Baogen Lu

Transcriptional targeting of tumors with a novel tumor-specific survivin promoter

It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. We employed a recombinant adenoviral vector (reAdGL3BSurvivin) in which a tumor-specific survivin promoter and a luciferase reporter gene were inserted into the E1-deleted region of adenovirus vector. Luciferase activity was measured in both multiple tumor cell lines and two primary melanoma cells infected with reAdGL3BSurvivin. Human fibroblast and mammary epithelial cell lines were used as negative controls. A reAdGL3CMV, containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity generated by the survivin promoter. Our data revealed that the survivin promoter showed high activity in both established tumor cell lines and the primary melanoma cells. In contrast, the in vivo studies indicated that the activities of survivin promoter were extremely low in the major mouse organs. The survivin promoter appears to be a promising tumor-specific promoter exhibiting a “tumor on” and “liver off” profile, and therefore, it may prove to be a good candidate for transcriptional targeting of cancer gene therapy in a wide variety of tumors.

Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter

Adenoviral vectors are considered to be good gene delivery vectors for cancer gene therapy due to their wide host tissue range and cell cycle-independent infectivity. However, the disadvantages include the lack of specificity for cancer cells and the high liver accumulation in vivo. The human CXCR4 gene is expressed at high levels in many types of cancers, but is repressed in the liver. We explored the CXCR4 promoter as a candidate to restrict adenoviral transgene expression to tumor cells with a low expression in host tissues. The luciferase activities in multiple cancer cell lines infected with recombinant adenovirus reAdGL3BCXCR4 or the control vector reAdGL3BCMV revealed that the CXCR4 promoter exhibited relatively high transcriptional activity in a breast cancer cell line, MDA-MB-361, and two ovarian cancer cell lines, OVCAR-3 and SKOV3. ip1, 65% (P=0.0087), 16.7% (P=0.1) and 20% (P=0.0079) compared to that of the CMV promoter, respectively, and low expression, 4.9 and 0.1%, respectively, in both normal cell lines HFBC and HMEC. In addition, CXCR4 had a low expression of luciferase (0.32%) compared to that of the CMV promoter in mouse liver in vivo. The data also revealed that the CXCR4 promoter was a stronger tumor-specific promoter (TSP) than the Cox-2M promoter in primary melanomas obtained from two patients. The CXCR4 promoter is shown to have a ‘tumor-on’ and ‘liver-off’ status in vitro and in vivo, and CXCR4 may prove to be a good candidate TSP for cancer gene therapy approaches for melanoma and breast cancers.

Evaluation of tumor-specific promoter activities in melanoma

Gene therapy is a novel therapy for melanoma. To date, however, there is still no powerful tumor specific promoter (TSP) to restrict the transgene expression in melanoma cells. In order to define a useful TSP for targeting in the context of melanoma gene therapy, four promoters, the cyclooxygenase-2 (Cox-2), α-chemokine SDF-1 receptor (CXCR4), epithelial glycoprotein 2 (EGP-2), and survivin, were tested in both established melanoma cell lines and primary melanoma cells. We employed recombinant adenoviral vectors (reAds) each with a candidate TSP (the Cox-2, CXCR4, EGP-2, or survivin), a reporter luciferase gene, and a poly-A signal, all of which were inserted into the E1-deleted region. A reAdGL3Bcytomegalovirus (CMV), containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity. Luciferase activity was measured in multiple tumor cell lines and two primary melanoma cell cultures after infection with reAds. Human epithelial melanocytes, HEM, were used as normal control. In contrast to three other promoters, the survivin promoter exhibited the highest activities within both melanoma cell lines and primary melanoma cells, but not in HEMs. Additionally, the survivin promoter exhibited very low activities in major mouse organs including the liver, in vivo. EGP-2 is not active in melanoma; messenger RNA expressions were correlated to promoter activities both in melanoma cell lines and primary cell cultures. Thus, these data suggest that the survivin promoter achieved a ‘tumor-on/liver-off’ profile, and thus represents a potentially useful tumor-specific promoter with applications for transcriptional targeting of Ad vector-based cancer gene therapy or oncolysis to melanoma.

Targeting Mesothelioma Using an Infectivity Enhanced Survivin-Conditionally Replicative Adenoviruses

Mesothelioma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard is the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; viral infectivity and tumor specificity have been poor. Herein we report on two CRAd agents, CRAd-S.RGD and CRAd-S.F5/3, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD or F5/3) in the adenovirus fiber region. These CRAd agents effectively target human mesothelioma cell lines, induce strong cytoxicity in these cells in vitro, and viral replication in a H226 murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting mesothelioma with low host toxicity. These agents should provide important insights into the identification of novel therapeutic strategies for mesothelioma.

Erratum: Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter (Gene Therapy (2004) vol. 11 (645-648) 10.1038/sj.gt.3302089)

Correction to: Gene Therapy (2004) 11, 645–648. doi:10.1038/sj.gt.3302089 The below author name was published incorrectly (shown above), the correct name is given below. M Yamamoto The authors would like to apologise for this mistake.

443. Incorporating the Survivin Promoter in an Infectivity Enhanced CRAd-Analysis of Oncolysis in Five Different Cancer Cell Lines

Top of pageAbstract Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculums. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect surrounding target cells, replicate and eradicate the infected tumor cells, leaving normal cells unaffected. Similar to adenoviral vectors, however, CRAd agents are limited in their use in cancer gene therapy for two main reasons: they have low infection efficacy due to paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR), and they lack specificity, thus infecting normal cells, which leads to the toxicity. In this study, we propose an improved CRAd agent to overcome these two obstacles. We constructed a novel CRAd agent, CRAd-Survivin, which overcomes the limitations described above and has the following characteristics: 1) The E1 gene of the adenovirus, which responses to adenoviral replication, is driven by a novel tumor specific promoter (tsp), the survivin promoter; 2) The vector maintains the E3 region containing the sequence coding the “death domain” to aid the cytocidal effect; 3) A modified RGD sequence (RGD4C) is incorporated into the HI loop of the fiber, which increases the infectivity in target cells; 4) We constructed the CRAd-Surivin by inserting a SV40 poly-A signal sequence between the LITR and tsp. This modification halts the transcription signal from LITR-Enh via the inserted SV40 poly-A signal. Five different cancer cell lines, including pancreatic cancer (PC-3), breast cancer (MDA-MB-361), melanoma (Mel-28), ovarian cancer (OV4), and glioma (D65), were infected with this improved CRAd agent. All cells were plated in a 24 well plate, and infected with 0, 0.1, 1.0, or 10 pfu/cell of CRAd-Survivin or a negative control, Ad-Survivin, which is a non-replicating agent. The cytocidal effect of the CRAd agent was detected by crystal violet stain, and the cell viability of tumor was measured by exclusion of Trypan Blue stain. The viabilities of D65 and MDA-MB-361 cells were 3.3% and 1.0%, respectively, 10 days post-infection at 0.1 pfu/cell of CRAd-Survivin. In contrast, the viabilities were 97% and 80% 10 days post-infection at 0.1 pfu/cell of Ad-Survivin, the non-replicative control. The viabilities of PC-3, Mel-28, and OV4 were 70.0%, 30%, and 95%, respectively, under the same experimental conditions. Further, strong cytocidal effects were observed in different glioma cell lines, including U118, U373, U87, and M59. These preliminary data indicate that an infectivity enhanced, tumor-specific CRAd agent, CRAd-Survivin, may be an excellent candidate for treatment of glioma and breast cancer by oncolysis.