New Oncolytic Adenoviruses with Hypoxia- and Estrogen Receptor-Regulated Replication

Oncolytic adenoviruses with restricted replication can be produced if the expression of crucial transcription units of the virus is controlled by tissue- or tumor-specific promoters. Here we describe a method for the rapid incorporation of exogenous promoters into the E1A and E4 regions of the human adenovirus type 5 genome. Using this system, we have generated AdEHT2 and AdEHE2F, two conditionally replicative adenoviruses for the treatment of breast cancer. The expression of the E1A gene in both viruses is controlled by a minimal dual-specificity promoter that responds to estrogens and hypoxia. The tight regulation of E1A expression correlated with the ability of these viruses to replicate and kill human cancer cells that express estrogen receptors, or are maintained under hypoxic conditions. The telomerase reverse transcriptase (TERT) promoter and the E2F-1 promoter are preferentially activated in cancer cells. They were introduced into the E4 region of AdEHT2 and AdEHE2F, respectively. The telomerase core promoter failed to block the replication of the virus in telomerase-negative cells. In contrast, AdEHE2F was attenuated in nontransformed quiescent cells growing under normoxic conditions, suggesting that an intact pRB pathway with low levels of E2F transcription factors acts as a negative modulator for the virus. These data indicate that the simultaneous regulation of E1A and E4 viral transcription units by the appropriate combination of promoters can increase the tumor selectivity of oncolytic adenoviruses.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63195/1/104303402760293574.pd

Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy

<p>Abstract</p> <p>Background</p> <p>Bioluminescent imaging (BLI) is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies.</p> <p>Results</p> <p>A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1) retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US), BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12). Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival.</p> <p>Conclusion</p> <p>We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.</p

A Novel, Conditionally Replicative Adenovirus for the Treatment of Breast Cancer That Allows Controlled Replication of E1a-Deleted Adenoviral Vectors

The efficiency of gene therapy strategies against cancer is limited by the poor distribution of the vectors in the malignant tissues. To solve this problem, a new generation of tumor-specific, conditionally replicative adenoviruses is being developed. To direct the replication of the virus to breast cancer, we have considered one characteristic present in a great proportion of these cancers, which is the expression of estrogen receptors (ERs). On the basis of the wild-type adenovirus type 5, we have constructed a conditionally replicative adenovirus (Ad5ERE2) in which the E1a and E4 promoters have been replaced by a portion of the pS2 promoter containing two estrogen-responsive elements (EREs). This promoter induces transcriptional activation of the E1a and E4 units in response to estrogens in cells that express the ERs. Ad5ERE2 is able to kill ER+ human breast cancer cell lines as efficiently as the wild-type virus, but has decreased capacity to affect ER- cells. By complementation of the E1a protein in trans, Ad5ERE2 allows restricted replication of a conventional E1adeleted adenoviral vector. When a virus expressing the proapoptotic gene Bc1-xs (Clarke et al., Proc. Natl. Acad. Sci. U.S.A. 1995;92:11024-11028) is used in combination with Ad5ERE2, the ability of both viruses to induce cell death is dramatically increased, and the effect can be modulated by addition of the antiestrogen tamoxifen.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63245/1/10430340050143435.pd

Intrapulmonary Expression of Macrophage Inflammatory Protein 1α (CCL3) Induces Neutrophil and NK Cell Accumulation and Stimulates Innate Immunity in Murine Bacterial Pneumonia

Macrophage inflammatory protein 1α (MIP-1α) (CCL3) is an important mediator of leukocyte recruitment and activation in a variety of inflammatory states, including infection. A recombinant human type 5 adenovirus containing the murine MIP-1α cDNA (AdMIP-1α) was constructed to determine the effect of transient intrapulmonary expression of MIP-1α on leukocyte recruitment, activation, and bacterial clearance in a murine model of Klebsiella pneumoniae pneumonia. The intratracheal administration of AdMIP-1α resulted in both time- and dose-dependent expression of MIP-1α mRNA and protein within the lung. Importantly, the intrapulmonary overexpression of MIP-1α resulted in a maximal 35- and 100-fold reduction in lung and blood bacterial burden, respectively, in animals cochallenged with K. pneumoniae, which was associated with a significant increase in neutrophil and activated NK cell accumulation. Furthermore, the transgenic expression of MIP-1α during bacterial pneumonia resulted in enhanced expression of gamma interferon mRNA, compared to that observed in Klebsiella-challenged animals pretreated with control vector. These findings indicate an important role for MIP-1α in the recruitment and activation of selected leukocyte populations in vivo and identify this cytokine as a potential immunoadjuvant to be employed in the setting of localized bacterial infection

A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling.

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals

<i>In vivo</i> activity of AAV8-3xIRF-ISRE-Luc in hamster in response to intravenous NDV-F3AA-GFP LaSota administration.

<p>Syrian hamsters were inoculated iv with 1x10¹¹ vg of the AAV8-3xIRF-ISRE-Luc vector. Three weeks later, animals received an iv administration of 1x10⁹ iu NDV-F3AA-GFP LaSota. A) <i>In vivo</i> luciferase activity was monitored at 10, 24, 48 and 72 hours after iv administration of the NDV virus. Each line represents an individual hamster. B) Image of a representative hamster before (basal) and 10 hours after the first NDV administration. C) Luciferase activity stimulation in hamsters receiving two doses of NDV-F3AA-GFP LaSota 6 weeks apart one from the other. D) Type I IFN activity in serum of hamsters before and 24 hours after second NDV administration, measured by bio-assay. E) NDV neutralizing antibodies in serum of hamsters before and 6 weeks after the first NDV administration. These data are from one experiment representative of two.</p

<i>In vitro</i> characterization of IFN-I reporters.

<p>A) Schematic representation of reporter plasmid constructs. B) Dual luciferase reporter assay in HuH-7 cells transfected with the indicated plasmids in response to 500 units/ml of human IFN-α for 24 hours. C) Luciferase activity (fold induction) in HuH-7 cells transfected with the indicated reporter plasmids and treated with 500 units/ml of human IFN-α for 24 hours. D) Dual luciferase reporter assay in Hepa 1.6 cells transfected with the indicated plasmids in response to Sendai Cantell virus (20 hemagglutination units, 24 hours). E) Fold induction of luciferase activity in cells transfected with the 3xIRF-ISRE reporter plasmids in the indicated cell lines treated during 24 hours with 500 units/ml of the corresponding species-specific IFN-α. F) Luciferase activity fold induction in murine cell lines transfected with the 3xIRF-ISRE reporter plasmid and treated with 500 units/ml murine IFN-α, 500 units/ml murine IFN-β, 50 μg/ml poly I:C or 1 μg/ml poly I:C mixed with 250 μg/ml DEAE-Dextran for 24 hours. G) Time course of luciferase activity in HuH-7 cells transfected with the 3xIRF-ISRE-luc reporter plasmids and treated with 500 units/ml of human IFN-α. These data are from one experiment representative of four. *** p<0.001, ns: not significant. TK: Thymidine kinase.</p

<i>In vivo</i> characterization of 3xIRF-ISRE-Luc reporter plasmid delivered to mouse liver by hydrodynamic injection.

<p>A) Mice received a hydrodynamic injection with 20 μg of 3xIRF-ISRE-Luc reporter plasmid through the tail vein. Once luciferase activity stabilized (one month after injection), mice were treated intraperitoneally with the indicated doses of murine IFN-β. Light emission was quantified by BLI 10 and 24 hours after treatment. Values correspond to fold luciferase activity, using baseline (pre-induction) activity as a reference. B) Quantitative RT-PCR of <i>OAS</i> and <i>Mx1</i> genes in peripheral blood lymphocytes of animals treated for 24 hours with different doses of recombinant murine IFN-β. C) Reporter activity re-induction in mice determined every week by intraperitoneally administration of 10,000 units of IFN-β. Each line represents an individual mouse. D) Representative BLI images of mice before and 10 hours after administration of 10,000 U of murine IFN-β. These data are from one experiment representative of three.** p<0.01 <i>vs</i> 3,000, 1,000 and 0 IFN-β units.</p