angel rivera - Academia.edu (original) (raw)

Papers by angel rivera

Research paper thumbnail of Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

Virology, 2004

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral... more The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdDE1AIL), the early gene E2B (AdDE2BIL), or the late fiber gene (AdDfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdDE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdDE1AIL) to six (AdDfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdDE2BIL, AdDfiberIL, and AdTL. AdDfiberIL and the parental virus, Ad5-D24, resulted in similar cytotoxicity, but AdDE2BIL and AdDE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdDE2BIL and AdDfiberIL, but substantial for AdDE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression. D 2004 Elsevier Inc. All rights reserved.

Research paper thumbnail of Retargeting of adenoviral infection to melanoma: Combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

International Journal of Cancer, 2004

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and ot... more Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA-mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild-type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma. © 2004 Wiley-Liss, Inc.

Research paper thumbnail of Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds

Journal of Molecular Medicine-jmm, 2007

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested... more Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-β1 and TGF-β2 precursor proteins, but an increase in expression of TGF-β3 precursor protein and TGF-β type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Research paper thumbnail of Genetic Replacement of the Adenovirus Shaft Fiber Reduces Liver Tropism in Ovarian Cancer Gene Therapy

Human Gene Therapy, 2004

Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their eff... more Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their efficacy and safety profile. Liver tropism is important with regard to potential clinical toxicity in humans. Ad5/3 chimeras in which the Ad5 knob is substituted by the Ad3 knob, such as Ad5/3luc1, have been recently shown to increase infectivity of ovarian cancer cell lines and primary tumor cells, which express low levels of the coxsackie-adenovirus receptor (CAR), without increasing infectivity of liver cells. A novel strategy to address the problem of liver uptake and improve the tumor/liver ratio is genetic replacement of the Ad fiber shaft. Ad5.Ad3.SH.luc1 is an Ad5-based vector that contains the fiber shaft from Ad serotype 3 but the fiber knob from Ad serotype 5. To compare tumor/liver of Ad5.Ad3.SH.luc1 and Ad5/3luc1 in vivo, we created three different tumor and treatment models of ovarian cancer in mice, simulating intraperitoneal and intravenous administration of tumors. Ad5.Ad3.SH.luc1 displayed the lowest liver tropism of all viruses in all models tested. Intravenous administration of all viruses resulted in higher tumor transduction rates compared to intraperitoneal administration. Genetic shortening of the Ad5 fiber shaft significantly increases relative tumor/liver gene transfer. This could improve the effective tumor dose and reduce side effects, thereby increasing the bioavailability of therapeutic agents.

Research paper thumbnail of The secretory leukoprotease inhibitor (SLPI) promoter for ovarian cancer gene therapy

Journal of Gene Medicine, 2003

Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their ... more Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their safety for the treatment of cancer has been established in clinical trials. However, one disadvantage is their promiscuous tropism. In this regard, tissue-specific promoters (TSPs) could be useful for directing transgene expression to target tissues and for reducing adverse effects in nontarget tissues. We hypothesize that selective adenovirus-mediated transgene expression could be achieved through the use of the secretory leukoprotease inhibitor (SLPI) promoter in the context of ovarian cancer.

Research paper thumbnail of A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy o... more Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver.

Research paper thumbnail of Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

Virology, 2004

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral... more The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdDE1AIL), the early gene E2B (AdDE2BIL), or the late fiber gene (AdDfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdDE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdDE1AIL) to six (AdDfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdDE2BIL, AdDfiberIL, and AdTL. AdDfiberIL and the parental virus, Ad5-D24, resulted in similar cytotoxicity, but AdDE2BIL and AdDE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdDE2BIL and AdDfiberIL, but substantial for AdDE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression. D 2004 Elsevier Inc. All rights reserved.

Research paper thumbnail of Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

Breast Cancer Research and Treatment, 2007

Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic ... more Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo. Experimental design HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival. Results Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone. Conclusion Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Research paper thumbnail of Retargeting of adenoviral infection to melanoma: Combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

International Journal of Cancer, 2004

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and ot... more Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA-mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild-type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma. © 2004 Wiley-Liss, Inc.

Research paper thumbnail of Transport across a polarized monolayer of Caco-2 cells by transferrin receptor-mediated adenovirus transcytosis

Virology, 2004

Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers ... more Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers such as the epithelium or endothelium. We report here the construction of an adenoviral vector that has the capability to be transported across polarized epithelial monolayers of Caco-2 cells (a colon carcinoma cell line) by transcytosis. This transcytosis is transferrin receptor (TfR)-mediated with use of a bifunctional adaptor, soluble coxsackie adenovirus receptor (sCAR)-Tf, and is both temperature and iron dependent. Under experimental conditions, the adenoviral transcytosis was inhibited by pretreatment of Caco-2 cells with colchicine, an inhibitor of transcytosis, and was not enhanced by pretreatment with Brefeldin A (BFA), an enhancer of transcytosis. In these Caco-2 cells, the transcytosis rate was 0.3 F 1.3% (SD). The transcytosed adenoviruses remain biologically functional. These data suggest the potential clinical benefit under conditions where drug delivery is a challenge, such as within the airway epithelium, at the bladder lumen urothelial cell interface, and across the blood -brain barrier for clinical treatment of lung, urogenital, and brain disorders, respectively, by adenoviral transcytosis of transgene delivery. D 2004 Elsevier Inc. All rights reserved.

Research paper thumbnail of Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds

Journal of Molecular Medicine-jmm, 2007

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested... more Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-β1 and TGF-β2 precursor proteins, but an increase in expression of TGF-β3 precursor protein and TGF-β type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Research paper thumbnail of Survivin-Driven and Fiber-Modified Oncolytic Adenovirus Exhibits Potent Antitumor Activity in Established Intracranial Glioma

Human Gene Therapy, 2007

The poor prognosis of patients with malignant gliomas necessitates the development of novel thera... more The poor prognosis of patients with malignant gliomas necessitates the development of novel therapies. Virotherapy, using genetically engineered adenovectors that selectively replicate in and kill neoplastic cells, represents one such strategy. In this study, we examined several oncolytic vectors with modified transcriptional and transductional control of viral replication. First, we incorporated the survivin promoter (S) to drive E1A gene expression. We then modified the adenovirus serotype 5 (Ad5) fiber protein via genetic knob switching or incorporation of peptide ligands to target the following glioma-associated receptors: the Ad3 attachment protein, or CD46, ␣ v ␤ 3 /␣ v ␤ 5 integrins, or heparan sulfate proteoglycans. The three conditionally replicative adenoviruses, CRAd-S-5/3, CRAd-S-RGD, and CRAd-S-pk7, were then examined in vitro with respect to transduction efficiency and tissue specificity. The most promising virus was then tested in vivo for evidence of tumor growth inhibition. CRAd-S-pk7 provided the highest level of viral replication and tumor oncolysis in glioma cell lines. At the same time, we observed minimal viral replication and toxicity in normal human brain. Injection of CRAd-S-pk7 inhibited xenograft tumor growth by more than 300% (p Ͻ 0.001). Sixty-seven percent of treated mice with intracranial tumors were long-term survivors (Ͼ110 days; p Ͻ 0.005). Analysis of tumor tissue indicated increased adenoviral infectivity, decreased mitotic activity, and enhanced tumor apoptosis. These findings demonstrate the effectiveness of CRAd-S-pk7 and provide the rationale for further development of this novel oncolytic virus for glioma gene therapy. 589

Research paper thumbnail of Genetic Replacement of the Adenovirus Shaft Fiber Reduces Liver Tropism in Ovarian Cancer Gene Therapy

Human Gene Therapy, 2004

Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their eff... more Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their efficacy and safety profile. Liver tropism is important with regard to potential clinical toxicity in humans. Ad5/3 chimeras in which the Ad5 knob is substituted by the Ad3 knob, such as Ad5/3luc1, have been recently shown to increase infectivity of ovarian cancer cell lines and primary tumor cells, which express low levels of the coxsackie-adenovirus receptor (CAR), without increasing infectivity of liver cells. A novel strategy to address the problem of liver uptake and improve the tumor/liver ratio is genetic replacement of the Ad fiber shaft. Ad5.Ad3.SH.luc1 is an Ad5-based vector that contains the fiber shaft from Ad serotype 3 but the fiber knob from Ad serotype 5. To compare tumor/liver of Ad5.Ad3.SH.luc1 and Ad5/3luc1 in vivo, we created three different tumor and treatment models of ovarian cancer in mice, simulating intraperitoneal and intravenous administration of tumors. Ad5.Ad3.SH.luc1 displayed the lowest liver tropism of all viruses in all models tested. Intravenous administration of all viruses resulted in higher tumor transduction rates compared to intraperitoneal administration. Genetic shortening of the Ad5 fiber shaft significantly increases relative tumor/liver gene transfer. This could improve the effective tumor dose and reduce side effects, thereby increasing the bioavailability of therapeutic agents.

Research paper thumbnail of Mesenchymal Stem Cells Effectively Deliver an Oncolytic Adenovirus to Intracranial Glioma

Stem Cells, 2008

Gene therapy represents a promising treatment alternative for patients with malignant gliomas. Ne... more Gene therapy represents a promising treatment alternative for patients with malignant gliomas. Nevertheless, in the setting of these highly infiltrative tumors, transgene delivery remains a challenge. Indeed, viral vehicles tested in clinical trials often target only those tumor cells that are adjacent to the injection site. In this study, we examined the feasibility of using human mesenchymal stem cells (hMSC) to deliver a replication-competent oncolytic adenovirus (CRAd) in a model of intracranial malignant glioma. To do so, CRAds with a chimeric 5/3 fiber or RGD backbone with or without CXCR4 promoter driving E1A were examined with respect to replication and toxicity in hMSC, human astrocytes, and the human glioma cell line U87MG by quantitative polymerase chain reaction and membrane integrity assay. CRAd delivery by virus-loaded hMSC was then evaluated in vitro and in an in vivo model of mice bearing intracranial U87MG xenografts. Our results show that hMSC are effectively infected by CRAds that use the CXCR4 promoter. CRAd-CXCR4-RGD had the highest replication, followed by CRAd-CXCR4–5/3, in hMSC, with comparable levels of toxicity. In U87MG tumor cells, CRAd-CXCR4–5/3 showed the highest replication and toxicity. Virus-loaded hMSC effectively migrated in vitro and released CRAds that infected U87MG glioma cells. When injected away from the tumor site in vivo, hMSC migrated to the tumor and delivered 46-fold more viral copies than injection of CRAd-CXCR4–5/3 alone. Taken together, these results indicate that hMSC migrate and deliver CRAd to distant glioma cells. This delivery strategy should be explored further, as it could improve the outcome of oncolytic virotherapy for glioma.Disclosure of potential conflicts of interest is found at the end of this article.

Research paper thumbnail of The secretory leukoprotease inhibitor (SLPI) promoter for ovarian cancer gene therapy

Journal of Gene Medicine, 2003

Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their ... more Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their safety for the treatment of cancer has been established in clinical trials. However, one disadvantage is their promiscuous tropism. In this regard, tissue-specific promoters (TSPs) could be useful for directing transgene expression to target tissues and for reducing adverse effects in nontarget tissues. We hypothesize that selective adenovirus-mediated transgene expression could be achieved through the use of the secretory leukoprotease inhibitor (SLPI) promoter in the context of ovarian cancer.

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

Cancer Gene Therapy, 2004

It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protei... more 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.

Research paper thumbnail of A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy o... more Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver.

Research paper thumbnail of The human survivin promoter: a novel transcriptional targeting strategy for treatment of glioma

Journal of Neurosurgery, 2006

ALIGNANT gliomas are the most common tumor in the central nervous system. Despite recent advances... more ALIGNANT gliomas are the most common tumor in the central nervous system. Despite recent advances in treatment, their prognosis remains poor. The 5-year survival rate in patients harboring a GBM, the most common and malignant glioma subtype, is less than 3%, whereas the 5-year survival rate for patients with a lower grade astrocytoma is 30%. 3,6,7,38 Therefore, the identification of novel therapeutic strategies for malignant gliomas merits a high priority. In this regard, gene therapy with adenoviral vectors is a promising new modality for the treatment of glioma. 9,10,23,26 Adenoviral vectors may be genetically designed to express therapeutic genes in cancer cells. To this end, tumor-selective transgene expression is critical and can be obtained by placing the transgene under the control of a TSP. Specifically, to achieve a high therapeutic index with adenoviral vectors in vivo, repression of TSPs in normal tissues, primarily those of the liver, emerges as the most critical predictor of a high tumor/normal tissue ratio. 12 Current TSP candidates for glioma target-

Research paper thumbnail of Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

Breast Cancer Research and Treatment, 2007

Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic ... more Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo. Experimental design HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival. Results Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone. Conclusion Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Research paper thumbnail of Transport across a polarized monolayer of Caco-2 cells by transferrin receptor-mediated adenovirus transcytosis

Virology, 2004

Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers ... more Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers such as the epithelium or endothelium. We report here the construction of an adenoviral vector that has the capability to be transported across polarized epithelial monolayers of Caco-2 cells (a colon carcinoma cell line) by transcytosis. This transcytosis is transferrin receptor (TfR)-mediated with use of a bifunctional adaptor, soluble coxsackie adenovirus receptor (sCAR)-Tf, and is both temperature and iron dependent. Under experimental conditions, the adenoviral transcytosis was inhibited by pretreatment of Caco-2 cells with colchicine, an inhibitor of transcytosis, and was not enhanced by pretreatment with Brefeldin A (BFA), an enhancer of transcytosis. In these Caco-2 cells, the transcytosis rate was 0.3 F 1.3% (SD). The transcytosed adenoviruses remain biologically functional. These data suggest the potential clinical benefit under conditions where drug delivery is a challenge, such as within the airway epithelium, at the bladder lumen urothelial cell interface, and across the blood -brain barrier for clinical treatment of lung, urogenital, and brain disorders, respectively, by adenoviral transcytosis of transgene delivery. D 2004 Elsevier Inc. All rights reserved.

Research paper thumbnail of Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

Virology, 2004

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral... more The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdDE1AIL), the early gene E2B (AdDE2BIL), or the late fiber gene (AdDfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdDE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdDE1AIL) to six (AdDfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdDE2BIL, AdDfiberIL, and AdTL. AdDfiberIL and the parental virus, Ad5-D24, resulted in similar cytotoxicity, but AdDE2BIL and AdDE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdDE2BIL and AdDfiberIL, but substantial for AdDE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression. D 2004 Elsevier Inc. All rights reserved.

Research paper thumbnail of Retargeting of adenoviral infection to melanoma: Combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

International Journal of Cancer, 2004

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and ot... more Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA-mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild-type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma. © 2004 Wiley-Liss, Inc.

Research paper thumbnail of Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds

Journal of Molecular Medicine-jmm, 2007

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested... more Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-β1 and TGF-β2 precursor proteins, but an increase in expression of TGF-β3 precursor protein and TGF-β type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Research paper thumbnail of Genetic Replacement of the Adenovirus Shaft Fiber Reduces Liver Tropism in Ovarian Cancer Gene Therapy

Human Gene Therapy, 2004

Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their eff... more Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their efficacy and safety profile. Liver tropism is important with regard to potential clinical toxicity in humans. Ad5/3 chimeras in which the Ad5 knob is substituted by the Ad3 knob, such as Ad5/3luc1, have been recently shown to increase infectivity of ovarian cancer cell lines and primary tumor cells, which express low levels of the coxsackie-adenovirus receptor (CAR), without increasing infectivity of liver cells. A novel strategy to address the problem of liver uptake and improve the tumor/liver ratio is genetic replacement of the Ad fiber shaft. Ad5.Ad3.SH.luc1 is an Ad5-based vector that contains the fiber shaft from Ad serotype 3 but the fiber knob from Ad serotype 5. To compare tumor/liver of Ad5.Ad3.SH.luc1 and Ad5/3luc1 in vivo, we created three different tumor and treatment models of ovarian cancer in mice, simulating intraperitoneal and intravenous administration of tumors. Ad5.Ad3.SH.luc1 displayed the lowest liver tropism of all viruses in all models tested. Intravenous administration of all viruses resulted in higher tumor transduction rates compared to intraperitoneal administration. Genetic shortening of the Ad5 fiber shaft significantly increases relative tumor/liver gene transfer. This could improve the effective tumor dose and reduce side effects, thereby increasing the bioavailability of therapeutic agents.

Research paper thumbnail of The secretory leukoprotease inhibitor (SLPI) promoter for ovarian cancer gene therapy

Journal of Gene Medicine, 2003

Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their ... more Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their safety for the treatment of cancer has been established in clinical trials. However, one disadvantage is their promiscuous tropism. In this regard, tissue-specific promoters (TSPs) could be useful for directing transgene expression to target tissues and for reducing adverse effects in nontarget tissues. We hypothesize that selective adenovirus-mediated transgene expression could be achieved through the use of the secretory leukoprotease inhibitor (SLPI) promoter in the context of ovarian cancer.

Research paper thumbnail of A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy o... more Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver.

Research paper thumbnail of Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

Virology, 2004

The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral... more The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdDE1AIL), the early gene E2B (AdDE2BIL), or the late fiber gene (AdDfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdDE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdDE1AIL) to six (AdDfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdDE2BIL, AdDfiberIL, and AdTL. AdDfiberIL and the parental virus, Ad5-D24, resulted in similar cytotoxicity, but AdDE2BIL and AdDE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdDE2BIL and AdDfiberIL, but substantial for AdDE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression. D 2004 Elsevier Inc. All rights reserved.

Research paper thumbnail of Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

Breast Cancer Research and Treatment, 2007

Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic ... more Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo. Experimental design HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival. Results Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone. Conclusion Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Research paper thumbnail of Retargeting of adenoviral infection to melanoma: Combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

International Journal of Cancer, 2004

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and ot... more Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA-mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild-type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma. © 2004 Wiley-Liss, Inc.

Research paper thumbnail of Transport across a polarized monolayer of Caco-2 cells by transferrin receptor-mediated adenovirus transcytosis

Virology, 2004

Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers ... more Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers such as the epithelium or endothelium. We report here the construction of an adenoviral vector that has the capability to be transported across polarized epithelial monolayers of Caco-2 cells (a colon carcinoma cell line) by transcytosis. This transcytosis is transferrin receptor (TfR)-mediated with use of a bifunctional adaptor, soluble coxsackie adenovirus receptor (sCAR)-Tf, and is both temperature and iron dependent. Under experimental conditions, the adenoviral transcytosis was inhibited by pretreatment of Caco-2 cells with colchicine, an inhibitor of transcytosis, and was not enhanced by pretreatment with Brefeldin A (BFA), an enhancer of transcytosis. In these Caco-2 cells, the transcytosis rate was 0.3 F 1.3% (SD). The transcytosed adenoviruses remain biologically functional. These data suggest the potential clinical benefit under conditions where drug delivery is a challenge, such as within the airway epithelium, at the bladder lumen urothelial cell interface, and across the blood -brain barrier for clinical treatment of lung, urogenital, and brain disorders, respectively, by adenoviral transcytosis of transgene delivery. D 2004 Elsevier Inc. All rights reserved.

Research paper thumbnail of Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds

Journal of Molecular Medicine-jmm, 2007

Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested... more Fibromodulin, a member of the small leucine-rich proteoglycan family, has been recently suggested as a biologically significant mediator of fetal scarless repair. To assess the role of fibromodulin in the tissue remodeling, we constructed an adenoviral vector expressing human fibromodulin cDNA. We evaluated the effect of adenovirus-mediated overexpression of fibromodulin in vitro on transforming growth factors and metalloproteinases in fibroblasts and in vivo on full-thickness incisional wounds in a rabbit model. In vitro, we found that Ad-Fibromodulin induced a decrease of expression of TGF-β1 and TGF-β2 precursor proteins, but an increase in expression of TGF-β3 precursor protein and TGF-β type II receptor. In addition, fibromodulin overexpression resulted in decreased MMP-1 and MMP-3 protein secretion but increased MMP-2, TIMP-1, and TIMP-2 secretion, whereas MMP-9 and MMP-13 were not influenced by fibromodulin overexpression. In vivo evaluation by histopathology and tensile strength demonstrated that Ad-Fibromodulin administration could ameliorate wound healing in incisional wounds. In conclusion, although the mechanism of scar formation in adult wounds remains incompletely understood, we found that fibromodulin overexpression improves wound healing in vivo, suggesting that fibromodulin may be a key mediator in reduced scarring.

Research paper thumbnail of Survivin-Driven and Fiber-Modified Oncolytic Adenovirus Exhibits Potent Antitumor Activity in Established Intracranial Glioma

Human Gene Therapy, 2007

The poor prognosis of patients with malignant gliomas necessitates the development of novel thera... more The poor prognosis of patients with malignant gliomas necessitates the development of novel therapies. Virotherapy, using genetically engineered adenovectors that selectively replicate in and kill neoplastic cells, represents one such strategy. In this study, we examined several oncolytic vectors with modified transcriptional and transductional control of viral replication. First, we incorporated the survivin promoter (S) to drive E1A gene expression. We then modified the adenovirus serotype 5 (Ad5) fiber protein via genetic knob switching or incorporation of peptide ligands to target the following glioma-associated receptors: the Ad3 attachment protein, or CD46, ␣ v ␤ 3 /␣ v ␤ 5 integrins, or heparan sulfate proteoglycans. The three conditionally replicative adenoviruses, CRAd-S-5/3, CRAd-S-RGD, and CRAd-S-pk7, were then examined in vitro with respect to transduction efficiency and tissue specificity. The most promising virus was then tested in vivo for evidence of tumor growth inhibition. CRAd-S-pk7 provided the highest level of viral replication and tumor oncolysis in glioma cell lines. At the same time, we observed minimal viral replication and toxicity in normal human brain. Injection of CRAd-S-pk7 inhibited xenograft tumor growth by more than 300% (p Ͻ 0.001). Sixty-seven percent of treated mice with intracranial tumors were long-term survivors (Ͼ110 days; p Ͻ 0.005). Analysis of tumor tissue indicated increased adenoviral infectivity, decreased mitotic activity, and enhanced tumor apoptosis. These findings demonstrate the effectiveness of CRAd-S-pk7 and provide the rationale for further development of this novel oncolytic virus for glioma gene therapy. 589

Research paper thumbnail of Genetic Replacement of the Adenovirus Shaft Fiber Reduces Liver Tropism in Ovarian Cancer Gene Therapy

Human Gene Therapy, 2004

Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their eff... more Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their efficacy and safety profile. Liver tropism is important with regard to potential clinical toxicity in humans. Ad5/3 chimeras in which the Ad5 knob is substituted by the Ad3 knob, such as Ad5/3luc1, have been recently shown to increase infectivity of ovarian cancer cell lines and primary tumor cells, which express low levels of the coxsackie-adenovirus receptor (CAR), without increasing infectivity of liver cells. A novel strategy to address the problem of liver uptake and improve the tumor/liver ratio is genetic replacement of the Ad fiber shaft. Ad5.Ad3.SH.luc1 is an Ad5-based vector that contains the fiber shaft from Ad serotype 3 but the fiber knob from Ad serotype 5. To compare tumor/liver of Ad5.Ad3.SH.luc1 and Ad5/3luc1 in vivo, we created three different tumor and treatment models of ovarian cancer in mice, simulating intraperitoneal and intravenous administration of tumors. Ad5.Ad3.SH.luc1 displayed the lowest liver tropism of all viruses in all models tested. Intravenous administration of all viruses resulted in higher tumor transduction rates compared to intraperitoneal administration. Genetic shortening of the Ad5 fiber shaft significantly increases relative tumor/liver gene transfer. This could improve the effective tumor dose and reduce side effects, thereby increasing the bioavailability of therapeutic agents.

Research paper thumbnail of Mesenchymal Stem Cells Effectively Deliver an Oncolytic Adenovirus to Intracranial Glioma

Stem Cells, 2008

Gene therapy represents a promising treatment alternative for patients with malignant gliomas. Ne... more Gene therapy represents a promising treatment alternative for patients with malignant gliomas. Nevertheless, in the setting of these highly infiltrative tumors, transgene delivery remains a challenge. Indeed, viral vehicles tested in clinical trials often target only those tumor cells that are adjacent to the injection site. In this study, we examined the feasibility of using human mesenchymal stem cells (hMSC) to deliver a replication-competent oncolytic adenovirus (CRAd) in a model of intracranial malignant glioma. To do so, CRAds with a chimeric 5/3 fiber or RGD backbone with or without CXCR4 promoter driving E1A were examined with respect to replication and toxicity in hMSC, human astrocytes, and the human glioma cell line U87MG by quantitative polymerase chain reaction and membrane integrity assay. CRAd delivery by virus-loaded hMSC was then evaluated in vitro and in an in vivo model of mice bearing intracranial U87MG xenografts. Our results show that hMSC are effectively infected by CRAds that use the CXCR4 promoter. CRAd-CXCR4-RGD had the highest replication, followed by CRAd-CXCR4–5/3, in hMSC, with comparable levels of toxicity. In U87MG tumor cells, CRAd-CXCR4–5/3 showed the highest replication and toxicity. Virus-loaded hMSC effectively migrated in vitro and released CRAds that infected U87MG glioma cells. When injected away from the tumor site in vivo, hMSC migrated to the tumor and delivered 46-fold more viral copies than injection of CRAd-CXCR4–5/3 alone. Taken together, these results indicate that hMSC migrate and deliver CRAd to distant glioma cells. This delivery strategy should be explored further, as it could improve the outcome of oncolytic virotherapy for glioma.Disclosure of potential conflicts of interest is found at the end of this article.

Research paper thumbnail of The secretory leukoprotease inhibitor (SLPI) promoter for ovarian cancer gene therapy

Journal of Gene Medicine, 2003

Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their ... more Background Adenoviruses allow efficient transduction of dividing and nondividing cells and their safety for the treatment of cancer has been established in clinical trials. However, one disadvantage is their promiscuous tropism. In this regard, tissue-specific promoters (TSPs) could be useful for directing transgene expression to target tissues and for reducing adverse effects in nontarget tissues. We hypothesize that selective adenovirus-mediated transgene expression could be achieved through the use of the secretory leukoprotease inhibitor (SLPI) promoter in the context of ovarian cancer.

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

Cancer Gene Therapy, 2004

It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protei... more 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.

Research paper thumbnail of A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy o... more Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver.

Research paper thumbnail of The human survivin promoter: a novel transcriptional targeting strategy for treatment of glioma

Journal of Neurosurgery, 2006

ALIGNANT gliomas are the most common tumor in the central nervous system. Despite recent advances... more ALIGNANT gliomas are the most common tumor in the central nervous system. Despite recent advances in treatment, their prognosis remains poor. The 5-year survival rate in patients harboring a GBM, the most common and malignant glioma subtype, is less than 3%, whereas the 5-year survival rate for patients with a lower grade astrocytoma is 30%. 3,6,7,38 Therefore, the identification of novel therapeutic strategies for malignant gliomas merits a high priority. In this regard, gene therapy with adenoviral vectors is a promising new modality for the treatment of glioma. 9,10,23,26 Adenoviral vectors may be genetically designed to express therapeutic genes in cancer cells. To this end, tumor-selective transgene expression is critical and can be obtained by placing the transgene under the control of a TSP. Specifically, to achieve a high therapeutic index with adenoviral vectors in vivo, repression of TSPs in normal tissues, primarily those of the liver, emerges as the most critical predictor of a high tumor/normal tissue ratio. 12 Current TSP candidates for glioma target-

Research paper thumbnail of Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma

Breast Cancer Research and Treatment, 2007

Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic ... more Purpose Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo. Experimental design HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival. Results Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone. Conclusion Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.

Research paper thumbnail of Transport across a polarized monolayer of Caco-2 cells by transferrin receptor-mediated adenovirus transcytosis

Virology, 2004

Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers ... more Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers such as the epithelium or endothelium. We report here the construction of an adenoviral vector that has the capability to be transported across polarized epithelial monolayers of Caco-2 cells (a colon carcinoma cell line) by transcytosis. This transcytosis is transferrin receptor (TfR)-mediated with use of a bifunctional adaptor, soluble coxsackie adenovirus receptor (sCAR)-Tf, and is both temperature and iron dependent. Under experimental conditions, the adenoviral transcytosis was inhibited by pretreatment of Caco-2 cells with colchicine, an inhibitor of transcytosis, and was not enhanced by pretreatment with Brefeldin A (BFA), an enhancer of transcytosis. In these Caco-2 cells, the transcytosis rate was 0.3 F 1.3% (SD). The transcytosed adenoviruses remain biologically functional. These data suggest the potential clinical benefit under conditions where drug delivery is a challenge, such as within the airway epithelium, at the bladder lumen urothelial cell interface, and across the blood -brain barrier for clinical treatment of lung, urogenital, and brain disorders, respectively, by adenoviral transcytosis of transgene delivery. D 2004 Elsevier Inc. All rights reserved.