[Show abstract][Hide abstract] ABSTRACT: Genetically engineered tumor-selective vaccinia virus (VV) has been demonstrated to be a highly effective oncolytic agent, but immune clearance may limit its therapeutic potential. As previously demonstrated, immunosuppression can lead to significant enhancement of viral recovery and therapeutic effect, but the magnitude of complement-mediated viral inactivation has not been fully elucidated and warrants further investigation. Using fluorescent microscopy and quantitative plaque assays, we have determined complement's key role in viral clearance and its multi-faceted means to pathogen destruction. Complement can lead to direct viral destruction and inhibition of viral uptake into cells, even in the absence of anti-vaccinia antibodies. Our data demonstrate C5 to be integral to the clearance pathway, and its inhibition by Staphylococcal superantigen-like protein leads to a 90-fold and 150-fold enhancement of VV infectivity in both the presence and absence of anti-VV antibodies, respectively. This study suggests that complement inhibition may reduce vaccinia viral neutralization and may be critical to future in vivo work.Cancer Gene Therapy advance online publication, 10 May 2013; doi:10.1038/cgt.2013.26.
[Show abstract][Hide abstract] ABSTRACT: Oncolytic poxviruses have demonstrated initial promising results in patients with cancer in clinical trials, yet further improvements are needed. It has been shown that a single point mutation in the A34R gene resulted in the production of more total progeny virus and more extracellular enveloped virus (EEV), a form that can be immune-evasive and with enhanced spread. We have genetically engineered a new oncolytic poxvirus (designated vA34R) by incorporating this mutated A34R gene into a viral backbone (vvDD) which was designed for tumor-selective replication. This rationally designed virus can evade neutralization from antipoxvirus antibodies and is highly cytotoxic to cancer cells. It demonstrates improved spread and increased replication within the peritoneal cavity resulting in improved antitumor effects in a peritoneal carcinomatosis (PC) model of MC38 colon cancer. Impressively, after carrier cell-mediated delivery in the preimmunized host, vA34R displayed high replication in tumor nodules yet low accumulation in normal tissues thus enhancing the therapeutic index leading to 70% long-term cures. These results demonstrate that vA34R gains an enhanced therapeutic index for PC via immune evasion, increased spread, and production of more progeny virus. Thus, vA34R may be a potent oncolytic virus (OV) for patients with PC, even after prior exposure to vaccinia virus (VV).Molecular Therapy (2013); doi:10.1038/mt.2013.27.
[Show abstract][Hide abstract] ABSTRACT: Promising phase II clinical results have been reported recently for several oncolytic viral therapeutics, including strains based on vaccinia virus. One reason for this has been an increased appreciation of the critical therapeutic importance of the immune response raised by these viruses. However, the most commonly used approaches to enhance these immunotherapeutic effects in oncolytic viruses, typically though expression of cytokine transgenes, often also result in a reduction in oncolytic activity and premature clearance of the virotherapy from the tumor. Approaches that enhance the immunotherapeutic effects while maintaining oncolytic activity would therefore be beneficial. Here, it is demonstrated that the expression of the chemokine CCL19 (ELC) from an oncolytic vaccinia virus (vvCCL19) results in increased antitumor effects in syngeneic mouse tumor models. This corresponded with increased t cell and dendritic cell infiltration into the tumor. However, vvCCL19 persisted in the tumor at equivalent levels to a control virus without CCL19, demonstrating that oncolytic activity was not curtailed. Instead, vvCCL19 was cleared rapidly and selectively from normal tissues and organs, indicating a potentially increased safety profile. The therapeutic activity of vvCCL19 could be further significantly increased through combination with adoptive transfer of therapeutic immune cells expressing CCR7, the receptor for CCL19. This approach therefore represents a means to increase the safety and therapeutic benefit of oncolytic viruses, used alone or in combination with immune cell therapies.
Neoplasia (New York, N.Y.) 12/2012; 14(12):1115-21. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intraperitoneal accumulation of mucinous ascites in pseudomyxoma peritonei (PMP) promotes an inflammatory/fibrotic reaction that progresses to bowel obstruction and eventual patient demise. Cytokines and inflammation-associated transcription factor binding sites, such as glucocorticoid response elements and COX-2, regulate secretory mucin, specifically MUC2, production. We hypothesized that anti-inflammatory drugs targeting inflammation-associated pathways may reduce mucin production and subsequent disease morbidity in PMP.
The effects of dexamethasone and Celebrex were assessed in mucin-secreting human colon cancer LS174T cells in vitro and murine xenograft models of LS174T and human appendiceal PMP in vivo by serial parametric measurements, MUC2 transcripts via real-time RT-PCR, and MUC2 protein expression via immunofluorescence assays.
Dexamethasone significantly inhibited basal MUC2 mRNA levels in LS174T cells, inhibited mucinous tumor accumulation in an intraperitoneal PMP xenograft model, and prolonged survival in a subcutaneous LS174T xenograft model. Celebrex significantly inhibited sodium butyrate-stimulated MUC2 mRNA levels in LS174T cells and demonstrated a statistically nonsignificant trend toward reduced mucinous tumor growth and prolonged survival in the xenograft models. MUC2 protein analysis by immunofluorescence demonstrated a dual effect of dexamethasone on mucin production and tumor cell count.
Inflammatory mediators are known to regulate mucin production and may promote overexpression of MUC2 by neoplastic cells with goblet cell phenotype in PMP. Anti-inflammatory drugs, dexamethasone and Celebrex, could inhibit extracellular mucin production in PMP by targeting inflammatory cascades and, therefore, may decrease compressive symptoms, increase the disease-free interval, and reduce the extent or frequency of morbid cytoreductive surgeries.
Annals of Surgical Oncology 02/2012; 19(5):1402-9. · 4.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor superfamily members, including Fas ligand and TRAIL, have been studied extensively for cancer therapy, including as components of gene therapy. We examined the use of FasL expression to achieve tumor-selective replication of an oncolytic poxvirus (vFasL), and explored its biology and therapeutic efficacy for FasR- and FasR+ cancers. Infection of FasR+ normal and MC38 cancer cells by vFasL led to abortive viral replication owing to acute apoptosis and subsequently showed both reduced pathogenicity in non-tumor-bearing mice and reduced efficacy in FasR+ tumor-bearing mice. Infection of FasR- B16 cancer cells by vFasL led to efficient viral replication, followed by late induction of FasR and subsequent apoptosis. Treatment with vFasL as compared with its parental virus (vJS6) led to increased tumor regression and prolonged survival of mice with FasR- cancer (B16) but not with FasR+ cancer (MC38). The delayed induction of FasR by viral infection in FasR- cells provides for potential increased efficacy beyond the limit of the direct oncolytic effect. FasR induction provides one mechanism for tumor-selective replication of oncolytic poxviruses in FasR- cancers with enhanced safety. The overall result is both a safer and more effective oncolytic virus for FasR- cancer.
Cancer gene therapy 11/2011; 19(3):192-201. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Homeobox genes murine Rhox5 and human RHOXF1 are expressed in early embryonic stages and then mostly restricted to germline tissues in normal adult, yet they are aberrantly expressed in cancer cells in vitro and in vivo . Here we study the epigenetic regulation and potential functions of Rhox5 gene.
In Rhox5-silenced or extremely low expresser cells, we observed low levels of active histone epigenetic marks (H3ac, H4ac and H3K4me2) and high levels of repressive mark H3K9me2 along with DNA hypermethylation in the promoter. In Rhox5 low expresser cells, we typically observed modest levels of both active and repressive histone marks along with moderate DNA methylation. In Rhox5 highly expressed CT26 cancer cells, we observed DNA hypomethylation along with high levels of both active and repressive histone marks. Epigenetic drugs (retinoic acid and MS-275) induced F9 cell differentiation with enhanced Rhox5 expression and dynamic changes of epigenetic marks. Finally, Rhox5 knockdown by small hairpin RNA (shRNA) in CT26 colon cancer decreased cell proliferation and migration in vitro and tumor growth in vivo .
Both DNA methylation and histone methylation/acetylation play key roles in modulating Rhox5 expression in various cell types. The stem cell-like "bivalent domain", an epigenetic feature originally identified in key differentiation genes within stem cells, exists in the Rhox5 gene promoter in not only embryonic stem cells but also cancer cells, cancer stem cells, and differentiated Sertoli cells. As Ras signaling-dependent Rhox5 expression promotes tumor growth, Rhox5 may be an ideal target for therapeutic intervention in cancer.
Molecular Cancer 05/2011; 10:63. · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor vaccines can induce robust immune responses targeting tumor antigens in the clinic, but antitumor effects have been disappointing. One reason for this is ineffective tumor infiltration of the cytotoxic T lymphocytes (CTLs) produced. Oncolytic viruses are capable of selectively replicating within tumor tissue and can induce a strong immune response. We therefore sought to determine whether these therapies could be rationally combined such that modulation of the tumor microenvironment by the viral therapy could help direct beneficial CTLs induced by the vaccine. As such, we examined the effects of expressing chemokines from oncolytic vaccinia virus, including CCL5 (RANTES), whose receptors are expressed on CTLs induced by different vaccines, including type-1-polarized dendritic cells (DC1). vvCCL5, an oncolytic vaccinia virus expressing CCL5, induced chemotaxis of lymphocyte populations in vitro and in vivo, and displayed improved safety in vivo. Interestingly, enhanced therapeutic benefits with vvCCL5 in vivo correlated with increased persistence of the viral agent exclusively within the tumor. When tumor-bearing mice were both vaccinated with DC1 and treated with vvCCL5 a further significant enhancement in tumor response was achieved which correlated with increased levels of tumor infiltrating lymphocytes. This approach therefore represents a novel means of combining biological therapies for cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: Pre-existing antipoxvirus immunity in cancer patients presents a severe barrier to poxvirus-mediated oncolytic virotherapy. We have explored strategies of immunosuppression (IS) and/or immune evasion for efficient delivery of an oncolytic double-deleted vaccinia virus (vvDD) to tumors in the pre-immunized mice. Transient IS using immunosuppressive drugs, including tacrolimus, mycophenolate mofetil and methylprednisolone sodium succinate, have been used successfully in organ transplantation. This drug cocktail alone did not enhance viral recovery from subcutaneous tumor after systemic viral delivery. Using B-cell knockout mice, we confirmed that the neutralizing antibodies had a significant role in preventing poxvirus infection. Using a MC38 peritoneal carcinomatosis model, we found that the combination of IS and tumor cells as carriers led to the most effective viral delivery, viral replication and viral spread inside the tumor mass. We found that our immunosuppressive drug cocktail facilitated recruitment of tumor-associated macrophages and conversion into an immunosuppressive M2 phenotype (interleukin (IL)-10(hi)/IL-12(low)) in the tumor microenvironment. A combination of IS and carrier cells led to significantly prolonged survival in the tumor model. These results showed the feasibility of treating pre-vaccinated patients with peritoneal carcinomatosis using an oncolytic poxvirus and a combined immune intervention strategy.
[Show abstract][Hide abstract] ABSTRACT: Vaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy.
[Show abstract][Hide abstract] ABSTRACT: We describe the clinical, pathologic and molecular characteristics of a xenograft model of metastatic mucinous appendiceal adenocarcinoma. Tumours from patients with mucinous appendiceal neoplasms were implanted in nude mice and observed for evidence of intraperitoneal tumour growth. Morphologic and immunohistochemical features, temporal growth characteristics relative to controls, and loss of heterozygosity (LOH) at multiple chromosomal alleles were assessed in a successfully engrafted tumour. Two of seventeen implanted tumours successfully engrafted and only one mucinous adenocarcinoma propagated throughout the course of the study. The successful xenograft is morphologically similar to the original tumour, produces abundant extracellular mucin and exhibits non-invasive growth on peritoneal surfaces. The temporal growth characteristics of the xenograft tumour relative to controls reveal that tumour burden can be followed indirectly by measuring the weight or abdominal girth of engrafted animals. The cytokeratin, mucin core protein, CDX2, Ki-67 and p53 expression patterns are identical in the xenograft and resected tumour and are consistent with the expected pattern of protein expression for mucinous adenocarcinoma of the appendix. LOH was found in 1 of 10 informative chromosomal loci (chromosome 10p23) in xenograft tumour cells. Although we were unable to engraft a low-grade appendiceal mucinous neoplasm, the engrafted adenocarcinoma will be useful for future evaluation of novel therapeutic strategies directed at mucinous appendiceal adenocarcinoma and evaluation of strategies for treating widespread, bulky, mucinous peritoneal surface neoplasms. Xenograft tumour enrichment can facilitate molecular studies of appendiceal epithelial neoplasia.
International Journal of Experimental Pathology 08/2010; 91(4):357-67. · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have explored a unique combination therapy for metastatic colorectal cancer. This strategy combines a potent and new oncolytic poxvirus expressing a membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or TNFSF10) and oxaliplatin (Ox) chemotherapy. We hypothesized that TRAIL expression would increase the efficacy of the oncolytic poxvirus, and that the therapeutic efficacy would be further enhanced by combination with chemotherapy. The cytotoxicity to cancer cells by Ox, oncolytic vaccinia virus (VV) and trail gene-armed VV alone or in combination was tested in vitro. The trail gene armed oncolytic VV-expressed high levels of TRAIL in infected cancer cells and had greater potency as a cytotoxic agent compared with the parent VV. Ox alone exerted concentration-dependent cytotoxicity. In vitro, the combination of the two agents applied at suboptimal concentrations for individual therapy displayed synergy in inducing cancer cells into enhanced levels of apoptosis/necrosis. Western blot analyses were consistent with the notion that TRAIL induced cancer cell death mainly through apoptosis, whereas Ox and vJS6 induced cell death more through non-apoptotic death pathways. In two aggressive colorectal carcinomatosis models derived from human HCT116 and murine MC38 cells, the combination therapy displayed synergistic or additive antitumor activity and prolonged the survival of the tumor-bearing mice compared with either Ox chemotherapy or vvTRAIL-mediated oncolytic gene therapy alone. This combination strategy may provide a new avenue to treating peritoneal carcinomatosis and other types of metastases of colorectal cancer.
[Show abstract][Hide abstract] ABSTRACT: Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2'-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.
[Show abstract][Hide abstract] ABSTRACT: In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer.
Cancer gene therapy 03/2008; 15(2):115-25. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To enhance further the safety and efficacy of oncolytic vaccinia virus, we have developed a new virus with targeted deletions of three viral genes encoding thymidine kinase and antiapoptotic/host range proteins SPI-1 and SPI-2 (vSPT). Infection of human and murine tumor cell lines yielded nearly equivalent or a log lower virus recovery in comparison to parental viruses. Viral infection activated multiple caspases in cancer cells but not in normal cells, suggesting infected cells may die via different pathways. In tumor-bearing mice, vSPT recovery from MC38 tumor was slightly reduced in comparison to two parental viruses. However, no virus was recovered from the brains and livers of mice injected with vSPT in contrast to control viruses. vSPT demonstrated significantly lower pathogenicity in nude mice. Systemic delivery of vSPT showed significant tumor inhibition in subcutaneous MC38 tumor, human ovarian A2780 and murine ovarian MOSEC carcinomatosis models; however, the tumor inhibition by vSPT was reduced compared with parental viruses. These results demonstrated that although deletion of these three viral genes further enhanced tumor selectivity, it also weakened the oncolytic potency. This study illustrates the complexity of creating a tumor-selective oncolytic virus by deleting multiple viral genes involved in multiple cellular pathways.
[Show abstract][Hide abstract] ABSTRACT: Background: Recombinant vaccinia virus is currently being explored as a potential replicating oncolytic virus for cancer virotherapy due to its exceptional ability to replicate in tumor cells. Vaccinia tropism is not limited to tumor tissues, but also other tissues including ovarian follicles, possibly due to the presence of angiogenic factors such as vascular endothelial growth factor (VEGF). We therefore investigated the utility of vaccinia virus as an oncolytic therapy in a murine model of ovarian cancer.Methods: A murine ovarian surface epithelial cell (MOSEC) line was employed for this model. Intraperitoneal inoculation of the MOSEC cell line leads to a highly malignant neoplasm containing both carcinomatous and sarcomatous components as well as production of hemorrhagic ascitic fluid. These mice were treated with a thymidine kinase (TK) and vaccinia growth factor (VGF) double-deleted virus with the cytokine deaminase (CD) gene inserted into the TK locus. This recombinant virus (vvddCD) was found to be equivalent to the native WR strain in tumor tropism with minimal systemic growth.Results: Following intraperitoneal (i.p.) inoculation with 7.5x106 cells, mice were treated with 109 pfu of the vvddCD strain of vaccinia virus i.p. on the same day (concurrent treatment). In vivo studies appeared to show complete inhibition of tumor growth as compared to the control group (median mouse weight with ascites at 88 days: 40.7+/-1.49 (control [n=10]) vs 27.43+/-1.12 (virus [n=10])) (p
[Show abstract][Hide abstract] ABSTRACT: The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.
Cancer Research 11/2005; 65(21):9991-8. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously studied vaccinia as an oncolytic virus and demonstrated enhanced tumor selectivity by deleting viral genes encoding thymidine kinase and vaccinia growth factor. Vaccinia virus encodes at least three anti-apoptosis genes, spi-1, spi-2 and F1L. Spi-2 is a homoloque of cowpox virus CrmA gene, whose product inhibits ICE and other caspase activity. Spi-1 is thought to inhibit the mitochondrial pathway of apoptosis by binding cathespin G. F1L is a newly characterized gene encoding a protein inhibiting apoptosis thorough stablization of the mitochondrial membrane. Cancer cells are well known to possess the ability to evade apoptosis. Therefore, the viral anti-apoptosis genes may be redundant for the virus to replicate in cancer cells, but essential for virus to replicate in normal cells. We have constructed mutant viruses deleted for either F1L alone (vF1L), for spi-1 and spi-2 (vSP), or for all three viral genes (vSPF). Infection of several tumor cell lines including murine colon adenocarcinoma (MC38), human colon adenocarcinoma (HT29) and human epithelial ovarian cancer cell (A2780) with vF1L, vSP, vSPF and a wild type vF13, yielded nearly equivalent viral recovery. The mechanism of cell death in infected cancer and normal cells was investigated by annexin V and PI staining utilizing flow cytometry. In the human colon carcinoma cells infected with WT virus, 11.5% were annexin V positive, while 30.4% were annexin V positive in vSPF-infected cells. However, both vF13L+ and vSPF induced more cell death and more apoptosis in human normal fibroblasts. The virus vF13L+ induced 34.5% and vSPF induced 58% of annexin V postive cells. These results suggest that normal cells are more sensitive to virus-induced apoptosis than cancer cells, and the cells-infected with vSPF are more prone to die via apoptosis.The biodistribution of these viruses were studied in nude mice bearing subcutaneous MC38 tumor. These mice were injected i.p. with either a mutant virus or WT at 107 plaque-forming units (pfu). The results demonstrated that vSPF reduced dramatically viral yields in brain and liver, as well as other normal tissues, while retaining similar yields in tumor. The deletion of F1L alone (vF1L) displayed significant reduction of virus in the liver. The triple deletion virus vSPF also displayed reduced pathogenicity. Naive nude mice receiving 108 pfu of vSPF i.p. lived more than 100 days without illness, whereas those receiving WT virus had median survivals of 7 days and died of viral pathogenicity. The efficiency for tumor regression is currently under study.
[Show abstract][Hide abstract] ABSTRACT: The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency.
To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A.
In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (P(hCMV*-1)) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original P(hCMV*-1) in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses.
The Journal of Gene Medicine 08/2004; 6(7):817-28. · 2.16 Impact Factor