX-007 or VRX-009. VRX-007-infected cells showed extensive CPE by 4 days p.i., whereas VRX-009 did not cause any CPE until 6 days p.i. (Fig. 7A)
. The replication of VRX-009 in HUVEC cells was assessed by extracting viruses from cells infected with 3 PFU/cell VRX-007 or VRX-009 at 4 days p.i. The burst size of VRX-009 was more than 2 orders of magnitude less than that of VRX-007 (Fig. 7B)
, confirming that the delay in the appearance of CPE is a result of delay in replication of VRX-009.
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. Over the course of the experiment, the vehicle-injected tumors grew approximately 40-fold, whereas the VRX-007- and VRX-009-injected tumors grew only approximately 8- and 4-fold, respectively (VRX-007 versus VRX-009, P = 0.29; VRX-007 versus mock, P = 0.001; VRX-009 versus mock, P = 0.002 at the conclusion of the experiment). In the A549 tumor xenografts, there was a significant difference between the efficacies of the two vectors (Fig. 8B)
; whereas VRX-007 completely repressed the tumors, VRX-009 was largely ineffective (VRX-007 versus VRX-009, P = 0.007; VRX-007 versus mock, P = 0.013; VRX-009 versus mock, P = 0.45 at the conclusion of the experiment).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have demonstrated that with VRX-009, the expression of E4ORF3, a representative E4 protein, is cell-line dependent; it is close to wt levels in SW480 colon cancer cells but undetectable in A549 lung cancer and DU145 prostate cancer cells. There were intermediate levels of E4 protein expression in LS513 colon cancer cells. The lack of E4 protein expression clearly limited vector replication; this was indicated by the lack of ADP synthesis in both cell lines that were not of colon cancer origin and by the observation that VRX-009 did not cause CPE in either A549 or DU145 cells at 48 h p.i. In the two colon cancer cell lines (SW480 and LS513), ADP synthesis was proportional to the expression of E4ORF3; i.e., VRX-009-infected SW480 cells expressed near wt amounts of ADP, whereas ADP expression in LS513 cells was somewhat diminished. SW480 cells have mutant APC (44) , and LS513 cells carry a mutation in the gene for the Axin protein (45) . Although both mutations result in the elevation of nuclear ß-catenin levels, the abundance of nuclear ß-catenin in these two cell lines relative to each other is not known. It was demonstrated before that a synthetic TCF promoter showed very high levels of activity in secondary colorectal cancer tissues (39) .
More direct evidence for cell line-specific replication of VRX-009 was obtained by determining the yield of VRX-007 and VRX-009 after synchronous infection of SW480, A549, and DU145 cells. The results clearly showed that VRX-009 replication is attenuated in A549 and DU145 cells, because in these cells an approximately 100 times lower yield was obtained compared with VRX-007 and Ad5. This was independently confirmed by the vector cell-to-cell spread assay in which VRX-009 behaved like its parental virus, VRX-007, in permissive SW480 cells, but spread much more slowly than VRX-007 in nonpermissive A549 cells. The lack of VRX-009 replication in A549 cells is especially significant because this is one of the most permissive cell lines for Ad replication. The immunofluorescence experiments revealed that a fraction (10–15%) of nonpermissive cells do progress into the late phase of infection. These cells express wt amount of fiber and probably account for the limited spread and virus yield observed in the experiments discussed above. We do not know what sets this subpopulation apart, clearly, more work is warranted on this topic.
From the prospect of selectivity, the behavior of VRX-009 in normal cells and tissues is of particular interest. In this regard, VRX-009 is partially attenuated in primary human bronchial epithelial cells (which are the natural host for Ads), as indicated by the lack of E4ORF3 and ADP expression in the immunoblot experiment and the decreased number of E4ORF3- and fiber-positive cells in the immunofluorescence experiment. VRX-009 is similarly attenuated in HUVEC cells, reaching less than one-hundredth of the burst size of the parental virus VRX-007.
We used the established animal model for testing oncolytic human Ad vectors, i.e., nude mice bearing human tumor xenografts, to evaluate the in vivo antitumor efficacy of VRX-009 in SW480 colon cancer cells that are permissive for VRX-009 replication. For comparison, we also tested the vector in nonpermissive lung cancer xenografts as a crude model of human lung tissue. As a control, we used VRX-007, the parental virus for VRX-009, which should be equally effective in both colon and lung tumors. VRX-009 caused a significant reduction in the growth of SW480 colon tumors, but it was largely ineffective against A549 lung tumors. On the other hand, VRX-007 was very effective against both types of tumors, practically stopping the growth of A549 tumors. These data indicate that VRX-009 retained the strong antitumor efficacy of VRX-007 in its target tumor type. The results with A549 tumors raise the possibility that VRX-009 may spare cells in vivo that do not have an activated wnt signaling pathway. Our data provide hope that VRX-009, or a vector similar in design, will be useful in battling colon cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: William S. M. Wold, Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63126. Phone: (314) 977-8857; Fax: (314) 977-8717; E-mail: woldws{at}slu.edu
Received 12/11/03. Revised 2/19/04. Accepted 3/ 9/04.
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