E1B-55K-Deleted Adenovirus Expressing E1A-13S by AFP-Enhancer/Promoter Is Capable of Highly Specific Replication in AFP-Producing Hepatocellular Carcinoma and Eradication of Established Tumor
Authors: Minoru Takahashi, Tsutomu Sato, Tamotsu Sagawa, Yue Lu, Yasushi Sato, Satoshi Iyama, Yasuyuki Yamada, Junki Fukaura, Sho Takahashi, Koji Miyanishi, Toshiharu Yamashita, Katsunori Sasaki, Katsuhisa Kogawa, Hirofumi Hamada, Junji Kato, Yoshiro Niitsu
Journal: Molecular Therapy (2002)

Here, we constructed a recombinant replication-competent adenovirus (rRCAd; AdAFPep/Rep) that expresses both E1A-13S driven by the α-fetoprotein (AFP) enhancer/promoter (AFPep) lacking any silencers in the 5′-flanking region of the AFP gene, and 55K-deleted E1B driven by the cytomegalovirus (CMV) promoter. We then examined the feasibility of gene therapy utilizing this virus for AFP-producing hepatocellular carcinoma (HCC). AdAFPep/Rep lysed all the AFP-producing HCC cell lines (HuH7, HepG2, PLC/PRF/5 (P5)) examined at a multiplicity of infection (MOI) as low as 0.1 and did not lyse primary human hepatocytes (Hc) at a MOI as high as 100, indicating that the rRCAd virus can lyse AFP-producing HCC cells with a higher specificity and potency than previously reported. Furthermore, this virus was capable of complete eradication of a preestablished HuH7-cell tumor by a single intratumoral injection of 108 plaque-forming units (pfu) of AdAFPep/Rep. Thus, AdAFPep/Rep may be applicable for clinical use. Here, we constructed a recombinant replication-competent adenovirus (rRCAd; AdAFPep/Rep) that expresses both E1A-13S driven by the α-fetoprotein (AFP) enhancer/promoter (AFPep) lacking any silencers in the 5′-flanking region of the AFP gene, and 55K-deleted E1B driven by the cytomegalovirus (CMV) promoter. We then examined the feasibility of gene therapy utilizing this virus for AFP-producing hepatocellular carcinoma (HCC). AdAFPep/Rep lysed all the AFP-producing HCC cell lines (HuH7, HepG2, PLC/PRF/5 (P5)) examined at a multiplicity of infection (MOI) as low as 0.1 and did not lyse primary human hepatocytes (Hc) at a MOI as high as 100, indicating that the rRCAd virus can lyse AFP-producing HCC cells with a higher specificity and potency than previously reported. Furthermore, this virus was capable of complete eradication of a preestablished HuH7-cell tumor by a single intratumoral injection of 108 plaque-forming units (pfu) of AdAFPep/Rep. Thus, AdAFPep/Rep may be applicable for clinical use. IntroductionHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide [1Deuffic S. Poynard T. Buffat L. Valleron A.J. Trends in primary liver cancer.Lancet. 1998; 351: 214-215Abstract Full Text Full Text PDF PubMed Google Scholar, 2El-Serag H.B. Mason A.C. Rising incidence of hepatocellular carcinoma in the United States.N. Engl. J. Med. 1999; 340: 745-750Crossref PubMed Scopus (2708) Google Scholar]. Despite recent advances in cancer treatment modalities, the efficacy of treatments for advanced stages of HCC is far from satisfactory [3Levin B. Amos C. Therapy of unresectable hepatocellular carcinoma.N. Engl. J. Med. 1995; 332: 1294-1296Crossref PubMed Scopus (82) Google Scholar]. With the expectation of increasing therapeutic efficacy, gene therapy has been introduced as the new direction in treatment modality [4Friedmann T. Progress toward human gene therapy.Science. 1989; 244: 1275-1281Crossref PubMed Scopus (358) Google Scholar, 5Strauss M. Liver-directed gene therapy: prospects and problems.Gene Ther. 1994; 1: 156-164PubMed Google Scholar].Strategies for cancer gene therapy can be largely divided into two groups: direct evocation of tumoricidal effects and indirect induction of antitumor effects, such as immunogene therapy [6Parmiani G. Rodolfo M. Melani C. Immunological gene therapy with ex vivo gene-modified tumor cells: a critique and a reappraisal.Hum. Gene Ther. 2000; 11: 1269-1275Crossref PubMed Scopus (73) Google Scholar]. Recent reports have demonstrated the effectiveness of treatments based on the latter strategy in animal models [7Barajas M. Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12.Hepatology. 2001; 33: 52-61Crossref PubMed Scopus (152) Google Scholar, 8Schmitz V. Adenovirus-mediated CD40 ligand gene therapy in a rat model of orthotopic hepatocellular carcinoma.Hepatology. 2001; 34: 72-81Crossref PubMed Scopus (58) Google Scholar]; however, no satisfactory results have been shown in clinical trials of immunogene therapy for HCC. That may be attributed to the generally poor immunogenicity of human HCC [9Tatsumi T. B7-1 (CD80)-gene transfer combined with interleukin-12 administration elicits protective and therapeutic immunity against mouse hepatocellular carcinoma.Hepatology. 1999; 30: 422-429Crossref PubMed Scopus (25) Google Scholar]. The feasibility of the former strategy has been examined in animal models by employing an adenoviral vector (AdV) that carries a cytocidal gene such as suicide gene [10Kanai F. Gene therapy for α-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.Hepatology. 1996; 23: 1359-1368Crossref PubMed Google Scholar, 11Krohne T.U. Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro.Hepatology. 2001; 34: 511-518Crossref PubMed Scopus (54) Google Scholar] or p53 gene [12Anklesaria P. Gene therapy: a molecular approach to cancer treatment.Curr. Opin. Mol. Ther. 2000; 2: 426-432PubMed Google Scholar]. However, it has never proved to be more than palliative, having low therapeutic efficacy due to the inability of the vectors either to transduce cytocidal genes specifically into tumors that often display multicentric distribution in the liver, or to achieve sufficiently high levels of gene expression in them.To overcome the low tumor specificity, we constructed viral vectors with transgenes driven either by the 5'-flanking sequence (5.5–7.6 kb) of the AFP gene containing silencers, enhancers, and promoter [13Huber B.E. Richards C.A. Regulated expression of artificial chimeric genes contained in retroviral vectors: implications for virus-directed enzyme prodrug therapy (VDEPT) and other gene therapy applications.J. Drug Target. 1996; 3: 349-356Crossref PubMed Scopus (8) Google Scholar, 14Ido A. Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human (-fetoprotein gene promoter.Cancer Res. 1995; 55: 3105-3109PubMed Google Scholar] or by the AFP promoter region (0.3 kb) alone [14Ido A. Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human (-fetoprotein gene promoter.Cancer Res. 1995; 55: 3105-3109PubMed Google Scholar, 15Ishikawa H. Utilization of variant-type of human (-fetoprotein promoter in gene therapy targeting for hepatocellular carcinoma.Gene Ther. 1999; 6: 465-470Crossref PubMed Scopus (42) Google Scholar]. We [16Igarashi T. Suzuki S. Takahashi M. Tamaoki T. Shimada T. A novel strategy of cell targeting based on tissue-specific expression of the ecotropic retrovirus receptor gene.Hum. Gene Ther. 1998; 9: 2691-2698Crossref PubMed Scopus (18) Google Scholar] and others [10Kanai F. Gene therapy for α-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.Hepatology. 1996; 23: 1359-1368Crossref PubMed Google Scholar, 17Mawatari F. Retrovirus-mediated gene therapy for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by human α-fetoprotein enhancer directly linked to its promoter.Cancer Gene Ther. 1998; 5: 301-306PubMed Google Scholar] made vectors that have a silencer-free AFPep and showed not only high tumor specificity, but also substantial activity even in a low AFP-producing tumor.Nevertheless, these tumor-specific promoters are generally not as powerful as nonspecific viral promoters currently used for clinical gene therapy; thus, when used for in vivo suicide gene therapy, they failed to induce complete regression of tumors even in animal models [18Kanai F. In vivo gene therapy for α-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene.Cancer Res. 1997; 57: 461-465PubMed Google Scholar, 19Hirano T. HVJ-liposome-mediated transfection of HSVtk gene driven by AFP promoter inhibits hepatic tumor growth of hepatocellular carcinoma in SCID mice.Gene Ther. 2001; 8: 80-83Crossref PubMed Scopus (25) Google Scholar].Therefore, to substantiate antitumor activity, Hallenbeck et al. [20Hallenbeck P.L. A novel tumor-specific replication-restricted adenoviral vector for gene therapy of hepatocellular carcinoma.Hum. Gene Ther. 1999; 10: 1721-1733Crossref PubMed Scopus (259) Google Scholar] recently generated a rRCAd (named AvE1a04i), which was constructed to encode the E1A gene, one of the genes essential for viral replication, under the control of an artificial AFP promoter. The modified Ad was intended to replicate in and lyse only AFP-producing HCC cell lines. However, as hepatocytes in hepatitis are known often to express AFP (though not at levels as high as hepatocytes in hepatoma), to prevent viral replication in AFP-expressing hepatocytes, these authors raised the tumor selectivity of rRCAd by intentionally inserting six copies of distal silencers in an artificial AFP promoter. Contrary to expectations, unfortunately, not all the AFP-producing cells underwent lysis with this virus; this might be due to the effects of excessive silencers, as silencers function not only in adult hepatocytes, but also in low AFP-producing HCC, as Hallenbeck et al. themselves have shown [21Nakabayashi H. A position-dependent silencer plays a major role in repressing α-fetoprotein expression in human hepatoma.Mol. Cell Biol. 1991; 11: 5885-5893Crossref PubMed Google Scholar].An alternative approach using the selectively replicable Ad mutant ONYX-015 to target tumor cells, originally constructed as dl1520 [22Barker D.D. Berk A.J. Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection.Virology. 1987; 156: 107-121Crossref PubMed Scopus (320) Google Scholar], was reported by Bischoff et al. [23Bischoff J.R. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.Science. 1996; 274: 373-376Crossref PubMed Scopus (1558) Google Scholar]. ONYX-015 is an Ad hybrid of serotypes 2 and 5 with a genome deletion in the E1B region, resulting in loss of expression of the viral 55-kDa protein (E1B-55K), and has been shown to bind wild-type p53 and to block p53-mediated transcriptional activation. Thus, the virus would specifically replicate in the tumor cells expressing mutated p53 [23Bischoff J.R. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.Science. 1996; 274: 373-376Crossref PubMed Scopus (1558) Google Scholar, 24Heise C. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents.Nat. Med. 1997; 3: 639-645Crossref PubMed Scopus (827) Google Scholar] and lyse them. However, it was later found that the mutated virus replicates not only in p53-mutated tumor cells, but also in some primary culture cells [25Rothmann T. Hengstermann A. Whitaker N.J. Scheffner M. zur Hausen H. Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells.J. Virol. 1998; 72: 9470-9478PubMed Google Scholar, 26Harada J.N. Berk A.J. p53-Independent and -dependent requirements for E1B-55K in adenovirus type 5 replication.J. Virol. 1999; 73: 5333-5344PubMed Google Scholar], although its lytic effects on the normal cells were found to be weaker than on the wild-type Ad [25Rothmann T. Hengstermann A. Whitaker N.J. Scheffner M. zur Hausen H. Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells.J. Virol. 1998; 72: 9470-9478PubMed Google Scholar]. In two other studies, a replicable Ad with mutated E1A was used [27Howe J.A. Evaluation of E1-mutant adenoviruses as conditionally replicating agents for cancer therapy.Mol. Ther. 2000; 2: 485-495Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. The Ad can effectively replicate in the quiescent cells because E1A induces S-phase entry by binding to and inactivating the retinoblastoma protein (pRb). The mutated E1A, however, is defective for binding to the pRb and therefore cannot induce infected, nondividing normal cells into S phase, but the Ad with mutated E1A would replicate in dividing tumor cells at S phase in which pRb is intrinsically inactivated. However, it is possible that this type of virus may replicate in and lyse dividing normal cells, such as intestinal epithelial cells, in which pRb is also intrinsically inactivated.In this investigation, we constructed a rRCAd with the following features. First, we used the previously reported AFPep, which is specifically regulated by AFP-transcriptional factor, to assure that the virus replicates only in AFP-producing cells and not in proliferative normal cells not producing AFP. Second, in this AFP promoter, we directly fused the enhancer and promoter regions and deleted silencers to sustain a high promoter activity, even in HCC producing low amounts of AFP. Third, because we surmised that the tumor-specific promoter activity would be less powerful if driving the whole E1A genome encoding 9S, 12S, and 13S, rather than minimal essential gene for replication, we used E1A-13S, which plays the most important role in human Ad5 DNA replication by trans-activating viral gene expression [28Schneider J.F. Fisher F. Goding C.R. Jones N.C. Mutational analysis of the adenovirus E1a gene: the role of transcriptional regulation in transformation.EMBO J. 1987; 6: 2053-2060PubMed Google Scholar, 29Turnell A.S. Grand R.J. Gallimore P.H. The replicative capacities of large E1B-null group A and group C adenoviruses are independent of host cell p53 status.J. Virol. 1999; 73: 2074-2083Crossref PubMed Google Scholar], as cDNA to be effectively expressed by AFPep. Fourth, we deleted E1B-55K from the E1B gene to ensure that the virus would not replicate in AFP-producing hepatocytes in hepatitis. By applying this rRCAd with doubleassured safety features, we examined both its in vitro cytocidal effects on AFP-producing HCC cells and on primary human hepatocytes, and its in vivo antitumor effect on inoculated AFP-producing HCC tumor in nude mice.ResultsHuman HCC Cells but Neither Colon Cancer Cells nor Primary Hepatocytes Secrete AFPAll three HCC cell lines used in this experiment were confirmed to secrete AFP with concentrations ranging between 734 and 129,753 ng/24 hours/106 cells. Both the human colon cancer cell line M7609, which produces both carcinoembryonic antigen (CEA), and primary human hepatocytes (Hc) secreted undetectable levels of AFP (Table 1).TABLE 1AFP secretion in cell cultureCellAFP secretion (ng/day/106 cells)aThese results are representative of four independent experiments.Primary human hepatocytes (Hc)N.D.M7609N.D.HepG26332 ± 588.4HuH7129753 ± 10047.2PLC/PRF/5734 ± 56.9The value represents the mean ± SD of five counts.N.D., not detectable.a These results are representative of four independent experiments. Open table in a new tab AdAFPep/Rep Is Capable of Specific Replication in AFP-producing HCC CellsExpression of Ad5 E1A was examined by immunotransblot in cells infected by AdAFPep/Rep (Fig. 1). Immunoreactive 43-kDa bands, corresponding to human Ad5 E1A, were observed in HepG2, HuH7, and P5, but not in Hc or in M7609 (Fig. 2).FIG. 2Selective replication capabilities of AdAFPep/Rep in AFP-producing HCC cell lines. All cells were infected at a MOI of 100 pfu/cell with AdAFPep/Rep. Cells were lysed at 7 days postinfection, and virus yields were quantified using 293 cells. The bars represent the mean of three independent experiments, and the upper range of the standard error is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cells infected with AdAFPep/Rep at a MOI of 100 per cell were tested for their viral production at day 7 by cytopathic effect (CPE) assay [16Igarashi T. Suzuki S. Takahashi M. Tamaoki T. Shimada T. A novel strategy of cell targeting based on tissue-specific expression of the ecotropic retrovirus receptor gene.Hum. Gene Ther. 1998; 9: 2691-2698Crossref PubMed Scopus (18) Google Scholar]. The viral yields of HepG2, HuH7, and P5 infected with AdAFPep/Rep were 6.7, 8.0, and 1.0 × 107 pfu/ml, respectively, but those of M7609 and Hc were below 1.0 × 102 pfu/ml.At day 7, we examined selective cytotoxicity of AdAFPep/Rep against AFP-producing HCC cell lines by the dye-uptake method, and showed that all the AFP-producing human HCC cell lines had undergone complete lysis at a MOI of 100, while Hc proved totally insensitive to this virus. On the other hand, AdCMVp/Rep lysed both HCC cells and Hc (Fig. 3).FIG. 3Cytotoxic effect of recombinant replication-competent adenovirus. (A) Selective cytotoxic effect of AdAFPep/Rep and (B) nonspecific cytotoxic effect of AdCMVp/Rep against Hc, non-AFP-producing tumor, or AFP-producing HCC cell lines. All cells were infected at a MOI of 100 pfu/cell with either AdAFPep/Rep or AdCMVp/Rep. Cytotoxicity was measured by dye-uptake assay at 7 days postinfection. The points on the ordinate correspond to the percentage of surviving cells compared with AdAFPep/nlacZ-infected cells as 100%. The values are represented as mean ± SD. Results represent three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Cytotoxic Effect of AdAFPep/Rep Is Retained with Lower MOI but Requires a Longer Cultivation PeriodTo investigate time- and dose-dependent cytotoxic effects, P5 cells were cultivated in the presence of AdAFPep/Rep at various MOIs (Fig. 4). Complete lysis was eventually observed in all of the cultures, irrespective of the MOI level (even as low as 0.1); however, the lower the MOI, the longer the required cultivation period.FIG. 4Emergence of cytotoxic effect against P5 infected at a lower MOI of AdAFPep/Rep after long-term observation. P5 cells cultured in 24-well plates were infected with AdAFPep/Rep at various MOIs. During a time course after infection, half of the medium was replaced with fresh culture medium every 3 days. Cytotoxicity was measured by dye-uptake assay. The data shown represent a single experiment in triplicate. The top two graphs represent data from P5 infected at a MOI of either 100 (closed circle) or 10 (triangle); the bottom two graphs represent data from P5 infected at a MOI of either 1 (square) or 0.1 (diamond). The points on the ordinate correspond to the percentage of surviving cells compared with AdAFPep/nlacZ-infected cells as 100%. P5, PLC/PRF/5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This indicates that AdAFPep/Rep multiplies via a self-replicating process in initially infected cells and spreads over noninfected cells, which eventually undergo lysis.Coculturing with AdAFPep/Rep-infected P5 Cells Brings About Lysis of Noninfected P5 CellsTo confirm the ability of the Ad released from infected cells to permeate neighboring noninfected cells, P5 cells that had been treated with the virus for 1 hour were mixed with noninfected cells at various ratios of infected/noninfected cells, and were then plated. Complete cell lysis was found at all tested ratios, although the higher the ratio of infected cells, the faster the complete lysis occurred (Fig. 5).FIG. 5Lysis of cocultured noninfected P5 cells by AdAFPep/Rep released from infected P5 cells. P5 cells were infected with AdAFPep/Rep or AdAFPep/nlacZ at a MOI of 100 and then mixed with uninfected cells in various proportions in 24-well plates. During a time course after infection, half of the medium was replaced with fresh culture medium every 3 days. Cytotoxicity was measured by dye-uptake assay. The data shown represent in a single experiment in triplicate. The values are represented as mean ± SD. Results represent three independent experiments. P5, PLC/PRF/5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Primary Human Hepatocytes Are Not Lysed by Coculture with AdAFPep/Rep-Infected HCC Cell LinesTo examine the effect of AdAFPep/Rep released from infected cells on neighboring normal hepatocytes, Hc were cultured with HepG2, HuH7 and P5 that had been preincubated for 1 hour with AdAFPep/Rep. The percentage of surviving cells in each coculture was exactly the same as that of Hc cells, indicating that Hc cells were resistant to AdAFPep/Rep in the microenvironment of AFP-producing HCC cell lines (Fig. 6).FIG. 6Survival of Hc in a microenvironment cocultured with AFP-producing HCC cell lines infected by AdAFPep/Rep, HepG2 (A), HuH7 (B), and P5 (C). AFP-producing HCC cell lines were cocultured with 0, 20, 40, 60, 80, or 100% of Hc, then the cocultured cells were infected with AdAFPep/Rep at a MOI of 100. Cytotoxicity was measured by dye-uptake assay at 7 days post infection. The points on the ordinate correspond to the percentage of surviving cells compared with primary human hepatocytes as 100%. The horizontal axis indicates the percentage of primary human hepatocytes in the mixed cells. The values are represented as mean ± SD. Results represent three independent experiments. P5, PLC/PRF/5; Hc, primary human hepatocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ONYX-015 Lyses Not Only HCC Cells but Also Primary Human HepatocytesWhen Hc cells were treated with ONYX-015 at a MOI of 100, almost 100% cell lysis was observed (Fig. 7). This finding is compatible with the previously reported observation that ONYX015 causes cytolysis of normal diploid fibroblasts or mammary epithelial cells [25Rothmann T. Hengstermann A. Whitaker N.J. Scheffner M. zur Hausen H. Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells.J. Virol. 1998; 72: 9470-9478PubMed Google Scholar].FIG. 7Comparison between AdAFPep/Rep and ONYX-015 on the cytotoxicity for Hc (A) and AFP-producing HCC cell lines, HepG2 (B), P5 (C), and HuH7 (D). Cells were infected at various MOIs with AdAFPep/Rep or ONYX-015. Cytotoxicity was measured by dye-uptake assay at 7 days postinfection. The bars represent the mean of three independent experiments, and the upper range of the standard error is shown. Hc, primary hepatocytes; P5, PLC/PRF/5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Single Injection of AdAFPep/Rep into P5 Tumor Results in Complete RegressionP5 cells (1 × 107) were inoculated s.c. into the backs of nude mice, and established tumors were injected with 100 µl of PBS, or 1 × 108 pfu/100 µl of either AdAFPep/nlacZ or AdAFPep/Rep. The tumor receiving AdAFPep/Rep on day 0 had undergone complete regression by day 21, while those receiving PBS or AdAFPep/nlacZ continued to grow exponentially. When tumors received three injections of AdAFPep/Rep, one on each of days 0, 3, and 6, complete regression occurred at day 10 (Fig. 8).FIG. 8Antitumor effects of AdAFPep/Rep against P5 established in athymic nude mice. P5 cells were inoculated into nude mice, and PBS (0.1 ml), AdAFPep/nlacZ (1 × 103 pfu/0.1 ml), or AdAFPep/Rep (1 × 108 pfu/0.1 ml) was administered into established tumors. Each experimental group contained six mice. One treatment group received intratumoral administration of AdAFPep/Rep (1 × 108 pfu/0.1 ml) on days 0, 3, and 6, and another group received AdAFPep/Rep on day 0, once. PBS or AdAFPep/nlacZ was injected on days 0, 3, and 6 for the nontreatment group. Tumor diameters were measured with calipers, and tumor volumes were calculated. P5, PLC/PRF/5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionIn the present study, we demonstrated that our virus, AdAFPep/Rep, was capable of lysing all the AFP-producing HCC cells examined, including those that a previously reported rRCAd, AvE1a04i, could not lyse [20Hallenbeck P.L. A novel tumor-specific replication-restricted adenoviral vector for gene therapy of hepatocellular carcinoma.Hum. Gene Ther. 1999; 10: 1721-1733Crossref PubMed Scopus (259) Google Scholar]. The lytic activity of our virus was only slightly lower than that of ONYX-015 in most cells and higher in some (Fig. 6). Furthermore, it was shown that a single intratumoral injection of 108 pfu AdAFPep/Rep was capable of complete abrogation of preestablished AFP-producing tumors inoculated into mice. Such complete eradication of tumors was not achieved by AvE1ai [17Mawatari F. Retrovirus-mediated gene therapy for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by human α-fetoprotein enhancer directly linked to its promoter.Cancer Gene Ther. 1998; 5: 301-306PubMed Google Scholar]. These comparatively strong antitumor effects may be ascribed to the following strategies. First, we deleted the silencers from the 5'-flanking region of the AFP gene to use it as a promoter to drive E1A-13S; second, considering that the AFP promoter is much less potent than the authentic viral promoter, we utilized E1A-13S cDNA, a minimal essential component of E1A, instead of the whole Ad E1A genome.As with ONYX-015, which does not by itself bring about complete regression of tumors when applied clinically, our virus may also need some combination modalities such as chemotherapy [30Khuri F.R. A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer.Nat. Med. 2000; 6: 879-885Crossref PubMed Scopus (993) Google Scholar] in future clinical trials. In this context, our virus may be preferable to ONYX-015 in terms of adverse effects. When ONYX-015 infects normal cells, E1A, which augments chemosensitivity or radiosensitivity by stabilizing p53 or downregulating Her-2/neu, would be fully expressed under the control of powerful adenoviral transcriptional elements [31Ueno N.T. Yu D. Hung M.C. Chemosensitization of HER-2/neu-overexpressing human breast cancer cells to paclitaxel (Taxol) by adenovirus type 5 E1A.Oncogene. 1997; 15: 953-960Crossref PubMed Scopus (94) Google Scholar, 32Kirn D. Replication-selective oncolytic adenoviruses: virotherapy aimed at genetic targets in cancer.Oncogene. 2000; 19: 6660-6669Crossref PubMed Scopus (124) Google Scholar], thereby allowing infected normal cells to be readily damaged. With our virus, on the other hand, infected normal cells would be much less sensitive to chemotherapy or radiotherapy, because the expression of E1A is suppressed under control of the AFP promoter.One of the most important issues of using a replication-competent virus is without doubt the specificity. Our virus, having the tumor-specific expression of E1A-13S by AFPep (Fig. 1), together with E1B-55K deletion, was designed to have double assurance of specificity, and it was indeed found to be highly specific for AFP-producing tumors, exerting no cytotoxicity to Hc, which express no detectable AFP (FIG. 3, FIG. 7). The tumor specificity was also confirmed by the experiment that showed no bystander effects [33Mesnil M. Yamasaki H. Bystander effect in herpes simplex virus—thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication.Cancer Res. 2000; 60: 3989-3999PubMed Google Scholar] of AdAFPep/Rep in a mixed culture of infected tumor cells and noninfected normal hepatocytes (Fig. 6). Incidentally, the fact that the ONYX-015 proved cytotoxic to normal diploid cells (Hc) indicates that the type of cell in which the virus can replicate is not restricted to the cells that have deficient or mutated p53, as had been initially speculated. Although application of rRCAd in practical use for HCC may be through the hepatic artery [34Habib N.A. Hodgson H.J. Lemoine N. Pignatelli M. A phase I/II study of hepatic artery infusion with wtp53-CMV-Ad in metastatic malignant liver tumours.Hum. Gene Ther. 1999; 10: 2019-2034Crossref PubMed Scopus (64) Google Scholar, 35Gerolami R. Evaluation of HSV-tk gene therapy in a rat model of chemically induced hepatocellular carcinoma by intratumoral and intrahepatic artery routes.Cancer Res. 2000; 60: 993-1001PubMed Google Scholar] to avoid the dissemination of the virus throughout the whole body, there is still a risk of replication in distant, dividing normal diploid cells such as gastrointestinal epithelial cells. AdAFPep/Rep, in this regard, is endowed with assured safety not to replicate in these dividing normal diploid cells because its replication is under the control of AFPep.Although the AFP promoter is indeed specific and potent, AFP-producing hepatocytes are quite often present in inflamed liver associated with hepatitis; therefore, if rRCAd equipp